發(fā)布時(shí)間：2018-08-03 09:10

【摘要】：第一章大鼠晶狀體上皮細(xì)胞的原代培養(yǎng) 目的：建立一種簡(jiǎn)單快速有效的體外培養(yǎng)大鼠晶狀體上皮細(xì)胞的方法,進(jìn)一步認(rèn)識(shí)其生長(zhǎng)、分化規(guī)律。 方法：取初生3-5天SD大鼠,在無(wú)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(xì)胞生長(zhǎng)規(guī)律及形態(tài)學(xué)特征；免疫熒光化學(xué)法對(duì)晶狀體上皮細(xì)胞進(jìn)行鑒定及分析其純度MTT法分析各代細(xì)胞的生長(zhǎng)速率及細(xì)胞的生長(zhǎng)曲線。 結(jié)果：酶消化法培養(yǎng)的大鼠晶狀體上皮細(xì)胞生長(zhǎng)迅速,呈扁平不規(guī)則多邊形,鑲嵌狀排列；第二代細(xì)胞開(kāi)始細(xì)胞呈單層貼壁生長(zhǎng),分布均勻；從第四代細(xì)胞開(kāi)始出現(xiàn)纖維化的趨勢(shì),細(xì)胞形態(tài)呈梭形成纖維細(xì)胞樣；第六代細(xì)胞開(kāi)始出現(xiàn)老化。從第一代細(xì)胞到第十代細(xì)胞幾乎100%表達(dá)aA-crystallin,從第五代細(xì)胞開(kāi)始胞漿內(nèi)aA-crystallin表達(dá)量下降；第四代第五代細(xì)胞生長(zhǎng)速率最大,第三代細(xì)胞次之；第三代細(xì)胞在培養(yǎng)約24h后達(dá)到對(duì)數(shù)期。 結(jié)論：酶學(xué)消化法是一種高效、實(shí)用、簡(jiǎn)便的晶狀體上皮細(xì)胞原代培養(yǎng)方法,第3代大鼠晶狀體上皮細(xì)胞是進(jìn)行細(xì)胞學(xué)及相關(guān)研究理想的實(shí)驗(yàn)對(duì)象。 第二章原代培養(yǎng)的大鼠晶狀體上皮細(xì)胞誘導(dǎo)去分化生成誘導(dǎo)多潛能干細(xì)胞的初步研究 目的：將第三代大鼠晶狀體上皮細(xì)胞在生長(zhǎng)對(duì)數(shù)期導(dǎo)入Oct4、c-Myc、Sox2、Klf4四個(gè)具有多能性的轉(zhuǎn)錄因子誘導(dǎo)去分化為誘導(dǎo)多潛能干細(xì)胞。 方法：用攜帶四個(gè)具有多能性的轉(zhuǎn)錄因子的仙臺(tái)病毒對(duì)處于對(duì)數(shù)期生長(zhǎng)的原代培養(yǎng)的晶狀體上皮細(xì)胞進(jìn)行誘導(dǎo)去分化,轉(zhuǎn)染后的第七天移植至飼養(yǎng)層細(xì)胞上,顯微鏡下觀察胚胎干細(xì)胞樣克隆團(tuán)塊的形成以及其生成的效率。將生成的胚胎干細(xì)胞樣克隆團(tuán)塊進(jìn)行免疫熒光細(xì)胞化學(xué)染色鑒定胚胎干細(xì)胞標(biāo)志性蛋白SSEA-1、OCT4的表達(dá),以及qRT-PCR鑒定胚胎干細(xì)胞標(biāo)志性基因NANOG、REX1、OCT4的表達(dá)。 結(jié)果：誘導(dǎo)后的晶狀體上皮細(xì)胞可生成RLEC-ES樣細(xì)胞克隆團(tuán)塊,轉(zhuǎn)染效率為0.034%±0.0092%,取RLEC-ES樣細(xì)胞克隆團(tuán)塊進(jìn)行胚胎干細(xì)胞標(biāo)志性蛋白SSEA-1以及OCT4免疫熒光組織化學(xué)染色呈陽(yáng)性；用qRT-PCR法檢測(cè)胚胎干細(xì)胞標(biāo)志性基因NANOG、 REX1、OCT4表達(dá)呈陽(yáng)性。 結(jié)論：大鼠晶狀體上皮細(xì)胞可由攜帶Oct4、c-Myc、Sox2、Klf4四個(gè)具有多能性轉(zhuǎn)錄因子的仙臺(tái)病毒誘導(dǎo)去分化生成RLEC-ES樣細(xì)胞克隆團(tuán)塊,并在形態(tài)學(xué)、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(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R776.1

【共引文獻(xiàn)】

相關(guān)會(huì)議論文 前2條

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相關(guān)博士學(xué)位論文 前10條

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本文編號(hào)：2161278