【摘要】：目的:1.采用CRISPR-Cas9基因編輯技術(shù),構(gòu)建與牙齒發(fā)育相關(guān)的MSX1基因敲除人胚胎干細(xì)胞(hESc)穩(wěn)定細(xì)胞系。2.研究MSX1基因敲除hESc系的多能性。3.探討MSX1基因敲除hESc的擬胚體(EB)向外、中、內(nèi)三胚層細(xì)胞分化的能力。方法:1.采用分子克隆的技術(shù)構(gòu)建pX330-MSX1KO-sgRNA載體、MSX1基因同源重組敲除載體(donor)。2.將pX330-MSX1KO-sgRNA載體以及donor共同電轉(zhuǎn)入hESc,PCR檢測和測序鑒定鑒定存活的克隆。3.對細(xì)胞進(jìn)行形態(tài)學(xué)觀察、染色體核型分析、流式細(xì)胞術(shù),檢測MSX1基因雙敲除hESc(H1-MSX1-DKO)的多能性。4.體外形成H1-MSX1-DKO的擬胚體(EB),并誘導(dǎo)EB向外、中、內(nèi)三胚層細(xì)胞分化,收集EB分化前后(D0和D8)細(xì)胞的總RNA,采用實(shí)時(shí)熒光定量PCR法檢測各組細(xì)胞三胚層細(xì)胞標(biāo)記物的表達(dá)情況。結(jié)果:1.質(zhì)粒測序結(jié)果顯示pX330-MSX1KO-sgRNA載體以及donor成功構(gòu)建,并成功獲得78株MSX1基因單敲除和2株MSX1基因雙敲除hESc系(H1-MSX1-SKO,H1-MSX1-DKO)。2.MSX1基因敲除hESc表現(xiàn)出典型的人胚胎干細(xì)胞的形態(tài);另外,細(xì)胞除了保持原有正常核型外,還高表達(dá)多能性標(biāo)記物OCT4和SSEA4。3.實(shí)時(shí)熒光定量PCR結(jié)果顯示分化D8的細(xì)胞外、中、內(nèi)三胚層細(xì)胞標(biāo)記物的表達(dá)水平都不同程度的高于未分化的細(xì)胞。結(jié)論:運(yùn)用CRISPR-Cas9基因編輯技術(shù)能夠?qū)崿F(xiàn)對人胚胎干細(xì)胞的特定的編輯;MSX1基因敲除hESc還能保持干細(xì)胞的多能性并向三胚層細(xì)胞分化。我們獲得的MSX1基因敲除hESc可以用于進(jìn)一步探討牙齒發(fā)育的分子機(jī)制,進(jìn)而為人類研究MSX1基因異常導(dǎo)致的先天缺牙等相關(guān)疾病的治療提供一定參考。
[Abstract]:Objective: 1. CRISPR-Cas9 gene editing technique was used to construct MSX1 gene knockout human embryonic stem cell (hESc) stable cell line. 2. 2. To study the pluripotency of MSX1 knockout hESc lines. To study the ability of MSX1 knockout hESc embryoid (EB) to differentiate into outer, mesoderm and endoderm cells. Methods: 1. PX330-MSX1KO-sgRNA vector was constructed by molecular cloning and MSX1 gene homologous recombination knockout vector (donor). 2. Electroporation of pX330-MSX1KO-sgRNA vector and donor into hESc,PCR detection and sequencing to identify the surviving clones. 3. 3. Morphological observation, karyotype analysis and flow cytometry were used to detect the pluripotency of MSX1 double knockout hESc (H1-MSX1-DKO). 4. The embryoid (EB), of H1-MSX1-DKO was formed in vitro and induced the differentiation of EB to outer, mesoderm and endodermal cells. The total RNA, of EB cells before and after (D0 and D8) differentiation were collected to detect the expression of the markers of tridermal cells in each group by real-time fluorescence quantitative PCR method. The result is 1: 1. The results of plasmid sequencing showed that pX330-MSX1KO-sgRNA vector and donor were successfully constructed and 78 strains of MSX1 gene single knockout and 2 MSX1 gene double knockout hESc lines (H1-MSX1-SKOH1-MSX1-DKO) were successfully constructed. 2.MSX1 gene knockout hESc showed typical morphology of human embryonic stem cells. In addition to maintaining normal karyotypes, cells also expressed high expression of multipotent markers OCT4 and SSEA4.3.. The results of real-time fluorescence quantitative PCR showed that the expression levels of markers in differentiated D8 cells were higher than those in undifferentiated cells to some extent. Conclusion: the specific editing of human embryonic stem cells can be achieved by using CRISPR-Cas9 gene editing technique, and MSX1 knockout hESc can also maintain the pluripotency of stem cells and differentiate into tridermal cells. The MSX1 knockout hESc we obtained can be used to further explore the molecular mechanism of tooth development and provide a certain reference for the study of the treatment of congenital tooth defects caused by abnormal MSX1 gene.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R78

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