构建间皮素（mesothelin，MSLN）RNAi重组慢病毒质粒，探讨其对卵巢癌OVCAR 3细胞 MSLN的表达及细胞增殖的影响。方法：根据 MSLN 基因信息,设计了4个小干扰序列和1个阴性对照序列，利用慢病毒质粒载体pRNAT U62/Lenti构建了5个重组质粒并进行了慢病毒包装，分别为LV MSLN negative、LV MSLN shRNA2、LV MSLN shRNA3、 LV MSLN shRNA4 ；感染OVCAR 3细胞后，Western blotting和荧光免疫组化检测干扰效率，选择干扰效率高的质粒载体进行慢病毒大量包装；感染卵巢癌细胞OVCAR 3后，用细胞增殖实验和平板克隆形成实验检测细胞增殖的变化。结果：测序结果证明5种质粒载体的插入序列完全正确，鉴定证明慢病毒包装成功。慢病毒感染OVCAR 3细胞后，Western blotting证实重组慢病毒LV MSLN shRNA4的干扰效率最高,对MSLN蛋白表达的抑制达90%。荧光免疫组化的共聚焦照片显示干扰组（OVC shRNA）定位于细胞膜的MSLN蛋白表达明显弱于阴性对照组（OVC neg）和空白对照组（OVC）。OVC shRNA组细胞增殖\[（11.2±1.3）×10 5\]显著慢于OVC neg组\[（20.5±2.5）×10 5\]和OVC组\[（21.9±2.3）×10 5\] ( P ＜0.05）。OVC shRNA组克隆形成率为(15.2±2.1)%，明显低于OVC neg组\[（27.9±2.5）%\]和OVC组\[（28.8±3.1）%\]（ P ＜0.05）。结论：成功构建MSLN RNAi重组慢病毒质粒LV MSLN shRNA4，它能有效抑制卵巢癌OVCAR 3细胞的MSLN表达及细胞增殖，为进一步研究MSLN应用于肿瘤基因治疗奠定了基础。

To construct a recombinant lentivirus plasmid of RNA interference targeting ( MSLN ) gene and to observe its effect on MSLN expression in human ovarian cancer cell line OVCAR 3 and its effect on cell proliferation. Methods: According to the Genbank information of MSLN, four RNA interfering sequences and a negative sequence were designed and inserted into plasmid pRNAT U6.2/Lenti and 5 kinds of plasmids were packaged: LV MSLN negative,LV MSLN shRNA1, LV MSLN shRNA2, LV MSLN shRNA3, and LV MSLN shRNA4; and they were used to transfect OVCAR 3 cells. Western blotting and indirect immunofluorescence were then used to investigate the interfering efficiency. The plasmid with high interfering efficiency was packaged. The cell proliferation test and clone forming test was used to assess the changes in cell proliferation. Results: DNA sequencing showed that the sequences of 5 recombinant lentivirus plasmids were correct. Lentivirus packaging was successfully done. Western blotting analysis confirmed that LV MSLN shRNA4 had the highest interfering efficiency (90%). MSLN specifically bound to cytomembrane of OVCAR 3 cells. Expression of MSLN in the interfered cells (OVC shRNA) was weaker than that in the control cells (OVC neg,OVC). OVC shRNA cells(\[11.2±1.3\]×10 5) grew slowly compared to OVC neg cells(\[20.5±2.5\]×10 5) and OVC cells(\[219±23\]×10 5) ( P ＜005）. There was a significant reduction in clone forming rate of OVC shRNA cells (152±2.1)% in comparison with OVC neg cells（\[27.9±2.5\]%) and OVC cells（\[288±31\]%）（ P ＜0.05）.Conclusions: We have successfully constructed MSLN RNAi recombination plasmid LV MSLN shRNA4, which can effectively inhibit MSLN expression and cell proliferation, which paves a way for studying MSLN function and gene therapy.

国家自然科学基金资助项目（No.30572093）; 河北省自然科学基金资助项目（No.C2005000804）