The Detection And Significance Of Survivin MRNA In The Transplanted Tumor Tissues Of Human Ovarian Cancer Cells In Nude Mice With The Treatment Of Estrogen And Cisplatin

ObjectiveTo investigate the expression of apoptosis inhibiting factor Survivin mRNA inhuman ovarian carcinoma cells in nude mice transplanted tumor tissues. To analyze thefunction of Survivin in the apoptosis and angiogenesis process after17β-E2applicated inovarian cancer xenografts in nude mice. Indicate the mechanism in that estrogen enhancescisplatin therapeutic. To explore the feasibility of tumor multidrug resistance and hormonereplacement therapy.To provide the theoretical basis for the use of HRT in patients withovarian cancer and improvement of postoperative quality of life.Methods(1) Cell culture: human ovarian cancer cell HO-8910frozen in liguid nitrogen wererevived in routine method, and inoculated in RPMI-1640blended with10％fetus cattleserum, cultured bottle. The bottle was put in the incubator with the condition of37℃,5％CO2. The growth of cells was observed everyday80％of the cells confluence weredigested.(2) Inoculability in nude mice: thirty female nude mice with weight from14g to16gand age4weeks were fed in the animal center of Tai Shan Medical University for5days.Cellls were cultured to logarithm phrase and digested with enzyme and then adjusteddensity of cells to1×107/ml. We mixed cells completely and inoculated0.2ml on the rightback of nude mice by hypodermic injection under the sterile condition.(3) Experment of the xenograft in nude mice: after subcutaneous implantation oftumor grew to the size military, we weighed the nude mice. The group marking methodwith cropped ears were divided into6groups. There were5mice in every group drugconcentration and specific groups are follows: A group (control group):0.2ml NS; B group (low dose17β-E2):0.2ml17β-E2with dose is1.3μg/d; C group(high dose17β-E2):0.2ml17β-E2with dose is3.9μg/d; D group(cisplatin group):0.2ml cisplatin with dose is5mg/kg;E group(cisplatin and low dose17β-E2):0.2ml cisplatin with dose is5mg/kg and17β-E21.3μg/d; F group(cisplatin and high dose17β-E2):0.2ml cisplatin with dose is5mg/kg and17β-E23.9μg/d.(4) After killing the nude mice and removing the tumor, use Real-time fluorescencequantitative RT-PCR technology to detect the expression of Survivin mRNA in17β-E2alone or in combination with cisplatin in ovarian carcinoma cells in nude mice transplantedtumor tissues.(5) Tumor tissue was detected by flow cytometry of apoptosis, tumor cell cycleconditions.(6) Immunohistochemical SP method to detect the tumor in nude mice inhibited theexpression of VEGF and CD34.Results(1) The expression relative weight of Survivin mRNA in the nude mice transplantedtumor tissues of group A, group B, group C, group D, group E, group F is1.000、0.429±0.014、0.210±0.018、0.090±0.007、0.045±0.004、0.021±0.001，The experimentalgroup compared with control group, P <0.05, the difference was statistically significant.17β-E2+cisplatin combined treatment group compared to the group of cisplatin alone, theexpression of Survivin mRNA in nude mice transplanted tumor tissues decreased, P <0.05,the difference was statistically significant.(2) Apoptosis: Apoptosis rate of the drug is higher the control group, the apoptosis rateof low-dose17β-E2compared with the saline group, the difference was not statisticallysignificant, the rest of the group compared with the saline group, the difference wasstatistically significant. The higher rate of apoptosis in the late treatment group.(3) Cell cycle: The number of G0/G1cell cycle in all treatment groups decreasedgradually, S phase cells increased block the cell cycle in phase of S phase.(4) Immunohistochemical detect the VEGF, CD34expression were significantly lowerin17β-E2combined with DDP treated xenograft tumors than17β-E2and DDP used alonexenograft tumors which had statistical difference (P<0.05).(5) The spearman rank correlation analysis indicated that the expression of SurvivinmRNA and apoptosis, VEGF and MVD in the nude mice transplanted tumor tissuesshow high positive correlation. Conclusions(1) The real time fluorescent quantitative RT-PCR technique can detecte theexpression of apoptosis inhibiting factor Survivin mRNA in human ovarian carcinomacells in nude mice transplanted tumor tissues sensitivly and specificly, and the method isaccurate and reliable, easy to operate.(2) The use of17β-E2alone or in combination with cisplatin in nude mice can reducethe expression of Survivin mRNA in every experimental group in nude mice transplantedtumor tissues.(3)17β-E2can improve the efficacy of cisplatin and two medicine has synergisticeffect. It may be associated with that Survivin plays an important role in apoptosis andangiogenesis.(4) Through the animal experiment, estrogen used in patients with ovarian cancer afteroperation and to improve the patients’ quality of life is feasible.