RNAi沉默PRDX Ⅲ基因表达诱导U251胶质瘤细胞凋亡的实验研究

目的探讨RNAi沉默PRDXⅢ基因表达诱导U251胶质瘤细胞凋亡。方法设计合成针对PRDXⅢ的siRNA,脂质体介导转染人U251多形胶质母细胞瘤细胞,RT-PCR法检测转染后24 h PRDXⅢmRNA的表达水平,Western-blotting检测转染后48 h PRDXⅢ蛋白的表达水平,筛选出有效的干扰序列;流式细胞仪、DNAladder、PI染色检测转染后48 hU251细胞的凋亡状况,MTT检测转染后48 h U251细胞的生长抑制率。结果 RT-PCR、Western Blotting检测显示空白对照组、空载体组和无关序列组PRDXⅢmRNA表达无明显变化,干扰序列1、3使PRDXⅢmRNA的表达明显降低。DNA ladder显示干扰组出现明显梯形电泳条带,空白对照组、空载体组和无关序列组细胞未见明显梯形电泳条带。PI染色结果显示空白对照组,空载体组和无关序列组细胞未见明显凋亡,而干扰组细胞核的染色质高度浓染,部分细胞核裂解为碎块,符合凋亡形态学的改变。FCM检测结果显示干扰组细胞凋亡率明显高于空白对照组、空载体组、无关序列组（P〈0.01）。MTT检测干扰组对U251细胞生长的抑制率为44.4%,明显减慢了细胞生长（P〈0.01）。结论 RNAi技术成功地抑制了U251细胞中PRDXⅢ基因的表达,并诱导U251细胞凋亡,表明PRDXⅢ基因与胶质瘤的凋亡有关,PRDXⅢ基因有可能成为胶质瘤基因治疗的靶基因。

Experimental study on the expression of PRDX Ⅲ in human gliomas and cell apoptosis of U251 gliomas cells induced by RNAi silencing PRDX Ⅲ gene

Objective To investigate the expression of PRDX Ⅲ in human gliomas and cell apoptosis of U251 gliomas cells induced by RNAi silencing PRDX Ⅲ gene.Methods The specific PRDX Ⅲ siRNAs were designed and transfected into U251 gliomas cells by liposome.The expression levels of PRDX Ⅲ mRNA and protein were detected by RT-PCR and Western Blotting 24 hours and 48 hours after the transfection,respectively.The apoptotic state 48 hours after the transfection was detected by flow cytometry(FCM),DNA ladder and PI staining,respectively,and the inhibition rate of U251 cell growth was determined by MTT 48 hours after transfection.Results The results of RT-PCR and Western Blotting showed that the expression of PRDX Ⅲ mRNA had no obvious changes in blank control group,empty carrier group and independent subsequence group,however,to interfere sequence 1,3 reduced significantly the expression of PRDXⅢ mRNA.The results of DNA ladder suggested that there appeared obvious ladder-shaped electrophoresis strip in interference group,but no obvious ladder-shaped electrophoresis strip was found in blank control group,empty carrier group and independent subsequence group.The results of PI staining showed that no obvious cell apoptotic changes were observed in blank control group,empty carrier group and independent subsequence group,however,some cellular nucleus were stained thickly and some of them were split into broken bits in interference group,which was in coincidence with apoptotic morphological changes.The results of FCM indicated that the cell apoptosis rate in interference group was significantly higher than that in blank control group,empty carrier group and independent subsequence group,respectively(P<0.01).The inhibition rate of U251 cell growth detected by MTT was 44.4% in interference group,which slowed down the cell growth(P<0.01).Conclusion RNAi technique can effectively inhibit the expression of PRDX Ⅲ gene in U251cells,and can induce cell apoptosis of U251,which suggests that PRDX Ⅲ gene is correlated with apoptosis of gliomas,and PRDX Ⅲ gene is possible to become the target gene in gene therapy for gliomas.