含hTERT启动子和Fcy::Fur基因重组质粒的构建及其对卵巢癌细胞的体外杀伤作用

Construction of recombinant plasmid containing hTERT promoter and Fcy::Fur gene and its killing effect on ovarian cancer cells

目的构建含人端粒酶逆转录酶(hTERT)启动子和融合型自杀基因Fcy::Fur的重组真核表达质粒并探讨它对卵巢癌细胞的体外杀伤作用。方法①从pORF5-Fcy::Fur质粒中PCR扩增Fcy::Fur片段,亚克隆进p2XEB质粒,构建含hTERT启动子和Fcy::Fur基因的重组真核表达质粒p2XEB-Fcy::Fur;同理,将Fcy::Fur亚克隆进pGL3-Promoter质粒,构建含猿猴病毒40(SV40)启动子和Fcy::Fur基因的重组质粒pGL3-Pro-Fcy::Fur,重组子经酶切鉴定、测序;②RT-PCR检测卵巢癌细胞SKOV3和人胚肺成纤维细胞MRC-5中hTERTmRNA的表达,荧光素酶活性分析检测hTERT启动子在2种细胞中活性;③用超声微泡作载体,将上述2种质粒分别转染SKOV3和MRC-5,与前药5-氟胞嘧啶(5-FC)共培养后,CCK-8测定转染细胞增殖抑制率;吖啶橙/溴化乙啶(AO/EB)染色观察细胞凋亡;RT-PCR检测Fcy::FurmRNA的表达。结果p2XEB-Fcy::Fur和pGL3-Pro-Fcy::Fur2种重组质粒酶切和测序结果与预期完全相符,hTERT启动子和Fcy::Fur片段测序与GenBank报道一致,且插入方向正确;SKOV3和MRC-5中hTERTmRNA表达分别为阳性和阴性,hTERT启动子在SKOV3中活性为26.2%,在MRC-5中为0.65%;p2XEB-Fcy::Fur/5-FC系统对SKOV3的增殖抑制率明显高于MRC-5(P=0.036),而pGL3-Pro-Fcy::Fur/5-FC系统对2种细胞的增殖抑制率无明显区别(P=0.87);转染pGL3-Pro-Fcy::Fur的SKOV3、MRC-5细胞以及转染p2XEB-Fcy::Fur的SKOV3细胞,均见大量凋亡细胞和Fcy::FurmRNA表达,而转染p2XEB-Fcy::Fur的MRC-5少见凋亡细胞,也无Fcy::FurmRNA表达,与前三者比较,差异有统计学意义(P<0.01)。结论含人端粒酶逆转录酶启动子和Fcy::Fur基因的重组质粒p2XEB-Fcy::Fur成功构建,p2XEB-Fcy::Fur/5-FC系统能靶向杀伤端粒酶逆转录酶阳性的卵巢癌细胞。

Objective To construct a recombinant expression plasmid containing Fur suicide gene and to explore its killing effect on ovarian cancer cells. Methods hTERT promoter and Fcy ①In order to construct recombinant plasmid p2XEB-Fcy： ：Fur and pGL3-Pro-Fcy： ：Fur, Fcy： ：Fur gene was amplified first from plasmid pORF5-Fcy： ：Fur using PCR and inserted into plasmid p2XEB and pGL3-promoter respectively. ②We detected the expression of hTERT at mRNA level in SKOV3 and MRC-5 cells by RT-PCR and analyzed its promoter activity using luciferase analysis method. ③We transfected the 2 recombinant plasmids into SKOV3 and MRC-5 cells by ultrasound destructing microbubble and co-cultured cells with 5-FC. The changes of cell proliferation and apoptosis were analyzed by CCK-8 and AO/EB dyeing. Fcy ： ： Fur mRNA was measured by RT- PCR. Results The plasmids of p2XEB-Fcy ： ： Fur and pGL3-Pro-Fcy ： ： Fur were constructed successfully and identified by restriction endonuclease and sequencing. SKOV3 and MRC-5 cells showed respectively positive and negative expression of hTERT mRNA, with hTERT promoter activity of 26.2% in SKOV3 cells and 0.65% in MRC-5 cells. Cell proliferation inhibitory rate was higher in p2XEB-Fcy： ： Fur/SKOV3 than that in p2XEB- Fcy： ：Fur/MRC-5 （P = 0. 036）, while the inhibitory rate was of no distinction in pGL3-Pro-Fcy： .Fur/SKOV3 and pGL3-Pro-Fcy： ： Fur/MRC-5 （P = 0.87）. Except in p2XEB-Fcy： ： Fur/MRC-5, large scale of cell apoptosis and Fcy ： ： Fur mRNA expression were found in the other three transfected groups （ P 〈 0. 01 ）. Gonclusion The recombinant plasmid of p2XEB-Fcy ： ： Fur is constructed successfully, and in the combination with 5-FC, it can kill targeted ovarian cancer cells.