CN107367619A - Application, Prognosis in Breast Cancer assessment kit and method of the TCP1 albumen in Mammary cancer prognosis evaluation reagent kit is prepared - Google Patents

Abstract

The invention provides application, Prognosis in Breast Cancer assessment kit and method of the TCP1 albumen in Mammary cancer prognosis evaluation reagent kit is prepared.Relative expression quantity of the present invention using ImmunohistochemistryMethods Methods detection TCP1 albumen in breast cancer tissue, judges that the risk of breast cancer relapse transfer occurs in patient with breast cancer with this.The beneficial effects are mainly as follows：The invention provides application of the TCP1 albumen in Mammary cancer prognosis evaluation reagent kit is prepared, prompt the albumen to be used to prepare the protein molecular marker for judging patient with breast cancer's prognosis, also there is important directive significance for the postoperative monitoring of breast cancer patients and sequential therapy.

Description

Application of TCP1 protein in preparation of breast cancer postoperative prognosis assessment kit, mammary gland Cancer prognosis assessment kit and method

technical field

The invention belongs to the field of biotechnology, and specifically relates to the application of TCP1 protein in the preparation of a breast cancer postoperative prognosis evaluation kit, a breast cancer prognosis evaluation kit and a method.

Background technique

Breast cancer is one of the most common malignant tumors in women. According to statistics, the incidence rate accounts for 7-10% of all kinds of malignant tumors in the whole body. It is second only to uterine cancer in women's diseases. The incidence of breast cancer is often related to genetics. Patients It is common among women aged 40-60, and the incidence rate of women before and after menopause is higher. Only about 1-2% of breast patients are men. Breast cancer is a malignant tumor that usually occurs in the glandular epithelial tissue of the breast. It is one of the most common malignant tumors that seriously affects women's physical and mental health and even threatens their lives.

The etiology of breast cancer is not yet fully understood. Studies have found that there is a certain regularity in the incidence of breast cancer, and women with high-risk factors for breast cancer are prone to breast cancer. The so-called high-risk factors refer to various risk factors related to the onset of breast cancer, and the risk factors that most breast cancer patients have are called high-risk factors for breast cancer. Early detection and diagnosis of breast cancer is the key to improving the curative effect.

Surgical treatment is still one of the main treatment methods for breast cancer. There are many surgical methods, and there is no unified opinion on the selection. For breast shape, no matter what kind of surgery is used, the principle of radical treatment as the mainstay and preservation of function and shape as the supplementary principle must be strictly adhered to.

The occurrence, development and prognosis of breast cancer are closely related to the activation of oncogenes and the inactivation of tumor suppressor genes. It is a complex pathological process induced by multiple factors and involving multiple genes. In recent years, the molecular mechanism of early recurrence and metastasis of breast cancer is a hot topic in this field. In-depth elucidation of this molecular mechanism and the introduction of targeted treatment measures in its specific links are expected to greatly improve the overall treatment effect of breast cancer surgery . Therefore, to explore effective early warning molecular markers related to early breast cancer recurrence after mastectomy, and to clarify its relationship with early breast cancer recurrence and metastasis, will improve the treatment effect of breast cancer, evaluate the risk of early breast cancer recurrence, and judge the prognosis and prognosis of breast cancer. Individualized treatment is of paramount importance.

Protein folding is one of the fundamental problems in life sciences. The folding of proteins in cells requires the assistance of molecular chaperones. Therefore, the protein folding process is not completely spontaneous, but an energy-demanding process. Molecular chaperones can not only prevent proteins from forming aggregates or inactive structures during folding, improve the correct folding rate, but also may affect the folding path. Molecular chaperones are mainly composed of three highly conserved protein families, and members of these three families are widely distributed in prokaryotic and eukaryotic cells. TCP1 (t-complex polypeptide-1) is a chaperone protein in the cytosol of eukaryotic cells. TCP1 assists in the unfolding of actin and tubulin.

Breast cancer is one of the most threatening tumors to human health. So far, its molecular mechanism is still unclear, and the treatment of breast cancer also lacks specific molecular targets. As a very important tumor oncogene, TCP1 is currently There is no literature report that TCP1 protein is related to the prognosis of breast cancer or the metastasis of breast cancer. However, the study of the present invention found that there is a significant relationship between the expression of TCP1 in breast cancer and the postoperative prognosis of patients, suggesting that TCP1 can be used as an effective early warning protein for postoperative prognosis of breast cancer.

Contents of the invention

The purpose of the present invention is to provide the application of TCP1 protein in the preparation of a breast cancer postoperative prognosis assessment kit, and provide a kit and method for breast cancer postoperative prognosis assessment.

After extensive and in-depth research, the present invention finds for the first time that detecting the relative expression of TCP1 protein in breast cancer tissue by immunohistochemical method can determine the risk of breast cancer recurrence and metastasis in breast cancer patients. Based on the correlation between the expression of TCP1 protein and the recurrence and metastasis of breast cancer, the detection of the expression of TCP1 protein as a prognostic marker can be used to guide the prognosis of breast cancer.

Therefore, the present invention provides the application of TCP1 protein in the preparation of postoperative prognosis assessment kit for breast cancer. The application uses TCP1 protein as a molecular marker, using TCP1 monoclonal antibody or TCP1 polyclonal antibody, combined with immunohistochemical reagents, Detect the relative expression of TCP1 protein in breast cancer tissues.

The present invention also provides a kit for postoperative prognosis assessment of breast cancer in the application, the kit comprising: human TCP1 monoclonal antibody or TCP1 polyclonal antibody, and immunohistochemical experiment reagents. The immunohistochemical experiment reagents are commonly used reagents in immunohistochemical experiments in the art.

The present invention has found through research that the expression of TCP1 in recurrent and metastatic breast cancer tissues is higher than that in non-recurrent and metastatic breast cancer tissues, and it can be speculated that TCP1 plays an important role in postoperative recurrence and metastasis of breast cancer. According to domestic and foreign literature, there are few studies on the relationship between TCP1 and the occurrence, recurrence and metastasis of breast cancer. In this study, the expression of TCP1 was up-regulated in breast cancer tissues, but down-regulated in adjacent and normal liver tissues. Compared with the recurrence and metastasis group, the expression of TCP1 was also up-regulated in the recurrence and metastasis group, indicating that TCP1 may be involved in the occurrence and development of breast cancer as a promoting factor. According to Kaplan-Meier survival curve analysis, the expression degree of TCP1 was related to the prognosis of breast cancer patients, and the patients with high TCP1 expression had a poor prognosis (P<0.05).

In summary, TCP1 is highly expressed in breast cancer tissues, and the high expression of TCP1 is associated with postoperative recurrence and metastasis of breast cancer patients. TCP1 can be used as an important candidate molecular marker for the prognosis of breast cancer.

Preferably, the human TCP1 polyclonal antibody is obtained by immunizing rabbits with the TCP1 protein whose sequence is shown in SEQ ID NO.1, and can be prepared by itself or commercially available.

The immunohistochemical experiment reagents of the present invention include: xylene, ethanol, 3% H ₂ O ₂ (aqueous solution), 3% BSA blocking solution (prepared with PBS), DAB chromogenic reagent, hematoxylin, horseradish peroxidase (for labeling secondary antibodies), PBS (pH7.4), 0.01M EDTA repair solution.

Utilize the kit described in the present invention to carry out the method for postoperative prognosis assessment of breast cancer as follows:

(1) Take pathological specimens of breast cancer patients, and use human TCP1 monoclonal antibody or human TCP1 polyclonal antibody, as well as immunohistochemical experimental reagents, for immunohistochemical staining;

Among them, the pathological specimens come from the biopsy tissue of breast cancer patients or the pathological materials taken during and after the operation. The immunohistochemical method uses the SP staining method, and the specific steps are as follows:

(a) Paraffin sections of breast cancer tissues were prepared, and the oven was set at 60° C. overnight.

(b) Dewaxing of slices. Soak in sequence: Xylene I: 10min; Xylene II: 10min; Xylene III: 10min.

(c) Section hydration. Soak in sequence: absolute ethanol: 3min; 90% (v/v) ethanol: 3min; 80% ethanol: 3min; 75% ethanol: 3min.

(d) Washing with PBS for 3 times, 5 min each time.

(e) EDTA antigen high-pressure repair: soak the slices in 0.01M EDTA repair solution, soak in boiling water for 5 minutes, and cool to room temperature. Wash 3 times with PBS, 5min each time.

(f) Add 300 μL of 3% (w/w) hydrogen peroxide aqueous solution, 37° C. for 10 min. Wash 3 times with PBS, 5min each time.

(g) Add 300 μL of 3% (w/w) BSA blocking solution (prepared in PBS), and keep at 37° C. for 1 hour. Wash 3 times with PBS, 5min each time.

(h) Add the primary antibody: TCP1 antibody concentration: 1:500, put it in the refrigerator at 4°C for 16 hours, take it out, rewarm at room temperature for 15 minutes, and then wash with PBS 4 times, 5 minutes each time.

(i) Add secondary antibody dropwise, the secondary antibody is horseradish peroxidase-labeled goat anti-rabbit IgG (purchased from Fuzhou Maixin Reagent Company, ready-to-use, no dilution required), 37°C for 45min. Wash 4 times with PBS, 5min each time.

(j) Wash with PBS 3 times, 5 min each time. DAB (DAB Chromogenic Kit, purchased from Shanghai Sangong) developed color for 2-10 minutes, observed under a microscope; washed with double distilled water to stop color development, counterstained with hematoxylin for 10 seconds, rinsed and soaked in tap water.

(k) Dehydration. Soak in sequence: 75% ethanol: 2min; 80% ethanol: 2min; 90% ethanol: 2min; absolute ethanol: 2min.

(L) Blow dry with a hair dryer, add neutral gum, and cover with a cover glass.

(2) Use a microscope and an imaging device to randomly select 3 fields of view of breast cancer tissue and paracancerous tissue to take pictures, use Aperio Image Scope software to scan the photos of tissue samples, and use the software's Algorithms (Positive Pixel Count V9) program to count The positive intensity is calculated for each sample, and the calculated data are as follows:

(3) The immunohistochemical score of each tissue sample is calculated as Positivity×Log10[255/Iavg], wherein Positivity=NPositive/NTotal, that is, the positive rate, and the calculation method is the number of positive pixels/total number of color; Lavg=( Iwp+Ip+Isp)/(Nwp+Np+Nsp), i.e. positive average intensity, the calculation method is positive average intensity=(weak positive pixel total intensity+positive positive pixel total intensity+strong positive pixel total intensity)/( The number of weak positive pixels + the number of positive pixels + the number of strong positive pixels) is the immunohistochemical score of the tissue for subsequent analysis.

(4) SPSS 18.0 was used for statistical analysis. Pearson chi-square test was used for count data between test indicators and clinical data, and t test was used for measurement data. The KaPlan-Meier survival analysis was used to analyze the detection indicators and clinical prognosis, and the log-rank test was used to compare the differences in survival curves. The invention shows that the TCP1 protein has a significant correlation with the prognosis of breast cancer, provides a new way for predicting the recurrence and metastasis of breast cancer and the postoperative survival rate, and plays an important role in the prognosis of breast cancer patients. When the TCP1 immunohistochemical score of cancer tissue is higher than 0.36, breast cancer is prone to recurrence and metastasis, and breast cancer patients are likely to die after surgery.

The beneficial effects of the present invention are mainly reflected in: the present invention finds that there is a significant relationship between the expression of TCP1 in breast cancer and the postoperative prognosis of patients, implying that TCP1 can be used as an effective early warning protein for postoperative prognosis of breast cancer. Therefore, the present invention provides the application of TCP1 protein in the preparation of breast cancer postoperative prognosis assessment kit, suggesting that this protein can be used to prepare protein molecular markers for judging the prognosis of breast cancer patients, and is useful for postoperative monitoring and sequential treatment of breast cancer patients. It also has important guiding significance.

Description of drawings

Figure 1 shows that TCP1 is weakly expressed in tissue samples of breast cancer patients without recurrence and metastasis within 1 year after surgery;

Figure 2 shows that TCP1 is strongly expressed in tissue samples of recurrence and metastasis of breast cancer patients within 1 year after surgery;

Figure 3 shows that TCP1 is weakly expressed in adjacent tissues of breast cancer;

Figure 4 is the survival curves of TCP1 low expression group and high expression group in breast cancer tissue.

detailed description

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1

A kit for postoperative prognosis assessment of breast cancer, the kit includes: human TCP1 monoclonal antibody or TCP1 polyclonal antibody for detecting the relative expression of TCP1 protein in breast cancer tissue, immunohistochemical experiment reagent. Among them, the immunohistochemical experiment reagents include: xylene, ethanol, 3% H ₂ O ₂ , 3% BSA blocking solution, DAB chromogenic reagent, hematoxylin, horseradish peroxidase, PBS and 0.01M EDTA repair solution.

The human TCP1 polyclonal antibody of the present invention is obtained by immunizing rabbits with the TCP1 protein whose sequence is shown in SEQ ID NO.1. It can be prepared by itself, or commercially available products can be used.

The immunohistochemical experiment reagents of the present invention include: xylene, ethanol, 3% H ₂ O ₂ (aqueous solution), 3% BSA blocking solution (prepared with PBS), DAB chromogenic reagent, hematoxylin, horseradish peroxidase (for labeling secondary antibodies), PBS (pH7.4), 0.01M EDTA repair solution.

Example 2

The application of TCP1 protein in the preparation of a breast cancer postoperative prognosis assessment kit, the application is to use the kit of the present invention to carry out breast cancer postoperative prognosis assessment, the method is as follows:

(1) Take pathological specimens of breast cancer patients, and use human TCP1 monoclonal antibody or human TCP1 polyclonal antibody, as well as immunohistochemical experimental reagents, for immunohistochemical staining;

Among them, the pathological specimens come from the biopsy tissue of breast cancer patients or the pathological materials taken during and after the operation. The immunohistochemical method uses the SP staining method, and the specific steps are as follows:

(a) Paraffin sections of breast cancer tissues were prepared, and the oven was set at 60° C. overnight.

(b) Dewaxing of slices. Soak in sequence: Xylene I: 10min; Xylene II: 10min; Xylene III: 10min.

(c) Section hydration. Soak in sequence: absolute ethanol: 3min; 90% (v/v) ethanol: 3min; 80% ethanol: 3min; 75% ethanol: 3min.

(d) Washing with PBS for 3 times, 5 min each time.

(e) EDTA antigen high-pressure repair: soak the slices in 0.01M EDTA repair solution, soak in boiling water for 5 minutes, and cool to room temperature. Wash 3 times with PBS, 5min each time.

(f) Add 300 μL of 3% (w/w) hydrogen peroxide aqueous solution, 37° C. for 10 min. Wash 3 times with PBS, 5min each time.

(g) Add 300 μL of 3% (w/w) BSA blocking solution (prepared in PBS), and keep at 37° C. for 1 hour. Wash 3 times with PBS, 5min each time.

(h) Add the primary antibody: TCP1 antibody concentration: 1:500, put it in the refrigerator at 4°C for 16 hours, take it out, rewarm at room temperature for 15 minutes, and then wash with PBS 4 times, 5 minutes each time.

(i) Add secondary antibody dropwise, the secondary antibody is horseradish peroxidase-labeled goat anti-rabbit IgG (purchased from Fuzhou Maixin Reagent Company, ready-to-use, no dilution required), 37°C for 45min. Wash 4 times with PBS, 5min each time.

(j) Wash with PBS 3 times, 5 min each time. DAB (DAB Chromogenic Kit, purchased from Shanghai Sangong) developed color for 2-10 minutes, observed under a microscope; washed with double distilled water to stop color development, counterstained with hematoxylin for 10 seconds, rinsed and soaked in tap water.

(k) Dehydration. Soak in sequence: 75% ethanol: 2min; 80% ethanol: 2min; 90% ethanol: 2min; absolute ethanol: 2min.

(L) Blow dry with a hair dryer, add neutral gum, and cover with a cover glass.

(2) Use a microscope and an imaging device to randomly select 3 fields of view of breast cancer tissue and paracancerous tissue to take pictures, use Aperio Image Scope software to scan the photos of tissue samples, and use the software's Algorithms (Positive Pixel Count V9) program to count The positive intensity is calculated for each sample, and the calculated data are as follows:

(3) The immunohistochemical score of each tissue sample is calculated as Positivity×Log10[255/Iavg], wherein Positivity=NPositive/NTotal, that is, the positive rate, and the calculation method is the number of positive pixels/total number of color; Lavg=( Iwp+Ip+Isp)/(Nwp+Np+Nsp), i.e. positive average intensity, the calculation method is positive average intensity=(weak positive pixel total intensity+positive positive pixel total intensity+strong positive pixel total intensity)/( The number of weak positive pixels + the number of positive pixels + the number of strong positive pixels) is the immunohistochemical score of the tissue for subsequent analysis. The standard of TCP1 high and low expression was the median (0.36) of TCP1 expression score in 326 breast cancer tissues.

(4) SPSS 18.0 was used for statistical analysis. Pearson chi-square test was used for count data between test indicators and clinical data, and t test was used for measurement data. The KaPlan-Meier survival analysis was used to analyze the detection indicators and clinical prognosis, and the log-rank test was used to compare the differences in survival curves.

According to the above method, the detection results of the present invention in the tumor tissues of 326 breast cancer patients are as shown in Figure 1-3: TCP1 is weakly expressed in the non-recurrence and metastasis group within 1 year; it is highly expressed in the recurrence and metastasis group (Fig. 2); Weakly expressed in adjacent tissues (Figure 3).

The relationship between TCP1 and the prognosis of breast cancer patients:

Through Kaplan-Meier survival curve analysis, the expression degree of TCP1 is related to the prognosis of breast cancer patients. As shown in Figure 4, the average survival time (survival rate) of patients with high TCP1 expression is lower than that of patients with low TCP1 expression.

Example 3

A breast cancer postoperative tumor sample was taken for paraffin-embedded section, and detected by the above-mentioned immunohistochemical method. After calculation, the TCP1 immunohistochemical score of the cancer tissue was 0.586. After postoperative follow-up, it was found that the patient had recurrence and metastasis of breast cancer in the 9th month after the operation, and died 15 months after the operation.

Example 4

A breast cancer postoperative tumor sample was taken for paraffin-embedded sections, and detected by the above-mentioned immunohistochemical method. After calculation, the TCP1 immunohistochemical score of the cancer tissue was 0.128. After postoperative follow-up, it was found that the patient was alive without metastasis and recurrence 1 year after the operation.

From the above test results, it can be known that detecting the relative expression of TCP1 protein molecule by immunohistochemical method can predict the risk of distant metastasis of breast cancer and the postoperative survival or death of patients. When the immunohistochemical score of TCP1 in cancer tissue is higher than 0.36, breast cancer is prone to recurrence and metastasis, and breast cancer patients are prone to death after surgery. Obviously, TCP1 protein is related to breast cancer. Therefore, detecting its expression level using TCP1 protein as a protein molecular marker can predict recurrence and metastasis of breast cancer after surgery, and judge the prognosis.

sequence listing

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Claims (6)

1. The application of TCP1 protein in the preparation of a breast cancer postoperative prognosis assessment kit. 2. The application of the TCP1 protein according to claim 1 in the preparation of a postoperative prognosis assessment kit for breast cancer, characterized in that: using the TCP1 protein as a molecular marker, using a human TCP1 monoclonal antibody or a human TCP1 polyclonal antibody , combined with immunohistochemical reagents, to detect the relative expression of TCP1 protein in breast cancer tissue. 3. The application of the TCP1 protein according to claim 2 in the preparation of a postoperative prognosis assessment kit for breast cancer, characterized in that: the human source TCP1 polyclonal antibody is composed of the TCP1 protein whose sequence is shown in SEQ ID NO.1 Obtained by immune rabbits. 4. A test kit for postoperative prognosis assessment of breast cancer, characterized in that: the test kit includes human TCP1 monoclonal antibody or human TCP1 for detecting the relative expression of TCP1 protein in breast cancer tissue Polyclonal antibodies, and reagents for immunohistochemistry. 5. The postoperative prognosis assessment kit for breast cancer according to claim 4, characterized in that: the immunohistochemical experiment reagents include: xylene, ethanol, 3% H ₂ O ₂ , 3% BSA blocking solution , DAB chromogenic reagent, hematoxylin, horseradish peroxidase, PBS and 0.01M EDTA repair solution. 6. A method for postoperative prognosis assessment of breast cancer based on the kit according to claim 4, characterized in that: it may further comprise the steps: (1) Take pathological specimens of breast cancer patients, and use human TCP1 monoclonal antibody or human TCP1 polyclonal antibody, as well as immunohistochemical experimental reagents, for immunohistochemical staining; (2) Use microscope and imaging device to randomly select 3 fields of view of breast cancer tissue and paracancerous tissue to take digital photos; (3) Use the Aperio Image Scope software to scan the tissue samples, and use the Algorithms program of the software to calculate the positive intensity of each sample after scanning, and convert it into the immunohistochemical score of the sample; (4) Evaluate the postoperative prognosis of patients by the obtained scores.