【摘要】：第一章大鼠晶狀體上皮細胞的原代培養(yǎng) 目的：建立一種簡單快速有效的體外培養(yǎng)大鼠晶狀體上皮細胞的方法,進一步認識其生長、分化規(guī)律。 方法：取初生3-5天SD大鼠,在無菌環(huán)境下撕取其晶狀體前囊膜和赤道周邊部囊膜,采用胰酶消化法培養(yǎng)。在顯微鏡下觀察細胞生長規(guī)律及形態(tài)學特征；免疫熒光化學法對晶狀體上皮細胞進行鑒定及分析其純度MTT法分析各代細胞的生長速率及細胞的生長曲線。 結(jié)果：酶消化法培養(yǎng)的大鼠晶狀體上皮細胞生長迅速,呈扁平不規(guī)則多邊形,鑲嵌狀排列；第二代細胞開始細胞呈單層貼壁生長,分布均勻；從第四代細胞開始出現(xiàn)纖維化的趨勢,細胞形態(tài)呈梭形成纖維細胞樣；第六代細胞開始出現(xiàn)老化。從第一代細胞到第十代細胞幾乎100%表達aA-crystallin,從第五代細胞開始胞漿內(nèi)aA-crystallin表達量下降；第四代第五代細胞生長速率最大,第三代細胞次之；第三代細胞在培養(yǎng)約24h后達到對數(shù)期。 結(jié)論：酶學消化法是一種高效、實用、簡便的晶狀體上皮細胞原代培養(yǎng)方法,第3代大鼠晶狀體上皮細胞是進行細胞學及相關(guān)研究理想的實驗對象。 第二章原代培養(yǎng)的大鼠晶狀體上皮細胞誘導去分化生成誘導多潛能干細胞的初步研究 目的：將第三代大鼠晶狀體上皮細胞在生長對數(shù)期導入Oct4、c-Myc、Sox2、Klf4四個具有多能性的轉(zhuǎn)錄因子誘導去分化為誘導多潛能干細胞。 方法：用攜帶四個具有多能性的轉(zhuǎn)錄因子的仙臺病毒對處于對數(shù)期生長的原代培養(yǎng)的晶狀體上皮細胞進行誘導去分化,轉(zhuǎn)染后的第七天移植至飼養(yǎng)層細胞上,顯微鏡下觀察胚胎干細胞樣克隆團塊的形成以及其生成的效率。將生成的胚胎干細胞樣克隆團塊進行免疫熒光細胞化學染色鑒定胚胎干細胞標志性蛋白SSEA-1、OCT4的表達,以及qRT-PCR鑒定胚胎干細胞標志性基因NANOG、REX1、OCT4的表達。 結(jié)果：誘導后的晶狀體上皮細胞可生成RLEC-ES樣細胞克隆團塊,轉(zhuǎn)染效率為0.034%±0.0092%,取RLEC-ES樣細胞克隆團塊進行胚胎干細胞標志性蛋白SSEA-1以及OCT4免疫熒光組織化學染色呈陽性；用qRT-PCR法檢測胚胎干細胞標志性基因NANOG、 REX1、OCT4表達呈陽性。 結(jié)論：大鼠晶狀體上皮細胞可由攜帶Oct4、c-Myc、Sox2、Klf4四個具有多能性轉(zhuǎn)錄因子的仙臺病毒誘導去分化生成RLEC-ES樣細胞克隆團塊,并在形態(tài)學、蛋白、RNA水平得到鑒定。
[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% 鹵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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R776.1

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