pcDNA3.1-Pax6载体的构建及其对胶质瘤细胞增殖的影响

Construction of Recombination Vector of pc DNA3. 1-Pax6 and the Effect of Pax6 on Proliferation of Glioma Cells

目的:构建pc DNA3.1-Pax6过表达质粒并观察其对胶质瘤U251细胞增殖的影响。方法:以K562细胞c DNA为模版,采用RT-PCR的方法扩增Pax6基因,将扩增产物酶切回收后与同样酶切回收的真核表达载体pc DNA3.1连接、转化,构建pc DNA3.1-Pax6重组质粒,再经菌落PCR、酶切及测序鉴定重组质粒;利用脂质体lipofectamine 2000转染重组质粒至U251细胞。Real-time PCR检测Pax6 mRNA的表达水平,Western blot检测PAX6蛋白的表达,MTT法检测U251细胞增殖。结果:PCR扩增获得长度为1 131 bp的阳性产物,经pc DNA3.1真核表达载体克隆、酶切鉴定及序列分析后,证实真核表达质粒pc DNA3.1-Pax6构建成功;Real-time PCR结果显示,Pax6过表达后的U251细胞Pax6 mRNA表达水平明显高于正常对照组;Western blot结果显示,PAX6蛋白表达亦明显高于正常对照组,差异具有统计学意义(P<0.05);MTT结果显示,与正常对照组细胞比较,转染pc DNA3.1-Pax6组的细胞,细胞增殖能力明显降低,差异亦具有统计学意义(P<0.05)。结论:成功构建人Pax6基因的pc DNA3.1-Pax6重组质粒,过表达Pax6基因抑制U251细胞的增殖。

Objective: To construct over-expression plasmid of pc DNA3. 1-Pax6 and investigate its impact on proliferation of glioma U251 cells. Methods: Pax6 gene was amplified from c DNA of K562 cells using RT-PCR. The Pax6 gene was restrictively digested and recycled,and then inserted into the eukaryotic expression vector pc DNA3. 1. The recombinant plasmid pc DNA3. 1-Pax6 was confirmed by colony PCR,restriction endonuclease digestion and sequencing. The recombinant plasmid was transfected into U251 cells through lipofectamine 2000. The expression of Pax6 at mRNA andprotein levels after transfection was identified by Real-time PCR and Western blotting,respectivly. The U251 cell proliferation was measured by MTT. Results: The purpose gene gained from PCR amplification had length of 1131 bp. Eukaryotic expression plasmid pc DNA3. 1-Pax6 was identificated by colony PCR,enzyme digestion and sequencing analysis. Real-time PCR showed that the expression of Pax6 mRNA was increased in U251 cells transfected with Pax6 gene compared to the normal group. Western blotting results showed that PAX6 protein levels in U251 cells transfected with Pax6 gene was also over-expressed compared with the normal group( P < 0. 05). MTT results showed that transfection of pc DNA3. 1-Pax6 inhibited the proliferation of the U251 cells compared to normal group( P < 0. 05). Conclusion: We successfully constructd the pc DNA3. 1-Pax6 recombinant plasmid. Over-expression of Pax6 gene could inhibit the proliferation of the U251 cells.