本文選題：胰島素樣生長因子1受體 + 心外膜祖細胞��； 參考：《重慶醫(yī)科大學》2017年博士論文

【摘要】：心外膜祖細胞被證實在心臟發(fā)育過程中起著重要作用,它能通過上皮間質(zhì)轉(zhuǎn)化(EMT)形成心外膜來源細胞(EPDCs),并在一系列的信號通路作用下最終分化為心臟成纖維細胞以及冠脈血管平滑肌細胞,參與心臟纖維骨架和冠脈血管的形成。因其具有多項分化潛能,心外膜祖細胞被認為是心臟的第三心生區(qū),是一個能特征性表達Tbx18、Wt1和Tcf21等基因的祖細胞池。成年心臟的心外膜細胞喪失心外膜祖細胞的特征,但有研究發(fā)現(xiàn)心肌梗死后梗死區(qū)周邊處于靜止期的心外膜細胞可再次激活,重新表達胚胎期心外膜祖細胞基因,如Tbx18和Wt1,并不斷增殖、EMT、遷移進入梗死區(qū)域參與心臟修復(fù)及血管再生。因此,研究心外膜祖細胞增殖以及EMT機制不僅對于深入探索心臟生長發(fā)育機制具有重要的意義,且有助于推進心外膜祖細胞在心臟再生領(lǐng)域的相關(guān)應(yīng)用。胰島素樣生長因子1型受體(IGF1R)介導(dǎo)的信號通路已經(jīng)被證實參與多種細胞的增殖以及EMT過程,并且有研究通過原位雜交的方式證實在E11.5-E14.5天心外膜上有IGF1R基因的表達,我們猜測此信號通路可能參與調(diào)控心外膜祖細胞的增殖以及EMT過程。IGF1R參與調(diào)控細胞增殖以及EMT的作用除了通過傳統(tǒng)經(jīng)典的PI3K/AKT以及MAPK/ERK途徑實現(xiàn)外,近年來發(fā)現(xiàn)其也可通過FAK途徑產(chǎn)生相似的生理或者病理作用,因此,我們通過本實驗研究探索IGF1R信號通路在心外膜祖細胞增殖以及EMT中的作用,以及探討其相關(guān)作用是否通過FAK介導(dǎo)實現(xiàn)。第一部分:IGF1R信號通路在小鼠胚胎心臟組織中的時空表達目的:探索IGF1R及其主要配體IGF1和IGF2在小鼠胚胎心臟組織中的時空表達情況。方法:雌性及雄性C57BL/6小鼠進行交配,取E11.5-E17.5天的胚胎組織進行冰凍切片,免疫熒光染色確定IGF1R及其配體IGF1和IGF2在E11.5-E17.5天胚胎心臟中的表達情況。結(jié)果:免疫熒光染色結(jié)果提示IGF1R在E11.5-E17.5天的胚胎心外膜上有明確表達。IGF1在E11.5-E13.5天的胚胎心外膜上表達量較少,在E14.5-E17.5天的胚胎心外膜上明顯表達。作為胚胎期生長因子,IGF2在胚胎心外膜的表達比IGF1更加明顯且高表達貫穿E11.5-E17.5天。結(jié)論:IGF1R及其配體IGF1和IGF2在E11.5-E17.5天的胚胎心外膜上均有表達,提示IGF1R信號通路可能參與調(diào)控小鼠心外膜祖細胞的發(fā)育過程。第二部分:IGF1R信號通路在心外膜祖細胞增殖中的作用及機制研究目的:建立小鼠心外膜祖細胞體外培養(yǎng)體系,并通過干預(yù)細胞IGF1R信號通路,研究IGF1R信號通路是否參與調(diào)控心外膜祖細胞的增殖,并深入探討其調(diào)控增殖作用是否通過FAK介導(dǎo)實現(xiàn)。方法:雌性及雄性C57BL/6小鼠進行交配,取E12.5d的胚胎心臟組織進行心外膜祖細胞培養(yǎng),使用免疫熒光染色技術(shù)檢測其特異性標志物Tbx18和Wt1的表達情況來進行細胞鑒定。同時也通過免疫熒光染色檢測IGF1R在體外培養(yǎng)的原代小鼠心外膜祖細胞中的表達情況。然后分別用不同濃度的Picropodophyllin(PPP)(0、0.2μM、0.4μM、0.8μm和1.6μM)干預(yù)原代心外膜祖細胞72小時,CCK-8(Cell Counting Kit-8)檢測細胞增殖情況。然后將體外培養(yǎng)的原代心外膜祖細胞隨機分為兩組:對照組和PPP組(給予0.4μM PPP干預(yù))。培養(yǎng)72小時后,使用免疫熒光染色檢測兩組細胞增殖標志物Ki67的表達以及FAK(p Y397)磷酸化水平;q RT-PCR測定Ki67、CCND1(細胞周期蛋白Cyclin D1編碼基因)以及Ptk2和Ptk2b(FAK編碼基因)在m RNA水平的表達情況,并通過流式細胞技術(shù)檢測兩組細胞之間細胞周期的變化�？紤]到血清之中含有胰島素樣生長因子,將原代心外膜祖細胞培養(yǎng)基中的胎牛血清(Fetal Bovine Serum,FBS)由10%降至1%。然后,分別用不同濃度的IGF1(0、5ng/ml、10ng/ml和50ng/ml)和IGF2(0、10ng/ml、50ng/ml和100ng/ml)干預(yù)細胞72h,CCK-8檢測細胞增殖情況。并將細胞隨機分為:對照組、IGF1組(50ng/ml)、IGF2組(100ng/ml)、IGF1+Y15組(50ng/ml IGF1+1n M Y15)和IGF2+Y15組(100ng/ml IGF2+1n M Y15)。CCK-8檢測各組細胞增殖能力,免疫熒光染色檢測Ki67表達以及FAK(p Y397)磷酸化情況,q RT-PCR檢測Ki67、CCND1、Ptk2和Ptk2在m RNA水平的表達情況,流式細胞技術(shù)檢測各組細胞周期變化。結(jié)果:培養(yǎng)出來的細胞在倒置細胞顯微鏡下呈現(xiàn)“鋪路石”樣外觀,形態(tài)規(guī)則、單一,細胞間連接緊密,細胞核內(nèi)特異性表達Tbx18及Wt1,且陽性細胞數(shù)高。細胞免疫熒光染色提示小鼠心外膜祖細胞膜上點狀分布IGF1R。CCK-8實驗結(jié)果提示隨著PPP濃度的增加,心外膜祖細胞增值能力逐漸降低。與對照組相比,免疫熒光染色顯示PPP組細胞Ki67表達、FAK(p Y397)磷酸化水平明顯下降,q RT-PCR提示干預(yù)組細胞Ki67、CCND1、Ptk2以及Ptk2b m RNA表達明顯降低,流式細胞周期檢查結(jié)果提示PPP組細胞中處于G1期的細胞明顯增加,S和G2期細胞則明顯減少。不同濃度的IGF1和IGF2干預(yù)細胞72后,隨著生長因子濃度的增加,心外膜祖細胞的增殖能力逐漸升高。與對照組相比,IGF1與IGF2組細胞Ki67表達以及FAK(p Y397)磷酸化水平明顯增高,Ki67、CCND1、Ptk2和Ptk2b m RNA的表達也明顯升高,G1期細胞減少,S、G2期細胞增多;使用Y15特異性阻斷FAK(p Y397)磷酸化后,Ptk2和Ptk2b在m RNA水平的表達不受影響,但引起Ki67、CCND1在m RNA水平的表達明顯降低,CCK-8顯示細胞增殖能力明顯受限,流式細胞周期檢查則提示細胞由G1期向S/G2期的轉(zhuǎn)化被明顯抑制。結(jié)論:我們成功建立了心外膜祖細胞的體外培養(yǎng)體系,并證實IGF1R信號通路可參與調(diào)控心外膜祖細胞的增殖,且此作用可通過FAK介導(dǎo)實現(xiàn)。第三部分:IGF1R信號通路在心外膜祖細胞上皮間質(zhì)轉(zhuǎn)化中的作用研究目的:探索IGF1R信號通路是否能夠影響心外膜祖細胞EMT過程。方法:同上,將體外培養(yǎng)的心外膜祖細胞隨機分為兩組:對照組和PPP組(給予0.4μM PPP干預(yù))。培養(yǎng)72小時后,使用免疫熒光染色檢測細胞上皮標志物ZO-1以及間質(zhì)標志物Vimentin的表達情況;q RT-PCR測定Cdh1(上皮標志物E-cadherin編碼基因)、Tjp1(ZO-1編碼基因)、Cdh2(間質(zhì)標志物N-cadherin編碼基因)以及Vim(Vimentin編碼基因)在m RNA水平的表達情況。將心外膜祖細胞的培養(yǎng)背景降低為1%FBS后,細胞隨機分為:對照組、IGF1組(50ng/ml)和IGF2組(100ng/ml)。免疫熒光染色檢測各組細胞ZO-1和Vimentin的表達情況,q RT-PCR檢測各組細胞Cdh1、Tjp1、Cdh2和Vim在m RNA水平的表達情況,以及Transwell實驗觀察三組細胞間遷移能力的差異。結(jié)果:細胞免疫熒光染色及q RT-PCR的結(jié)果提示,對照組與PPP組細胞不論是ZO-1和Vimentin的表達還是Cdh1、Tjp1、Cdh2和Vim在m RNA水平的表達均無明顯差異。同樣地,IGF1與IGF2干預(yù)處理細胞也不能引起所檢測的EMT相關(guān)標志物表達的明顯改變。Transwell試驗提示IGF1及IGF2并不影響細胞的遷移能力。結(jié)論:IGF1R信號通路不影響心外膜祖細胞的EMT過程。
[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.

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R54

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