HIF-1α在奶牛胎盘滋养层细胞迁移及侵袭变化中的机制研究

The Mechanism of HIF-1αin Migration and Invasion of Dairy Cow Placental Trophoblast Cells

为探究HIF-1α在CoCl_(2)影响奶牛胎盘滋养层细胞迁移及侵袭变化中的作用,揭示奶牛胎盘在缺氧条件下的形成机制,从早期妊娠奶牛胎盘中分离纯化奶牛胎盘滋养层细胞(BTCs),用吉姆萨染色法观察细胞形态,免疫荧光法检测上皮细胞标志蛋白角蛋白7(CK7)、Thy/CD90蛋白表达,后将pCI-neo-hTERT质粒转染至BTCs,用qPCR和Western Blot法检测细胞TERT mRNA及蛋白表达,用CCK8法测定细胞增殖率,用ELISA法检测细胞分泌胎盘催乳素(PL)的能力,进一步利用siRNA沉默细胞缺氧诱导因子-1α(HIF-1α)的表达,ELISA法检测细胞分泌血管内皮生长因子(VEGFA)能力,通过划痕试验及Transwell检测细胞迁移及侵袭能力。结果显示,分离纯化的BTCs为典型上皮样细胞,存在一定数量的双核细胞,并表达上皮细胞标志蛋白CK7,未见Thy/CD90表达,转染后的细胞可稳定表达端粒酶(TERT)mRNA及蛋白,连续传代50代未见细胞老化现象,且增殖率显著高于原代BTCs(P<0.05),分泌PL能力较原代BTCs无显著差异(P>0.05)。经CoCl_(2)处理后发现,BTCs细胞活力随CoCl_(2)处理浓度及时间的增加而降低,HIF-1αmRNA及蛋白表达随CoCl_(2)浓度的增加而减少(P<0.05),400μmol/L CoCl_(2)作用于BTCs 24 h建立缺氧模型,VEGFA分泌和迁移侵袭能力在BTCs缺氧状态下显著升高(P<0.05),而将HIF-1α基因沉默后显著降低了BTCs分泌VEGFA和迁移侵袭能力(P<0.05)。总之,通过外源性TERT基因转导建立了永生化的BTCs(Bovine trophoblast cells),永生化后的细胞具有与原代细胞相似的生物学特性,BTCs在缺氧条件下具有更强的VEGFA分泌能力及迁移侵袭能力,且与HIF-1α基因表达的上调有关。

To investigate the effect of HIF-1αon the migration and invasion of trophoblast cells in placenta of dairy cows affected by cobalt dichloride(CoCl_(2)),and reveal the formation mechanism of placenta of dairy cows under hypoxia conditions,bovine placental trophoblast cells(BTCs)were isolated and purified from the placenta of early pregnancy cows.The cell morphology was observed by giemsa staining method,and the expression of cell markers cytokeratin 7(CK7)and Thy/CD90 were detected by immunofluorescence method.pCI-neo-hTERT plasmid was then transfected into BTCs,qPCR and Western Blot methods were used to detect the TERT mRNA and protein expression in BTCs.Proliferation rate and placental lactogen(PL)secretory ability were analyzed with CCK8 method and ELISA,respectively.siRNA was further used to silence the expression of Hypoxia inducible factor-1α(HIF-1α),vascular endothelial growth factor A(VEGFA)secretion ability in BTCs was detected by ELISA and the ability of migration and invasion of BTCs was detected by scratch test and transwell.According to the above all results,the isolated and purified BTCs were typical epithelioid cells with a certain number of binucleated cells and expressed epithelial marker protein CK7,but Thy/CD90 expression was not observed.The transfected cells could stably express telomerase reverse transcriptase(TERT)mRNA and protein.No cell aging was observed after 50 generations of continuous passage,and the proliferation rate was significantly higher than these primary BTCs(P<0.05),and the PL secretion ability showed no significantly different from that of primary BTCs(P>0.05).The cell viability of BTCs treated with CoCl_(2)decreased with the increase of CoCl_(2)concentration and time(P<0.05),and HIF-1αmRNA and protein expression decreased with the increase of CoCl_(2)concentration(P<0.05).BTCs was treated with 400μmol/L CoCl_(2)for 24 h to establish a hypoxia model,and VEGFA secretion and invasion ability were significantly increased under BTCs hypoxia(P<0.05),while HIF-1αsilencing decreased VEGFA secretion and invasion ability of BTCs(P<0.05).In conclusion,immortalized BTCs was established through exogenous TERT gene transduction.The immortalized BTCs had similar biological characteristics to the primary cells,and have stronger VEGFA secretion and invasion ability under hypoxia condition,and were associated with HIF-1αup-regulation.