溶血磷脂促人脐带间充质干细胞增殖的机理及对表面标记物表达影响
发布时间：2018-09-11 09:59
【摘要】：间充质干细胞(MSCs)是一类具有自我更新和多向分化潜能的成体干细胞,可分化成骨细胞、软骨细胞、脂肪细胞等。在细胞治疗及组织工程等方面显示出巨大的应用前景。人脐带间充质干细胞(hUC-MSCs)因其免疫原性低,获取方便,不涉及伦理问题的优点已成为干细胞治疗研究热点。然而,制约hUC-MSCs临床应用的一个关键问题是如何获取足够数量的细胞满足临床治疗需要。 目前研究提示,溶血磷脂(Lysophospholipids, LPs)中两个重要成员—鞘氨醇-1-磷酸(Sphingosine-1-phosphate, S1P)和溶血磷脂酸(Lysophosphatidic acid,LPA),具有促细胞增殖作用。但对hUC-MSCs的促增殖作用和分化及其机制方面却未见报导。所以,该研究对S1P、LPA是否促进hUC-MSCs增殖并维持表面标记物表达及机制进行了深入研究。为解决hUC-MSCs慢周期性提供一种研究思路,促进hUC-MSCs在细胞治疗及组织工程中的应用；对无血清培养基的开发也有潜在的应用价值。 本研究用MTT法证实了S1P、LPA对hUC-MSCs具有促增殖作用,并确定了其具体信号通路,其中Real time PCR检测hUC-MSCs中溶血磷脂受体表达。探讨受体拮抗剂、Gi蛋白、ERK抑制剂处理对S1P、LPA诱导hUC-MSCs增殖影响。采用蛋白印迹技术检测ERK1/2磷酸化。流式细胞术检测hUC-MSCs表面标记物表达情况。 结果显示,S1P、LPA均能促进hUC-MSCs增殖且呈剂量依赖性。在hUC-MSCs中优势表达S1PR1-3。S1PR1/3拮抗剂完全抑制而S1PR3拮抗剂部分抑制S1P诱导hUC-MSCs增殖、S1PR2拮抗剂对此没有明显影响。LPAR1/3拮抗剂完全抑制LPA诱导hUC-MSCs增殖。Western blot显示S1P、LPA均能促进hUC-MSCs中ERK1/2磷酸化。Gi蛋白、ERK抑制剂完全抑制S1P、LPA诱导hUC-MSCs增殖及ERK1/2磷酸化,而PI3K抑制剂对S1P诱导hUC-MSCs增殖没有明显作用。流式细胞仪检测发现S1P、LPA对hUC-MSCs表面标记物表达没有明显影响。 结论：S1P、LPA具有促进hUC-MSCs增殖作用,S1P是通过S1PR1/3、Gi偶联蛋白并进一步活化ERK1/2信号通路促进hUC-MSCs增殖。LPA通过LPAR1、Gi偶联蛋白及ERK1/2信号通路促进hUC-MSCs增殖。S1P、LPA对表面标记物表达均没有明显影响,表明可以维持hUC-MSCs的多潜能性(不分化)。该研究对于hUC-MSCs的临床应用和无血清培养基开发均有潜在的应用价值。
[Abstract]:Mesenchymal stem cell (MSCs) is a kind of adult stem cells with self-renewal and multi-differentiation potential, which can differentiate osteoblasts, chondrocytes, adipocytes and so on. It shows great application prospect in cell therapy and tissue engineering. Human umbilical cord mesenchymal stem cells (hUC-MSCs) have become a research hotspot in stem cell therapy because of their low immunogenicity, easy access and no ethical problems. However, a key problem in clinical application of hUC-MSCs is how to obtain a sufficient number of cells to meet the needs of clinical treatment. The present studies suggest that two important members of lysophosphatidylcholine (Lysophospholipids, LPs), Sphingosine-1-phosphate, S1P and lysophosphatidic acid (Lysophosphatidic acid,LPA), can promote cell proliferation. However, the proliferation and differentiation of hUC-MSCs and its mechanism have not been reported. Therefore, the mechanism and mechanism of S1PnLPA promoting hUC-MSCs proliferation and maintaining the expression of surface markers were studied. In order to solve the slow periodicity of hUC-MSCs and promote the application of hUC-MSCs in cell therapy and tissue engineering, it has potential application value for the development of serum-free medium. In this study, MTT method was used to confirm that S1PnLPA could promote the proliferation of hUC-MSCs and determine its specific signal pathway. Real time PCR was used to detect the expression of lysophosphatidylcholine receptor in hUC-MSCs. Objective: to investigate the effects of receptor antagonist Gi protein ERK inhibitor on the proliferation of hUC-MSCs induced by LPA. ERK1/2 phosphorylation was detected by Western blot. The expression of hUC-MSCs surface markers was detected by flow cytometry. The results showed that S-1 PPA could promote the proliferation of hUC-MSCs in a dose-dependent manner. Expression of S1PR1-3.S1PR1/3 antagonist in hUC-MSCs was completely inhibited, while S1PR3 antagonist partly inhibited S1P-induced proliferation of hUC-MSCs. S1PR2 antagonist had no significant effect on this. LPAR1 / 3 antagonist completely inhibited hUC-MSCs proliferation induced by LPA. Western blot showed that S1PnLPA could promote ERK1/2 phosphoric acid in hUC-MSCs. Gi inhibitor completely inhibited the proliferation of hUC-MSCs and the phosphorylation of ERK1/2 induced by S1PnLPA. However, PI3K inhibitor had no obvious effect on S 1P induced hUC-MSCs proliferation. Flow cytometry showed that S1PnLPA had no effect on the expression of hUC-MSCs surface markers. Conclusion S1P can promote the proliferation of hUC-MSCs through S1PR1 / 3Gi coupling protein and further activate ERK1/2 signaling pathway to promote hUC-MSCs proliferation. LPA has no significant effect on the expression of surface markers by promoting hUC-MSCs proliferation through LPAR1,Gi coupling protein and ERK1/2 signaling pathway. It is suggested that the multipotential (indifferentiation) of hUC-MSCs can be maintained. This study has potential application value for clinical application of hUC-MSCs and development of serum-free medium.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
[Abstract]:Mesenchymal stem cell (MSCs) is a kind of adult stem cells with self-renewal and multi-differentiation potential, which can differentiate osteoblasts, chondrocytes, adipocytes and so on. It shows great application prospect in cell therapy and tissue engineering. Human umbilical cord mesenchymal stem cells (hUC-MSCs) have become a research hotspot in stem cell therapy because of their low immunogenicity, easy access and no ethical problems. However, a key problem in clinical application of hUC-MSCs is how to obtain a sufficient number of cells to meet the needs of clinical treatment. The present studies suggest that two important members of lysophosphatidylcholine (Lysophospholipids, LPs), Sphingosine-1-phosphate, S1P and lysophosphatidic acid (Lysophosphatidic acid,LPA), can promote cell proliferation. However, the proliferation and differentiation of hUC-MSCs and its mechanism have not been reported. Therefore, the mechanism and mechanism of S1PnLPA promoting hUC-MSCs proliferation and maintaining the expression of surface markers were studied. In order to solve the slow periodicity of hUC-MSCs and promote the application of hUC-MSCs in cell therapy and tissue engineering, it has potential application value for the development of serum-free medium. In this study, MTT method was used to confirm that S1PnLPA could promote the proliferation of hUC-MSCs and determine its specific signal pathway. Real time PCR was used to detect the expression of lysophosphatidylcholine receptor in hUC-MSCs. Objective: to investigate the effects of receptor antagonist Gi protein ERK inhibitor on the proliferation of hUC-MSCs induced by LPA. ERK1/2 phosphorylation was detected by Western blot. The expression of hUC-MSCs surface markers was detected by flow cytometry. The results showed that S-1 PPA could promote the proliferation of hUC-MSCs in a dose-dependent manner. Expression of S1PR1-3.S1PR1/3 antagonist in hUC-MSCs was completely inhibited, while S1PR3 antagonist partly inhibited S1P-induced proliferation of hUC-MSCs. S1PR2 antagonist had no significant effect on this. LPAR1 / 3 antagonist completely inhibited hUC-MSCs proliferation induced by LPA. Western blot showed that S1PnLPA could promote ERK1/2 phosphoric acid in hUC-MSCs. Gi inhibitor completely inhibited the proliferation of hUC-MSCs and the phosphorylation of ERK1/2 induced by S1PnLPA. However, PI3K inhibitor had no obvious effect on S 1P induced hUC-MSCs proliferation. Flow cytometry showed that S1PnLPA had no effect on the expression of hUC-MSCs surface markers. Conclusion S1P can promote the proliferation of hUC-MSCs through S1PR1 / 3Gi coupling protein and further activate ERK1/2 signaling pathway to promote hUC-MSCs proliferation. LPA has no significant effect on the expression of surface markers by promoting hUC-MSCs proliferation through LPAR1,Gi coupling protein and ERK1/2 signaling pathway. It is suggested that the multipotential (indifferentiation) of hUC-MSCs can be maintained. This study has potential application value for clinical application of hUC-MSCs and development of serum-free medium.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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