IGF1R信号通路在心外膜祖细胞增殖和上皮间质转化中的作用及机制研究

发布时间：2018-04-27 05:12

本文选题：胰岛素样生长因子1受体 + 心外膜祖细胞　；参考：《重庆医科大学》2017年博士论文

【摘要】：心外膜祖细胞被证实在心脏发育过程中起着重要作用,它能通过上皮间质转化(EMT)形成心外膜来源细胞(EPDCs),并在一系列的信号通路作用下最终分化为心脏成纤维细胞以及冠脉血管平滑肌细胞,参与心脏纤维骨架和冠脉血管的形成。因其具有多项分化潜能,心外膜祖细胞被认为是心脏的第三心生区,是一个能特征性表达Tbx18、Wt1和Tcf21等基因的祖细胞池。成年心脏的心外膜细胞丧失心外膜祖细胞的特征,但有研究发现心肌梗死后梗死区周边处于静止期的心外膜细胞可再次激活,重新表达胚胎期心外膜祖细胞基因,如Tbx18和Wt1,并不断增殖、EMT、迁移进入梗死区域参与心脏修复及血管再生。因此,研究心外膜祖细胞增殖以及EMT机制不仅对于深入探索心脏生长发育机制具有重要的意义,且有助于推进心外膜祖细胞在心脏再生领域的相关应用。胰岛素样生长因子1型受体(IGF1R)介导的信号通路已经被证实参与多种细胞的增殖以及EMT过程,并且有研究通过原位杂交的方式证实在E11.5-E14.5天心外膜上有IGF1R基因的表达,我们猜测此信号通路可能参与调控心外膜祖细胞的增殖以及EMT过程。IGF1R参与调控细胞增殖以及EMT的作用除了通过传统经典的PI3K/AKT以及MAPK/ERK途径实现外,近年来发现其也可通过FAK途径产生相似的生理或者病理作用,因此,我们通过本实验研究探索IGF1R信号通路在心外膜祖细胞增殖以及EMT中的作用,以及探讨其相关作用是否通过FAK介导实现。第一部分:IGF1R信号通路在小鼠胚胎心脏组织中的时空表达目的:探索IGF1R及其主要配体IGF1和IGF2在小鼠胚胎心脏组织中的时空表达情况。方法:雌性及雄性C57BL/6小鼠进行交配,取E11.5-E17.5天的胚胎组织进行冰冻切片,免疫荧光染色确定IGF1R及其配体IGF1和IGF2在E11.5-E17.5天胚胎心脏中的表达情况。结果:免疫荧光染色结果提示IGF1R在E11.5-E17.5天的胚胎心外膜上有明确表达。IGF1在E11.5-E13.5天的胚胎心外膜上表达量较少,在E14.5-E17.5天的胚胎心外膜上明显表达。作为胚胎期生长因子,IGF2在胚胎心外膜的表达比IGF1更加明显且高表达贯穿E11.5-E17.5天。结论:IGF1R及其配体IGF1和IGF2在E11.5-E17.5天的胚胎心外膜上均有表达,提示IGF1R信号通路可能参与调控小鼠心外膜祖细胞的发育过程。第二部分:IGF1R信号通路在心外膜祖细胞增殖中的作用及机制研究目的:建立小鼠心外膜祖细胞体外培养体系,并通过干预细胞IGF1R信号通路,研究IGF1R信号通路是否参与调控心外膜祖细胞的增殖,并深入探讨其调控增殖作用是否通过FAK介导实现。方法:雌性及雄性C57BL/6小鼠进行交配,取E12.5d的胚胎心脏组织进行心外膜祖细胞培养,使用免疫荧光染色技术检测其特异性标志物Tbx18和Wt1的表达情况来进行细胞鉴定。同时也通过免疫荧光染色检测IGF1R在体外培养的原代小鼠心外膜祖细胞中的表达情况。然后分别用不同浓度的Picropodophyllin(PPP)(0、0.2μM、0.4μM、0.8μm和1.6μM)干预原代心外膜祖细胞72小时,CCK-8(Cell Counting Kit-8)检测细胞增殖情况。然后将体外培养的原代心外膜祖细胞随机分为两组:对照组和PPP组(给予0.4μM PPP干预)。培养72小时后,使用免疫荧光染色检测两组细胞增殖标志物Ki67的表达以及FAK(p Y397)磷酸化水平;q RT-PCR测定Ki67、CCND1(细胞周期蛋白Cyclin D1编码基因)以及Ptk2和Ptk2b(FAK编码基因)在m RNA水平的表达情况,并通过流式细胞技术检测两组细胞之间细胞周期的变化。考虑到血清之中含有胰岛素样生长因子,将原代心外膜祖细胞培养基中的胎牛血清(Fetal Bovine Serum,FBS)由10%降至1%。然后,分别用不同浓度的IGF1(0、5ng/ml、10ng/ml和50ng/ml)和IGF2(0、10ng/ml、50ng/ml和100ng/ml)干预细胞72h,CCK-8检测细胞增殖情况。并将细胞随机分为:对照组、IGF1组(50ng/ml)、IGF2组(100ng/ml)、IGF1+Y15组(50ng/ml IGF1+1n M Y15)和IGF2+Y15组(100ng/ml IGF2+1n M Y15)。CCK-8检测各组细胞增殖能力,免疫荧光染色检测Ki67表达以及FAK(p Y397)磷酸化情况,q RT-PCR检测Ki67、CCND1、Ptk2和Ptk2在m RNA水平的表达情况,流式细胞技术检测各组细胞周期变化。结果:培养出来的细胞在倒置细胞显微镜下呈现“铺路石”样外观,形态规则、单一,细胞间连接紧密,细胞核内特异性表达Tbx18及Wt1,且阳性细胞数高。细胞免疫荧光染色提示小鼠心外膜祖细胞膜上点状分布IGF1R。CCK-8实验结果提示随着PPP浓度的增加,心外膜祖细胞增值能力逐渐降低。与对照组相比,免疫荧光染色显示PPP组细胞Ki67表达、FAK(p Y397)磷酸化水平明显下降,q RT-PCR提示干预组细胞Ki67、CCND1、Ptk2以及Ptk2b m RNA表达明显降低,流式细胞周期检查结果提示PPP组细胞中处于G1期的细胞明显增加,S和G2期细胞则明显减少。不同浓度的IGF1和IGF2干预细胞72后,随着生长因子浓度的增加,心外膜祖细胞的增殖能力逐渐升高。与对照组相比,IGF1与IGF2组细胞Ki67表达以及FAK(p Y397)磷酸化水平明显增高,Ki67、CCND1、Ptk2和Ptk2b m RNA的表达也明显升高,G1期细胞减少,S、G2期细胞增多;使用Y15特异性阻断FAK(p Y397)磷酸化后,Ptk2和Ptk2b在m RNA水平的表达不受影响,但引起Ki67、CCND1在m RNA水平的表达明显降低,CCK-8显示细胞增殖能力明显受限,流式细胞周期检查则提示细胞由G1期向S/G2期的转化被明显抑制。结论:我们成功建立了心外膜祖细胞的体外培养体系,并证实IGF1R信号通路可参与调控心外膜祖细胞的增殖,且此作用可通过FAK介导实现。第三部分:IGF1R信号通路在心外膜祖细胞上皮间质转化中的作用研究目的:探索IGF1R信号通路是否能够影响心外膜祖细胞EMT过程。方法:同上,将体外培养的心外膜祖细胞随机分为两组:对照组和PPP组(给予0.4μM PPP干预)。培养72小时后,使用免疫荧光染色检测细胞上皮标志物ZO-1以及间质标志物Vimentin的表达情况;q RT-PCR测定Cdh1(上皮标志物E-cadherin编码基因)、Tjp1(ZO-1编码基因)、Cdh2(间质标志物N-cadherin编码基因)以及Vim(Vimentin编码基因)在m RNA水平的表达情况。将心外膜祖细胞的培养背景降低为1%FBS后,细胞随机分为:对照组、IGF1组(50ng/ml)和IGF2组(100ng/ml)。免疫荧光染色检测各组细胞ZO-1和Vimentin的表达情况,q RT-PCR检测各组细胞Cdh1、Tjp1、Cdh2和Vim在m RNA水平的表达情况,以及Transwell实验观察三组细胞间迁移能力的差异。结果:细胞免疫荧光染色及q RT-PCR的结果提示,对照组与PPP组细胞不论是ZO-1和Vimentin的表达还是Cdh1、Tjp1、Cdh2和Vim在m RNA水平的表达均无明显差异。同样地,IGF1与IGF2干预处理细胞也不能引起所检测的EMT相关标志物表达的明显改变。Transwell试验提示IGF1及IGF2并不影响细胞的迁移能力。结论:IGF1R信号通路不影响心外膜祖细胞的EMT过程。
[Abstract]:Epicardial progenitor cells have been proved to play an important role in the development of the heart. It can form the epicardial source cells (EPDCs) through epithelial mesenchymal transformation (EMT), and ultimately differentiate into cardiac fibroblasts and coronary vascular smooth muscle cells under a series of signaling pathways, and participate in the formation of the cardiac fibrous skeleton and coronary vessels. Because of its multiple differentiation potential, epicardial progenitor cells are considered to be the third cardiac region of the heart, a progenitor cell that can feature genes such as Tbx18, Wt1 and Tcf21. The epicardial cells of the epicardial cells of the adult heart lose the epicardial progenitor cells, but there is a study of the epicardium that is at rest at the rest of the infarct area after the death of the myocardial infarction The cell can reactivate and reexpress the embryonic epicardial progenitor cells, such as Tbx18 and Wt1, and continue to proliferate, EMT, and migrate into the infarct area to participate in cardiac repair and vascular regeneration. Therefore, the study of epicardial progenitor cell proliferation and the mechanism of EMT are not only important to the deep exploration of the long heart development mechanism, but also contribute to the advancement of the cardiac growth mechanism. The application of epicardial progenitor cells in the field of cardiac regeneration. The signaling pathway mediated by insulin like growth factor 1 receptor (IGF1R) has been proved to be involved in multiple cell proliferation and EMT processes, and the expression of the IGF1R gene in the E11.5-E14.5 epicardium is confirmed by in situ hybridization. We speculate this signal The pathway may participate in the regulation of the proliferation of epicardial progenitor cells and the EMT process.IGF1R participates in the regulation of cell proliferation and the role of EMT in addition to the traditional classical PI3K/AKT and MAPK/ERK pathway. In recent years, it is found that it can also produce similar physiological or pathological effects through FAK pathway. Therefore, we explore I through this experimental study. The role of GF1R signaling pathway in the proliferation of epicardial progenitor cells and in EMT, and to explore whether its related role is mediated by FAK. Part 1: the temporal and spatial expression of IGF1R signaling pathway in mouse embryonic cardiac tissue: To explore the temporal and spatial expression of IGF1R and its main ligand IGF1 and IGF2 in rat embryonic cardiac tissue. Methods: female and male C57BL/6 mice were copulated, frozen section of embryonic tissues from E11.5-E17.5 days, and immunofluorescence staining was used to determine the expression of IGF1R and its ligand IGF1 and IGF2 in the heart of E11.5-E17.5 days embryo. Results: the results of immunofluorescence staining suggested that IGF1R clearly expressed.IGF1 on the epicardium on E11.5-E17.5 days embryo. The expression on the epicardium of E11.5-E13.5 days is less, and it is clearly expressed on the epicardial membrane of E14.5-E17.5 days. As an embryonic growth factor, the expression of IGF2 in the epicardium is more obvious and higher than that of IGF1 through E11.5-E17.5 days. Conclusion: IGF1R and its ligand IGF1 and IGF2 are expressed in the epicardium of the embryo of E11.5-E17.5 days. The IGF1R signaling pathway may be involved in regulating the development of epicardial progenitor cells in mice. Second part: the role and mechanism of the IGF1R signaling pathway in the proliferation of epicardial progenitor cells: to establish an in vitro culture system of epicardial progenitor cells in mice and to investigate whether the IGF1R signaling pathway is involved in the modulation by interfering with the IGF1R signaling pathway. The proliferation of epicardial progenitor cells was controlled and whether the regulation of its proliferation was mediated by FAK. Methods: female and male C57BL/6 mice were copulated and E12.5d embryonic cardiac tissue was cultured for epicardial progenitor cells. Immunofluorescence staining was used to detect the expression of its specific markers, Tbx18 and Wt1. At the same time, the expression of IGF1R in the epicardial progenitor cells cultured in vitro was also detected by immunofluorescence staining. Then the primary epicardial progenitor cells were treated with different concentrations of Picropodophyllin (PPP) (0,0.2 M, 0.4, 0.8, and 1.6 mu M) for 72 hours, and CCK-8 (Cell Counting Kit-8) was used to detect cell proliferation. The original epicardial progenitor cells were randomly divided into two groups: control group and PPP group (0.4 M PPP intervention). After 72 hours culture, immunofluorescence staining was used to detect the expression of Ki67 and FAK (P Y397) phosphorylation level, Q RT-PCR determined Ki67, CCND1 (cyclin Cyclin encoding gene) and The expression of Ptk2 and Ptk2b (FAK encoding gene) at m RNA level and the change of cell cycle between two groups of cells were detected by flow cytometry. The fetal bovine serum (Fetal Bovine Serum, FBS) in the primary epicardial progenitor cell culture medium was reduced from 10% to 1%. and then to 1%.. The concentration of IGF1 (0,5ng/ml, 10ng/ml and 50ng/ml) and IGF2 (0,10ng/ml, 50ng/ml and 100ng/ml) intervened cell 72h, and the proliferation of cells was detected by CCK-8. The expression of Ki67 and FAK (P Y397) phosphorylation were detected by force and immunofluorescence staining. Q RT-PCR was used to detect the expression of Ki67, CCND1, Ptk2 and Ptk2 in M RNA. Cell cycle changes were detected by flow cytometry. Results: the cultured cells showed "pave" appearance under the inverted cell microscope, form rule, single, cell Tbx18 and Wt1 were specifically expressed in the nucleus, and the number of positive cells was high. Cell immunofluorescence staining suggested that the IGF1R.CCK-8 experiment on the membrane of epicardial progenitor cells showed that the value of epicardial progenitor cells increased gradually with the increase of PPP concentration. Compared with the control group, the immunofluorescence staining showed that the PPP group was fine. The phosphorylation level of FAK (P Y397) decreased obviously, and Q RT-PCR indicated that the expression of Ki67, CCND1, Ptk2 and Ptk2b m RNA cells decreased obviously. The results of flow cytometry showed that the cells in the cell were obviously increased, and the cells of P Y397 decreased obviously. The proliferation ability of epicardial progenitor cells increased gradually. Compared with the control group, the expression of Ki67 and FAK (P Y397) phosphorylation in the IGF1 and IGF2 groups increased significantly, and the expression of Ki67, CCND1, Ptk2 and Ptk2b m RNA increased significantly. After acidification, the expression of Ptk2 and Ptk2b at the level of M RNA was not affected, but the expression of Ki67, CCND1 at m RNA level was obviously reduced, CCK-8 showed that the cell proliferation ability was obviously limited, and the flow cytometry showed that the transformation of the cell from G1 to S/G2 phase was obviously inhibited. Conclusion: we successfully established the culture of epicardial progenitor cells in vitro. The system, and confirmed that IGF1R signaling pathway can participate in the regulation of the proliferation of epicardial progenitor cells, and this effect can be mediated by FAK. Third part: the role of IGF1R signaling pathway in the epithelial mesenchymal transition of epicardial progenitor cells: To explore whether IGF1R signaling pathway can affect the EMT process of epicardial progenitor cells. Method: in the same, in vitro The cultured epicardium cells were randomly divided into two groups: control group and PPP group (0.4 M PPP intervention). After 72 hours culture, immunofluorescence staining was used to detect the expression of cell epithelial markers ZO-1 and interstitial marker Vimentin; Q RT-PCR was used to determine Cdh1 (E-cadherin coding gene), Tjp1 (ZO-1 coding gene), Cdh2 (interstitial) The expression of N-cadherin encoding gene) and Vim (Vimentin encoding gene) at m RNA level. After the culture background of epicardial progenitor cells was reduced to 1%FBS, the cells were randomly divided into the control group, the IGF1 group (50ng/ml) and the IGF2 group (100ng/ml). The expression of Cdh1, Tjp1, Cdh2 and Vim at m RNA level and the difference between the three groups of cell migration ability in the Transwell experiment were observed. Results: the results of cell immunofluorescence staining and Q RT-PCR showed that the expression of ZO-1 and Vimentin in the control group and the PPP group was not obvious. Similarly, IGF1 and IGF2 intervention cells did not cause significant changes in the expression of the detected EMT related markers..Transwell test suggested that IGF1 and IGF2 did not affect cell migration ability. Conclusion: IGF1R signaling pathway does not affect the EMT process of epicardial progenitor cells.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R54

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