TGF-β1对人蜕膜基质细胞趋化因子及其受体表达的影响
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摘要
为研究转化生长因子-β（TGF-β）与蜕膜基质细胞分泌的趋化因子之间的关系、探讨其在蜕膜基质细胞中发挥免疫调节作用的机制，本试验采用胰酶-胶原酶-DNaseⅠ联合消化的方法分离人滋养层细胞和人蜕膜基质细胞，对细胞进行体外培养及鉴定；运用RT-PCR方法对不同浓度TGF-β处理后人蜕膜基质细胞中趋化因子及其受体mRNA水平进行分析，经Western-blot方法检测TGF-β对人蜕膜基质细胞中趋化因子及其受体蛋白水平的影响，结果如下：
1.通过胰蛋白酶、胶原酶Ⅳ和DNaseⅠ酶联合消化法成功分离到人绒毛滋养层细胞和人蜕膜基质细胞，对两种细胞体外培养体系进行了优化，经本培养体系培养得到的人绒毛膜滋养层细胞呈片状铺展生长，具上皮样形态，细胞角蛋白（Cytokeratin）染色阳性；人蜕膜基质细胞呈细长纤维状，E2和P4诱导后细胞形态逐渐变为钝圆形，细胞轮廓清晰，胞浆颗粒丰富；波形蛋白和催乳素染色阳性。
2.分别以0ng/mL、1ng/mL、5ng/mL和10ng/mL的TGF-β1处理人蜕膜基质细胞，RT-PCR和Western-blot检测结果表明趋化因子配体CXCL12、CXCL16和CX3CL1mRNA和蛋白的表达均有所下降，而趋化因子受体CXCR4、CXCR6和CX3CR1mRNA和蛋白的表达则升高。在mRNA水平，各浓度的TGF-β1均能极显著抑制CX3CL1的表达（P＜0.01）；10ng/mL的TGF-β1对CXCL12具有显著的抑制作用（P＜0.05）；5ng/mL的TGF-β1对CXCL16有显著抑制效应（P＜0.05）。与趋化因子配体不同，10ng/mL的TGF-β1能够显著促进趋化因子受体CXCR4和CXCR6的表达（P＜0.05）。在蛋白水平，10ng/mL的TGF-β1能够极显著的抑制CX3CL1的表达（P＜0.01），显著抑制CXCL16的表达（P＜0.05）；显著促进CXCR6的表达（P＜0.05）。
结论：本研究建立的人绒毛滋养层细胞和人蜕膜基质细胞的分离、体外培养和纯化方法为进一步在体外研究两种细胞参与蜕膜化过程的分子机制研究打下了基础。TGF-β1能够下调蜕膜基质细胞中趋化因子配体CX3CL1、CXCL12和CXCL16的表达，上调趋化因子受体CXCR4和CXCR6的表达。结果说明TGF-β1对趋化因子配体/受体有显著的调节作用，并通过趋化因子参与母胎界面的免疫调节。
In order to study the relationship between the transforming growth factor β (TGF-β) andchemokine which secreted by human stromal cells and to explore the mechanism of itsimmunomodulatory effects in human decidual stromal cells, In this experiment,we isolatedhuman trophoblast cells and human decidual stromal cells by the way of digestion with thetrypsin-collagenase-DNaseⅠ, the cells were cultured and identified in vitro. The expressionof chemokines and its receptors in early human decidual stromal cells after treatment withfour different concentrations of TGF-β1were detected by RT-PCR and Western-blot. Theresult showed that:
1. Human trophoblast cells and human decidual stromal cells were isolated successfullyby the way of the digestion with trypsin, collagenase Ⅳa nd DNase Ⅰ. The cell culturesystem of two kinds of cells had been optimized, It is observing that human trophoblast cellswere a typical epithelial-like cell morphology, spreading growth by sheet, The staining ofcytokeratin was positive. Human decidual stromal cells were a fibroblast-like cellmorphology, the cell morphology gradually became blunt round after inducible with E2andP4.The cells outline were clear and the cytoplasmic particles were rich. The staining ofvimentin and prolactin were positive.
2. The mRNA and protein level of chemokines(CXCL12, CXCL16and CX3CL1) weredecreased by RT-PCR and Western-blot after treatment with four different concentrations ofTGF-β1(0ng/mL,1ng/mL,5ng/mL and10ng/mL). On the contrary, the mRNA and proteinlevel of chemokine receptors were increased.At the mRNA level. Four concentrations ofTGF-β1can inhibit the expression of CX3CL1significantly(P＜0.01),10ng/mL TGF-β1caninhibit the expression of CXCL12significantly(P＜0.05),5ng/mL TGF-β1can inhibit theexpression of CXCL16significantly(P＜0.05),10ng/mL TGF-β1can promote the expressionof CXCL16significantly(P＜0.05). At the protein level,10ng/mL TGF-β1can inhibit theexpression of CX3CL1(P＜0.01) and CXCL16(P＜0.05) significantly, but can promote theexpression of CXCR6(P＜0.05).
Conclusion: The method of separation,cultured and purification of human trophoblastcell and human decidual stromal cell laid the foundation for further study the molecular mechanism of decidualization in the study. TGF-β1can down-regulated the expression ofthe chemokine(CXCL12,CXCL16and CX3CL1) and up-regulated the expression of thereceptors(CXCR4and CXCR6). This study suggests that TGF-β1plays a significant role inregulating chemokine ligand/receptor expression and participate in immune tolerance ofmaternal-fetal interface by chemokines.
1.通过胰蛋白酶、胶原酶Ⅳ和DNaseⅠ酶联合消化法成功分离到人绒毛滋养层细胞和人蜕膜基质细胞，对两种细胞体外培养体系进行了优化，经本培养体系培养得到的人绒毛膜滋养层细胞呈片状铺展生长，具上皮样形态，细胞角蛋白（Cytokeratin）染色阳性；人蜕膜基质细胞呈细长纤维状，E2和P4诱导后细胞形态逐渐变为钝圆形，细胞轮廓清晰，胞浆颗粒丰富；波形蛋白和催乳素染色阳性。
2.分别以0ng/mL、1ng/mL、5ng/mL和10ng/mL的TGF-β1处理人蜕膜基质细胞，RT-PCR和Western-blot检测结果表明趋化因子配体CXCL12、CXCL16和CX3CL1mRNA和蛋白的表达均有所下降，而趋化因子受体CXCR4、CXCR6和CX3CR1mRNA和蛋白的表达则升高。在mRNA水平，各浓度的TGF-β1均能极显著抑制CX3CL1的表达（P＜0.01）；10ng/mL的TGF-β1对CXCL12具有显著的抑制作用（P＜0.05）；5ng/mL的TGF-β1对CXCL16有显著抑制效应（P＜0.05）。与趋化因子配体不同，10ng/mL的TGF-β1能够显著促进趋化因子受体CXCR4和CXCR6的表达（P＜0.05）。在蛋白水平，10ng/mL的TGF-β1能够极显著的抑制CX3CL1的表达（P＜0.01），显著抑制CXCL16的表达（P＜0.05）；显著促进CXCR6的表达（P＜0.05）。
结论：本研究建立的人绒毛滋养层细胞和人蜕膜基质细胞的分离、体外培养和纯化方法为进一步在体外研究两种细胞参与蜕膜化过程的分子机制研究打下了基础。TGF-β1能够下调蜕膜基质细胞中趋化因子配体CX3CL1、CXCL12和CXCL16的表达，上调趋化因子受体CXCR4和CXCR6的表达。结果说明TGF-β1对趋化因子配体/受体有显著的调节作用，并通过趋化因子参与母胎界面的免疫调节。
In order to study the relationship between the transforming growth factor β (TGF-β) andchemokine which secreted by human stromal cells and to explore the mechanism of itsimmunomodulatory effects in human decidual stromal cells, In this experiment,we isolatedhuman trophoblast cells and human decidual stromal cells by the way of digestion with thetrypsin-collagenase-DNaseⅠ, the cells were cultured and identified in vitro. The expressionof chemokines and its receptors in early human decidual stromal cells after treatment withfour different concentrations of TGF-β1were detected by RT-PCR and Western-blot. Theresult showed that:
1. Human trophoblast cells and human decidual stromal cells were isolated successfullyby the way of the digestion with trypsin, collagenase Ⅳa nd DNase Ⅰ. The cell culturesystem of two kinds of cells had been optimized, It is observing that human trophoblast cellswere a typical epithelial-like cell morphology, spreading growth by sheet, The staining ofcytokeratin was positive. Human decidual stromal cells were a fibroblast-like cellmorphology, the cell morphology gradually became blunt round after inducible with E2andP4.The cells outline were clear and the cytoplasmic particles were rich. The staining ofvimentin and prolactin were positive.
2. The mRNA and protein level of chemokines(CXCL12, CXCL16and CX3CL1) weredecreased by RT-PCR and Western-blot after treatment with four different concentrations ofTGF-β1(0ng/mL,1ng/mL,5ng/mL and10ng/mL). On the contrary, the mRNA and proteinlevel of chemokine receptors were increased.At the mRNA level. Four concentrations ofTGF-β1can inhibit the expression of CX3CL1significantly(P＜0.01),10ng/mL TGF-β1caninhibit the expression of CXCL12significantly(P＜0.05),5ng/mL TGF-β1can inhibit theexpression of CXCL16significantly(P＜0.05),10ng/mL TGF-β1can promote the expressionof CXCL16significantly(P＜0.05). At the protein level,10ng/mL TGF-β1can inhibit theexpression of CX3CL1(P＜0.01) and CXCL16(P＜0.05) significantly, but can promote theexpression of CXCR6(P＜0.05).
Conclusion: The method of separation,cultured and purification of human trophoblastcell and human decidual stromal cell laid the foundation for further study the molecular mechanism of decidualization in the study. TGF-β1can down-regulated the expression ofthe chemokine(CXCL12,CXCL16and CX3CL1) and up-regulated the expression of thereceptors(CXCR4and CXCR6). This study suggests that TGF-β1plays a significant role inregulating chemokine ligand/receptor expression and participate in immune tolerance ofmaternal-fetal interface by chemokines.
引文
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