CN101893630A - A method for detecting the expression level of annexin A3 - Google Patents

Abstract

The invention generally relates to the field of medicament resistance of chemotherapeutic medicaments, in particular to a method for detecting the expression level of annexin A3 in the supernate of the culture medium of platinum-based chemotherapeutic medicament resisting tumor cells, a method for adjusting the excretion amount of tumor cell annexin A3, a method for evaluating the medicament resistance of patients with tumor to the platinum-based chemotherapeutic medicament, a method for adjusting the platinum releasing of the platinum-based chemotherapeutic medicament resisting tumor cells, a method for combining deoxyribonucleic acid (DNA) and the platinum in the platinum-based chemotherapeutic medicament resisting tumor cells and a method for adjusting the quantity of vesicles in the platinum-based chemotherapeutic medicament resisting tumor cells.

Description

A method for detecting the expression level of annexin A3

technical field

The present invention generally relates to the field of chemotherapeutic drug resistance, and specifically relates to the following aspects: a method for detecting the expression level of annexin A3 (Annexin A3) in the culture medium supernatant of platinum-based chemotherapeutic drug resistant tumor cells, a A method for regulating the amount of tumor cell annexin A3 exocytosis, a method for assessing the resistance of tumor patients to platinum-based chemotherapy drugs, a method for regulating the release of platinum from platinum-based chemotherapy drug-resistant tumor cells, a method for regulating platinum-based chemotherapy Methods for DNA binding to platinum in drug-resistant tumor cells, and a method for modulating the number of vesicles in platinum-based chemotherapeutic drug-resistant tumor cells.

Background technique

Ovarian cancer is the first fatal disease of gynecological tumors. The 5-year survival rate of advanced patients has always hovered at 15%-20%. The resistance of tumor cells to platinum-based chemotherapy drugs is one of the main reasons for this situation. Platinum chemotherapeutic drugs include cisplatin, carboplatin, oxalate platinum, nedaplatin, raplatin, etc., which are the most commonly used first-line chemotherapy drugs for the treatment of ovarian cancer. They are also used for the treatment of other solid tumors including breast cancer, lung cancer, testicular tumors, head and neck cancer Commonly used drugs for cancer, osteosarcoma, melanoma, esophageal cancer, etc.

Platinum chemotherapeutic drugs combine with DNA to form cross bonds, thereby destroying the function of DNA and making it impossible to replicate, and can also inhibit the synthesis of RNA and protein at high concentrations. However, when platinum-based chemotherapy drugs are used to treat the above-mentioned solid tumors, drug resistance occurs, that is, the tumor is well controlled in the early stage of treatment, but relapse occurs in the later stage of treatment or there is no response in the early stage of treatment.

The resistance of ovarian cancer to platinum-based chemotherapy drugs has been clearly recognized and defined clinically. At the same time, research on the mechanism of cisplatin resistance has also achieved certain results, such as MDR-1, LRP-1, MRP-1, GST-pi, etc. have become well-known resistance-related genes.

However, many research results show that the expression of multiple drug resistance-related genes in ovarian cancer cannot predict the drug resistance and prognosis of tumors well, and the inconsistency between the abundance of mRNA and its corresponding protein level may be one of the main reasons. DNA is the carrier of genetic information, and protein is the executor of functions. The present inventors compared the expression of proteins in platinum-based chemotherapy-sensitive cells and corresponding platinum-based chemotherapy-resistant cells through comparative proteomics technology, and found five platinum-based chemotherapy-resistance-related proteins, which are Annexin A3 (Annexin A3), IDHc, cofilin 1, GSTO1-1, destrin, and through RT-PCR and Western blot techniques, platinum-based chemotherapy-resistant and sensitive cells were verified at the RNA and protein levels (Yan XD , Pan LY, et al. J Proteome Res. 2007, 6(2): 772-780). According to the degree of difference between the five proteins, Annexin A3 with the most obvious difference was selected for the relevant platinum-resistant mechanism exploration experiment, and the tumor cells were preferably ovarian cancer cells.

Annexin A3 is a member of the annexin superfamily, which has the functions of inhibiting phospholipase A2 and anticoagulation. It can also cut off inositol 1, 2-cyclic phosphate to form inositol 1-phosphate, which plays a role in controlling cell proliferation (Ross TS, et al. J Biol Chem. 1991, 266(14): 9086-9092.). Annexin A3 can bind to phosphatidylserine in vesicles at a certain Ca ²⁺ concentration, and mediate membrane-to-membrane interactions such as fusion of phagosomes and lysosomes and granule disappearance. As calnexin, annexin A3 has a series of extracellular functions, including membrane transport, lymphocyte migration, cell motility, calcium efflux and signal transduction. One of the important functions of annexin A3 is one of the important functions of annexin A3, which is one of the important functions of annexin A3 to produce calcium-dependent swollen vesicles bound to negatively charged membrane phospholipids by high-abundance annexin A3 (Gerke V, Moss SE. Physiol. Rev. 2002, 82: 331-371).

A series of experiments have shown that annexin A3 is one of the mechanisms of chemoresistance to platinum-based chemotherapy drugs in ovarian cancer. The present inventor reduces the expression of annexin A3 in ovarian cancer cells by experimental means, and the sensitivity of ovarian cancer to platinum-based chemotherapy drugs increases; Reduced drug sensitivity. This experimental result has been effectively verified in human ovarian cancer tissue and animal experiments (YanXD, Pan LY, et al.J Proteome Res.2007, 6(2): 772-780; XuedongYan, Jie Yin, Huiyu Yao , Ning Mao, Yili Yang, Lingya Pan. CancerRes, 2010, 70(4): OF1-9.).

In addition, high levels of annexin A3 were found concentrated in membrane-bound regions in about two-thirds of colon cancer patients' cancer tissues (Madoz-Gurpide J, Lopez-Serra P, et al. Mol Cell Proteomics. 2006, 5: 1471-1483). Recently, in the urine of prostate cancer patients at different stages, the varying levels of annexin A3 can be detected by Western blot method, and compared with the specific marker prostate specific antigen currently used clinically, it is found that annexin A3 Protein A3 suggests that prostate cancer is more prevalent at an early stage.

In summary, Annexin A3 has the hope of becoming a human tumor marker. Based on the correlation between annexin A3 and platinum-based chemotherapeutic drug resistance, the present invention intends to clinically evaluate the sensitivity of tumor platinum-based chemotherapeutic drugs through the level of annexin A3 in human body fluid, which is still a new standard in the field of tumor chemotherapeutic drug resistance. first.

In view of the current lack of clinical protein markers that can predict tumor platinum chemotherapy drugs in advance, in order to give full play to the advantages of translational medicine, on the basis of discovering annexin A3, a protein associated with platinum chemotherapy drug resistance, the inventors further passed The experiment affirmed its status as a specific marker of platinum-based chemotherapy drug resistance, and explored its molecular mechanism leading to platinum-based chemotherapy drug resistance. At the same time, on the basis of the discovery that tumor cells (such as ovarian cancer cells) secrete annexin A3, further explore its exocrine pathway, and build an enzyme-linked immunosorbent assay protocol for specific detection of human annexin A3 protein, successfully The presence of annexin A3 was detected in the serum of patients with ovarian cancer, and on this basis, the levels of annexin A3 in the serum of patients with ovarian cancer platinum-based chemotherapy drug resistance and the serum levels of annexin A3 in patients with ovarian cancer platinum-based chemotherapy drug-sensitive were evaluated. Differences in levels of Annexin A3. A large number of clinical samples have proved that high levels of annexin A3 in serum can accurately predict the occurrence of resistance to platinum-based chemotherapy drugs. Early detection of resistance to platinum-based chemotherapy drugs can change the tumor treatment plan in time, increase the 5-year survival rate, and improve the prognosis.

Contents of the invention

One aspect of the present invention is to provide a method for detecting the expression level of annexin A3. The method uses the culture supernatant of platinum-based chemotherapeutic drug-resistant tumor cells as the sample to be detected.

Wherein, preferably, the platinum chemotherapy drug is cisplatin; preferably, the tumor cells are ovarian cancer cells; preferably, the medium is a serum-free medium, and more preferably DMEM; preferably, the Said detection is preferably immunological detection, and more preferably Western blot or ELISA detection.

In one embodiment of the present invention, when using Western blot to detect, described method comprises the steps:

1) Collecting the culture supernatant of platinum-based chemotherapeutic drug-resistant tumor cells and the culture supernatant of platinum-based chemotherapeutic drug-sensitive tumor cells of the same type, and concentrating them respectively. The concentration ratios are both 75-100 times to obtain two concentrated liquids;

2) detecting the expression level of annenxin A3 in the two concentrated liquids;

3) Comparing the expression levels of annenxin A3 in the two concentrated liquids, and semi-quantifying the expression levels of annexin A3 in the culture supernatant of platinum-based chemotherapeutic drug-resistant tumor cells.

Another aspect of the present invention is to provide a method for regulating the amount of tumor cell annexin A3 exocytosis, the method includes the step of changing the expression level of annenxin A3 in tumor cells, wherein, preferably, the platinum chemotherapy drug is cis Platinum; preferably, the tumor cells are ovarian cancer cells.

In one embodiment of the present invention, the tolerance of tumor cells to platinum-based chemotherapy drugs is increased by increasing the expression of annexin A3 in tumor cells, preferably, by expressing annexin A3 gene in tumor cells. Annexin A3 expression in cells, more preferably, the level of Annexin A3 in said cells is increased by using a plasmid expressing a positive-sense Annexin A3 gene.

In one embodiment of the invention, the resistance of tumor cells to platinum-based chemotherapy drugs is reduced by reducing and/or inhibiting the expression of annexin A3 in tumor cells, preferably by expressing annexin in tumor cells An antisense nucleic acid to A3 and/or a ribozyme specific for Annexin A3 reduces and/or inhibits Annexin A3 expression in the cell, more preferably, by using a DNA construct expressing antisense Annexin A3 or Expression vectors to reduce Annexin A3 levels in cells.

Another aspect of the present invention is to provide a method for assessing the drug resistance of tumor patients to platinum-based chemotherapy drugs, comprising the following steps:

1) detecting the level of annexin A3 in the patient's body fluid sample;

2) If the level of annexin A3 in step 1) is higher than 1.34ng/ml, it is judged that the tumor patient is resistant to platinum chemotherapy drugs;

Wherein, preferably, the body fluid is serum; preferably, the detection in step 1) is ELISA detection. The cancer is a cancer that can be treated with platinum-based chemotherapy drugs, such as ovarian cancer, breast cancer, lung cancer, testicular tumor, head and neck cancer, osteosarcoma, melanoma, esophageal cancer, etc., preferably ovarian cancer.

In the study of the present invention, the inventors used enzyme-linked immunosorbent assay to detect and quantify the content of annexin A3 in human serum for the first time. The expression level of annexin A3 was obtained, and the expression level of annexin A3 predicting the best sensitivity and specificity of platinum-based chemotherapy drug resistance was obtained. Establish a clinical platform for prompting platinum-based chemotherapy resistance by detecting serum annexin A3 levels.

First of all, through further experiments, it was proved that Annexin A3 is a specific marker protein of platinum-based chemotherapy drug resistance, and the detection of intracellular platinum content shows that the mechanism of annexin A3 causing platinum-based chemotherapy drug resistance may be through intracellular Platinum release is achieved. Platinum-based chemotherapeutic drug-sensitive tumor cells after upregulation of intracellular annexin A3 expression levels by transfection of sense annexin A3 showed enhanced resistance to cisplatin, but resistance to paclitaxel and epirubicin was not altered , which shows that annexin A3 is a specific protein associated with resistance to platinum-based chemotherapy drugs, and the expression level of annexin A3 determines the sensitivity of tumor cells to platinum-based chemotherapy drugs. An ideal marker is able to be secreted into peripheral blood or other body fluids of the human body for easy detection. The present inventors collected culture supernatants of platinum-based chemotherapeutic drug-sensitive and drug-resistant tumor cells to detect whether the tumor cells can secrete annexin A3 and the regulation method of protein secretion. In order to avoid the influence of high-abundance proteins, the medium is preferably serum-free medium. The liquid in the culture medium was concentrated by centrifugal ultrafiltration technology, and the content of annexin A3 in the concentrated liquid was detected by immunoblotting technology and semi-quantified. It was found that tumor cells (ovarian cancer cells) resistant to platinum-based chemotherapy drugs had significantly stronger ability to secrete annexin A3 than drug-sensitive cells. Changing the amount of annexin A3 exocytosis can be achieved by changing the content of annexin A3 in cells. This shows that the level of annexin A3 in the external fluid can accurately reflect the expression level of annexin A3 in the cells, and can determine the sensitivity of the cells to platinum-based chemotherapy drugs.

Secondly, the inventors discovered and verified the exocrine phenomenon of annexin A3 in ovarian cancer cells and the exocrine pathway of annexin A3, and a good marker for predicting resistance to platinum-based chemotherapy drugs is the body fluid that can be secreted in the human body Or in the blood to detect this substance, and the expression level of this substance in human body fluid has a certain correlation with the sensitivity of platinum chemotherapy drugs. The human annexin A3 enzyme-linked immunosorbent assay kit (ANXA3 ELISAkit) was used to successfully detect and quantify the content of annexin A3 in human serum. The average expression level of serum annexin A3 was 1.6898±2.6563, Mann-Whitney test was performed between the two groups, P<0.0001. All ovarian cancer patients were divided into platinum-based chemotherapeutic drug-resistant group and platinum-based chemotherapeutic drug-sensitive group. Group comparison P=0.0003<0.05. When the level of annexin A3 is greater than 1.13ng/ml, the sensitivity of this protein to predict ovarian cancer platinum chemotherapy drug resistance reaches 63%, and the specificity reaches 80%. Serum from patients with ovarian cancer was collected before chemotherapy and was not affected by chemotherapy. All patients with ovarian cancer received platinum-based chemotherapy after surgery. In conclusion, the expression level of annexin A3 in the serum of tumor patients can well predict the sensitivity of ovarian cancer patients to platinum-based chemotherapy drugs.

Another aspect of the present invention is to provide a method for regulating the release of platinum from platinum-based chemotherapy drug-resistant tumor cells, the method comprising the step of changing the expression level of annenxin A3 in tumor cells, wherein, preferably, the platinum-based chemotherapy drug is cisplatin; preferably, the tumor cells are ovarian cancer cells.

Another aspect of the present invention is to provide a method for regulating the combination of DNA and platinum in platinum-based chemotherapeutic drug-resistant tumor cells, the method includes the step of changing the expression level of annenxin A3 in tumor cells, wherein, preferably, the platinum The chemotherapeutic drug is cisplatin; preferably, the tumor cells are ovarian cancer cells.

Another aspect of the present invention is to provide and a method for regulating the number of vesicles in platinum-based chemotherapeutic drug-resistant tumor cells, the method includes the step of changing the expression level of annenxin A3 in tumor cells, wherein, preferably, the platinum The chemotherapeutic drug is cisplatin; preferably, the tumor cells are ovarian cancer cells.

In order to better illustrate that tumor cells (ovarian cancer cells) can persistently secrete annexin A3 protein, the applicant of the present invention observed the exocrine pathway of annexin A3 by electron microscopy. Two cisplatin-resistant ovarian cancer cells, SKOV3/Cis and A2780/Cis, and two cisplatin-sensitive ovarian cancer cells, SKOV3 and A2780, were transfected with annexin A3 sense plasmid to upregulate annexin in cisplatin-sensitive cells A3-expressing cells SKOV3/Ann and A2780/Ann, two kinds of cells transfected with annexin A3 antisense plasmid down-regulate the expression of annexin A3 in cisplatin-resistant cells SKOV3/Cis/R and A2780/Cis/R are The research object was to observe the distribution of annexin A3 protein in the submicrostructure of cells by immunofluorescence and immunoelectron microscopy, and to explore the pathway of annexin A3 exocytosis. Under the transmission electron microscope, the submicrostructure of the above-mentioned cisplatin-sensitive and drug-resistant ovarian cancer cells was observed. Compared with the cisplatin-sensitive cells, it was found that there were more vesicle-like structures in the cytoplasm of the cisplatin-resistant cells, which were scattered in the In the cytoplasm, the surface is smooth, different from the rough endoplasmic reticulum and Golgi body, and there is no mitochondria-like ridge in the vesicle, and some unidentified granular substances exist in some vesicles, which is different from the homogeneous lysosome quality. By observing the vesicles along the cell membrane, it can be observed that some vesicles are very close to the cell membrane, some fused with the cell membrane, and some have broken through the cell membrane. Using transmission electron microscopy to observe the up-regulation of annexin A3 levels in cisplatin-sensitive cells after transfection of annexin A3 sense plasmid, it was found that there were many similar cisplatin-resistant cells in the cytoplasm of sensitive cells compared with before transfection On the contrary, after the annexin A3 antisense plasmid was transfected to down-regulate the level of annexin A3 in cisplatin-resistant cells, the intracytoplasmic vesicles in cisplatin-resistant cells were greatly reduced. Upregulated Annexin A3 can lead to the generation of intracellular vesicle structures. Using immunofluorescence technique to specifically stain the annexin A3 protein in the nucleus and cytoplasm of the above eight types of cells, it was found that the annexin A3 of ovarian cancer cell types was mainly distributed in the cytoplasm, and a few were distributed in the nucleus. cytoplasmic protein. Using immunoelectron microscopy, the distribution of annexin A3 cells was localized to subcellular organelles, and the above eight types of annexin A3 were immunostained, and colloidal gold particles were used to label annexin A3 proteins, and it was found that annexin A3 was distributed in large quantities in The structure of the inner membrane of the cytoplasm, including the membrane surface of the vesicles found under the transmission electron microscope, and the presence of annexin A3 can be found in individual vesicles, especially the vesicles near the membrane, indicating that annexin A3 passes through the vesicle secreted in the form of vesicles.

The above-mentioned method for regulating the release of platinum from platinum-based chemotherapeutic drug-resistant tumor cells, the method of regulating the combination of DNA and platinum in platinum-based chemotherapeutic drug-resistant tumor cells, and the method of regulating the number of vesicles in platinum-based chemotherapeutic drug-resistant tumor cells, All include the step of changing the expression level of annenxinA3 in tumor cells, which can be achieved by the following methods, for example:

This is achieved by increasing the expression of Annexin A3 in the tumor cells, preferably by expressing the Annexin A3 gene in the tumor cells to increase the expression of Annexin A3 in the cells, more preferably by expressing a positive sense Annexin A3 A plasmid of the gene to increase the content of annexin A3 in the cell; or

Achieved by reducing and/or inhibiting the expression of Annexin A3 in the tumor cells, preferably by expressing in the tumor cells an antisense nucleic acid for Annexin A3 and/or a ribozyme specific for Annexin A3 to reduce and/or Or inhibit the expression of Annexin A3 in cells, more preferably, reduce the content of Annexin A3 in cells by using a DNA construct or expression vector expressing antisense Annexin A3.

In all aspects of the invention, if applicable, it is preferred that said tumor cells include, but are not limited to, cells of cancers such as ovarian cancer, breast cancer, lung cancer, testicular tumors, head and neck cancer, osteosarcoma, melanoma, esophagus Cells such as cancer, preferably ovarian cancer cells.

In all aspects of the present invention, if applicable, preferably, the platinum-based chemotherapeutics include but not limited to cisplatin, carboplatin, oxalateplatin, nedaplatin, roplatin, etc., preferably cisplatin.

In the present invention, the term "drug-resistant cells" refers to cells that are less likely to accept certain stimuli and undergo corresponding changes compared with similar parental cells. In the present invention, it corresponds to cells that are less likely to accept the stimulation of platinum-based chemotherapeutic drugs and undergo corresponding changes.

In the present invention, the term "sensitive cell" refers to a cell that easily accepts some stimuli and changes accordingly. In the present invention, it corresponds to cells that are prone to change correspondingly to the stimulation of platinum-based chemotherapy drugs.

In the present invention, the term "drug resistance" means that the effect of the drug decreases with time, or the dose needs to be increased to ensure that the effect of the drug does not decrease. The present invention refers to the drug resistance of cells to platinum-based chemotherapeutic drugs, which is usually expressed by the median lethal dose of the drug (IC50) and drug resistance index (RI).

In the present invention, the term "sensitivity", when compared with specificity or as a statistical concept, refers to the percentage of "true positive" test results; "Relative concept.

In the present invention, the term "specificity" refers to the percentage of "true negative" test results.

In the present invention, unless otherwise specified, SKOV3 and A2780 are SKOV3 and A2780 transfected with an empty plasmid, SKOV3/Cis is a subline of SKOV3 cisplatin-resistant cells transfected with an empty plasmid, and A2780/Cis is a subline of cisplatin-resistant cells transfected with an empty plasmid. A2780 cisplatin-resistant cell subline, the above empty plasmid is pcDNA3.1(+) plasmid.

Beneficial Effects of the Invention

For the first time, the present invention detects the expression level of annexin A3 through the medium supernatant of platinum-based chemotherapeutic drug-resistant tumor cells, and for the first time provides a method for evaluating the sensitivity of tumor patients to platinum-based chemotherapeutic drugs, enabling early detection Platinum chemotherapeutic drug resistance, change the tumor treatment plan in time, increase the 5-year survival rate, and improve the prognosis.

Description of drawings

In the figures below, SKOV3 and A2780 are SKOV3 and A2780 cells transfected with empty pcDNA3.1(+) plasmid; SKOV3/Cis are SKOV3 cisplatin-resistant cells transfected with empty pcDNA3.1(+) plasmid Subline, A2780/Cis is A2780 cisplatin-resistant cell line transfected with empty pcDNA3.1(+) plasmid; SKOV3/Ann and A2780/Ann cell lines are SKOV3 and A2780 transfected with positive-sense Annexin A3 plasmid, respectively SKOV3/Cis/R and A2780/Cis/R are stable transfection clones obtained after SKOV3/Cis and A2780/Cis were transfected with antisense Annexin A3 plasmid.

Figure 1: Comparison of platinum concentrations in eight ovarian cancer cells, ^* P<0.01, ^** P<0.001.

Figure 2: Comparison of Pt excretion rates in SKOV3 and SKOV3/Ann cells.

Figure 3: Comparison of Pt-DNA concentrations in eight ovarian cancer cells, ^* P<0.05, ^** P<0.01.

Figure 4: Detection of annexin A3 in the culture supernatant of ovarian cancer cells. Parental represents parental cells, that is, tumor cells not transfected with plasmids; Transfectant represents tumor cells transfected with plasmids. Panel A: Both SKOV3 and A2780 were transfected with the sense Annexin A3 plasmid, and the intracellular Annexin A3 level was up-regulated; Panel B: Both SKOV3/Cis and A2780/Cis were transfected with the antisense Annexin A3 plasmid, and the intracellular Annexin A3 level was upregulated. The expression level of protein A3 was down-regulated; β-actin was actin, which was used as the internal reference of this experiment.

Figure 5: A: Changes in the submicrostructure of cisplatin-sensitive ovarian cancer cells (SKOV3 and A2780) and their cisplatin-resistant cells (SKOV3/Cis and A2780/Cis) under a transmission electron microscope (50,000x). B: Extending the cell membrane to observe the relationship between the increased vesicle structure in the cytoplasm and the cell membrane (100,000x). C: Changes of intracytoplasmic vesicles in ovarian cancer cells after transfection of Annexin A3 positive and antisense plasmids. Arrows indicate abnormal vesicle structures in the cytoplasm.

Figure 6: Detection of the localization of annexin A3 in ovarian cancer cells by immunofluorescence technique.

Figure 7: Immuno-electron microscopy detection of Annexin A3 subcellular organelle localization (comparative observation at 50,000x and 100,000x). A: control (PBS instead of annexin A3 antibody); B, C, and D show the localization of annexin A3 on the surface and inside of vesicles. The black particles in the cytoplasm are colloidal gold particles, and each particle represents annexin A3 protein; the small arrow indicates the position of annexin A3 protein. Large arrows indicate vesicles. The 100,000x image is a magnification of the box in the 50,000x image.

Figure 8: The expression of annexin A3 exosome (a form of intracellular vesicles released to the outside of the cell) in the culture supernatant of drug-resistant cells SKOV3/Cis. Figure A: The shape of the exosome observed by transmission electron microscopy, mostly round or oval, with a diameter between 40-100nm; Figures B and C: The expression of annexin A3 in the exosome observed by immunoelectron microscopy; Figure D: DMEM1 represents normal SKOV3/Cis culture supernatant concentrate (concentration factor 75x), DMEM2 represents the exosome removed SKOV3/Cis culture supernatant concentrate (concentration factor 75x), sucrose represents the exosome upper layer sucrose solution during the exosome extraction process, and Hsp70 represents the exosome Refer to (exosome expresses more Hsp70).

Figure 9: Panel A: scatter plot of the expression levels of annexin A3 in the serum of 30 chemotherapy-resistant ovarian cancer patients, 20 chemotherapy-sensitive ovarian cancer patients, and 30 normal women; panel B: the expression level of annexin A3 in serum Ovarian cancer patients with a level greater than 1.13ng/ml were classified as group A, and ovarian cancer patients with a level of less than 1.13ng/ml were classified as group B. The Kaplan Meier curve was drawn, and the platinum-free interval of the two groups (the first course of platinum-containing drugs There was a significant statistical difference in the length of time from the end of combined chemotherapy to ovarian cancer recurrence (P=0.009<0.05). IQR: interquartile range number.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. Those who do not indicate specific techniques or conditions in the embodiments, according to the techniques or conditions described in the literature in this field (for example, refer to J. Sambrook et al., "Molecular Cloning Experiment Guide" translated by Huang Peitang, the third edition, Science Press) or follow the product instructions. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1: In vitro study on the mechanism of annexin A3 regulating the drug resistance of ovarian cancer epithelial cells to platinum-based chemotherapy drugs

1. Experimental method

1) Cell lines and their culture conditions

Human epithelial ovarian cancer cell SKOV3 cell line was purchased from the Basic Medical Cell Center of the Chinese Academy of Medical Sciences. SKOV3/Cis is the SKOV3 cisplatin-resistant cell subline induced by the inventor (refer to Yan Xuedong, Zhang Mingwei, Pan Lingya. Basic Medicine and Clinical, 2006, 26(7):739-744). This experiment also used A2780 and its cisplatin-resistant cell line A2780/Cis (refer to LuanYZ, Li L. et al. Zhonghua Fu Chan Ke Za Zhi, 2004, 39(6): 403-407.). These cells were all transfected with an empty pcDNA3.1(+) plasmid. SKOV3/Ann and A2780/Ann cell lines are stable transfection clones obtained after SKOV3 and A2780 were transfected with positive-sense Annexin A3 plasmids, respectively, and SKOV3/Cis/R and A2780/Cis/R are SKOV3/Cis and A2780/Cis transfected clones. Stably transfected clones obtained after transfection of the antisense Annexin A3 plasmid (refer to Xuedong Yan, Jie Yin, Huiyu Yao, Ning Mao, Yili Yang, Lingya Pan. Cancer Res, 2010, 70(4): OF1-9. ), all stably transfected clones have been verified by Western blot technology. At the same time, cells with empty plasmids were transfected with SKOV3 and A2780 (refer to Xuedong Yan, Jie Yin, Huiyu Yao, Ning Mao, Yili Yang, Lingya Pan. Cancer Res, 2010, 70(4): OF1-9.) as a control.

All the above-mentioned cell lines were grown adherently in DMEM (HG) medium containing 10% fetal bovine serum, cultured in an incubator with 5% CO ₂ saturated humidity at 37°C, and used 0.25 % Trypsinization and passaging.

2) Drug sensitivity test

The sensitivity of cells to cisplatin (Cisplatin, CDDP), carboplatin (Carboplatin, CBP), paclitaxel (Taxol) and epirubicin (epirubicin) was detected.

Cells in the exponential growth phase were taken and digested with trypsin to make a single cell suspension. According to 2000/well/100 μL, 6 parallel wells were set up for each concentration of each drug, and 12 control wells. No drug was added, of which 6 wells were Negative control (add cells, no drug), 6 wells are blank control (only add culture medium). After culturing at 37° _C and 5% _CO2 for 24 hours, add chemotherapeutic drugs, dilute into 7 gradients, and continue culturing for 72 hours. 100 μL lysate (containing 20% SDS, 50% NN dimethylformamide, pH 4.7), overnight, the automatic microplate reader measures the absorbance (OD value) at 540nm, and the average absorbance value of each drug concentration can be calculated Value, calculate cell viability and inhibition rate, according to drug concentration and inhibition rate, use SPSS11.5 software to calculate IC50 (drug concentration that inhibits 50% cell growth) and drug resistance index (RI).

Inhibition rate = [(OD control - OD experiment)/OD control] × 100%

RI = IC50 of drug-resistant cell line/IC50 of sensitive cell line

3) Determination of intracellular platinum (Pt) concentration

After each cell grows to the logarithmic growth phase, digest and count, seed 2× ¹⁰⁶ cells on a 100mm culture dish, add CDDP to a final concentration of 10 μM after 24 hours, and culture in a 37°C, 5% _CO2 saturated humidity incubator for 24 hours. Hours (no drug was added to the control group), the medium was discarded, washed 3 times with PBS, the cells were scraped off with a cell scraper, and collected into Ep tubes. Use 50-100 μL of 70% nitric acid to lyse the cells, put them in a 65°C water bath for 2 hours (take out part of the lysate at this time and use the BCA method to detect the protein concentration), add distilled water to the lysate, and dilute it to 5% nitric acid concentration. The platinum (Pt) content in cells was detected by inductively coupled plasma mass spectrometry. In order to detect the discharge of CDDP, add CDDP to the cells to a final concentration of 10 μM, culture in a 37°C, 5% _CO2 saturated humidity incubator for 24 hours, discard the medium, replace it with drug-free medium and incubate for 2 hours, collect as in the previous step And treat the cells, detect the Pt content. Pt excretion rate=(1-intracellular Pt concentration for 2 hours of drug-free culture/intracellular Pt concentration for 24 hours after drug addition)×100%.

4) Detection of DNA-bound Pt

After each cell grows to the logarithmic growth phase, digest and count, seed 2× ¹⁰⁶ cells on a 100mm culture dish, add CDDP to a final concentration of 10 μM after 24 hours, and culture in a 37°C, 5% _CO2 saturated humidity incubator for 24 hours. Hours (no drug was added to the control group), the medium was discarded, washed 3 times with PBS, the cells were scraped off with a cell scraper, and collected into Ep tubes. Extract DNA according to the instructions of the Qiagen DNA extraction kit, lyse the cells with 300 μL Buffer FG1, mix by pipetting, add 300 μL Buffer FG2/protease, mix by inverting for 3 times, put in a water bath at 65°C for 10 minutes, add 600 μL of isopropanol, and mix by inverting centrifuge at 10,000g for 3 minutes, discard the supernatant, invert the Ep tube on filter paper to blot the water, add 600μl 70% ethanol, vortex for 5s, centrifuge at 10,000g for 3 minutes, discard the supernatant , put the Ep tube upside down on the filter paper to blot the water, air-dry the DNA precipitation until the liquid evaporates, add 50-100 μL of 70% nitric acid to lyse the cells, and put them in a water bath at 65°C for 2 hours (take out part of the lysate at this time and use a spectrophotometer to detect the DNA concentration), distilled water was added to the lysate to dilute it to a 5% nitric acid concentration. The content of Pt bound to DNA was detected by inductively coupled plasma mass spectrometry.

5) Statistical analysis

The experimental data were processed by SPSS11.5 statistical software, and statistical analysis was carried out by t test and variance test (Oneway Anova).

2. Experimental results

1) Detection of cell resistance to cisplatin, carboplatin, paclitaxel and epirubicin

The CDDP-resistant cells SKOV3/Cis and A2780/Cis were not only resistant to CDDP, but also developed the same resistance to CBP (Table 1). In addition, the sensitivity of each transfected cell to paclitaxel and epirubicin was tested. We can see that compared with sensitive cells SKOV3 and A2780, drug-resistant cells SKOV3/Cis and A2780/Cis not only developed resistance to platinum, but also cross-resistance to paclitaxel and epirubicin, their RI are shown in Table 2, respectively. However, the sensitivity to paclitaxel and epirubicin was not significantly changed in cells overexpressing or not expressing Annexin A3 compared with their controls (cells transfected with empty plasmid) (Table 2).

Table 1 Comparison of the resistance of platinum-resistant cells to cisplatin and carboplatin

^* P<0.05, ^** P<0.01, compared with parental cells

Table 2 Drug resistance of stably transfected cells and their control cells to paclitaxel and epirubicin.

2) Determination of Pt concentration

After each cell was treated with 10 μM CDDP for 24 hours, the concentration of Pt in the cell was detected. Pt concentrations were significantly decreased (P<0.001, P=0.002) in cells overexpressing Annexin A3 (SKOV3/Ann, A2780/Ann) compared to sensitive cells transfected with empty plasmid (SKOV3, A2780). Compared with drug-resistant cells transfected with empty plasmids (SKOV3/Cis, A278/Cis), Pt concentrations were significantly increased in cells with downregulated expression of annexin A3 (SKOV3/Cis/R, A2780/Cis/R) (P=0.008, P=0.009) (Figure 1). In the experiment of detecting Pt excretion rate, SKOV3 cells were (31.4±4.0)% and SKOV3/Ann cells were (59.2±2.2)%. Pt excretion in SKOV3/Ann cells was significantly increased (P<0.001) (Figure 2). In addition, in the results of detecting Pt-DNA binding, it was found that in SKOV3/Ann and A2780/Ann, the concentration of Pt bound to DNA decreased (P=0.007, P=0.011). In SKOV3/Cis/R, A2780/Cis/R, the concentration of Pt bound to DNA was significantly increased (P=0.003, P=0.026) ( FIG. 3 ).

3. Conclusion

The reduction of intracellular drug accumulation is one of the important mechanisms causing platinum drug resistance, and an important function of annexin A3 is to mediate the contact between membranes, which can bind to the plasma membrane through phosphatidylserine, so that The permeability of the cell membrane changes and participates in activities such as phagocytosis and secretion of cells. We therefore examined whether there is an altered accumulation of platinum in Annexin A3 overexpressed and Annexin A3 downregulated cells. The experimental results showed that in the annexin A3 overexpression cells, the platinum concentration was significantly reduced, and the Pt-DNA binding was significantly reduced, while in the cells with annexin A3 downregulation, the same results were obtained, indicating that intracellular platinum The reduction of drug accumulation is an important factor for annexin A3 to lead to platinum resistance. Platinum drugs damage DNA by forming adducts with DNA, and the recognition of adducts by the mismatch repair system can lead to activation of p53, activation of the MAPK pathway, induction of BAX expression, and ultimately apoptosis (Hartwell LH, et al.Science, 1994, 266(5192):1821-1828). This experiment also confirmed this point. Compared with the parental cells transfected with an empty pcDNA3.1(+) plasmid, in Annexin A3 overexpressing cells, after treatment with cisplatin, the platinum concentration was significantly reduced, Pt-DNA binding was significantly reduced, and p53 was increased. The degree is smaller than that of parental cells, so apoptosis is reduced, leading to drug resistance. Since the peak plasma concentration of CDDP chemotherapy dose in clinical treatment is 10 μM, this dose is used for the effect concentration of CDDP in this part of experiments.

Example 2: Identification of Exocrine Annexin A3 in Epithelial Ovarian Cancer Cells

1. Experimental method

1) Cell lines and cell culture

With embodiment 1.

2) Collection and concentration of epithelial ovarian cancer cell line culture supernatant

Take 2×10 ⁵ cells and inoculate them in a 25cm ² cell culture flask for adherent growth. When the growth reaches 70%, replace with serum-free DMEM, give 5ml medium for each 1×10 ⁶ cells, and place them in a cell culture incubator for 48 hours. Then collect this serum-free DMEM in a 10ml centrifuge tube and centrifuge at 1000g for 10 minutes to remove the formed components in DMEM. After centrifugation, carefully collect the supernatant and place it in an Amicon Ultra-15 ultrafiltration tube (Millipore, USA). , 3000g horizontal centrifugation for 15 minutes, collect the concentrated liquid in Ep tube, and store at -70°C.

3) Semi-quantitative detection of the content of annexin A3 in the medium by Western blot

a. Preparation of test samples

The concentrated cell culture supernatant and total intracellular protein were the samples to be tested in this experiment. The culture supernatant sample was mixed with laemmli loading buffer (Bio-Rad, USA) at 2:1 to make a loading sample, and placed on ice. In a water bath, a volume of about 20 µl is good. Take non-drug-resistant and drug-resistant cells in the logarithmic growth phase, digest with trypsin, centrifuge at 1500rpm for 5 minutes, discard the supernatant, add sterile PBS (pH 7.4) to resuspend, perform cell counting, centrifuge at 1500rpm for 5 minutes, discard the supernatant Wash the cells twice with cold PBS and aspirate the supernatant. About 10 ⁶ cells were added with laemmli lysate (Bio-Rad, USA). When the cells were lysed, other cells were placed in an ice-water bath. After adding the lysate, boil at 99°C for 10 minutes, centrifuge at 10,000g at 4°C for 10 minutes, take it out and bathe in ice water. After taking out the sample for protein concentration determination, add 5 μl of 2-ME (2-mercaptoethanol) to the remaining sample per 100 μl, centrifuge at 10000 g, 4°C for 10 minutes, and store at -20°C.

b.BCA method to measure total protein concentration in cells

Take the BCA Protein Quantification Kit (Pierce) A solution and B solution and mix it at 50:1 (v:v), take the above sample and add it to 500 μl AB mixed solution, mix well, add 2 μl lysate solution to 500 μl AB mixed solution for the blank control , in a water bath at 37°C for 30 minutes, set to zero with a blank control, and measure the OD value at a wavelength of 562nm on a Smartspec ^TM plus spectrophotometer (Bio-Rad). Use Excel to draw a standard curve and calculate the sample concentration.

c. SDS-PAGE electrophoresis

Gel preparation: first prepare 5ml of 10% separating gel: 1.9ml of deionized water, 1.7ml of 30% acrylamide, 1.3ml of 1.5% Tris (pH 8.8), 0.05ml of 10% SDS, 0.05ml of 10% ammonium persulfate, TEMED 0.002 ml. After the separation gel solidified for 30 minutes, prepare 2ml of 5% stacking gel: 1.4ml of deionized water, 0.33ml of 30% acrylamide, 0.25ml of 1.0% Tris (pH 6.8), 0.02ml of 10% SDS, 10% ammonium persulfate 0.02ml, TEMED0.002ml. Adding samples: After the concentrated gel is solidified, take 20 μl of the concentrated medium loading sample and 30 μg of total intracellular protein and add it to the sample tank. Electrophoresis: add sufficient amount of Tris-glycine electrophoresis buffer (25mmol/L Tris, 250mmol/L glycine, 0.1% SDS) 80-100v, electrophoresis for 2 hours.

d. Transfer film

Prepare transfer buffer: 48 mM Tris-HCl, 39 mM glycine, 0.037% SDS, 20% methanol. Cut 6 pieces of filter paper with the same size as the gel and a PVDF membrane (Millipore, USA). The filter paper was soaked in the transfer buffer for 15 minutes. The PVDF membrane was activated with methanol for 10 seconds and then soaked in the transfer buffer. The filter paper, gel and PVDF membrane are stacked in the order of three layers of filter paper-PVDF membrane-gel-three layers of filter paper. The whole operation process is completed in the transfer buffer, and no air bubbles should be taken care of. bottom right corner mark. 270mA, electrotransfer in an ice-water bath for 1 hour.

e. Western blot

Take off the PVDF membrane on which the protein is transferred, with the protein side facing up, shake and wash 3 times with TBST (200mM Tris-HCl, 1.5mM NaCl, 0.05% Tween-20), after 5 minutes each time, in TBST containing 5% skimmed milk powder After blocking in medium for 1 hour, take it out, add primary antibody (rabbit anti-human annexin A3 specific polyclonal antibody) prepared in blocking solution, and shake gently overnight at 4°C. Shake and wash 3 times with TBST, each time for 5 minutes, then add HRP-labeled secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG antibody) prepared for blocking, incubate at room temperature for 1 hour, shake and wash three times with TBST, and The chemiluminescence substrate solution ECL (Thermo Company of the United States) A and B solutions were mixed in a volume ratio of 1:1, and the PVDF membrane was acted on for 5 minutes, suggesting that the X-ray film was exposed and developed.

2. Experimental results

The protein content of tumor cell serum-free medium is very low. After the supernatant is concentrated by ultrafiltration tubes, the volume can be concentrated from 15ml to 500μl, which greatly increases the protein content in the medium. provides the basis. Annexin A3 was successfully detected in the concentrated medium supernatant by Western blot technology, and annexin A3 in the medium supernatant of the above eight ovarian cancer cell lines was semi-quantified. The semi-quantitative comparison is mainly to compare the thickness of the annexin A3 protein band in western blotting. In the above two cisplatin-resistant ovarian cancer cell lines, the expression levels of annexin A3 in the culture supernatant and cells were higher than those of the corresponding cisplatin-sensitive cell lines, especially the expression level of annexin A3 in the supernatant increased more obvious. After the transfection of annexin A3 antisense plasmid down-regulated the expression level of annexin A3 in cisplatin-resistant cells (SKOV3/Cis/R and A2780/Cis/R cell lines), the level of annexin A3 in the cell culture medium decreased Significantly decreased. In contrast, the expression level of Annexin A3 was significantly upregulated in the culture medium of SKOV3/Ann and A2780/Ann cells transfected with the positive-sense Annexin A3 plasmid ( FIG. 4 ).

3. Conclusion

The supernatant of ovarian cancer cell culture medium contains a certain amount of annexin A3, indicating that ovarian cancer cells have the function of secreting annexin A3. Through semi-quantitative comparison, it shows that the amount of annexin A3 secreted by ovarian cancer cells is comparable to that in cells. The content of annexin A3 was positively correlated, and the high level of annexin A3 in the culture medium could accurately reflect the drug resistance of ovarian cancer cells to cisplatin. Based on the above, Annexin A3 has the basis to be an effective biomarker for predicting resistance to platinum-based chemotherapy drugs.

Example 3: The Exocrine Pathway of Annexin A3 in Ovarian Cancer Cells

1. Experimental method

1) Cell lines and culture

The cells used in this experiment and their culture conditions are completely consistent with those in "Example 2", and will not be repeated here.

2) Immunofluorescence and its laser confocal imaging

Seed 2×10 ⁴ cells into a 24-well cell culture plate that has been laid on glass slides, culture overnight in a 37°C, 5% CO ₂ saturated humidity incubator, discard the medium, wash 3 times with PBS, 4% Paraformaldehyde was fixed for 20 minutes, washed 3 times with PBS, permeabilized with 0.5% TritonX-100 for 20 minutes, washed 3 times with PBS, blocked with 2% BSA for 30 minutes, 1:50 rabbit anti-human annexin A3 polyclonal antibody (primary antibody ) at 4°C overnight. Wash 3 times with PBS, incubate with goat anti-rabbit FITC secondary antibody for 1 hour, wash 3 times with PBS, incubate in 1:1000 PI staining solution containing 0.1% RNase for 20 minutes at room temperature, wash 3 times with PBS, cover with PBS containing 90% glycerol . Observed and photographed under a Radiance 2100 laser confocal microscope.

3) Transmission electron microscope

a. For cells growing in logarithmic phase (total number of cells ≥ 1×10 ⁶ ), discard the medium and wash with PBS for 2 times.

b. Add 10ml of PBS and send it to the Electron Microscopy Room of the Instrument Testing Center of the Academy of Military Medical Sciences (No. 27 Taiping Road, Haidian District, Beijing) to prepare cell transmission electron microscope specimens.

c. The specimen preparation process is as follows: grow the cells to the logarithmic growth phase, wash them twice with PBS, scrape off the cells with a cell brush, centrifuge at 1000g for 10 minutes, 3% glutaraldehyde (1/15M PBS pH 7.4), at 4°C Fix for 2 hours, rinse with 1/15M PBS+0.19M sucrose buffer for 15 minutes at 4°C. Gradient dehydration with ethanol at 4°C (50% ethanol, 70% ethanol, 90% ethanol, 90% ethanol+90% acetone, 90% acetone, 100% acetone for 10 minutes each), and 100% acetone for 10 minutes at room temperature. 100% acetone: embedding agent (1:1) was soaked at room temperature for 30 minutes, and pure embedding agent was soaked overnight. For embedding: the polymerization time is 35°C, 12 hours; 45°C, 12 hours; 60°C, 24 hours. ULTRACUT E/S microtome (RMC Company, USA) was used for ultra-thin sectioning, stained with uranyl acetate staining solution in the dark for 10 minutes, and stained with lead citrate for 10 minutes.

d. Voltage 75kV, observe with EM400T electron microscope (Philips, Netherlands), choose ×8000, ×22000 electron microscope to take pictures, observe cell submicrostructure.

4) Immuno-electron microscopy

a. For cells growing in logarithmic phase (total number of cells ≥ 1×10 ⁶ ), the medium was discarded and washed twice with PBS.

b. Add 10ml of PBS and send to the Electron Microscope Room of Chinese Academy of Medical Sciences to prepare cell ultrathin sections (70-80nm thick). The process is as follows: the cells grow to the logarithmic growth phase, washed twice with PBS, scraped off the cells with a cell brush, centrifuged at 1000g for 10 minutes, fixed solution mixed with 4% paraformaldehyde phosphate and 0.1% glutaraldehyde 1:1, 4 The cells were fixed at ℃ for 4-6 hours, and the cells were embedded with special white resin (Sigma, USA) after gradient ethanol dehydration. After the embedding agent was coagulated, ultrathin sections were performed with an ULTRACUT E/S microtome (RMC Company, USA).

c. Immunostaining: Rinse the ultrathin sections three times with deionized water, place in 1% BSA blocking solution for 30 minutes, and incubate overnight in a 4°C wet box with 1:200 rabbit anti-human annexin A3 polyclonal antibody, 0.1M Wash 3 times with PBS, incubate with 1:40 colloidal gold particle-labeled donkey anti-rabbit secondary antibody at room temperature for 1 hour, and wash 3 times with deionized water.

d. Staining observation: After immunostaining, the ultrathin sections of the cells were stained with uranyl acetate staining solution in the dark for 10 minutes, and then stained with lead citrate for 10 minutes. The annexin A3 protein labeled with colloidal gold particles was observed under an electron microscope, and photographed under a 50000× electron microscope.

5) Exosome extraction, observation and identification of Annexin A3 expression in the culture supernatant of SKOV3/Cis cell line

a. Take about 5×10 ⁶ SKOV3/Cis cells and inoculate them on average in 2 T75 cell culture flasks. After the cells adhere to the wall, replace with serum-free medium (DMEM) and culture in a 5% CO ₂ cell incubator 48 hours. Collect about 60ml of culture supernatant.

b. Centrifuge the collected medium supernatant at 1000×g for 10 minutes to remove cells and debris, collect and centrifuge to get the supernatant. The supernatant was then placed in a Centricon Plus-20 filter capsule (Millipore, USA), centrifuged at 4000×g for 30 minutes, and finally the 60ml supernatant was concentrated into 1.5ml concentrated solution, and then the concentrated solution was diluted to 15ml with PBS and placed in 100kDa Amicon Ultra-15 (Milipore, USA), centrifuged at 4000×g for 30 minutes, concentrated to 200 μl concentrate.

c. Collect 200 μl of concentrated solution, dilute to 5ml with PBS, and transfer to 5ml ultracentrifuge tube, meanwhile add 300μl 30% sucrose/D ₂ O to the bottom, centrifuge at 100,000×g, 4°C for 40 minutes, collect 350μl bottom glucose cushion, and Diluted to 15ml with PBS, concentrated again to 200μl with 100kDa Amicon Ultra-15 (Milipore, USA) (this is the liquid containing exosome). At the same time, 350 μl of low-concentration glucose solution (hereinafter referred to as “sucrose” for this sample) was collected from the upper layer as a control solution without exosome, stored at -20°C, and used for western blot analysis. Store 100 μl of exosome-containing liquid in a -20°C freezer. When the low-concentration glucose solution and exosome-containing liquid were analyzed by western blot, the liquid sample was first mixed with laemmli loading buffer (Bio-Rad, USA) at a ratio of 1:1, and the expression of annexin A3 in sucrose and exosome was detected ( The western blot method has been described in detail in Example 2, and will not be described here again).

d. The 200μl concentrated solution contains a large amount of exosomes. Drop the concentrated solution onto a 400-mesh nickel grid covered with carbon film, and let it dry naturally. At this time, the exosome has been adsorbed on the nickel grid. 1% glutaraldehyde is used to fix the exosome, and then 1% glutaraldehyde is used to fix the exosome. Uranyl acetate was negatively stained, and the exosome morphology was observed under a transmission electron microscope.

e. During immuno-electron microscope observation, the exosome was first adsorbed to the nickel mesh, washed with PBS repeatedly 3 times, incubated overnight in a 4°C wet box with 1:200 rabbit anti-human annexin A3 polyclonal antibody, washed 3 times with 0.1MPBS, 1 :40 colloidal gold particle-labeled donkey anti-rabbit secondary antibody was incubated at room temperature for 1 hour, then fixed with 1% glutaraldehyde, and observed under a transmission electron microscope.

6) Collection of cell culture supernatant without exosome

a. Inoculate 5×10 ⁶ SKOV3/Cis cells evenly into 2 T75 cell culture flasks, replace the serum-free medium after the cells adhere to the wall, and collect about 30ml of the medium supernatant after continuing to culture for 48 hours.

b. Centrifuge the collected medium supernatant at 1000×g for 10 minutes, and collect 30 ml of the supernatant.

c. Divide the collected supernatant into two equal parts, one part is centrifuged at 4000×g for 30 minutes with 100kDa Amicon Ultra-15 (Millipore, USA), and the lower non-concentrated liquid is collected with a volume of about 15ml, and then 15ml of the liquid is used with 10kDa AmiconUltra-15 (Millipore, USA) was centrifuged under the same conditions for 30 minutes, and about 200 μl of the concentrated solution in the upper layer was collected. This concentrated solution was the supernatant from which the exosome was removed, and the sample was named DMEM2.

d. Another part of the supernatant that was equally divided was centrifuged at 4000×g for 30 minutes with 10kDa Amicon Ultra-15 (Millipore, USA), and about 200 μl of the concentrated liquid in the upper layer was collected. This liquid was the concentrated supernatant containing exosome. The sample Name it DMEM1.

e. All samples were stored in a refrigerator at -20°C, mixed with laemmli loading buffer (Bio-Rad, USA) 1:1, and the expression level of annexin A3 was detected by western blot method (western blot experimental steps can be Refer to Example 2).

2. Experimental results

Under the transmission electron microscope, observe the submicrostructure of the above-mentioned cisplatin-sensitive and drug-resistant ovarian cancer cells. Compared with the cisplatin-sensitive cells, it is found that there are more vesicle-like structures in the cytoplasm of the cisplatin-resistant cells, and the structures are scattered. In the cytoplasm, the surface is smooth, different from the rough endoplasmic reticulum and Golgi body, and there is no mitochondria-like ridge in the vesicles, and some unknown granular substances can be seen in some vesicles, different from lysozyme homogeneity in the body. When the vesicles were observed along the cell membrane, some vesicles could be observed very close to the cell membrane, some fused with the cell membrane, and some had broken through the cell membrane (Fig. 5A-B). Using transmission electron microscopy to observe the up-regulation of annexin A3 levels in cisplatin-sensitive cells after transfection of annexin A3 sense plasmid, it was found that there were many similar cisplatin-resistant cells in the cytoplasm of sensitive cells compared with before transfection On the contrary, after transfecting annexin A3 antisense plasmid to down-regulate the level of annexin A3 in cisplatin-resistant cells, the intracytoplasmic vesicles of cisplatin-resistant cells were greatly reduced (Fig. 5C), The above shows that the up-regulated annexin A3 in cells can lead to the generation of intracellular vesicle structures. Using immunofluorescence technique to specifically stain the annexin A3 protein in the nucleus and cytoplasm of the above eight types of cells, it was found that the annexin A3 of ovarian cancer cell types was mainly distributed in the cytoplasm, and a few were distributed in the nucleus. Cytoplasmic proteins (Figure 6). Using immunoelectron microscopy, the distribution of annexin A3 cells was localized to subcellular organelles, and the above eight types of annexin A3 were immunostained, and colloidal gold particles were used to label annexin A3 proteins, and it was found that annexin A3 was distributed in large quantities in On the structure of the inner membrane of the cytoplasm, including the membrane surface of the vesicles found under the transmission electron microscope, at the same time, the presence of annexin A3 can be found in individual vesicles, especially the vesicles near the membrane (Fig. 7), indicating that the annexin A3 Protein A3 is secreted in the form of vesicles. Using the western blot method, the difference in the expression of annexin A3 between DMEM1 and DMEM2 was detected, indicating that the exosome carried a large amount of annexin A3 protein. At the same time, the level of annexin A3 between exosome and sucrose was detected to illustrate the degree of purification of exosome ( FIG. 8 ).

3. Conclusion

Annexin A3 exists in the cytoplasm and nucleus of ovarian cancer cells, but mainly exists in the cytoplasm, and is a cytoplasmic protein. The up-regulated Annexin A3 can generate vesicle structure in the cytoplasm of ovarian cancer cells, which is neither like the endoplasmic reticulum nor the Golgi apparatus and lysosome. For Annexin A3 protein, observe the vesicles along the cell membrane, and it can be found that the vesicles have a tendency to fuse with the cell membrane and protrude from the cell, which can indicate that Annexin A3 is secreted out of the cell through this pathway. This secretion mechanism provides a more solid theoretical basis for annexin A3, as a secreted protein, to become a clinical predictor of platinum-based chemotherapeutic drug resistance biological markers in ovarian cancer.

Example 4: Detection of the expression level of annexin A3 in human peripheral blood

1. Experimental method

1) Collection of clinical case data

From 2007 to 2009, healthy women (30 cases) who received physical examination at Peking Union Medical College Hospital of Chinese Academy of Medical Sciences and Peking Union Medical College Hospital and patients with ovarian cancer (50 cases) who were treated in obstetrics and gynecology before operation (or before postoperative chemotherapy) were all included in this experiment. research object. Healthy women need to meet the following conditions: (1) women in childbearing age, perimenopause and menopause; (2) not suffering from any chronic diseases, infectious diseases and benign or malignant tumor diseases; (3) not taking any drugs in the past week and healthcare products. Serum collected from healthy women was placed in Ep tubes and stored at -70°C until testing. Ovarian cancer patients selected for this experiment need to meet the following requirements: (1) Histopathological diagnosis of primary ovarian epithelial malignant tumor after surgery; (2) After the first tumor cytoreductive surgery, all patients received regular platinum-based therapy Chemotherapy drugs (cisplatin or carboplatin) as a single drug or combined chemotherapy; (3) After surgery and after the first chemotherapy, all patients received regular follow-up, and the follow-up time was about 6 days after the first chemotherapy was completed. months to 1 year. Patients were followed up once a month in the first year after surgery, every 3 months in the second year, and every 6 months after the third year. Peripheral blood CA125 was detected at each follow-up, gynecological examination, corresponding imaging examination, and biopsy when necessary. The studies were included according to the inclusion criteria, and the age, pathological type, stage, grade, and chemotherapy of the patients were collected (Table 3). Platinum chemotherapy drug resistance is defined as complete remission (CR) after satisfactory tumor cytoreductive surgery and standard chemotherapy, and the duration is <6 months; the best curative effect of the tumor during chemotherapy is partial remission (PR), stable ( SD) or progression (PD). Sensitivity to platinum-based chemotherapy was defined as clinical complete remission (CR) lasting >6 months after satisfactory cytoreductive surgery and standard chemotherapy. According to the above criteria, 30 of the 50 patients with ovarian cancer met the drug resistance criteria after follow-up, and the remaining 20 were classified as the sensitive group (Table 3).

Table 3 Statistics of clinical case data of 50 patients with ovarian cancer. a and b: Both pathological and clinical stages are staged according to international uniform standards.

2) ELISA (enzyme-linked immunosorbent assay)

a. Experimental specimens: Sera from healthy women and ovarian cancer patients who meet the above enrollment conditions are the detection objects of this experiment. Collect blood samples in 5ml collection tubes. After the blood coagulates naturally, centrifuge at 3000g for 5 minutes, collect supernatant (serum), store in Ep tubes at -70°C.

b. Human annexin A3 ELISA detection kit (Cusabio, USA) was used to quantitatively detect the expression level of annexin A3 in serum. The kit contains ELISA plates, standards, sample diluent, biotin-labeled antibody diluent, horseradish peroxidase-labeled avidin diluent, biotin-labeled antibody, horseradish peroxidase-labeled avidin , substrate solution, wash solution and stop solution. Specifically follow the instructions of the ELISA detection kit, and the steps are as follows:

(1) Dilute the standard. Concentrations are 0ng/ml, 0.4ng/ml, 0.8ng/ml, 1.6ng/ml, 3.2ng/ml, 6.2ng/ml, 12.5ng/ml and 25ng/ml, and the standard is prepared 15 minutes before the experiment;

(2) Add sample. Set blank wells, standard wells and sample wells to be tested respectively. Add the sample to be tested and the standard substance to the ELISA plate that has been embedded with annxin A3 antibody, 100 microliters per well, add 100 microliters of standard dilution solution to the blank well, and set up duplicate holes for each well. After adding the sample, cover the microplate with film, place it in a wet box, and react at 37°C for 120 minutes;

(3) mark. Discard the liquid, spin dry, without washing, add 100 microliters of biotin-labeled antibody working solution to each well, 37°C, 60 minutes; then discard the liquid, spin dry, wash the plate 3 times with the washing solution, each time for 2 minutes, each time Well 200 microliters, spin dry, add horseradish peroxidase-labeled avidin working solution 100 microliters, 37 ° C, 60 minutes;

(4) Color development. After incubation, discard the liquid, dry it, wash the plate 5 times, add 90 microliters of substrate solution to each well in turn, and develop color at 37°C in the dark for 30 minutes, the color of the liquid in the well turns blue. Microliter stop solution, at this time the liquid turns yellow;

(5) Detection. After adding the stop solution, detect on a microplate reader within 15 minutes, set the absorption wavelength to 450nm, and record the OD value of each well.

c. Data processing: Use Excel to make a standard curve with the OD value on the Y axis and the concentration of 1g on the X axis to obtain the trend line equation, calculate the average OD value of each sample and put it into the formula to get the inside of each sample Concentration values of Annexin A3.

3) Statistical analysis

The data were analyzed using GraphPad Prism 5.0 software. All the data were made according to the normal group and the ovarian cancer group (divided into platinum-based chemotherapeutic drug-resistant group and sensitive group). Whitney analysis, when P>0.05, the difference was statistically significant.

3. Experimental results

The human annexin A3 enzyme-linked immunosorbent assay kit (ANXA3 ELISAkit) was used to successfully detect and quantify the content of annexin A3 in human serum, and the expression level of annexin A3 in human peripheral blood is relatively low. The average expression level of serum annexin A3 in normal women was 0.8590±0.0744, and the average expression level of serum annexin A3 in patients with ovarian cancer was 1.6898±2.6563. Mann-Whitney test was performed between the two groups, P<0.0001. All ovarian cancer patients were divided into platinum-based chemotherapeutic drug-resistant group and platinum-based chemotherapeutic drug-sensitive group. Group comparison P=0.0003<0.05 (FIG. 9). When the level of annexin A3 is greater than 1.13ng/ml, the sensitivity of this protein to predict the drug resistance of platinum chemotherapy drugs in ovarian cancer reaches 63.3%, and the specificity reaches 80%. When it is greater than 1.34ng/ml, the sensitivity and specificity of this protein to predict ovarian cancer platinum-based chemotherapeutic drugs both reach 100% (Table 4).

4 Conclusion

Annexin A3, as a platinum-based chemotherapeutic drug resistance-related protein, has been confirmed to have exocrine function in ovarian cancer cells by in vitro experiments, and annexin A3 was successfully detected in human peripheral blood by ELISA. And comparing the expression levels of annexin A3 in the drug-resistant group and the sensitive group, the expression level of annexin A3 in the drug-resistant group was significantly higher than that in the sensitive group. When the annexin A3 level was higher than 1.13 ng/ml, it predicted platinum chemotherapy The sensitivity and specificity of drug resistance are more than 50%, and when it is higher than 1.34ng/ml, the sensitivity and specificity reach 100%. In this experiment, human serum was selected as the research object because it is relatively easy to obtain and is a commonly used clinical detection object. In theory, other human secretions such as ascites, urine, saliva, and leucorrhea can also be used as detection objects. In this experiment, for the sake of quality control, all sample tests were completed by a single person at one time, and the data analysis was performed by a double-blind method.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims (10)

1. a method that detects expression level of annexin A 3 is characterized in that, uses platinum-based chemotherapy medicine drug-resistant tumor cell culture medium supernatant as detected sample,

Wherein, preferably, described platinum-based chemotherapy medicine is a cis-platinum; Preferably, described tumour cell is an ovarian cancer cell; Preferably, described nutrient culture media is a serum free medium, and DMEM more preferably; Preferably, described detection is preferably immunology detection, and Westernblot or ELISA detection more preferably.

2. the regulate tumor cell annexin A 3 method of the amount of secreting outward, described method comprises the step that changes annenxin A3 expression in the tumour cell,

Wherein, preferably, described platinum-based chemotherapy medicine is a cis-platinum; Preferably, described tumour cell is an ovarian cancer cell.

3. method according to claim 2, improve the tolerance of tumour cell by the annexin A 3 expression that improves in the tumour cell to the platinum-based chemotherapy medicine, preferably, express by the annexin A 3 that expression annexin A 3 gene in tumour cell improves in the cell, more preferably, by using the plasmid of expressing just annexin A 3 gene to improve the content of the annexin A 3 in the described cell.

4. method according to claim 2, reduce the tolerance of tumour cell by the annexin A 3 expression that reduces and/or suppress in the tumour cell to the platinum-based chemotherapy medicine, preferably, antisensenucleic acids by expressing annexin A 3 in tumour cell and/or the ribozyme that is specific to annexin A 3 reduce and/or suppress annexin A 3 expression in the cell, more preferably, DNA construct by using the antisence annexin A 3 or the expression vector content that reduces the annexin A 3 in the cell.

5. a method of assessing tumor patient to the platinum-based chemotherapy drug resistance comprises the steps:

1) detects annexin A 3 level in patient's humoral sample;

2), judge that tumor patient is to platinum-based chemotherapy medicine resistance if the annexin A 3 level in the step 1) is higher than 1.34ng/ml;

Wherein, preferably, described body fluid is serum; Preferably, the detection in the step 1) is that ELISA detects.

6. regulate the method that platinum-based chemotherapy medicine drug-resistant tumor cell discharges platinum for one kind, described method comprises the step that changes annenxin A3 expression in the tumour cell,

Wherein, preferably, described platinum-based chemotherapy medicine is a cis-platinum; Preferably, described tumour cell is an ovarian cancer cell.

7. regulate the method that DNA combines with platinum in the platinum-based chemotherapy medicine drug-resistant tumor cell for one kind, described method comprises the step that changes annenxin A3 expression in the tumour cell,

Wherein, preferably, described platinum-based chemotherapy medicine is a cis-platinum; Preferably, described tumour cell is an ovarian cancer cell.

8. method of regulating vesica quantity in the platinum-based chemotherapy medicine drug-resistant tumor cell, described method comprise the step that changes annenxin A3 expression in the tumour cell,

Wherein, preferably, described platinum-based chemotherapy medicine is a cis-platinum; Preferably, described tumour cell is an ovarian cancer cell.

9. according to each described method in the claim 6 to 8, described method is expressed realization by the annexin A 3 that improves in the tumour cell, preferably, express by the annexin A 3 that expression annexin A 3 gene in tumour cell improves in the cell, more preferably, by using the plasmid of expressing just annexin A 3 gene to improve the content of the annexin A 3 in the described cell.

10. according to each described method in the claim 6 to 8, described method is expressed realization by the annexin A 3 that reduces and/or suppress in the tumour cell, preferably, antisensenucleic acids by expressing annexin A 3 in tumour cell and/or the ribozyme that is specific to annexin A 3 reduce and/or suppress annexin A 3 expression in the cell, more preferably, DNA construct by using the antisence annexin A 3 or the expression vector content that reduces the annexin A 3 in the cell.

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