Livin靶向SiRNA协同5-氟尿嘧啶对大肠癌细胞的化疗增敏作用

The effect of enhancement chemotherapy sensitivity by combining siRNA recombinant expression vector targeting Livin gene with 5-Fu To human colon cancer cells

目的构建Livin靶向SiRNA重组表达载体，研究其对人大肠癌细胞生长的抑制作用和对5-氟尿嘧啶（5-Fu）的化疗增敏作用。方法构建Livin靶向siRNA重组表达载体并转染大肠癌细胞；通过逆转录一聚合酶链反应（RT—PCR）和Western blot方法检测Livinsi RNA对大肠癌细胞Livin mRNA和蛋白表达的抑制率；采用噻唑蓝（MTT）比色法分别于转染后24、48、72h用自动酶标读数仪（波长490nm）测定每孔A值来计算大肠癌细胞抑制率；采用流式细胞仪于转染后48h在488nm波长下检测细胞的凋亡率的变化。结果经酶切鉴定和测序结果证实Livin靶向SiRNA重组表达载体构建成功。RT—PCR检测和Western blot方法检测Livin siRNA对大肠癌细胞Livin mRNA和蛋白表达的抑制率为分别为30．18％和28．88％，可以协同5-Fu增强对大肠癌细胞生长的抑制。结论Livin靶向siRNA重组表达载体构建成功，它能有效抑制Livin基因表达并能协同5-Fu增强对大肠癌细胞的杀伤作用和诱导凋亡作用。

Objective To construct SiRNA recombinant expression vector targeting Livin gene and research the effect of it in inhibition of human colon cancer cells proliferation and enhancement of chemotherapy sensitivity. Methods SiRNA recombinant expression vector targeting Livin gene was constructed and transfeeted into human colon cancer cell. The effect of SiRNA recombinant expression vector was detected by RT-PCR and Western blot. Using auto-enzyme-labeled reading device with the 490nm wave,the value of A in each foramen was respectively measured after 24,48,72 hr of transfection by MTT reduction assay, and then calculated the inhibitor rate of tumor cell. After 48 hours of transfection, the change of the apoptosis rate was detected under the 488nm wave by flow cytometry. Results It was confirmed by restriction endonuelease and sequence analysis that SiRNA recombinant expression vector targeting Livin gene was constructed successfully. Inhibition ratio of Livin SiRNA at mRNA and protein levels were 30.18% and 28.88%. It could enhance chemotherapy sensitivity by combining 5-Fu. Conclusion The SiRNA recombinant expression vector targeting Livin gene has been constructed successfully and can inhibite the expression of Livin gene. It also can enhance chemotherapy sensitivity and apoptosis induction effect by combining 5-Fu in human colon cancer cells significantly.