【摘要】：間充質(zhì)干細(xì)胞(MSCs)是一類具有自我更新和多向分化潛能的成體干細(xì)胞,可分化成骨細(xì)胞、軟骨細(xì)胞、脂肪細(xì)胞等。在細(xì)胞治療及組織工程等方面顯示出巨大的應(yīng)用前景。人臍帶間充質(zhì)干細(xì)胞(hUC-MSCs)因其免疫原性低,獲取方便,不涉及倫理問題的優(yōu)點(diǎn)已成為干細(xì)胞治療研究熱點(diǎn)。然而,制約hUC-MSCs臨床應(yīng)用的一個(gè)關(guān)鍵問題是如何獲取足夠數(shù)量的細(xì)胞滿足臨床治療需要。 目前研究提示,溶血磷脂(Lysophospholipids, LPs)中兩個(gè)重要成員—鞘氨醇-1-磷酸(Sphingosine-1-phosphate, S1P)和溶血磷脂酸(Lysophosphatidic acid,LPA),具有促細(xì)胞增殖作用。但對hUC-MSCs的促增殖作用和分化及其機(jī)制方面卻未見報(bào)導(dǎo)。所以,該研究對S1P、LPA是否促進(jìn)hUC-MSCs增殖并維持表面標(biāo)記物表達(dá)及機(jī)制進(jìn)行了深入研究。為解決hUC-MSCs慢周期性提供一種研究思路,促進(jìn)hUC-MSCs在細(xì)胞治療及組織工程中的應(yīng)用；對無血清培養(yǎng)基的開發(fā)也有潛在的應(yīng)用價(jià)值。 本研究用MTT法證實(shí)了S1P、LPA對hUC-MSCs具有促增殖作用,并確定了其具體信號通路,其中Real time PCR檢測hUC-MSCs中溶血磷脂受體表達(dá)。探討受體拮抗劑、Gi蛋白、ERK抑制劑處理對S1P、LPA誘導(dǎo)hUC-MSCs增殖影響。采用蛋白印跡技術(shù)檢測ERK1/2磷酸化。流式細(xì)胞術(shù)檢測hUC-MSCs表面標(biāo)記物表達(dá)情況。 結(jié)果顯示,S1P、LPA均能促進(jìn)hUC-MSCs增殖且呈劑量依賴性。在hUC-MSCs中優(yōu)勢表達(dá)S1PR1-3。S1PR1/3拮抗劑完全抑制而S1PR3拮抗劑部分抑制S1P誘導(dǎo)hUC-MSCs增殖、S1PR2拮抗劑對此沒有明顯影響。LPAR1/3拮抗劑完全抑制LPA誘導(dǎo)hUC-MSCs增殖。Western blot顯示S1P、LPA均能促進(jìn)hUC-MSCs中ERK1/2磷酸化。Gi蛋白、ERK抑制劑完全抑制S1P、LPA誘導(dǎo)hUC-MSCs增殖及ERK1/2磷酸化,而PI3K抑制劑對S1P誘導(dǎo)hUC-MSCs增殖沒有明顯作用。流式細(xì)胞儀檢測發(fā)現(xiàn)S1P、LPA對hUC-MSCs表面標(biāo)記物表達(dá)沒有明顯影響。 結(jié)論：S1P、LPA具有促進(jìn)hUC-MSCs增殖作用,S1P是通過S1PR1/3、Gi偶聯(lián)蛋白并進(jìn)一步活化ERK1/2信號通路促進(jìn)hUC-MSCs增殖。LPA通過LPAR1、Gi偶聯(lián)蛋白及ERK1/2信號通路促進(jìn)hUC-MSCs增殖。S1P、LPA對表面標(biāo)記物表達(dá)均沒有明顯影響,表明可以維持hUC-MSCs的多潛能性(不分化)。該研究對于hUC-MSCs的臨床應(yīng)用和無血清培養(yǎng)基開發(fā)均有潛在的應(yīng)用價(jià)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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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