本文選題：誘導(dǎo)多能干細(xì)胞　切入點(diǎn)：心肌細(xì)胞　出處：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：[目的] 體外構(gòu)建誘導(dǎo)多能干細(xì)胞與原代心肌細(xì)胞的融合細(xì)胞，初步探討融合細(xì)胞體外生物學(xué)特性。 [方法] 1.iPSCs細(xì)胞株(IP14D-102)購自中國科學(xué)院動(dòng)物研究所(北京干細(xì)胞庫)，iPSCs接種在事先經(jīng)絲裂霉素C處理過的MEF上，，培養(yǎng)基為DMEM-H,包含10%FBS、1000IU/ml LIF、0.1mmol/Lβ-巰基乙醇、0.1mmol/L L-谷氨酰胺、1%非必需氨基酸。傳代時(shí)先將重懸液差異貼壁1小時(shí)去除MEF，然后吸取上清液重新接種到新的MEF飼養(yǎng)層上。 2.采用0.1%Ⅱ型膠原酶和0.1%胰蛋白酶分次消化乳鼠心肌組織，分離新生小鼠心肌細(xì)胞，體外觀察心肌細(xì)胞生物學(xué)特點(diǎn)，用免疫熒光法鑒定心肌細(xì)胞。 3. iPSCs與原代心肌細(xì)胞通過PEG-4000誘導(dǎo)融合，免疫熒光法鑒定融合細(xì)胞中是否同時(shí)表達(dá)干細(xì)胞和心肌細(xì)胞特異性蛋白。融合細(xì)胞按照標(biāo)準(zhǔn)iPSCs培養(yǎng)條件培養(yǎng)，并在體外觀察融合細(xì)胞生長特點(diǎn)變化情況，以及體外形成擬胚體的能力檢測。 4. RT-PCR和免疫熒光法檢測干細(xì)胞、心肌細(xì)胞特異性基因及蛋白在融合細(xì)胞中的表達(dá)情況。染色體核型分析，確定融合細(xì)胞是否發(fā)生細(xì)胞核的融合及程度，以及融合細(xì)胞堿性磷酸酶染色。 [結(jié)果] 1.飼養(yǎng)層細(xì)胞呈長梭形狀貼壁生長，iPSCs克隆團(tuán)在飼養(yǎng)層細(xì)胞上呈圓形或者橢圓形島狀，細(xì)胞團(tuán)周圍折光性強(qiáng),其內(nèi)的細(xì)胞間排列緊密，界限和形態(tài)不易區(qū)分。熒光顯微鏡下可見與光學(xué)顯微鏡下相對應(yīng)的綠色細(xì)胞團(tuán)(Oct-4-GFP+)。 2.心肌細(xì)胞呈三角形、多邊形或者扁平不規(guī)則形態(tài)。培養(yǎng)第3d起，心肌細(xì)胞伸出的偽足相互接觸交織成網(wǎng)，并可見局部呈同步性收縮的心肌細(xì)胞，搏動(dòng)頻率30-50/分鐘。第12天大部分心肌細(xì)胞停止搏動(dòng)，胞漿出現(xiàn)空泡，細(xì)胞皺縮、脫落。細(xì)胞免疫熒光染色后，胞漿內(nèi)可見發(fā)紅色熒光的cTnT。 3. PEG-4000能夠介導(dǎo)iPSCs與心肌細(xì)胞融合，融合后第4天開始出現(xiàn)成集落狀生長的細(xì)胞團(tuán)，整個(gè)實(shí)驗(yàn)期間，均未見到心肌細(xì)胞樣的融合細(xì)胞出現(xiàn)。染色體核型分析顯示，超過65%的融合細(xì)胞表現(xiàn)出四倍體或者近似四倍體的核型。 4.不同時(shí)間點(diǎn)的融合細(xì)胞AKP陽性率不完全相同，而融合細(xì)胞的AKP陽性率明顯低于同時(shí)間點(diǎn)的iPSCs AKP陽性率；P5代以前的融合細(xì)胞同時(shí)表達(dá)干細(xì)胞特異性基因(Oct-4、Nanog)和心肌細(xì)胞特異性基因(α-MHC、β-MHC)，之后的融合細(xì)胞只表達(dá)干細(xì)胞特異性基因；融合細(xì)胞初期Oct-4蛋白與cTnT蛋白均表達(dá)陽性，P4代以后的融合細(xì)胞Oct-4表達(dá)陽性，而cTnT未見表達(dá)。 [結(jié)論] 1. PEG-4000能夠介導(dǎo)二倍體iPSCs與二倍體原代心肌細(xì)胞發(fā)生細(xì)胞融合，融合細(xì)胞早期表現(xiàn)出雙向重建，隨著時(shí)間的延長，P5代以后的融合細(xì)胞表現(xiàn)出干細(xì)胞特點(diǎn)的單向重建。 2.通過本實(shí)驗(yàn)尚不能解釋融合細(xì)胞中干細(xì)胞特異性基因與蛋白的表達(dá)來源：親本干細(xì)胞基因組表達(dá)的延續(xù)，或者親本心肌細(xì)胞中原本沉默的干細(xì)胞基因被激活而重新表達(dá)。
[Abstract]:[Objective]
In vitro, the fusion cells were constructed to induce the pluripotent stem cells and the primary cardiomyocytes, and the biological characteristics of the fused cells in vitro were preliminarily discussed.
[method]
1.iPSCs cells (IP14D-102) were purchased from the animal research institute (Beijing China stem cell bank), iPSCs vaccine in advance by mitomycin C treated MEF, medium for DMEM-H, including 10%FBS, 1000IU/ml LIF, 0.1mmol/L mercaptoethanol, 0.1mmol/L L- glutamine, 1% non essential amino acids were first. Re suspension of differential adherence for 1 hours to remove MEF, then the supernatant was re inoculated into the new MEF feeder layer.
2., we used 0.1% type collagenase and 0.1% trypsin to digest the neonatal rat cardiac muscle, and isolated neonatal mouse cardiomyocytes. The biological characteristics of cardiomyocytes were observed in vitro, and cardiomyocytes were identified by immunofluorescence.
3. iPSCs with primary myocardial cells induced by PEG-4000 fusion, immunofluorescence identification of fusion cells and determine the expression of stem cell and myocardial cell specific protein. Cell fusion in accordance with the standard iPSCs cultured in vitro, and to observe the fusion cell growth characteristics change situation, and the ability to form embryoid bodies in vitro detection.
4. RT-PCR and immunofluorescence method were used to detect the expression of stem cells, cardiomyocyte specific genes and proteins in fusion cells. Karyotype analysis was used to determine whether fusion cells had nuclear fusion and degree, and fusion cells alkaline phosphatase staining.
[results]
1. feeder cells were spindle shaped adherent growth, iPSCs clone group showed round or oval shaped island on the feeder cells, cell clusters around the high refractive index, the cell in the compact form, boundary and not easy to distinguish. Green cells were observed under fluorescence microscope and optical microscope (corresponding to the Oct-4-GFP+).
2. myocardial cells were triangular, polygonal or irregular shape. The culture of 3D, myocardial cell out pseudopodia contacts with each other into a network, and a visible local systolic synchrony of myocardial cells, pulse frequency 30-50/ minutes. Twelfth days most of the myocardial cells stopped beating, cytoplasmic vacuoles, cell shrinkage, cell shedding. After immunofluorescence staining, cTnT. visible red fluorescence in the cytoplasm
3. PEG-4000 can mediate iPSCs and myocardial cell fusion, fusion appeared fourth days after a colony like growth of cells, the whole experiment period, there were no myocardial cell like cells. Fusion of chromosome karyotype analysis showed that more than 65% of the fusion cells showed tetraploid karyotype or similar tetraploid.
4. different time points of fusion cells AKP positive rate is not exactly the same, but the positive rate of AKP fusion cells was significantly lower than that of the positive rate of iPSCs AKP at the same time; P5 generation before fusion cells expressed stem cell specific genes (Oct-4, Nanog) and myocardial cell specific gene (-MHC alpha, beta -MHC). After the fusion cells only expressed stem cell specific gene; fusion cell early Oct-4 protein and cTnT protein expressions were Oct-4 P4 fusion cell generations after positive expression and no cTnT expression.
[Conclusion]
1. PEG-4000 can mediate the fusion of diploid iPSCs and diploid primary cardiomyocytes, and the fusion cells show bidirectional reconstruction at the early stage. With the extension of time, the fusion cells after P5 generation show the unidirectional reconstruction of stem cell characteristics.
2., we cannot explain the source of stem cell specific gene and protein expression in the fusion cells: the continuation of the genome expression of parental stem cells or the re expression of the original silent stem cell gene in the parental cardiomyocytes.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 王濤,余志斌,謝滿江,車紅磊;新生大鼠心肌細(xì)胞培養(yǎng)技巧[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2003年02期

2 胡顯文,陳昭烈,黃培堂;人胚胎干細(xì)胞的研究[J];生物技術(shù)通訊;2000年02期

3 趙志強(qiáng);鄭小林;張思杰;韋曉蘭;;細(xì)胞融合技術(shù)[J];生物學(xué)通報(bào);2005年10期

本文編號：1659405