Kir 4.1基因沉默对U-251细胞周期的影响

Effect of Kir 4.1 gene knockdown on cell cycle of U-251 cells

摘要:

目的：针对人内向整流性钾离子通道（inwardly rectifying potassium channel protein，Kir）4.1基因构建siRNA干扰载体，探讨其对U-251细胞增殖、生长的影响，为临床治疗及开发新的抗肿瘤药物提供实验依据。方法：采用细胞培养、质粒转染、流式分选、RT-PCR、Western blot及流式细胞仪对构建的质粒进行鉴定并观察对照组和干扰组细胞周期的变化。结果：对DH5α中抽提的重组载体测序验证结果和插入序列一致；成功筛选出一个有效质粒；质粒转染U-251细胞后，细胞周期中的G2期细胞数量由19.39%降至1.5%，明显减少（P=0.018），S期细胞数量由36.32%升至51.22%，明显增多（P=0.027）。结论：成功构建针对人Kir 4.1基因的siRNA干扰载体；Kir 4.1干扰载体可能是通过抑制U-251细胞周期中的G2期，使U-251细胞的生长停滞在S期，从而达到干扰U-251细胞的增殖能力。

Abstract:

Objective：To construct the siRNA expression vector which targeted the human Kir 4.1 gene，to study the impact of the Kir 4.1 on the proliferation of U-251 by RNAi and to explore the new method in astrocytoma treatment. Methods：Cell culturing，transforming，flow cytometry screening，RT-PCR and Western blot were used to detect recombinant plasmid and investigate the cell cycle change（G1 phase，S phase and G2 phase） of U-251 cell in control group，negative control group and transformed cells. Results：Three interference vectors were constructed successfully. A plasmid which didn’t induce Kir 4.1 mRNA production was obtained. Flow cytometry screening showed that the cell number in each phase of negative control group had no apparent difference while in the transformed group the cell number in G2 phase decreased from 19.39% to 1.5%（P=0.018），the cell number in S phase increased obvi－ously from 36.32% to 51.22%（P=0.027）. Conclusion：The siRNA expression vector which targeted the human Kir 4.1 is constructed successfully. Kir 4.1 gene is silenced in the transformed U-251 cells by siRNA expression vector. U-251 cell stops at S phase proba－bly by inhibiting the G2 phase，which can decrease cell proliferation.