诱导多能干细胞与心肌细胞融合的实验研究

发布时间：2018-03-24 18:00

本文选题：诱导多能干细胞　切入点：心肌细胞　出处：《重庆医科大学》2012年硕士论文

【摘要】：[目的] 体外构建诱导多能干细胞与原代心肌细胞的融合细胞，初步探讨融合细胞体外生物学特性。 [方法] 1.iPSCs细胞株(IP14D-102)购自中国科学院动物研究所(北京干细胞库)，iPSCs接种在事先经丝裂霉素C处理过的MEF上，，培养基为DMEM-H,包含10%FBS、1000IU/ml LIF、0.1mmol/Lβ-巯基乙醇、0.1mmol/L L-谷氨酰胺、1%非必需氨基酸。传代时先将重悬液差异贴壁1小时去除MEF，然后吸取上清液重新接种到新的MEF饲养层上。 2.采用0.1%Ⅱ型胶原酶和0.1%胰蛋白酶分次消化乳鼠心肌组织，分离新生小鼠心肌细胞，体外观察心肌细胞生物学特点，用免疫荧光法鉴定心肌细胞。 3. iPSCs与原代心肌细胞通过PEG-4000诱导融合，免疫荧光法鉴定融合细胞中是否同时表达干细胞和心肌细胞特异性蛋白。融合细胞按照标准iPSCs培养条件培养，并在体外观察融合细胞生长特点变化情况，以及体外形成拟胚体的能力检测。 4. RT-PCR和免疫荧光法检测干细胞、心肌细胞特异性基因及蛋白在融合细胞中的表达情况。染色体核型分析，确定融合细胞是否发生细胞核的融合及程度，以及融合细胞碱性磷酸酶染色。 [结果] 1.饲养层细胞呈长梭形状贴壁生长，iPSCs克隆团在饲养层细胞上呈圆形或者椭圆形岛状，细胞团周围折光性强,其内的细胞间排列紧密，界限和形态不易区分。荧光显微镜下可见与光学显微镜下相对应的绿色细胞团(Oct-4-GFP+)。 2.心肌细胞呈三角形、多边形或者扁平不规则形态。培养第3d起，心肌细胞伸出的伪足相互接触交织成网，并可见局部呈同步性收缩的心肌细胞，搏动频率30-50/分钟。第12天大部分心肌细胞停止搏动，胞浆出现空泡，细胞皱缩、脱落。细胞免疫荧光染色后，胞浆内可见发红色荧光的cTnT。 3. PEG-4000能够介导iPSCs与心肌细胞融合，融合后第4天开始出现成集落状生长的细胞团，整个实验期间，均未见到心肌细胞样的融合细胞出现。染色体核型分析显示，超过65%的融合细胞表现出四倍体或者近似四倍体的核型。 4.不同时间点的融合细胞AKP阳性率不完全相同，而融合细胞的AKP阳性率明显低于同时间点的iPSCs AKP阳性率；P5代以前的融合细胞同时表达干细胞特异性基因(Oct-4、Nanog)和心肌细胞特异性基因(α-MHC、β-MHC)，之后的融合细胞只表达干细胞特异性基因；融合细胞初期Oct-4蛋白与cTnT蛋白均表达阳性，P4代以后的融合细胞Oct-4表达阳性，而cTnT未见表达。 [结论] 1. PEG-4000能够介导二倍体iPSCs与二倍体原代心肌细胞发生细胞融合，融合细胞早期表现出双向重建，随着时间的延长，P5代以后的融合细胞表现出干细胞特点的单向重建。 2.通过本实验尚不能解释融合细胞中干细胞特异性基因与蛋白的表达来源：亲本干细胞基因组表达的延续，或者亲本心肌细胞中原本沉默的干细胞基因被激活而重新表达。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】