CN106405096A - Application of nucleolin to preparation of diagnostic reagent and therapeutic for gastric cancer - Google Patents

Abstract

The invention belongs to the field of biotechnology, and relates to a multifunctional non-ribosomal protein nucleolin and its use, in particular to the application of nucleolin as a human gastric cancer molecular marker and its preparation or kit for detecting or diagnosing human gastric cancer and Use in therapeutic medicine. In the present invention, the ELISA detection of nucleolin is carried out on the plasma samples of gastric cancer patients and normal controls, and the results show that the level of nucleolin in the plasma of gastric cancer patients is significantly higher than that of normal controls, indicating that the nucleolin is highly expressed in gastric cancer and secreted into the blood Therefore, nucleolin can be used as a non-invasive marker for the diagnosis of gastric cancer. Further, the use of nucleolin in the preparation of gastric cancer diagnostic reagents and kits, and a drug for treating gastric cancer targeting nucleolin are provided. The invention provides a new auxiliary judgment means for the early diagnosis and early warning of gastric cancer.

Description

Application of nucleolin in the preparation of gastric cancer diagnostic reagents and therapeutic drugs

technical field

The invention belongs to the field of biotechnology, and relates to a multifunctional non-ribosomal protein nucleolin and its use, in particular to the application of nucleolin as a human gastric cancer molecular marker and its preparation or kit for detecting or diagnosing human gastric cancer and Use in therapeutic medicine.

Background technique

The prior art discloses that gastric cancer is one of the most common malignant tumors, with about 1 million new cases of gastric cancer each year, and its mortality rate is second only to lung cancer in the world, ranking second in the cancer mortality rate. Gastric cancer is the leading cause of death in digestive tract tumors every year in my country. Studies have shown that the prognosis of gastric cancer is closely related to the stage of the disease, and early diagnosis and treatment can significantly improve the prognosis and prolong the survival period. In current clinical practice, serum blood tests are more common and routine tests. Serum CEA, CA19-9, CA50, CA125 and other tumor-associated antigens can be elevated in patients with gastric cancer, but their sensitivity and specificity are not high. At the same time, most patients with gastric cancer have no obvious clinical symptoms in the early stage, which has caused certain difficulties in the early diagnosis of gastric cancer; therefore, it has become a researcher in this field to find effective markers of gastric cancer and accurately judge the clinical stage and prognosis of the disease. focus of attention.

Nucleolin is a multifunctional non-ribosomal protein, which is mainly distributed in the nucleus, and a small amount is distributed in the nucleoplasm, cytoplasm and cell membrane. It is involved in many aspects of DNA metabolism, and is also widely involved in the regulation of RNA, such as transcription, ribosome assembly, translation of mRNA and maintenance of its stability, and processing of microRNA. Nucleolin affects disease progression at the cellular level by regulating post-transcriptional target mRNAs, promoting cell proliferation and resisting apoptosis. Especially in tumors and viral infections, nucleolin is closely involved in the pathological progression of diseases by regulating the function of RNA.

It has been reported that in the cytoplasm, nucleolin binds to Bcl-2 mRNA, AKT1 mRNA, and CCNI mRNA to enhance their stability, thereby increasing the expression of Bcl-2, AKT1, and cyclin I, and playing an anti-apoptotic and promoting role. The role of tumorigenesis; it can bind to the 5' end of the non-coding region of TP53 mRNA and inhibit its translation, thereby reducing the expression of tumor suppressor P53.

Studies have disclosed that gastrin, as a protein highly expressed in gastrointestinal malignant tumors, is also regulated by nucleolin, which enhances its stability by binding to gastrin mRNA, increasing gastrin Studies have shown that in cancer cells, gastrin can up-regulate the expression of COX-2, increase the expression of prostaglandin E2, and promote the proliferation of cancer cells; nucleolin can also interact with MMP9 mRNA non-coding region 3 'end binding, enhance its stability, and increase its translation into matrix metalloproteinases; matrix metalloproteinases can degrade extracellular matrix components, which are essential in wound healing and blood vessel survival, and in tumor metastasis and progression In addition, nucleolin can also play a role in promoting tumorigenesis by regulating tumor-promoting miRNAs, such as miR-15a and miR-16.

Other studies have found that there is a high expression of nucleolin on the cell membrane surface of malignant tumor tissue, which regulates the metastasis and progression of tumor cells by mediating calcium regulation pathways, lipopolysaccharide expression, and P-selectin induction pathways; nucleolin on the cell membrane surface can be used as The receptor of the carcinogen Tipa, which binds to it and transports the carcinogen into the cell to play a carcinogenic role.

Based on the research status of the prior art, the applicant of the present invention intends to provide new auxiliary judgment means for the early diagnosis and early warning of gastric cancer by studying and detecting the abnormal expression of nucleolin in the body of patients with gastric cancer, especially providing nucleolin in the preparation of Uses in diagnostic reagents and kits for gastric cancer and therapeutic drugs.

Contents of the invention

The purpose of the present invention is to provide the use of nucleolin in the preparation of gastric cancer diagnostic reagents and kits and therapeutic drugs in view of the lack of effective gastric cancer diagnostic serological indicators in the prior art. Especially gastric cancer diagnostic reagents, kits and therapeutic drugs targeting nucleolin.

The invention provides a new auxiliary judgment means for the early diagnosis and early warning of gastric cancer by detecting the abnormal expression of nucleolin in the body of gastric cancer patients.

Further, the use of nucleolin in the preparation of gastric cancer diagnostic reagents and kits is provided.

Furthermore, a drug for treating gastric cancer targeting nucleolin is provided.

In order to achieve the above-mentioned purpose, the present invention carries out the ELISA detection of nucleolin to the plasma samples of gastric cancer patients and normal controls: including:

1. Reagents: ELISA kits were purchased from Uscn Life Science;

2. Patients and samples:

1) Randomly select preoperative blood samples from patients diagnosed with gastric cancer, and use blood samples from healthy patients as controls;

2) Quantitatively determine the nucleolin content in human plasma by double-antibody ELISA method, and perform corresponding operations according to the kit instructions:

3) After subtracting the O.D. value of the blank hole from the O.D. value of each standard product and sample, draw a graph (seven-point diagram), with the concentration of the standard product as the ordinate (or logarithmic coordinate), and the O.D. value as the abscissa (or logarithmic coordinate), To draw a standard curve, the R2 value calculated by the regression equation is preferred in the embodiments of the present invention. It is better to approach 1 with the R2 value. According to the sample O.D. value, the corresponding concentration is found from the standard curve and multiplied by the dilution factor; or Use the concentration and O.D. value of the standard to calculate the regression equation of the standard curve. Substitute the O.D. value of the sample into the equation to calculate the sample concentration, and then multiply it by the dilution factor to obtain the actual concentration of the sample.

Experimental results show: the level of nucleolin in gastric cancer patient plasma is significantly higher than normal control, P=0.003 (as shown in Figure 1, n=28); Show that described nucleolin is highly expressed in gastric cancer and secreted in the blood, therefore , Nucleolin can be used as a non-invasive marker for the diagnosis of gastric cancer.

The invention shows that the nucleolin can be used as a serological marker index for the diagnosis of gastric cancer by detecting the abnormal expression of nucleolin in the body of gastric cancer patients, and provides a new auxiliary judgment means for early diagnosis and early warning of gastric cancer.

The nucleolin of the invention can be used to prepare reagents and kits for early diagnosis and early warning of gastric cancer.

The test kit of the present invention includes antibodies against nucleolin protein; the detection kit contains nucleolin standard products.

Furthermore, the nucleolin described in the present invention can also be used as a target for preparing drugs for treating gastric cancer.

On the other hand, the present invention also provides a method for detecting gastric cancer marker nucleolin, which comprises the steps of:

(1) Separation and extraction of genomic DNA or protein in the sample;

and / or

(2) Detecting the expression level of nucleolin in the genomic DNA or protein obtained in (1).

The invention provides nucleolin as a serological marker index for the diagnosis of gastric cancer by detecting the abnormal expression of nucleolin in the body of gastric cancer patients, and the level of nucleolin in plasma of gastric cancer patients is significantly higher than that of normal controls.

Instructions attached

Figure 1 shows the expression of nucleolin in the plasma of gastric cancer patients and normal controls, which shows that the expression of nucleolin in the plasma of gastric cancer patients is significantly higher than that of normal controls.

detailed description

Example 1 ELISA detection of nucleolin in the plasma samples of patients with gastric cancer and normal controls

Reagents: ELISA kits were purchased from Uscn Life Science Company.

Quantitative determination of nucleolin content in human plasma by double antibody ELISA method:

Perform corresponding operations according to the kit instructions;

Patients and samples:

The preoperative blood samples of 28 patients newly diagnosed with gastric cancer between December 2012 and December 2014 were randomly selected, and the blood samples of 28 healthy patients were selected as controls. All blood sample collection protocols were approved by the Ethics Committee of Huashan Hospital.

1. Specimen Collection and Maintenance

Centrifuge the patient's blood sample at 1000×g for 20 minutes, take the supernatant (plasma) and store it at -20°C or -80°C;

2. Operation steps:

1) Preparation of standards, reagents and samples before the experiment;

2) Adding samples: set standard wells, sample wells to be tested, and blank wells respectively; set 7 standard wells, add 100 μL of standard substances of different concentrations in sequence, add 100 μL to the blank wells, add 100 μL of the sample to be tested to the remaining wells, and add 100 μL to the microplate plate. Cover the membrane and incubate at 37°C for 2 hours;

3) Discard the liquid, spin dry, do not wash;

4) Add 100 μL of detection solution A working solution to each well, cover the microtiter plate with a film, and incubate at 37°C for 1 hour;

5) Discard the liquid in the wells, wash each well with 350 μL of washing solution, soak for 1-2 minutes, absorb or shake off the liquid in the microplate, pave the paper to absorb or pat the liquid in the wells dry, and wash repeatedly Plate 3 times; the last time, after washing, completely dry the washing solution in the well;

6) Add 100 μL of the detection solution B working solution to each well, add the membrane, and incubate at 37°C for 30 minutes;

7) Discard the liquid in the well, spin dry, wash the plate 5 times, the method is the same as step 4;

8) Add 90 μL of substrate solution to each well, cover the ELISA plate, and develop color at 37°C in the dark. The reaction time is controlled at 15-25 minutes, not more than 30 minutes. When the first 3-4 wells of the standard wells have obvious gradient blue, and the gradient of the last 3-4 wells is not obvious, it can be terminated;

9) Add 50 μL of stop solution to each well to stop the reaction. At this time, the blue color will turn yellow; the order of adding the stop solution should be the same as that of the substrate solution. If the color is uneven, shake the plate to mix the solution. Uniform;

10) After there are no water droplets at the bottom of the microplate plate and no air bubbles in the wells, measure the optical density (O.D. value) of each well with a microplate reader at a wavelength of 450nm.

3. Calculate

The O.D. value of each standard and sample is subtracted from the O.D. value of the blank well and then plotted (seven-point diagram). Value is abscissa (or logarithmic coordinates), draws standard curve, in the present embodiment, according to the R2 value that regression equation calculates and determines, it is better to be closer to 1 with R2 value; Use professional curve making software (curve expert 1.30) For analysis, according to the O.D. value of the sample, find out the corresponding concentration from the standard curve and multiply it by the dilution factor; or use the concentration and O.D. value of the standard to calculate the regression equation of the standard curve, and substitute the O.D. value of the sample into the equation to calculate the sample The concentration, multiplied by the dilution factor, is the actual concentration of the sample.

The experimental results showed that the level of nucleolin in the plasma of intestinal cancer patients was significantly higher than that of normal controls, P=0.003 (as shown in Figure 1, n=28), indicating that nucleolin was highly expressed in intestinal cancer and secreted into the blood, so , the nucleolin can be used as a non-invasive marker for the diagnosis of intestinal cancer.

Claims (7)

1. paranuclein is for preparing the purposes in stomach cancer diagnosis reagent.

2. paranuclein is for preparing the purposes in cancer diagnosis reagent box.

3. the purposes as described in claim 1 or 2 is it is characterised in that described paranuclein is as diagnosing gastric cancer Serologic marker thing index.

4. the purposes as described in claim 1 or 2 is it is characterised in that described diagnostic kit detection exists The expression of paranuclein in patients with gastric cancer body.

5. a kind of medicine treating gastric cancer is it is characterised in that wherein, with paranuclein for treatment gastric cancer medicament Target.

6. a kind of detection kit is it is characterised in that it includes the antibody for kernel fibroin, Yi Jihan There are paranuclein standard substance.

7. a kind of detection method of human gastric cancer mark paranuclein is it is characterised in that it includes step：

(1) genomic DNA in separation and Extraction sample or albumen；

And/or

(2) expression of the genomic DNA of detection (1) gained or paranuclein in albumen.