自杀基因联合化疗干预卵巢癌的动物实验研究

目的探讨单纯疱疹病毒胸腺激酶（herpes simplex virus thymidine kinase,HSV-TK）基因/更昔洛韦（ganciclovir,GCV）系统联合化疗药物拓扑替康（Topotecan）对卵巢癌细胞NuTu-19杀伤作用及自杀基因系统与化疗药物不同的给药次序的疗效差异。为临床治疗提供最佳方案。方法常规方法培养PA317/TK细胞、NuTu-19细胞;耐药集落形成法测定病毒滴度;自杀基因转导,PCR方法检测TK基因阳性克隆细胞;测定NuTu-tk细胞对GCV敏感性;建立动物模型：接种NuTu-19细胞及同时接种PA317/TK细胞和NuTu-19细胞的大鼠成瘤后应用化疗药物（Topotecan）,GCV及GCV联合化疗药物,测量肿瘤大小并计算抑瘤率。结果经PCR检测证实PA317/TK已经导入NuTu-19中;GCV浓度在0.01-30时对NuTu–tk细胞均有明显的杀伤作用,GCV对NuTu–tk细胞的杀伤率与其浓度呈明显正相关（r=0.612,t=2.04,P〈0.01）;大鼠接种肿瘤细胞后10-12天局部长出约0.4×0.5cm大小的肿瘤,然后局部逐渐呈膨胀性生长,质硬,活动,与腹壁不粘连,呈球形或椭圆形或多分叶状实体瘤,表面可见丰富皮下血管网;HSV-TK/GCV系统、Topotecan及HSV-TK/GCV联合Topotecan对大鼠皮下移植肿瘤有抑制作用;先给自杀基因组较先给化疗药物组疗效明显,均有统计学意义。结论 HSV-TK/GCV与拓扑替康联合杀伤卵巢癌细胞有协同作用;先用HSV-TK/GCV后用拓扑替康治疗效果最佳。自杀基因联合化疗治疗卵巢癌的动物实验研究为HSV-TK自杀基因系统的临床应用提供了实验基础,也为复发性、难治性卵巢癌患者治疗开辟新的途径。

The animal model study of suicide gene combined with chemotherapy in the treatment of ovarian cancer

Objective To investigate the killing effect of herpes simplex virus thymidine kinase gene（HSV-TK）/ ganciclovir（GCV） system combined with chemotherapy topology Topotecan to ovarian cancer cell NuTu-19and the efficiency difference of different administered order of suicide gene system and chemotherapy drugs.This will provide a better treatment for clinic.Methods PA317 / TK cells and NuTu-19cells were cultured with conventional methods;The virus titer was determined with resistant colony-forming method;Suicide gene transduction and PCR were used to detect the TK gene-positive clones cells;NuTu-tk cells to GCV sensitivity were determined;And animal models were established ： the logarithmic growth phase PA317 / TK cells and PA317 / TK with NuTu-19cells were inoculated in rats abdomen.After tumor bearing rats were treated with different drugs ： chemotherapy drugs（Topotecan）,GCV and GCV with Topotecan.After treatment,tumor size was measured and tumor inhibitory rate was calculated.Results PA317 / TK imported into NuTu-19cells was confirmed by PCR detection;GCV has significant killing effect on NuTutk cells at concentration of 0.01-30.Destruction rate of GCV on NuTu-tk cells and its concentration was positively correlated（r=0.612,t=2.04,P0.01）;0.4×0.5cm tumors were grown 12days after tumor cells inoculation.Then tumors were expansively growth and turn to harder,spherical,oval or the multisection lobulated solid tumors,not adhesive to abdominal wall,and the vascular network were visible on the surface of tumor;HSV-TK / GCV system,Topotecan and HSV-TK / GCV combined with Topotecan all have inhibitive effects on rat subcutaneous transplantation tumors;The effect of suicide gene system group was better than the chemotherapy drugs group.Conclusion HSVTK / GCV and topotecan have synergistic effect on killing ovarian cancer cells;Using topotecan followed HSV-TK / GCV treatment provide the best effect in.Ovarian cancer animal models studies of suicide gene combined with chemotherapy provides an experimental basis for clinical application,and also open new avenues for recurrent,refractory o-varian cancer treatment.