利用大鼠iPSCs和囊胚互补技术再生肺器官的研究
发布时间：2019-03-25 08:43
【摘要】：目的：本课题旨在利用大鼠的诱导多功能干细胞(rat induced pluripotent stem cells, rat iPSCs)技术和囊胚互补技术在TTF-1基因敲除的小鼠模型上验证异种间肺器官再生的可行性。并为最终在猪、猴等大动物体内培育出人源化的肺器官奠定基础。方法：(1)利用N2B27+2iPD0325901, CHIR99021)的培养体系培养和维持EGFP标记的大鼠iPSCs。(2)采用碱性磷酸酶染色、RT-PCR,细胞免疫荧光、畸胎瘤形成和类胚体形成实验等验证大鼠iPSCs的干性。(3)鉴定并繁育TTF-1小鼠,采集TTF-1+/-基因型小鼠杂交的囊胚。应用显微操作技术,将状态较好和干性较强的大鼠iPSCs作为供体细胞进行种间囊胚互补实验。(4)利用PCR对嵌合小鼠进行基因型鉴定；通过H/E染色对嵌合鼠的肺进行形态组织学观测；利用免疫组化检测EGFP、TTF-1阳性细胞在嵌合小鼠的肺及其它脏器中的分布；利用免疫组化检测CCSP、SP-C和Foxj1表达,观测再生肺的发育和分化情况。结果：(1)大鼠iPSCs碱性磷酸酶染色呈强阳性；RT-PCR和细胞免疫荧光均检测到干细胞多能性因子在大鼠iPSCs中的表达；畸胎瘤形成和类胚体形成实验等证明大鼠iPSCs具有三胚层分化的能力。(2)应用显微操作技术,我们成功进行了三次囊胚注射实验,共注射191枚胚胎,小鼠出生44只,44只出生小鼠中发生嵌合5只。(3)利用小鼠特异的TTF-1引物对获得的5只嵌合小鼠进行了基因型鉴定,确定其中一只为TTF-1双敲小鼠,H/E结果表明TTF-1双敲小鼠有再生的肺组织；EGFP、TTF-1免疫组化结果表明TTF-1双敲小鼠再生的肺组织来源于大鼠iPSCs; CCSP, SP-C和Foxj1免疫组化的结果表明再生的肺有克拉拉细胞、肺泡Ⅱ型细胞和终末细支气管上皮细胞的分化。结论：应用大鼠iPSCs结合囊胚互补技术成功在TTF-1敲除的肺发育缺陷的小鼠模型中再生出大鼠iPSCs来源的肺。我们的实验结果表明利用诱导多功能干细胞和囊胚互补技术能够实现种间器官的再生,为最终在猪等大动物体内制造可供移植的人肺器官打下了理论基础。
[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，
本文编号：2446822
[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，
本文编号：2446822
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