| Part 1The study of angiogenesis and MVD in cervical lesionsObjective:To investigate the expression of VEGF Protein and microvessel density (MVD) in normal, cervical intraepithelial neoplasia (CIN) and cervical cancer (CC) in tissue sections, compared the VEGF expression in degree of cervical lesions and determined the relationship between microvessel protein density and VEGF. Methods: 40 patients with different degree of cervical lesions as the research group,10 cases of normal cervix women as the control group. Used immunohistochemistry to detect the expression of VEGF protein and microvessel density. Results:1 microvessel density in normal control, CINⅠ-Ⅲand cervical cancer were 22.74±6.34,24.57±3.48,32.66±4.16,43.55±6.31,60.01±12.29. CIN I and normal controls have no significant difference in MVD. Normal control group, CIN I and CIN II, CINⅢ、CC were significantly different (P<0.05), CIN II, CIN III and cervical cancer also has significant differences (P<0.05). From normal to cervical cancer MVD is gradually increasing trend.2 VEGF protein could be detected in different degrees of cervical lesions and normal controls by immunostaining. staining for VEGF in normal is weak positive, but in cervical lesions is mostly strongly positive, and increased and enhanced with the histological grade level. VEGF expression in CIN IⅡ,Ⅲand cervical cancer compared to normal control and CIN I the difference was significant (P<0.05).Conclusions:1 MVD increased from normal controls to CIN、cervical cancer. Angiogenesis exists in cervical lesions,and has an important role in the development of CIN to cervical cancer.2 VEGF expression increased with the degree of cervical lesions gradually increased.3 VEGF expression and MVD was significantly related. The study shows that VEGF was closely related.in angiogenesis in cervical lesionsPart 2The study of rAAV-Endostatin treatment in the model of human cervical cancer by nude mice Objective:To establish human cervical cancer model (hela cell) in nude mice to observe the situation of tumor angiogenesis and to explore the rAAV-Es in the role of tumor treatment. Methods:cervical cancer models of nude mice were established by inoculating hela cell suspension (5×106/ml) subcutaneously into peritoneal surface. Observing subcutaneous tumor growths to 2-3mm after 1 week. Nude mice were randomly divided into 3 groups of 10 each group, injection the 0.1ml of PBS, rAAV, rAAV-Es in/or the nude mice peritumora tumor. After the treatment 4 weeks the mice were killed to remove specimens. We observed the growth of the transplants in nude mice and detect them by immunohistochemical SP method.The endothelial cells or microvessel density(MVD) was tracted by CD31 marker, concentrations of Endostatin and VEGF in nude mice serum were detected by Elisa,and observed changes in nude heart, lung, liver, kidney and other tissue by HE staining. Results: The cervical cancer model in nude mouse was successfully established:tumor formation was 100%.We can see angiogenesis peripheral and central tumor. rAAV-Es group’s the tumor size、tumor weight were lower than the PBS group, rAAV group (P<0.05), and nude body weight was no significant difference among the groups. The positive cells for CD31 were endothelial cells situated inside the vascular wall. Endostatin levels in serum of nude mice detected by Elisa were 133.78±13.09 ng/ ml,130.31±9.96 ng/ml,204.64±25.97 ng/ml, rAAV-Es group was higher than the two control groups, the difference was statistically significant; VEGF concentrations of the three groups were 264.28±49.82 ng/ml,282.36±37.18 ng/ ml,193.03±28.46 ng/ml, rAAV-Es group was lower than the two control groups, the difference was statistically significant. There were no inflammatory、degeneration and necrosis in pathological slices of heart、liver、lung、spleen and kidney of treatment group at 4 weeks after treatment. Conclusions:Cervical cancer model in nude mouse with hela cell is successfully established, observing the angiogenesis by surface of tumor. rAAV-Es gene therapy is safe and reliable in nude mice. Endostatin protein can be detected in blood of mice long-term and stability; inhibit the expression of VEGF to inhibit the tumor growing; Mice’s heart, liver, spleen, kidneys and lungs have not obvious morphological change. The gene therapy method is safe. Part 3Comparative study of endostatin gene therapy mediated by recombinant adeno-associated virus vector on cervical cancer of animal modelObjective:To study the therapeutic effect of recombinant adeno-associated virus cairying human endostatin gene therapy alone or with cisplatin on cervical cancer xenografts in nude mice model. To evaluate the gene therapy’s effectiveness and safety in the treatment of in nude mice model,and discussed the possible mechanism in anti-angiogenesis therapy. Methods:50 cervical cancer models of nude mice were divided into 5 groups treated with rAAV-Es/rAAV-Es+cisplatin or cisplatin alone intraperitoneally; control group including 20 mice injected with rAAV and with phosphate buffered saline(PBS),observing the tumor growing. VEGF, KDR, MMP-2 and microvessel density (MVD) expression were detected by immunohistochemistry in tumor; Endostatin, VEGF expression was tracted by Elisa assay in nude mice serum after treatment; PI3K signaling was detected in tumor expression by situ hybridization(ISH) and for statistical analysis. Results:(1)The endostatin gene was transferred into nude mice successfully and expressed effectively. Alone rAAV-Es/ cisplatin group and the combined treatment of cervical cancer xenografts significantly inhibited the growth of nude mice tumor, it has statistically significant differences between control groups (p<0.05). And the antitumor effect of combined treatment group is most clear. (2) In tumor VEGF, KDR, MMP-2 expression decreased in rAAV-Es group, rAAV-Es+cisplatin and cisplatin group,the result compared with PBS, empty vector control group was statistically significant (p<0.05); In the experiment three groups, the combined treatment group decreased most obviously in expression of three proteins.the rAAV-Es group has no significant difference with cisplatin group. Decreased expression of VEGF and its receptor KDR trend was synchronized. (3) The tumor angiogenesis was significantly inhibited in, rAAV-Es group, rAAV-Es+ cisplatin group and cisplatin group, MVD in rAAV-Es+cisplatin group is smallest statistically significant difference between the groups. (4) Endostatin secretion raised in mice serum in rAAV-Es groups, while the cisplatin group and the control group have no difference (p<0.05). VEGF secretion decreased in rAAV-Es group, rAAV-Es+cisplatin group and cisplatin group, and it decreased more significantly in application of rAAV-Es’groups than in cisplatin group(p<0.05). (5) The expression of PI3K decreased statistically significant in rAAV-Es group, rAAV-Es+cisplatin group and cisplatin group compared with the control group.In the three test groups combined treatment group decreased expression is most obvious, and it’s statistically significant compared rAAV-Es group and cisplatin group. Conclusions:The recombinant adeno-associated virus carrying human endostatin gene therapy could inhibit angiogenesis in cervical cancer of nude mice model. This therapy could significantly inhibited tumor growth and reduce the side effects of cisplatin. We suspect its mechanism may be related to inhibition of angiogenesis factor and its receptor, to reduce the basement membrane degradation. In gene standard endostatin may inhibit the cell signaling pathway PI3K/AKT to induce endothelial cell apoptosis. Through the above mechanism of endostatin induced apoptosis in vascular endothelial cells, made the tumor cells hungry-. promoted cancer cell apoptosis and inhibit metastasis of tumor cells. Antiangiogenic therapy combined with chemotherapy cisplatin enhanced anti-tumor effect and had slightly side effects. The clinical treatment of cervical cancer is expected to become a new way with a antiangiogenic therapy combined with chemotherapy cisplatin. |
PDF Full Text Request