TGF-β1通过p38MAPK信号通路调节大鼠睾丸支持细胞闭锁蛋白表达的研究
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摘要
目的探讨TGF-β1介导的p38MAPK信号通路对大鼠睾丸支持细胞闭锁蛋白表达的作用及意义。方法体外分离培养大鼠睾丸支持细胞,随机分为空白对照组、刺激组、受体阻滞剂组、受体阻滞剂+刺激组、抑制因子组和抑制因子+刺激组6组;测定各组细胞增殖情况、p38MAPK mRNA和闭锁mRNA的表达水平以及各组细胞闭锁蛋白和P-p38MAPK蛋白表达水平。结果组间比较差异有统计学意义(F=4.86,P<0.05)。刺激组细胞增殖活性升高,差异有统计学意义(P<0.05);刺激组闭锁蛋白mRNA表达比空白对照组降低,p38MAPK mRNA表达升高;抑制因子组p38MAPK mRNA表达降低,差异均有统计学意义(P<0.05)。受体阻滞剂+刺激组闭锁蛋白mRNA表达比刺激组升高,p38MAPK mRNA表达降低;抑制因子+刺激组闭锁蛋白mRNA表达升高,p38MAPK mRNA表达降低,差异均有统计学意义(P<0.05)。刺激组闭锁蛋白表达比对照组降低,P-p38MAPK蛋白表达升高;抑制因子组P-p38MAPK蛋白表达降低,差异有统计学意义(P<0.05)。受体阻滞剂+刺激组闭锁蛋白表达比刺激组升高,P-p38MAPK蛋白表达降低;抑制因子+刺激组闭锁蛋白表达升高,P-p38MAPK蛋白表达降低差异均有统计学意义(P<0.05)。结论 TGF-β1可能通过p38MAPK信号通路调控大鼠睾丸支持细胞闭锁蛋白的表达。
Objective To investigate the role of TGF-β1 mediated p38MAPK signaling pathway in the expression of occludin protein in rat sertoli cell. Methods The rats sertoli cells were isolated and cultured in vitro,and then randomly assigned to blank control group,stimulus group,receptor blockers group,receptor blockers + stimulus group,inhibitory factor group and inhibitory factor + stimulus group. MTT method was used to determine the proliferation of rat sertoli cells in each group.RT-PCR was used to determine the expression of p38MAPK mRNA and occludin mRNA in each group. Occludin protein and p-p38MAPK protein were detected by Western-blot. Results The results of MTT showed that the difference between groups was statistically significant( F = 4. 86,P < 0. 05). The cell proliferation activity of stimulus group significantly increased compared with the other five groups( P < 0. 05). In stimulus group,the expression of occludin mRNA significantly decreased,and p38MAPK mRNA significantly increased,compared with the blank control group. In inhibitory factor group,p38MAPK mRNA significantly decreased compared with the blank control group( P < 0. 05). In the stimulus group,occludin mRNA significantly increased,with the difference statistically significant( P < 0. 05). mRNA increased but p38MAPK mRNA decreased in receptor blockers + stimulus group,with the differences statistically significant( P < 0. 05). Compared with the control group,p38MAPK mRN increased in the stimulus group; in inhibitory group,p38MAPK mRN decreased,with the differences statistically significant( P < 0. 05). Compared with the stimulus group,P-p38MAPK protein significantly decreased in receptor blockers + stimulus group. Compared with the stimulus group,P-p38MAPK protein significantly decreased in inhibitory factor + stimulus group,with the differences statistically significant( P < 0. 05). Conclusion TGF-β1 may regulate the expression of occludin protein in rat sertoli cell by p38MAPK signaling pathway.
Objective To investigate the role of TGF-β1 mediated p38MAPK signaling pathway in the expression of occludin protein in rat sertoli cell. Methods The rats sertoli cells were isolated and cultured in vitro,and then randomly assigned to blank control group,stimulus group,receptor blockers group,receptor blockers + stimulus group,inhibitory factor group and inhibitory factor + stimulus group. MTT method was used to determine the proliferation of rat sertoli cells in each group.RT-PCR was used to determine the expression of p38MAPK mRNA and occludin mRNA in each group. Occludin protein and p-p38MAPK protein were detected by Western-blot. Results The results of MTT showed that the difference between groups was statistically significant( F = 4. 86,P < 0. 05). The cell proliferation activity of stimulus group significantly increased compared with the other five groups( P < 0. 05). In stimulus group,the expression of occludin mRNA significantly decreased,and p38MAPK mRNA significantly increased,compared with the blank control group. In inhibitory factor group,p38MAPK mRNA significantly decreased compared with the blank control group( P < 0. 05). In the stimulus group,occludin mRNA significantly increased,with the difference statistically significant( P < 0. 05). mRNA increased but p38MAPK mRNA decreased in receptor blockers + stimulus group,with the differences statistically significant( P < 0. 05). Compared with the control group,p38MAPK mRN increased in the stimulus group; in inhibitory group,p38MAPK mRN decreased,with the differences statistically significant( P < 0. 05). Compared with the stimulus group,P-p38MAPK protein significantly decreased in receptor blockers + stimulus group. Compared with the stimulus group,P-p38MAPK protein significantly decreased in inhibitory factor + stimulus group,with the differences statistically significant( P < 0. 05). Conclusion TGF-β1 may regulate the expression of occludin protein in rat sertoli cell by p38MAPK signaling pathway.
引文
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[12]赖琼,贺丽萍,谢远杰,等.p-p38MAPK在SD大鼠隐睾中的表达[J].中国计划生育学杂志,2004,12(12):735-737.
[13]BakkebM,Huse K,VI Hilden,et al.TGF-b-induced growth inhibition in B-cell lymphomacorrelates with Smad1/5 signalling and constitutively active p38 MAPK[J].Bio Med Central Immunol,2010,11(1):1-10.
[14]张奇,白晓东,付小兵.p38MAPK信号通路研究进展[J].感染、炎症、修复,2005,6(2):121-123.
[15]Cheng CY,Mruk DD.The blood-testis barrier and its implications for male contraception[J].Pharmacological Reviews,2012,64(1):16-64.
[16]Fijak M,Meinhardt A.The testis in immune privilege[J].Immunological Reviews,2006,213(1):66.
[17]Pérez CV,Sobarzo CM,Jacobo PV,et al.Loss of occludin expression and impairment of blood-testis barrier permeability in rats with autoimmune orchitis:effect of interleukin 6 on Sertoli cell tight junctions[J].Biol Reprod,2012,87(5):122.
[18]Vivian W.Phosphorylation of occludin correlates with occludin localization and function at the tight junction[J].Ame J Physiol,1997,273(1):1859-1867.
[2]Liu WY,Wang CH,Mruk D.TGF-β3 regulates the blood-testis barrier dynamics via the p38 mitogen-activated protein(MAP)kinase pathway:an in vivo study[J].Endocrinology,2003,144(4):1139-1142.
[3]Yi C,Liu D,Fong CC,et al.Gold nanoparticles promote osteogenic differentiation of mesenchymal stem cells through p38 MAPK pathway[J].ACS Nano,2010,4(11):6439-6449.
[4]Zhou C,Zhou J,Sheng F,et al.The heme oxygenase-1 inhibitor Zn PPIX induces non-canonical,Beclin 1-independent,autophagy through p38 MAPK pathway[J].Acta Biochim Biophys Sin(Shanghai),2012,44(10):815-822.
[5]Du QC,Zhang DZ,Chen XJ,et al.The effect of p38MAPK on cyclic stretch in human facial hypertrophic scar fibroblast differentiation[J].PLo S One,2013,8(10):e75635.
[6]Morales MG,Vazquez Y,Acu1a MJ,et al.Angiotensin II-induced pro-fibrotic effects require p38MAPK activity and transforming growth factor beta 1 expression in skeletal muscle cells[J].Int J Biochem Cell Biol,2012,44(11):1993-2002.
[7]丁云天,张明,杨洋,等.大鼠睾丸支持细胞的分离培养及鉴定[J].南昌大学学报,2016,40(5):493-497.
[8]侯武刚,张远强,赵洁,等.转化生长因子β及其受体在小鼠精子发生过程中的表达[J].解剖学报,2006,37(2):226-231.
[9]Gonzalez CR,Matzkin ME,Frungieri MB,et al.Expression of the TGF-beta1 system in human testicular pathologies[J].Reproduct Biol Endocrinol,2010,8(1):148.
[10]Wendy V,Ingman S,Robertson A.Transforming Growth Factor-1Null Mutation Causes Infertility in Male Mice Associated with Testosterone Deficiency and Sexual Dysfunction[J].Endocrinology,2007,148(8):4032-4043.
[11]Wrighton KH,Lin X,Feng XH.Phospho-control of TGF-betasuperfamily signaling[J].Cell Res,2009,19(1):8.
[12]赖琼,贺丽萍,谢远杰,等.p-p38MAPK在SD大鼠隐睾中的表达[J].中国计划生育学杂志,2004,12(12):735-737.
[13]BakkebM,Huse K,VI Hilden,et al.TGF-b-induced growth inhibition in B-cell lymphomacorrelates with Smad1/5 signalling and constitutively active p38 MAPK[J].Bio Med Central Immunol,2010,11(1):1-10.
[14]张奇,白晓东,付小兵.p38MAPK信号通路研究进展[J].感染、炎症、修复,2005,6(2):121-123.
[15]Cheng CY,Mruk DD.The blood-testis barrier and its implications for male contraception[J].Pharmacological Reviews,2012,64(1):16-64.
[16]Fijak M,Meinhardt A.The testis in immune privilege[J].Immunological Reviews,2006,213(1):66.
[17]Pérez CV,Sobarzo CM,Jacobo PV,et al.Loss of occludin expression and impairment of blood-testis barrier permeability in rats with autoimmune orchitis:effect of interleukin 6 on Sertoli cell tight junctions[J].Biol Reprod,2012,87(5):122.
[18]Vivian W.Phosphorylation of occludin correlates with occludin localization and function at the tight junction[J].Ame J Physiol,1997,273(1):1859-1867.