杆状病毒介导的DNMT1 siRNA对人胰腺癌裸鼠移植瘤的抑制作用

Effects of baculovirus-mediated siRNA of DNMT1 on inhibition of pancreatic cancer xenografts in nude mice

目的:观察介导DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)基因RNA干扰(RNAi)的杆状病毒载体(Bac-si-DNMT1-D)对人胰腺癌裸鼠移植瘤生长的影响及毒性反应。方法:建立人胰腺癌SW1990细胞裸鼠移植瘤模型,随机分为4组:空载体组、生理盐水组、低浓度病毒组及高浓度病毒组进行实验。TUNEL法检测肿瘤细胞凋亡;实时荧光定量PCR检测DNMT1 mRNA表达;蛋白质印迹法检测DNMT1、Bax和Bcl-2蛋白的表达;病理学及血清学检测裸鼠心、肝、肾功能变化。结果:病毒组肿瘤生长较对照组明显被抑制(P<0.05),凋亡指数显著高于两对照组(P<0.01)。病毒组的DNMT1 mRNA表达量显著低于两对照组(P<0.05)。病毒组DNMT1及Bcl-2蛋白表达低于两对照组,但Bax蛋白表达高于两对照组(P<0.01)。各组间裸鼠心、肝及肾未见异常。结论:杆状病毒介导的DNMT1 siRNA载体可以有效降低人胰腺癌裸鼠移植瘤内DNMT1基因表达,抑制肿瘤生长,诱导肿瘤细胞凋亡,且对裸鼠无明显毒性作用。

Objective： To observe the effects of baculovirus-mediated RNA interference of DNMT1（Bac-si-DNMT1-D） on the growth of pancreatic cancer xenografts and the toxic effect in nude mice.Methods： Human pancreatic cancer cell line SW1990 cells were inoculated into nude mice to establish transplant tumor model.The model mice were randomly divided into 4 groups： High Dose recombinant baculovirus group,Low Dose recombinant baculovirus group,Carrier control group and Physiologic saline control group.The apoptosis of tumor xerographs was measured by TUNEL assay.The expression of DNMT1 mRNA was detected by real time quantitative PCR.Western blotting methods was used to detect the change of expression of DNMT1,Bax and Bcl-2.To measure the toxic effect,the morphological change of heart,liver and kidneys were pathological tested and blood serum was collected.Results： The inhibition of tumors in two recombinant baculovirus groups were significant than the other two control groups（P〈0.05）.TUNEL showed the apoptotic rate in two recombinant baculovirus groups were significantly higher than that in the other two control groups（P〈0.01）.The expression of DNMT1 mRNA in two recombinant baculovirus groups were significantly lower than the other two control groups（P〈0.05）.Compared with two control groups,the protein expression of DNMT1 and Bcl-2 in two recombinant baculovirus groups were obviously decreased,however,the expression of Bax in two recombinant baculovirus groups were obviously more than in other two control groups（P〈0.01）.Among groups there was no significant difference between heart,liver and kidney function.Conclusion：Intratumor injection of Bac-si-DNMT1-D which was baculovirus-mediated RNA interference of DNMT1 could down-regulation DNMT1,inhibit growth and induce apoptosis in of human pancreatic cancer xenografts in nude mice.It was also no obvious toxic effect that treated on nude mice.