TFPI-2对缺氧大鼠星形胶质细胞生物学特性及机制的研究

目的:研究组织因子途径抑制物-2(TFPI-2)对缺氧大鼠星形胶质细胞生物学特性的影响,探讨其对大鼠星形胶质细胞缺氧保护的机制。方法:从大鼠脑组织中分离培养星形胶质细胞,将细胞作缺氧处理,分别采用qRT-PCR和Western blot法检测星形胶质细胞中TFPI-2表达水平。在星形胶质细胞中转染TFPI-2 siRNA后,采用qRT-PCR和Western blot法检测TFPI-2基因沉默效果,MTT检测星形胶质细胞增殖,流式细胞术检测星形胶质细胞凋亡情况,qRT-PCR检测星形胶质细胞中β-连环蛋白(β-catenin)、c-myc、Bcl-2相关X蛋白(Bax)和剪切的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)mRNA表达水平。结果:Hypoxia组星形胶质细胞中TFPI-2 mRNA水平和蛋白水平明显高于Control组星形胶质细胞,转染TFPI-2 siRNA后,si-TFPI-2组星形胶质细胞中TFPI-2 mRNA和蛋白表达水平明显低于si-control组细胞,差异有统计学意义(P<0. 05)。缺氧星形胶质细胞存活率降低,细胞凋亡率升高,细胞中β-catenin、c-myc、Bax、Cleaved Caspase-3蛋白表达明显升高,与正常培养的星形胶质细胞比较,差异有统计学意义(P<0. 05)。对缺氧星形胶质细胞敲减TFPI-2后,细胞存活率升高,细胞凋亡率和细胞中β-catenin、c-myc、Bax、Cleaved Caspase-3蛋白水平降低,与缺氧星形胶质细胞比较,差异有统计学意义(P<0. 05)。结论:下调TFPI-2可以促进缺氧星形胶质细胞增殖,抑制细胞凋亡,其作用机制与抑制Wnt/β-catenin信号通路的激活有关。

Study on biological characteristics and mechanisms of TFPI2 on astrocytes in hypoxtic rats

Objective:To investigate the effect of tissue factor pathway inhibitor-2 (TFPI-2) on the biological characteristics of rat astrocytes under hypoxia,and to explore the mechanism of hypoxia protection on rat astrocytes. Methods: Primary astrocytes were isolated and cultured from rat brain tissue.The cells were treated with hypoxia.qRT-PCR and Western blot were used to detect the expression level of TFPI-2 in astrocytes.After transfected with TFPI-2 siRNA in astrocytes under hypoxia,the knock down effect of TFPI-2 siRNA was detected by qRT-PCR and Western blot.The proliferation of astrocytes was detected by MTT assay.Apoptosis of astrocytes was deteated by flow cytometry.Expression of β-catenin,c-myc,Bcl-2 associated X protein (Bax),and cleavage of cysteine by Cleaved Caspase-3 in astrocytes was detected by Western blot. Results: The level of TFPI-2 mRNA and protein in astrocytes of Hypoxia group was significantly higher than that of control astrocytes.After transfection of TFPI-2 siRNA,TFPI-2 mRNA and protein expression levels in astrocytes of si-TFPI-2 group were significantly lower than the si-control group ( P <0.05).Under hypoxia,the survival rate of astrocytes decreased,the rate of apoptosis increased,and the protein expression of β-catenin,c-myc,Bax,and Cleaved Caspase-3 increased significantly,compared with normal cultured astrocytes.The difference was statistically significant ( P <0.05).When TFPI-2 was knocked down in astrocytes under hypoxia,the survival rate of the cells was increased,and the rate of apoptosis and β-catenin,c-myc,Bax,Cleaved Caspase-3 protein expression levels in the cells were decreased,compared with normal astrocytes under hypoxia.The difference was statistically significant ( P <0.05). Conclusion: Down-regulation of TFPI-2 can promote the proliferation of astrocytes under hypoxia and inhibit apoptosis,which is related to the inhibition of Wnt/β-catenin signaling pathway activation.