Influence Of RAAV2-hEndo On Tumor Growth And Angiogenesis Of Implanted Human Breast Cancer In Nude Mice

Objective:To investigate the therapeutical effect and its mechanism of recombinate adeno-associated viral vectors2(rAAV2) mediated human endostatin(ES) gene on implanted human breast cancer in nude mice,and check the dependability and security of rAAV2 as genetic carrier.These results would be hoped to elicit a novel approach to the clinical treatment of human breast cancer.Methods:MCF-7 human breast cancer cells were anabiosised and cultured in 5%CO₂ air and humidity-controlled condition.Total 15 female nude mice of 4-6 weeks were breeded,5×10⁶ human breast cancer MCF-7 cells suspended in 0.1ml PBS were injected subcutaneouly into the left groin of each nude mouse.Animal models were established in the nude mice bearing human breast carcinoma.When the transplanted tumor grew for averaged diameter 4mm,15 nude mice were divided into 2 groups randomly,experiment group10 mice,control group 5 mice.The experiment group animals received three injections of rAAV2-hEndo-EGFP(containing5×10⁹vg) on days 12,19,and 26,while in blank control group,animals only received three blank rAAV2 injections at the same time.The body weights of mice and the volume of tumors were evaluated at one time every 2 days.Then the growth curves were drawn to observe the growth of breast cancer implanted in nude mice.After first injected rAAV2-hEndo for 28 days,total 15 nude mice were killed by overdose anesthesia.Then 15 tumor tissues were collected.One part of fourth of every tumor was made for the frozen sections to observe the information of gene transfection. Other part of every tumor was fixed in 10%neutral formalin,embedded in paraffin, sectioned,and stained with HE routinely.And immunohistochemistry method was used to detect the effects of endostatin on microvesel density(MVD) and the expression of VEGF protein on breast cancer tissues implanted in nude mice. Statistical software SPSS 11.5 was used in analysis.P value less than 0.05 was considered as statistically significant.Results: (1) MCF-7 human breast cancer cells cultured in vitro had been grown well,to show typical epithelial cell appearance;and possess malignant characteristics.(2) Animal models were established in nude mice bearing human breast cancer.The general state of nude mice was fine.After injected MCF-7 human breast cancer cells for 10 days,a mass could be touched obviously in inoculated place of all nude mice. The ratio of tumor appearance was 100%.The average time of tumor appearance was 8 days.(3) Before injected rAAV2-hEndo,the difference of tumorvolume between experiment group and control group had no statistically significant(P＞0.05).After injected rAAV2-hEndo for 7 days,the tumor volume of experiment group was decreased significantly compared with the control group(P＜0.05).Along with the time of therapy extending,the difference of tumor volume between experiment group and control group was increasing.At the end of animal experiment the tumor inhibition rate was 66.09%.(4) During the period of animal experiment,no nude mice had an accidental death and any viral infection symptom.(5) Under fluorescence microscope,the most tumor cells could be stimulated to send out flavo-green fluorescence.This result hinted that rAAV2-hEndo-EGFP had been transfected to the target cells.(6) Transplanted tumor observed in general,the average tumor volume of experiment group was 672.40±232.14 mm³,and the control group was 1982.76±257.91mm³.The difference of tumor volume between experiment group and control group was seen significantly(P=0.000).The histomorphology of transplanted tumors present typical characteristics of human breast infitrating ductal carcinoma.(7) The results of immunohistochemistry indicated that the microvesel density and VEGF expression of experiment group was statistically significantly decreased compared with the control group(all of P＜0.05).Both in experiment group and control group,the expression of VEGF was opsitive correlated with MVD significantly(r=0.75,0.92 respectively,all of P＜0.05).Conclusions: (1) The system of MCF-7 human breast cancer cells/ nude mice was one of ideal animal models.(2) Transfection efficiency of adeno-associated viral vectors2 was high and it's dependability and security were seen relatively.It may be one of ideal genetic carriers for tumor's gene therapy.(3) Adeno-associated viral vectors2(rAAV2) mediated human endostatin(ES) gene may inhibite the growth and angiogenesis of transplantation human breast tumor,and down regulation the expression level of VEGF in transplantation tumors.These results hinted that endostatin gene would be a new approach for clinical therapy of breast cancer.