人组氨酸磷酸酶蛋白PHP14的过表达及其对NIH-3T3细胞体外增殖和非锚定依赖性生长的影响

The Overexpression of PHP14 and Its Effect on NIH-3T3 Cells Proliferation in vitro and Anchor Independent Growth

目的:获取人组氨酸磷酸酶蛋白PHP14基因,并构建其N端和C端GFP融合的真核表达载体,建立过表达PHP14的NIH-3T3细胞系,并观察其对NIH-3T3细胞体外增殖和非锚定依赖性生长的影响。方法:以HeLa cDNA为模板,PCR扩增PHP14的全长编码基因,分别克隆到pEGFP-N2和pEGFP-C3载体中,构建pEGFP-N2-PHP14和pEGFP-C3-PHP14真核表达载体,利用脂质体将构建的载体转染到NIH-3T3细胞中,MTT法检测细胞增殖,软琼脂成集落法检测体外非锚定依赖性生长能力。结果:成功构建了过表达PHP14的真核表达载体pEGFP-N2-PHP14和pEGFP-C3-PHP14,并在NIH-3T3细胞中检测到了目的基因的过表达,PHP14在NIH-3T3细胞中过表达并不影响NIH-3T3细胞的体外增殖,但赋予了NIH-3T3细胞非锚定依赖性生长的能力。结论:成功构建了过表达PHP14的NIH-3T3细胞模型,在NIH-3T3细胞中过表达PHP14并不影响NIH-3T3细胞的体外增殖,但赋予了NIH-3T3细胞非锚定依赖性生长能力。

Objective： To amplify human PHP14 gene, and construct the N-terminal and C-terminal GFP fused vectors, establish the overexpress PHP14 NIH-3T3 cell line to investigate its effect on NIH-3T3 cell proliferation and anchor independent growth. Methods： The cDNA sequence of PHP14 gene was amplified and sub-cloned into pEGFP-N and pEGFP-C3 vectors. Transfecting of those vectors into NIH-3T3 cells and investigating the cell proliferation and anchor independent growth in PHP14 overexpressed NIH-3T3 cells using MTT and Soft agar colony assay. Results： The pEGFP-N2-PHP14 and pEGFP-C3-PHP14 prokaryotic expression vectors were constructed successfully and the expression of those vectors were detected in NIH-3T3 ceils. Overexpression of PHP14 in NIH-3T3 cells did not affect the cell proliferation of NIH-3T3 cells, but gave the NIH-3T3 cells the ability of anchor independent growth. Conclusion： PHP14 prokaryotic expression vectors were constructed successfully and overexpression of PHP14 in NIH-3T3 cells did not affect the cell proliferation of NIH-3T3 cells, but gave the NIH-3T3 cells the ability of anchor independent growth.