缺氧环境下HSulf-1,HIF-1α在HaCaT细胞中的表达及HSulf-1过表达载体的构建

Expression of HSulf-1 and HIF-1α in Human HaCaT Keratinocytes Under Hypoxia and Construction of HSulf-1-overexpressed Vector

目的探讨HSulf-1,HIF-1α在缺氧时人角质形成细胞株HaCaT细胞中的表达并构建HSulf-1基因过表达载体。方法 Western blot法检测不同缺氧浓度下HaCaT细胞中HSulf-1,HIF-1α蛋白的表达;构建重组质粒p CDH-HSulf-1;Western blot法筛选HSulf-1过表达稳转HaCaT细胞株。结果 HSulf-1在缺氧条件下低表达,与HIF-1α表达呈负相关;成功筛选过表达HaCaT稳定细胞株。结论 HSulf-1低表达,与肿瘤细胞或肿瘤组织中表达一致,过表达可抑制肿瘤细胞增殖。推测过表达HSulf-1可能抑制角质形成细胞的增殖,构建HSulf-1基因过表达载体,便于进一步体内外研究与角质形成细胞增殖的影响。

Objective To investigative the expression of HSulf-1 and HIF-1 alpha in human HaCaT keratinocytes under hypoxia and construct the overexpression vector of HSulf-1. Methods HSulf-1 and HIF-1 alpha protein expression were detected by using Western Blotting. Human HaCaT keratinocytes were subjected to different concentrations of hypoxia. The HSulf-1 gene was inserted into adenovirus genome to generate recombinant adenovirus pCDH-HSulf-1. HSulf-1 protein in HaCaT keratinocytes transfected by this overexpression vector was examined by using Western blotting. Results HSnlf-1 protein expressed in low level in HaCaT keratinocytes, and was negatively correlated with HIF-1 alpha under hypoxic condition. The stable Hsulf-overex- pressed cell line was successfully screened. Conclusion There was a low level of HSulf-1 expression in HaCaT keratinocytes under hypoxia, which was consistent with its expression in tumor cells or tumor tissues. The overexpression of HSulf-1 can inhibit the proliferation of tumor cells. It was hypothesized in this study that overexpression of HSulf-1 can inhibit the proliferation of keratinocytes. Therefore, construction of the overexpressed vector of HSulf-1 would facilitate the further investigation into the effects of HSulf-1 expression on the proliferation of keratinocytes in vivo and in vitro.