| Background:With high malignant degree, difficulty of early diagnosis, poor drug therapy effect and high mortality rate, Gallbladder adenocarcinoma is the most common bile duct system malignant tumor. The joint action of a multiple genes leads to the occurrence and development of Gallbladder adenocarcinoma. Continuous research in the various involved cancer genes and tumor-suppressor genes, and search for the more effective methods to early diagnose, treat and judge prognosis on gallbladder adenocarcinoma have become hot topics nowadays.More and more data show that the abnormality of cell apoptosis, just like malignant cell proliferation and differentiation hindering, plays an important part in cancer development. Normally, apoptosis can destroy DNA-damaged cells or cycle-disordered cells. The proliferation rate of tumor cells is not always higher than that of normal cells, which shows that escaping apoptosis helps the accumulation of tumor cells. The disorder of regulation in apoptosis may prolong the survival of cells aberrantly, being subject to accumulation of mutation which leads to transformation, and thereby involves in carcinogenesis. Therefore, tumor is not only due to the disorder of cell proliferation and differentiation but also owing to the abnormality of cell apoptosis.Livin and Survivin are the most effective inhibitor factors up to now. Survivin and Livin were newly discovered the members of inhibitor of apoptosis protein family and play important role in the regulation of apoptosis. As the novel apoptosis inhibitory proteins, Survivin and Livin play the important role in the regulation of apoptosis. Survivin and Livin were highly expressed in human embryonic tissues and involved in tissue differentiation and organ formation during embryonic development, had not detected in terminally differentiated adult tissues but widely expressed in a variety of tumor tissue. Research shows that they were strong expressed in many solid tumors such as lung cancer, colorectal cancer, pancreatic cancer, prostate cancer and gastrointestinal cancer and hematopoietic malignancies.In recent years, the researches of Livin and Survivin in digestive tumors have made great progress, but study on gallbladder cancer was few, especially the research about Livin and gallbladder adenocarcinoma had not been reported.Objective:This paper applied the method of immunohistochemical to detect expressions of Livin, Survivin and Ki-67in gallbladder adenocarcinoma organization, and discussed and analyzed the relationship between the patient’s clinical features. Methods:1. Collecting specimen:All specimens were selected from Guangzhou military area commands Wuhan general hospital during2007and2010, and only those that after surgical resection pathological diagnosis were clear, and preoperative did not do radiation and chemotherapy were chosen to be preserved file organization of wax. In the chosen40patients with gallbladder adenocarcinoma, there were11male cases,29female patients,17cases of less than55years old,23cases of more than or equal to 55years old,16cases of cancer<2cm in diameter and24cases of cancer≥2cm in diameter.TNM staging:4cases in Ⅰ preiod,13in Ⅱ period,11cases Ⅲ period, and12in Ⅳ period.25patients have stone,15cases of no stone. Pathology classification:senior differentiation6cases, intermediate differentiation10cases, low differentiation24cases; Lymph node metastasis,19negative cases while21positive cases. Random20cases for the same period the gallbladder surgery to remove polyps of the gallbladder organization (to take the2cm disease extent and pathology without inflammation, im and proliferative lesion) as a control group. This experiment selection of the cases had made the patient and the family’s informed consent, and informed agreement signed.2. HE experimental method:Paraffin wax specimen were sliced with4u m thickness, the slice contained gallbladder mucous epithelial, submucosal interstitium, muscle layer. HE dyeing and immunohistochemical staining were performed. HE dyeing:line gradient wax lost and hydration, sappanwood staining5min, then red dyeing2min, conventional dehydration, transparent, sealing piece.3. Immunohistochemical experimental method:After gradient wax lost and hydration, the slice antigen was repaired by heat, then cooled to room temperature after washing5min, and3%hydrogen peroxide incubation to block the endogenous peroxidase; Phosphate buffer (PBS) washed2min with a total of three times; Add nonimmunologic animal serum to block nonspecific binding,4℃moisturizing, overnight; PBS wash5min, a total of three times, adding second antibody, keep in room temperature for30min; PBS washed2min, a total of three times, and then stained with DAB, water flushing for5min, sappanwood restain, step-down dehydration, transparent, and then sealing with neutral gum.4. Immunohistochemical image analysis:The expression of Livin and Survivin in the organization in the gallbladder judgment in cytoplasm and (or) the nucleus appear tan dyeing as positive, Ki-67positive expressed as the nucleus dyed brown color. After immunohistochemical stains success, through the HMIAS-2000high-resolution colour medical graphic analysis system observation, every example each index in tumor randomly selected area five at high magnification, the vision (x400) gathering pictures, application immunohistochemical measurement procedures measure three index Livin, Survivin and Ki-67specific express parameters. The average unit values positive (PU) selected to compare the specimens of positive expression differences in this experiment.5. Statistical analysis:The measured results of clinical data and establish database, SPSS13.0statistics software package was used for the statistical data processing. The differences between groups Compared by independent T test samples, and the connections between indexes by Pearson correlation analysis. P<0.05, the difference was statistically significant.Results:1. The expresses of Livin, Survivin and Ki-67in adenocarcinoma and normal tissue of the gallbladderThe expression of Livin protein in the gallbladder tissue:Livin show a strong positive expression in gall bladder adenocarcinoma specimens, and was mainly distributed in the nucleus week’s cytoplasm. These uniform tan particles were in a shape of diffuse distribution except for few in the shape of focal distribution. The positive unit value of Livin in gall bladder adenocarcinoma is40.89±11.20, but in normal tissues in the gallbladder only28.32+6.08, and the difference was statistically significant (P<0.01);The expression of Survivin protein in gallbladder tissue:in gallbladder adenocarcinoma of the specimen Survivin positive expression, was mainly distributed in the cell plasma, the nucleus and cell membrane accidentally visible dyeing. Gallbladder adenocarcinoma of the unit value of positive Survivin38.90±7.97, and the normal gallbladder25.12±5.54, and the significant difference was a statistically significant (P<0.01);The expression of Ki-67protein in gallbladder tissue:of all the specimens were visible in Ki-67express positive signal, mainly distributed in the cell nucleus, visible tan particles diffuse distribution. Ki-67in gall bladder adenocarcinoma of the unit value of positive43.25±12.42than normal gallbladder group27.30±3.40, the difference was statistically significant (P<0.01).2. The relationship between the expressions of Livin, Survivin and Ki-67in gallbladder adenocarcinoma tissue and clinical pathology characteristicThe relationship between the expressions of Livin, Survivin, Ki-67and gender and age of patients, tumor size, degrees of differentiation, and presence of stones was of no statistically significant correlation (P>0.05). The expressions of Livin, Survivin, Ki-67were related with the stages of tumor TNM, the positive expression of patients in Ⅲ period was higher (P<0.05); The expressions in positive patients of Lymph node metastasis were higher than those in the patients without the transfer of Lymph node metastasis significantly higher (P<0.01).3. The correlation analysis between the expressions of Livin, Survivin and Ki-67in gall bladder adenocarcinoma organizationThe relation between the expressions of Survivin and Livin were significantly positive (Pa=0.006), that if a patient showed high positive expression of Livin, his expression of Survivin was high also, and vice versa. The expressions of Ki-67were positively related with that of Livin and Survivin (Pb=0.000, Pc=0.001), which suggested the corresponding high expressions of Livin and Survivin in the patients with the high cell proliferation activity.Conclusion:The results showed that the positive expression of Livin in the gallbladder adenocarcinoma obviously stronger than that in the normal gallbladder organization and the positive expression of Survivin protein in gall bladder adenocarcinoma was stronger than that in the normal gallbladder wall organization, and the latter was consistent with the most reported documents. It could be inferred that there may be a close relationship between the high expression of Survivin and Livin in gallbladder adenocarcinoma organization and the occurrence and development of adenocarcinoma. Though both were members of apoptosis inhibiting protein, there was a slight difference in expression, as the expression of Livin was stronger than that of Survivin maybe related with the possible Livin RING zinc finger structure about domain.The fact that Livin and Survivin highly expressed in adenocarcinoma organization and they were positively related suggested that there maybe existed some internal contact between the expressions of Livin and Survivin in gallbladder adenocarcinoma and these jointly high expressions promoted the development and occurrence of adenocarcinoma of the gallbladder, which was similar to the cooperative expression of Livin and Survivin in colon cancer. There maybe existed some common signal transduction pathways in the cooperative expression of Livin and Survivin, but the specific mechanism was still unknown and further research was needed.There were statistical differences among groups devided by other different clinicopathological parameters, the patients gender, age of onset, tumor size, differentiation degree and presence of stone were irrelevant (P>0.05), but the tumor TNM staging and lymph node metastasis condition were related, and these expressions were similar to those in other cancer tissue. With the increase of tumor TNM staging, the positive expresses of Survivin and Livin were stronger, which showed that Survivin and Livin played important roles in the development of malignant tumors. The expressions of Survivin and Livin in the bodies of lymph node metastasis positive patients were stronger than those in the patients without lymph node metastasis, which suggested Livin and Survivin were closely related with cancer transfer and their high expressions maybe indicated highly aggressive and strong ability to transfer of cancer.As Ki-67was the protein related with the nuclei and proliferation of cell division, it was selected as reference index to identify malignant degrees of gallbladder.The experimental results showed that the average positive expression of Ki-67in gallbladder adenocarcinoma was higher than that of Livin and Survivin, and the expression of ki-67was closely related with TNM stage and lymph node metastasis of patients and can objectively evaluate the malignant degrees of gallbladder adenocarcinoma. That the expressions of Livin and Survivin and Ki-67were positive related suggested that there was close relationship between the high expressions of Survivin and Livin and high hyperplastic of tumor.To sum up, over-active Survivin and Livin in the gallbladder adenocarcinoma might played an important role in promoting occurrence and development of the gallbladder adenocarcinoma, and their positive expressions were stronger with the increase of TNM classification of tumor and lymph node transfer. Deep research of Survivin and Livin was not only helped to further understand the biological characteristics of gallbladder of adenocarcinoma, but also maybe provided certain basis for gallbladder adenocarcinoma gene therapy targets choice and further clinical treatments. |
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