摘要
目的构建缺氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)基因的短发夹状干扰RNA(short hair-pin RNA,shRNA)表达质粒,探讨其在低氧条件下对BeWo细胞转化生长因子3β(transforming growth factor3β,TGF-3β)基因表达的抑制作用。方法根据小干扰RNA(small interference RNA,siRNA)设计原则,针对HIF-1α基因设计并合成两条寡聚DNA片段,退火后克隆入pSilencer2.1-U6-neo质粒,应用脂质体将重组质粒转染BeWo细胞。缺氧培养后,应用Western blot法检测滋养细胞HIF-1α和TGF-3β蛋白表达水平,应用real time PCR法检测滋养细胞HIF-1α和TGF-3βmRNA表达水平。结果成功构建HIF-1α的shRNA真核表达载体,转染BeWo细胞。低氧条件下,细胞HIF-1α蛋白和mRNA表达下降;同时细胞TGF-3β蛋白和mRNA表达也降低。结论构建的HIF-1α基因shRNA表达质粒在低氧条件下明显抑制滋养细胞TGF-3β基因的表达。
Objective： To construct eukaryotic vector expressing short hairpin RNA （shRNA） of hypoxia - inducible factor lot （HIF - 1 α）, and observe its effect on the expression of transforming growth factor 3β （TGF -3 β） in BeWo cells under hypoxic condition. Methods： The shRNA templates was designed based on HIF - 1α gene sequence and was cloned into pSilencer2. 1 -U6 - neo vector. The resultant plasmid was transfected into BeWo cells with Lipofectamine 2000. The cells transfected were incubated under hy- poxic condition. The protein levels of HIF - 1α and TGF -3β were detected by Western blot, and the mRNA levels of HIF - 1α and TGF -3 were measured by real time PCR. Results： The plasmid pSilencer2. 1 - U6 - HIF - lot was successfully constructed and transfected into BeWo cells. The expressions of HIF - lot and TGF -3β in BeWo cells decreased under hypoxic condition. Conclu- sions： The HIF -1α siRNA plasmid could suppress the expression of TGF -3β in trophoblasts under hypoxic condition.
出处
《中国优生与遗传杂志》
2009年第1期18-20,共3页
Chinese Journal of Birth Health & Heredity
