诱导大鼠晶状体上皮细胞去分化生成诱导多潜能干细胞的初步研究
发布时间：2018-08-03 09:10
【摘要】：第一章大鼠晶状体上皮细胞的原代培养 目的：建立一种简单快速有效的体外培养大鼠晶状体上皮细胞的方法,进一步认识其生长、分化规律。 方法：取初生3-5天SD大鼠,在无菌环境下撕取其晶状体前囊膜和赤道周边部囊膜,采用胰酶消化法培养。在显微镜下观察细胞生长规律及形态学特征；免疫荧光化学法对晶状体上皮细胞进行鉴定及分析其纯度MTT法分析各代细胞的生长速率及细胞的生长曲线。 结果：酶消化法培养的大鼠晶状体上皮细胞生长迅速,呈扁平不规则多边形,镶嵌状排列；第二代细胞开始细胞呈单层贴壁生长,分布均匀；从第四代细胞开始出现纤维化的趋势,细胞形态呈梭形成纤维细胞样；第六代细胞开始出现老化。从第一代细胞到第十代细胞几乎100%表达aA-crystallin,从第五代细胞开始胞浆内aA-crystallin表达量下降；第四代第五代细胞生长速率最大,第三代细胞次之；第三代细胞在培养约24h后达到对数期。 结论：酶学消化法是一种高效、实用、简便的晶状体上皮细胞原代培养方法,第3代大鼠晶状体上皮细胞是进行细胞学及相关研究理想的实验对象。 第二章原代培养的大鼠晶状体上皮细胞诱导去分化生成诱导多潜能干细胞的初步研究 目的：将第三代大鼠晶状体上皮细胞在生长对数期导入Oct4、c-Myc、Sox2、Klf4四个具有多能性的转录因子诱导去分化为诱导多潜能干细胞。 方法：用携带四个具有多能性的转录因子的仙台病毒对处于对数期生长的原代培养的晶状体上皮细胞进行诱导去分化,转染后的第七天移植至饲养层细胞上,显微镜下观察胚胎干细胞样克隆团块的形成以及其生成的效率。将生成的胚胎干细胞样克隆团块进行免疫荧光细胞化学染色鉴定胚胎干细胞标志性蛋白SSEA-1、OCT4的表达,以及qRT-PCR鉴定胚胎干细胞标志性基因NANOG、REX1、OCT4的表达。 结果：诱导后的晶状体上皮细胞可生成RLEC-ES样细胞克隆团块,转染效率为0.034%±0.0092%,取RLEC-ES样细胞克隆团块进行胚胎干细胞标志性蛋白SSEA-1以及OCT4免疫荧光组织化学染色呈阳性；用qRT-PCR法检测胚胎干细胞标志性基因NANOG、 REX1、OCT4表达呈阳性。 结论：大鼠晶状体上皮细胞可由携带Oct4、c-Myc、Sox2、Klf4四个具有多能性转录因子的仙台病毒诱导去分化生成RLEC-ES样细胞克隆团块,并在形态学、蛋白、RNA水平得到鉴定。
[Abstract]:Chapter 1 Primary culture of rat lens epithelial cells objective: to establish a simple, rapid and effective method to culture rat lens epithelial cells in vitro, and to further understand the growth and differentiation of rat lens epithelial cells. Methods: the anterior capsule of lens and the periequatorial capsule of SD rats were avulsed in aseptic environment and cultured by trypsin digestion. The growth law and morphological characteristics of lens epithelial cells were observed under microscope and the growth rate and cell growth curve of each generation were analyzed by immunofluorescence method and the purity of lens epithelial cells was analyzed by MTT method. Results: the rat lens epithelial cells cultured by enzyme digestion grew rapidly with flat irregular polygons and mosaics and the second generation cells began to grow as monolayer adherent cells and distributed evenly. From the fourth generation cells began to appear the tendency of fibrosis, the morphology of the cells appeared fusiform fibroblasts, and the sixth generation cells began to aging. From the first generation to the tenth generation, almost 100% of the cells expressed aA-crystallin, the expression of aA-crystallin in the cytoplasm decreased from the fifth generation to the fifth generation, the growth rate of the fourth generation cells was the largest, the third generation cells took the second place, and the third generation cells reached the logarithmic phase after 24 hours of culture. Conclusion: enzymatic digestion is an effective, practical and simple method for primary culture of lens epithelial cells. The third passage of rat lens epithelial cells is an ideal experimental object for cytology and related research. Primary study of rat lens epithelial cells induced by dedifferentiation and induction of pluripotent stem cells in the second chapter objective: to introduce the third generation rat lens epithelial cells into Oct4nc-Mycnc-Sox2Klf4 in logarithmic phase of growth Pluripotent transcription factors induce dedifferentiation into induced pluripotent stem cells. Methods: Sendai virus carrying four pluripotent transcription factors was used to induce dedifferentiation of primary cultured lens epithelial cells in logarithmic phase and transplanted to feeder layer cells on the 7th day after transfection. The formation and efficiency of embryonic stem cell like colony were observed under microscope. The expression of embryonic stem cell iconic protein SSEA-1OCT4 was identified by immunofluorescence cytochemical staining, and the expression of NANOGN REX1OCT4 was identified by qRT-PCR. Results: the induced lens epithelial cells could produce RLEC-ES like cell clones, and the transfection efficiency was 0.034% 卤0.0092%. The RLEC-ES like cell clones were taken for SSEA-1 and OCT4 immunocytochemical staining. The qRT-PCR assay was used to detect the expression of the signature gene NANOG4 and REX1 + OCT4. Conclusion: rat lens epithelial cells can be induced to dedifferentiate into RLEC-ES like cell clone blocks by four Sendai viruses carrying Oct4c-Myctssox2Klf4 with multipotent transcription factors, and can be identified at the level of morphology and protein.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R776.1
[Abstract]:Chapter 1 Primary culture of rat lens epithelial cells objective: to establish a simple, rapid and effective method to culture rat lens epithelial cells in vitro, and to further understand the growth and differentiation of rat lens epithelial cells. Methods: the anterior capsule of lens and the periequatorial capsule of SD rats were avulsed in aseptic environment and cultured by trypsin digestion. The growth law and morphological characteristics of lens epithelial cells were observed under microscope and the growth rate and cell growth curve of each generation were analyzed by immunofluorescence method and the purity of lens epithelial cells was analyzed by MTT method. Results: the rat lens epithelial cells cultured by enzyme digestion grew rapidly with flat irregular polygons and mosaics and the second generation cells began to grow as monolayer adherent cells and distributed evenly. From the fourth generation cells began to appear the tendency of fibrosis, the morphology of the cells appeared fusiform fibroblasts, and the sixth generation cells began to aging. From the first generation to the tenth generation, almost 100% of the cells expressed aA-crystallin, the expression of aA-crystallin in the cytoplasm decreased from the fifth generation to the fifth generation, the growth rate of the fourth generation cells was the largest, the third generation cells took the second place, and the third generation cells reached the logarithmic phase after 24 hours of culture. Conclusion: enzymatic digestion is an effective, practical and simple method for primary culture of lens epithelial cells. The third passage of rat lens epithelial cells is an ideal experimental object for cytology and related research. Primary study of rat lens epithelial cells induced by dedifferentiation and induction of pluripotent stem cells in the second chapter objective: to introduce the third generation rat lens epithelial cells into Oct4nc-Mycnc-Sox2Klf4 in logarithmic phase of growth Pluripotent transcription factors induce dedifferentiation into induced pluripotent stem cells. Methods: Sendai virus carrying four pluripotent transcription factors was used to induce dedifferentiation of primary cultured lens epithelial cells in logarithmic phase and transplanted to feeder layer cells on the 7th day after transfection. The formation and efficiency of embryonic stem cell like colony were observed under microscope. The expression of embryonic stem cell iconic protein SSEA-1OCT4 was identified by immunofluorescence cytochemical staining, and the expression of NANOGN REX1OCT4 was identified by qRT-PCR. Results: the induced lens epithelial cells could produce RLEC-ES like cell clones, and the transfection efficiency was 0.034% 卤0.0092%. The RLEC-ES like cell clones were taken for SSEA-1 and OCT4 immunocytochemical staining. The qRT-PCR assay was used to detect the expression of the signature gene NANOG4 and REX1 + OCT4. Conclusion: rat lens epithelial cells can be induced to dedifferentiate into RLEC-ES like cell clone blocks by four Sendai viruses carrying Oct4c-Myctssox2Klf4 with multipotent transcription factors, and can be identified at the level of morphology and protein.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R776.1
【共引文献】
相关会议论文 前2条
1 周灿权;李宇彬;;研究中的生殖医学技术[A];中华医学会生殖医学分会第二次全国生殖临床学术研讨会论文汇编[C];2012年
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相关博士学位论文 前10条
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3 吴月红;诱导新生狨猴皮肤成纤维细胞来源的多潜能干细胞的研究[D];西北农林科技大学;2010年
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