本文選題：誘導多能干細胞　切入點：心肌細胞　出處：《重慶醫(yī)科大學》2012年碩士論文

【摘要】：[目的] 體外構建誘導多能干細胞與原代心肌細胞的融合細胞，初步探討融合細胞體外生物學特性。 [方法] 1.iPSCs細胞株(IP14D-102)購自中國科學院動物研究所(北京干細胞庫)，iPSCs接種在事先經絲裂霉素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。傳代時先將重懸液差異貼壁1小時去除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胞，體外觀察心肌細胞生物學特點，用免疫熒光法鑒定心肌細胞。 3. iPSCs與原代心肌細胞通過PEG-4000誘導融合，免疫熒光法鑒定融合細胞中是否同時表達干細胞和心肌細胞特異性蛋白。融合細胞按照標準iPSCs培養(yǎng)條件培養(yǎng)，并在體外觀察融合細胞生長特點變化情況，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胞、心肌細胞特異性基因及蛋白在融合細胞中的表達情況。染色體核型分析，確定融合細胞是否發(fā)生細胞核的融合及程度，以及融合細胞堿性磷酸酶染色。 [結果] 1.飼養(yǎng)層細胞呈長梭形狀貼壁生長，iPSCs克隆團在飼養(yǎng)層細胞上呈圓形或者橢圓形島狀，細胞團周圍折光性強,其內的細胞間排列緊密，界限和形態(tài)不易區(qū)分。熒光顯微鏡下可見與光學顯微鏡下相對應的綠色細胞團(Oct-4-GFP+)。 2.心肌細胞呈三角形、多邊形或者扁平不規(guī)則形態(tài)。培養(yǎng)第3d起，心肌細胞伸出的偽足相互接觸交織成網，并可見局部呈同步性收縮的心肌細胞，搏動頻率30-50/分鐘。第12天大部分心肌細胞停止搏動，胞漿出現空泡，細胞皺縮、脫落。細胞免疫熒光染色后，胞漿內可見發(fā)紅色熒光的cTnT。 3. PEG-4000能夠介導iPSCs與心肌細胞融合，融合后第4天開始出現成集落狀生長的細胞團，整個實驗期間，均未見到心肌細胞樣的融合細胞出現。染色體核型分析顯示，超過65%的融合細胞表現出四倍體或者近似四倍體的核型。 4.不同時間點的融合細胞AKP陽性率不完全相同，而融合細胞的AKP陽性率明顯低于同時間點的iPSCs AKP陽性率；P5代以前的融合細胞同時表達干細胞特異性基因(Oct-4、Nanog)和心肌細胞特異性基因(α-MHC、β-MHC)，之后的融合細胞只表達干細胞特異性基因；融合細胞初期Oct-4蛋白與cTnT蛋白均表達陽性，P4代以后的融合細胞Oct-4表達陽性，而cTnT未見表達。 [結論] 1. PEG-4000能夠介導二倍體iPSCs與二倍體原代心肌細胞發(fā)生細胞融合，融合細胞早期表現出雙向重建，隨著時間的延長，P5代以后的融合細胞表現出干細胞特點的單向重建。 2.通過本實驗尚不能解釋融合細胞中干細胞特異性基因與蛋白的表達來源：親本干細胞基因組表達的延續(xù)，或者親本心肌細胞中原本沉默的干細胞基因被激活而重新表達。
[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.

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

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