| Objective:This study uses nude mice as experimental models for cervical cancer, to investigate the inhibition and the mechanism of recombinant adeno-associated virus-endostatin(rAAV-ES)on the growth of transplanted tumor of nude mice,through detecting the expressions of Bcl-2, Bax and Caspase-3 in the transplanted tumor.Methods:Sixty mature, female nude mice of cervical cancer model were established by left anterior subcutaneous inoculation. After 1 week of transplantation, model mice were randomly divided into three groups (twenty in each), including PBS control group, Empty virus group and rAAV-ES group.The method of administration is as follows:the PBS group was injected the PBS 0.1ml in and around the tumor; empty virus group was injected the adenovirus without endostatin 5*1010 v.g./0.1ml in and around the tumor; the experimental group was injected the adenovirus with the endostatin 5*1010 v.g./0.1ml in and around the tumor. The weight of nude mice and tumor size were measured every other day. Calculate the tumor volume. After 3 weeks of treatment, mice were killed by cervical dislocation, the tumor was removed and the volume and weight of tumor were measured. The tumor inhibition rate was calculated. HE staining was used to observe the tumor cells morphology. The expressions of Bcl-2, Bax and Caspase-3 in the transplanted tumor were detected by immunohistochemistry.Results:(1) Nude Mice model of cervical cancer were established successfully with an achievement ratio of 100%.(2) Compared with the control group and Empty virus group, the tumor growth slow down significantly in rAAV-ES group (P<0.001), the tumor inhibition rate was 62.55%.No differences in early treatment (P>0.05),the tumor size in rAAV-ES group decreased significantly than other two groups at 14th day and 21th day (P<0.01). The average necrosis rate of tumor cells in in rAAV-ES group is significantly higher than the control group and empty virus group (P<0.01), the control group and the empty virus group was no statistically differences (P>0.05) The rAAV-ES group appear significant apoptosis morphological changes:cytoplasmic shrinkage,nuclear chromatin was concentrated,the formation of apoptotic bodies,were scattered around without inflammatory infiltration.(3)The expression of Bcl-2,Bax and Caspase-3 were localized in the cytoplasm of cancer cells,showing brown yellow granules. After treatment, the positive rate of Bcl-2 expression was 35% in endostatin group, much lower than that of the other two groups(85% and 80%, P< 0.01).The positive rate of Bax expression had no significant change among the three groups (P>0.05). The positive rate of Caspase-3 expression was 70% in endostatin group, much higher than that of other two groups(25% and 20%, P< 0.01); The expression of Bcl-2 is negatively correlated with that of Caspase-3 (r=-0.653, P<0.01)Conclusions:(1)The method of establishing the model of cervical cancer in nude mice is convenient and reliable, and with a high achievement ratio. The species is near human and the results of the experiment are reliable.(2)After the treatment by rAAV-ES the volume of tumor were significantly reduced compared with the control group and empty virus group, the average necrosis rate of tumor cells significantly increased, significant apoptosis morphological changes appeared.(3) After the treatment by rAAV-ES,the expression of Bcl-2 decreased, the expression of Casepase-3 increased, the inhibition of tumor growth and the apoptosis inducement are not the empty virus,but the results of endostatin gene therapy.(4)rAAV-ES can inhibit tumor's growth, and the mechanism may be through down-regulating Bcl-2 protein and activating the downstream Caspase-3 to induce cell apoptosis. Endostatin gene therapy is likely to become a new mehtod for cervical cancer. |
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