TFPI-2对缺氧大鼠星形胶质细胞生物学特性及机制的研究
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摘要
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。
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,cmyc,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.
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,cmyc,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.
引文
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[13] Wu W,Wei W,Lu M,et al. Neuroprotective effect of chitosan oligosaccharide on hypoxic-ischemic brain damage in neonatal rats[J]. Neurochem Res,2017,42(11):3186-3198.
[14] Ma Q,Dasgupta C,Li Y,et al. Inhibition of microRNA-210provides neuroprotection in hypoxic-ischemic brain injury in neonatal rats[J]. Neurobiol Dis,2016,89:202-212.
[15] Revuelta M,Arteaga O,Alvarez A,et al. Characterization of gene expression in the rat brainstem after neonatal hypoxic-ischemic injury and antioxidant treatment[J]. Mol Neurobiol,2017,54(2):1129-1143.
[16] Wang K,Ren Y,Liu Y,et al. Tumor necrosis factor(TNF)-α-induced protein 8-like-2(TIPE2)inhibits proliferation and tumorigenesis in breast cancer cells[J]. Oncol Res,2017,25(1):55-63.
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[18] Liu T,Zhang X,Ke B,et al. F-127-PEI co-delivering docetaxel and TFPI-2 plasmid for nasopharyngeal cancer therapy[J]. Mater Sci Eng C Mater Biol Appl,2016,61:269-277.
[19] Gao S,Lin Z,Li C,et al. TFPI2AS1,a novel lncRNA that inhibits cell proliferation and migration in lung cancer[J]. Cell Cycle,2017,16(23):2249-2258.
[20] Yang Z,Geng Y,Yao Z,et al. Spatiotemporal expression of Bcl-2/Bax and neural cell apoptosis in the developing lumbosacral spinal cord of rat fetuses with anorectal malformations[J].Neurochem Res,2017,42(11):3160-3169.
[21] Chen B,Wang G,Li W,et al. Memantine attenuates cell apoptosis by suppressing the Calpain-Caspase-3 pathway in an experimental model of ischemic stroke[J]. Exp Cell Res,2017,351(2):163-172.
[22] Ni HM,Mc Gill MR,Chao X,et al. Caspase inhibition prevents tumor necrosis factor-α-induced apoptosis and promotes necrotic cell death in mouse hepatocytes in vivo and in vitro[J]. Am J Pathol,2016,186(10):2623-2636.
[23] Dong Y,Tan Q,Tao L,et al. Hypermethylation of TFPI2 correlates with cervical cancer incidence in the Uygur and Han populations of Xinjiang,China[J]. Int J Clin Exp Pathol,2015,8(2):1844-1854.
[24] DiRenzo DM,Chaudhary MA,Shi X,et al. A crosstalk between TGF-β/Smad3 and Wnt/β-catenin pathways promotes vascular smooth muscle cell proliferation[J]. Cell Signal,2016,28(5):498-505.
[25] Zhou T,Zu G,Zhang X,et al. Neuroprotective effects of ginsenoside Rg1 through the Wnt/β-catenin signaling pathway in both in vivo and in vitro models of Parkinson's disease[J]. Neuropharmacol,2016,101:480-489.
[26]周金敬,毕晓,徐勇,等.组织因子途径抑制物-2表达与肝癌细胞体外侵袭能力的实验研究[J].肿瘤,2009,29(10):976-979.Zhou JJ,Bi X,Xu Y,et al. The expression of tissue factor pathway inhibitor-2 and invasive cability of hepatocelluar carcinoma in vitro[J]. Tumor,2009,29(10):976-979.
[27] Zhao XP,He SW,Yue B,et al. Molecular characterization,expression analysis,and bactericidal activity of the derivative peptides of TFPI-1 and TFPI-2 in half-smooth tongue sole,Cynoglossus semilaevis[J]. Fish Shellfish Immunol,2016,58:563-571.
[2] Karve IP,Taylor JM,Crack PJ. The contribution of astrocytes and microglia to traumatic brain injury[J]. Br J Pharmacol,2016,173(4):692-702.
[3]林祺,高亮,秦松.星形胶质细胞激活与创伤性颅脑损伤研究进展[J].中国临床神经科学,2017,25(1):81-87.Lin Q,Gao L,Qin S. Progress of astrocyte activation and traumatic brain injury research[J]. Chin J Clin Neurosci,2017,25(1):81-87.
[4] Becerra-Calixto A,Cardona-Gómez GP. Neuroprotection induced by transplanted CDK5 knockdown astrocytes in global cerebral ischemic rats[J]. Mol Neurobiol,2017,54(9):6681-6696.
[5] Fry J,Kato S,Abarzua L,et al. Down-regulation of TFPI-2 in the progression of ovarian cancer[J]. Eur J Cancer,2016,61(1):S60.
[6] Wang G,Zeng Y,Chen S,et al. Localization of TFPI-2 in the nucleus modulates MMP-2 gene expression in breast cancer cells[J]. Sci Rep,2017,7(1):13575.
[7]秦晓林,徐勇,范晓卿,等. TFPI-2对人肝癌细胞生长增殖、凋亡及AFP合成的影响[J].中国生物工程杂志,2011,31(12):33-38.Qin XL,Xu Y,Fan XQ,et al. Effects of TFPI-2 on growth,apoptosis and AFP synthesis of human hepatoma cells[J]. China J Biotechnol,2011,31(12):33-38.
[8]韩东兴,刘兵,潘卉,等. TFPI-2在结直肠癌及正常结肠黏膜组织中的表达差异[J].中国肿瘤生物治疗杂志,2015,22(3):385-387.Han DX,Liu B,Pan H,et al. Differential expression of TFPI-2protein in colorectal cancer and normal mucosa tissues[J]. Chin J Cancer Biother,2015,22(3):385-387.
[9] VaitkienèP,SkiriutèD,Skauminas K,et al. Associations between TFPI-2 methylation and poor prognosis in glioblastomas[J].Medicina,2012,48(7):345-349.
[10] Zhang M,Yue B,Zhang AH,et al. TC38,a teleost TFPI-2 peptide that kills bacteria via penetration of the cell membrane and interaction with nucleic acids[J]. Fish Shellfish Immunol,2017,64:104-110.
[11]丁娟,丁敬宾,马小虎,等.一种改进的大鼠大脑皮质星形胶质细胞的培养方法[J].神经解剖学杂志,2017,33(3):349-353.Ding J,Ding JB,Ma XH,et al. An improved method for culture of rat cerebral cortical astrocytes[J]. J Neuroanatomy,2017,33(3):349-353.
[12] Farmer WT,Abrahamsson T,Chierzi S,et al. Neurons diversify astrocytes in the adult brain through sonic hedgehog signaling[J]. Science,2016,351(6275):849-854.
[13] Wu W,Wei W,Lu M,et al. Neuroprotective effect of chitosan oligosaccharide on hypoxic-ischemic brain damage in neonatal rats[J]. Neurochem Res,2017,42(11):3186-3198.
[14] Ma Q,Dasgupta C,Li Y,et al. Inhibition of microRNA-210provides neuroprotection in hypoxic-ischemic brain injury in neonatal rats[J]. Neurobiol Dis,2016,89:202-212.
[15] Revuelta M,Arteaga O,Alvarez A,et al. Characterization of gene expression in the rat brainstem after neonatal hypoxic-ischemic injury and antioxidant treatment[J]. Mol Neurobiol,2017,54(2):1129-1143.
[16] Wang K,Ren Y,Liu Y,et al. Tumor necrosis factor(TNF)-α-induced protein 8-like-2(TIPE2)inhibits proliferation and tumorigenesis in breast cancer cells[J]. Oncol Res,2017,25(1):55-63.
[17]董永强,梁江水,殷桂林,等. TFPI-2、VEGF在非小细胞肺癌中的表达及相关性研究[J].临床肺科杂志,2013,18(9):1625-1627.Dong YQ,Liang JS,Yin GL,et al. Expression and correlation of TFPI-2 and VEGF in non-small cell lung cancer[J]. J Clin Pulmonary Med,2013,18(9):1625-1627.
[18] Liu T,Zhang X,Ke B,et al. F-127-PEI co-delivering docetaxel and TFPI-2 plasmid for nasopharyngeal cancer therapy[J]. Mater Sci Eng C Mater Biol Appl,2016,61:269-277.
[19] Gao S,Lin Z,Li C,et al. TFPI2AS1,a novel lncRNA that inhibits cell proliferation and migration in lung cancer[J]. Cell Cycle,2017,16(23):2249-2258.
[20] Yang Z,Geng Y,Yao Z,et al. Spatiotemporal expression of Bcl-2/Bax and neural cell apoptosis in the developing lumbosacral spinal cord of rat fetuses with anorectal malformations[J].Neurochem Res,2017,42(11):3160-3169.
[21] Chen B,Wang G,Li W,et al. Memantine attenuates cell apoptosis by suppressing the Calpain-Caspase-3 pathway in an experimental model of ischemic stroke[J]. Exp Cell Res,2017,351(2):163-172.
[22] Ni HM,Mc Gill MR,Chao X,et al. Caspase inhibition prevents tumor necrosis factor-α-induced apoptosis and promotes necrotic cell death in mouse hepatocytes in vivo and in vitro[J]. Am J Pathol,2016,186(10):2623-2636.
[23] Dong Y,Tan Q,Tao L,et al. Hypermethylation of TFPI2 correlates with cervical cancer incidence in the Uygur and Han populations of Xinjiang,China[J]. Int J Clin Exp Pathol,2015,8(2):1844-1854.
[24] DiRenzo DM,Chaudhary MA,Shi X,et al. A crosstalk between TGF-β/Smad3 and Wnt/β-catenin pathways promotes vascular smooth muscle cell proliferation[J]. Cell Signal,2016,28(5):498-505.
[25] Zhou T,Zu G,Zhang X,et al. Neuroprotective effects of ginsenoside Rg1 through the Wnt/β-catenin signaling pathway in both in vivo and in vitro models of Parkinson's disease[J]. Neuropharmacol,2016,101:480-489.
[26]周金敬,毕晓,徐勇,等.组织因子途径抑制物-2表达与肝癌细胞体外侵袭能力的实验研究[J].肿瘤,2009,29(10):976-979.Zhou JJ,Bi X,Xu Y,et al. The expression of tissue factor pathway inhibitor-2 and invasive cability of hepatocelluar carcinoma in vitro[J]. Tumor,2009,29(10):976-979.
[27] Zhao XP,He SW,Yue B,et al. Molecular characterization,expression analysis,and bactericidal activity of the derivative peptides of TFPI-1 and TFPI-2 in half-smooth tongue sole,Cynoglossus semilaevis[J]. Fish Shellfish Immunol,2016,58:563-571.