【摘要】：目的：本課題旨在利用大鼠的誘導(dǎo)多功能干細(xì)胞(rat induced pluripotent stem cells, rat iPSCs)技術(shù)和囊胚互補(bǔ)技術(shù)在TTF-1基因敲除的小鼠模型上驗(yàn)證異種間肺器官再生的可行性。并為最終在豬、猴等大動(dòng)物體內(nèi)培育出人源化的肺器官奠定基礎(chǔ)。方法：(1)利用N2B27+2iPD0325901, CHIR99021)的培養(yǎng)體系培養(yǎng)和維持EGFP標(biāo)記的大鼠iPSCs。(2)采用堿性磷酸酶染色、RT-PCR,細(xì)胞免疫熒光、畸胎瘤形成和類胚體形成實(shí)驗(yàn)等驗(yàn)證大鼠iPSCs的干性。(3)鑒定并繁育TTF-1小鼠,采集TTF-1+/-基因型小鼠雜交的囊胚。應(yīng)用顯微操作技術(shù),將狀態(tài)較好和干性較強(qiáng)的大鼠iPSCs作為供體細(xì)胞進(jìn)行種間囊胚互補(bǔ)實(shí)驗(yàn)。(4)利用PCR對(duì)嵌合小鼠進(jìn)行基因型鑒定；通過H/E染色對(duì)嵌合鼠的肺進(jìn)行形態(tài)組織學(xué)觀測(cè)；利用免疫組化檢測(cè)EGFP、TTF-1陽性細(xì)胞在嵌合小鼠的肺及其它臟器中的分布；利用免疫組化檢測(cè)CCSP、SP-C和Foxj1表達(dá),觀測(cè)再生肺的發(fā)育和分化情況。結(jié)果：(1)大鼠iPSCs堿性磷酸酶染色呈強(qiáng)陽性；RT-PCR和細(xì)胞免疫熒光均檢測(cè)到干細(xì)胞多能性因子在大鼠iPSCs中的表達(dá)；畸胎瘤形成和類胚體形成實(shí)驗(yàn)等證明大鼠iPSCs具有三胚層分化的能力。(2)應(yīng)用顯微操作技術(shù),我們成功進(jìn)行了三次囊胚注射實(shí)驗(yàn),共注射191枚胚胎,小鼠出生44只,44只出生小鼠中發(fā)生嵌合5只。(3)利用小鼠特異的TTF-1引物對(duì)獲得的5只嵌合小鼠進(jìn)行了基因型鑒定,確定其中一只為TTF-1雙敲小鼠,H/E結(jié)果表明TTF-1雙敲小鼠有再生的肺組織；EGFP、TTF-1免疫組化結(jié)果表明TTF-1雙敲小鼠再生的肺組織來源于大鼠iPSCs; CCSP, SP-C和Foxj1免疫組化的結(jié)果表明再生的肺有克拉拉細(xì)胞、肺泡Ⅱ型細(xì)胞和終末細(xì)支氣管上皮細(xì)胞的分化。結(jié)論：應(yīng)用大鼠iPSCs結(jié)合囊胚互補(bǔ)技術(shù)成功在TTF-1敲除的肺發(fā)育缺陷的小鼠模型中再生出大鼠iPSCs來源的肺。我們的實(shí)驗(yàn)結(jié)果表明利用誘導(dǎo)多功能干細(xì)胞和囊胚互補(bǔ)技術(shù)能夠?qū)崿F(xiàn)種間器官的再生,為最終在豬等大動(dòng)物體內(nèi)制造可供移植的人肺器官打下了理論基礎(chǔ)。
[Abstract]:Aim: to investigate the feasibility of heterologous lung organ regeneration in TTF-1 knockout mice using (rat induced pluripotent stem cells, rat iPSCs) and blastocyst complementation techniques. It lays a foundation for breeding humanized lung organs in large animals such as pigs and monkeys. Methods: (1) N2B27 2iPD0325901 (CHIR99021) was used to culture and maintain EGFP labeled rat iPSCs. (2) by alkaline phosphatase staining and RT-PCR, cell immunofluorescence. Teratoma formation and embryoid formation tests were used to verify the dryness of rat iPSCs. (3) TTF-1 mice were identified and bred, and blastocysts of TTF-1 /-genotype mice were collected. Interspecific blastocyst complementation test was carried out using rat iPSCs as donor cells using micromanipulation technique. (4) Genotype identification of chimeric mice was carried out by PCR. The morphology and histology of the lungs of chimeric mice were observed and the distribution of EGFP,TTF-1 positive cells in the lungs and other organs of the chimeric mice were detected by immunohistochemical staining, and the distribution of the positive cells in the lungs and other organs of the chimeric mice was detected by immunohistochemistry. The expression of CCSP,SP-C and Foxj1 was detected by immunohistochemistry, and the development and differentiation of regenerated lung were observed. Results: (1) the expression of stem cell pluripotency factor in rat iPSCs was detected by RT-PCR and cellular immunofluorescence, and the expression of iPSCs alkaline phosphatase was strongly positive. Teratoma formation and embryoid body formation test proved that rat iPSCs had the ability of triembryonic differentiation. (2) We successfully carried out three blastocyst injection experiments, in which 44 mice were born, 3 times blastocyst injection was carried out. Chimerism occurred in 5 of 44 newborn mice. (3) Genotype identification of 5 chimeric mice was carried out using mouse-specific TTF-1 primers, and one of them was identified as TTF-1 double knock mice. The results of Hue showed that TTF-1 double knockout mice had regenerated lung tissue. The results of EGFP,TTF-1 immunohistochemistry showed that the regenerated lung tissue of TTF-1 double knock mice originated from iPSCs; of rats. The results of CCSP, SP-C and Foxj1 immunohistochemistry showed that the regenerated lung was differentiated into Clara cells, alveolar type 鈪，

本文編號(hào)：2446822