【摘要】：目的特納綜合征(Turner Syndrome,TS)是一條X染色體全部或部分缺失所致的性染色體異常綜合征,其具體發(fā)病機(jī)制目前并不清楚。誘導(dǎo)多能干細(xì)胞(Induced pluripotent stem cells,iPS cells)是通過人為方法過表達(dá)胚胎干細(xì)胞主要轉(zhuǎn)錄因子基因,重編程成體細(xì)胞而獲得的一類“類胚胎干細(xì)胞”細(xì)胞,為人們探討疾病發(fā)病機(jī)理提供了新的手段。本研究擬建立兩種細(xì)胞(絨毛細(xì)胞及外周血單個核細(xì)胞)來源的45,XO誘導(dǎo)多能干細(xì)胞系,比較兩種組織來源的細(xì)胞建系效率,并同時研究45,XO誘導(dǎo)多能干細(xì)胞從全能性細(xì)胞向神經(jīng)細(xì)胞誘導(dǎo)分化的過程。為探討特納綜合征的發(fā)病機(jī)制及X染色體功能提供較好的細(xì)胞模型。方法1.采用仙臺病毒載體法將人Oct,Sox2,Klf4,c-Myc 4個基因轉(zhuǎn)入到染色體核型為45,XO的絨毛細(xì)胞和外周血單個核細(xì)胞中,使其重編程為誘導(dǎo)多能干細(xì)胞。2.對誘導(dǎo)多能干細(xì)胞進(jìn)行核型鑒定,用堿性磷酸酶染色、免疫熒光染色、畸胎瘤實驗等對兩株iPS細(xì)胞系的多能性和體內(nèi)外分化能力進(jìn)行鑒定。3.通過比較兩種細(xì)胞重編程后出現(xiàn)干細(xì)胞克隆的時間長短以及克隆數(shù)目對兩種細(xì)胞來源的建系效率進(jìn)行比較。4.對iPSCs進(jìn)行體外神經(jīng)干細(xì)胞的誘導(dǎo)分化并比較誘導(dǎo)效率,進(jìn)行免疫熒光染色;獲得神經(jīng)干細(xì)胞后進(jìn)一步誘導(dǎo)其向神經(jīng)元分化,進(jìn)行免疫熒光染色鑒定。結(jié)果1.45,XO絨毛細(xì)胞在感染后第5天形態(tài)上發(fā)生變化,感染第15天有干細(xì)胞樣克隆形成。45,XO外周血單個核細(xì)胞感染第9天有干細(xì)胞樣克隆形成。機(jī)械法挑選建系,這些細(xì)胞能夠長期在體外穩(wěn)定傳代并保持核型45,XO,維持自我更新。2.兩株45,XO iPS細(xì)胞系分別傳代進(jìn)行堿性磷酸酶染色呈強(qiáng)陽性,免疫熒光染色顯示SSEA3,SSEA4,Oct4陽性;誘導(dǎo)多能干細(xì)胞在體外自然分化可形成三胚層來源細(xì)胞;將克隆細(xì)胞注射于SCID小鼠皮下形成的畸胎瘤內(nèi)出現(xiàn)三胚層樣結(jié)構(gòu)。3.1X10~5個感染后的兩種類型的細(xì)胞接種在飼養(yǎng)層細(xì)胞上,在感染后25天進(jìn)行堿性磷酸酶染色,外周血單個核細(xì)胞來源的得到300個陽性克隆,效率為0.300%。絨毛細(xì)胞得到25個克隆,效率為0.025%。經(jīng)χ2檢驗P0.05,差異有統(tǒng)計學(xué)意義。4.對兩株45,XO-iPSCs與一株正常人46,XX-iPSCs進(jìn)行神經(jīng)誘導(dǎo)分化,兩株45,XO-iPSCs與一株46,XX-iPSCs體外分化神經(jīng)干細(xì)胞免疫熒光結(jié)果顯示神經(jīng)干細(xì)胞特異性標(biāo)記Nestin呈陽性,比較誘導(dǎo)效率無顯著差異。繼續(xù)將神經(jīng)干細(xì)胞誘導(dǎo)分化為神經(jīng)元細(xì)胞系,細(xì)胞免疫熒光檢測結(jié)果顯示神經(jīng)元細(xì)胞特異性標(biāo)記Tuj1、NeuN的表達(dá)為陽性。結(jié)論1.建立了兩株分別來源于絨毛細(xì)胞及外周血單個核細(xì)胞的ips細(xì)胞系,兩株ips細(xì)胞系在體外長期傳代中能維持異常核型及干細(xì)胞多能性,可作為特納綜合征研究的細(xì)胞模型;2.比較兩種細(xì)胞建系的效率發(fā)現(xiàn)利用外周血單個核細(xì)胞建立45,XO-i PSCs效率高于絨毛細(xì)胞,外周血單個核細(xì)胞可作為制備45,XO-iPSCs的理想細(xì)胞來源;3.兩株45,XO-iPSCs在神經(jīng)分化過程中,分化效率與正常的iPSCs相比無顯著差異。
[Abstract]:Objective Turner Syndrome (TS) is a sex chromosome abnormality syndrome caused by complete or partial deletion of an X chromosome. The pathogenesis of TS is unclear. Induced pluripotent stem cells (iPS cells) are recombined by artificially overexpressing the major transcription factor genes of embryonic stem cells. A class of "embryonic stem cell-like" cells derived from adult cells provides a new way to explore the pathogenesis of diseases. In this study, we intend to establish 45, XO-induced pluripotent stem cell lines derived from two kinds of cells (villous cells and peripheral blood mononuclear cells), compare the efficiency of cell line-building from two kinds of tissue sources, and study the induction of 45, XO at the same time. Methods 1. Human Oct, Sox2, Klf4, c-Myc genes were transfected into chromosome 45, XO villous cells and peripheral blood mononuclear cells by Sendai virus vector method. Cells were reprogrammed as induced pluripotent stem cells. 4. Induction and differentiation of neural stem cells from iPSCs in vitro were compared and immunofluorescence staining was performed. After obtaining neural stem cells, they were further induced to differentiate into neurons and identified by immunofluorescence staining. On the fifth day of infection, stem cell-like clones were formed on the fifteenth day of infection, and stem cell-like clones were formed on the ninth day of infection of XO peripheral blood mononuclear cells. Phosphatase staining was strongly positive, immunofluorescence staining showed SSEA3, SSEA4, Oct4 positive; induced pluripotent stem cells differentiated spontaneously in vitro to form triembryonic cells; cloned cells were injected into the teratoma of SCID mice subcutaneously to form a triembryonic layer-like structure. After 25 days of infection, 300 positive clones were obtained from peripheral blood mononuclear cells with an efficiency of 0.300%. 25 clones were obtained from chorionic villi with an efficiency of 0.025%. By_2 test, P 0.05, the difference was statistically significant. 4. Two strains of 45, XO-iPSCs and a normal 46, XX-iPSCs were induced to differentiate into neurons, and two strains were 45. XO-iPSCs and a strain of 46,XX-iPSCs differentiated neural stem cells in vitro immunofluorescence results showed that the specific marker Nestin of neural stem cells was positive, and there was no significant difference in induction efficiency. Two iPS cell lines derived from chorionic villi and peripheral blood mononuclear cells were established. two iPS cell lines maintained abnormal karyotype and stem cell pluripotency during long-term passage in vitro, which could be used as cell models for Turner's syndrome. 2. comparing the efficiency of two cell lines, we found that peripheral blood mononuclear cells were used for the study of Turner's syndrome. The efficiency of XO-i PSCs was higher than that of chorionic villus cells. Peripheral blood mononuclear cells could be used as an ideal cell source for the preparation of 45,XO-iPSCs.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R596

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1 徐龍勇;陳德桂;;組蛋白去甲基化酶研究進(jìn)展[J];生命科學(xué);2010年02期

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