重型α-地中海贫血胎儿羊水来源的诱导多能干细胞系的建立及鉴定

发布时间：2018-07-03 06:37

本文选题：血红蛋白Bart’s胎儿水肿综合征 + 羊水细胞　；参考：《广州医科大学》2017年硕士论文

【摘要】：α-地中海贫血(α-地贫)是一组由于α-珠蛋白基因缺失或突变使α-珠蛋白链合成不足或缺乏而引起的遗传性溶血性贫血,是人类最常见的单基因疾病之一。临床上将α-地中海贫血分为静止型携带者、α-地中海贫血特性、Hb H病和血红蛋白Bart’s胎儿水肿综合征。血红蛋白Bart’s胎儿水肿综合征又称重型α-地中海贫血,主要由于缺少4个α-基因引起。因α-珠蛋白链合成完全缺乏,从而导致胎儿严重贫血。若不进行宫内治疗,胎儿多在孕晚期死亡或出生后数小时死亡。重型α-地中海贫血是一个重要的全球性健康问题。诱导多能干细胞(i PSCs)是可以通过在已分化的体细胞中同时表达在胚胎干细胞(ESC)中通常以高水平表达的几种转录因子而产生的一种多能干细胞。该干细胞与胚胎干细胞相似,具有自我更新和向三个胚层分化的能力。因此,诱导多能干细胞的产生可以避免使用胚胎干细胞带来的伦理问题,同时建立个体化疾病特异性i PSCs来治疗遗传性和退行性疾病可以避免免疫排斥的风险。一、目的建立羊水细胞来源、无外源基因整合的血红蛋白Bart’s胎儿水肿综合征患儿特异性诱导多能干细胞,为基因治疗研究及实现个性化宫内治疗提供模型。二、方法1、获得知情同意后,胎儿被诊断为血红蛋白Bart’s胎儿水肿综合征(--SEA/--SEA)的孕妇于孕16+周在广州市妇女儿童医疗中心产前诊断中心行超声引导下的羊膜腔穿刺术,抽取10毫升羊水用于本研究。2、利用非整合型仙台病毒介导Klf4、Oct3/4、Sox2及c-Myc 4种转录因子将血红蛋白Bart’s胎儿水肿综合征胎儿的羊水细胞重编程为诱导多能干细胞。标记为h-AF-Se V-i PSCs。3、通过碱性磷酸酶试验,免疫荧光标记等对h-AF-Se V-i PSCs进行多能性鉴定。通过拟胚体形成和自发分化实验、畸胎瘤形成实验对h-AF-Se V-i PSCs在体外及体内向3个胚层细胞分化的能力进行鉴定。4、将h-AF-Se V-i PSCs第10代及第20代的细胞进行G显带染色体核型分析,检测细胞经重编程及传代培养后遗传物质的稳定性。将h-AF-Se V-i PSCs及原代羊水细胞提取DNA,进行缺失型α-地贫基因检测,对突变基因型进行验证,同时排除细胞污染。5、6×104个仙台病毒转导后的羊水细胞接种在饲养层细胞上,观察出现克隆的时间,并计算ES样克隆(碱性磷酸酶染色阳性)的个数。三、结果1、成功将血红蛋白Bart’s胎儿水肿综合征胎儿的羊水细胞利用仙台病毒重编程为非整合型诱导性多能干细胞(h-AF-Se V-i PSCs)。2、h-AF-Se V-i PSCs碱性磷酸酶染色(AP)强阳性。免疫荧光染色提示h-AF-Se Vi PSCs表达胚胎干细胞特异性蛋白Oct4、Sox2、SSEA-4和Tra-1-81。3、将h-AF-Se V-i PSCs注射到7周龄SPF级NOD/SCID雄性小鼠腹股沟皮下,i PS细胞注射10周后摘除肿块,并进行固定、包埋、切片、HE染色及免疫组化染色,HE染色和免疫组化染色结果均提示畸胎瘤内含有内胚层、中胚层及外胚层组织。h-AF-Se V-i PSCs悬浮培养后可形成囊性拟胚体,贴壁培养后同样能向三个胚层分化。4、h-AF-Se V-i PSCs第10代及第20代的细胞G显带染色体核型分析提示均为正常核型(46,XY)。h-AF-Se V-i PSCs及其原代羊水细胞提DNA行Gap-PCR检查提示其地中海贫血基因型均为--SEA/--SEA。5、仙台病毒转导后第4天,羊水细胞中出现胚胎干细胞样的克隆。转导后第7天,将羊水细胞接种到饲养层细胞上,转导后第9天,可见克隆样生长的细胞。共出现ES样克隆30个,碱性磷酸酶染色均阳性,其转导效率大约为0.05%。四、结论1、血红蛋白Bart’s胎儿水肿综合征胎儿的羊水细胞在体外可被仙台病毒诱导为无外源基因整合的i PSCs。2、h-AF-Se V-i PSCs具有自我更新和向三个胚层分化的能力,且羊水细胞经过重编程及在体外长期传代培养仍能维持其遗传物质的稳定,因此,h-AF-Se V-i PSCs可作为研究血红蛋白Bart’s胎儿水肿综合征宫内治疗的理想细胞模型。3、羊水细胞可作为是i PSCs的理想细胞来源。
[Abstract]:Alpha thalassemia (alpha ground poverty) is a group of hereditary hemolytic anemia caused by the deficiency or mutation of alpha globin gene, which causes deficiency or lack of alpha globin chain. It is one of the most common human monogenic diseases. Alpha thalassemia is divided into static carriers, alpha thalassemia characteristics, Hb H disease and hemoglobin B Art 's fetal edema syndrome. Hemoglobin Bart' s fetal edema syndrome, also known as severe alpha thalassemia, is mainly due to the lack of 4 alpha gene causes. A complete lack of alpha globin chain synthesis leads to severe anaemia in the fetus. Anaemia is an important global health problem. Induced pluripotent stem cells (I PSCs) are pluripotent stem cells that can be produced by simultaneous expression of several transcription factors expressed in embryonic stem cells (ESC) in differentiated somatic cells. The stem cells are similar to embryonic stem cells and have self renewal and are self renewing. The ability to differentiate into three germ layers. Therefore, induction of pluripotent stem cells can avoid the ethical problems caused by embryonic stem cells, and the establishment of individualized disease specific I PSCs to treat hereditary and degenerative diseases can avoid the risk of immune rejection. Specific induction of pluripotent stem cells in children with erythrop Bart 's fetal edema syndrome, providing a model for gene therapy and individualized intrauterine treatment. Two, method 1, after obtaining informed consent, pregnant women who were diagnosed with hemoglobin Bart' s fetal edema syndrome (--SEA/--SEA) were in Guangzhou women and children's medical treatment at 16+ weeks. Amniocentesis guided by ultrasound was performed by the center of prenatal diagnosis. 10 ml of amniotic fluid was used in this study for.2. 4 transcription factors of Klf4, Oct3/4, Sox2 and c-Myc were used to induce amniotic fluid in Bart 's fetal edema syndrome to induce pluripotent stem cells with non integrated Sendai virus. It was labeled as h-AF-Se V-i PSC. S.3, h-AF-Se V-i PSCs was identified by alkaline phosphatase test, immunofluorescence marker and so on. Through the embryogenesis and spontaneous differentiation experiment, the teratoma formation experiment was used to identify the ability of h-AF-Se V-i PSCs to differentiate into 3 germ cells in vitro and in vivo. H-AF-Se V-i PSCs tenth and 20 generation cells were carried out G. The chromosome karyotype analysis was used to detect the stability of genetic material after reprogramming and subculture. DNA was extracted from h-AF-Se V-i PSCs and primary amniotic fluid cells to detect the deletion type alpha poor gene, to verify the mutant genotype, and to exclude the cell contamination of.5,6 * 104 Sendai virus transduced amniotic fluid cells inoculated in the feed. On the cultured cells, the time of cloning was observed and the number of ES like clones (alkaline phosphatase staining) was calculated. Three, the result 1, successfully reprogrammed the amniotic fluid cells of the hemoglobin Bart 's fetal edema syndrome by Sendai virus to the non integrated inducible pluripotent stem cells (h-AF-Se V-i PSCs).2, h-AF-Se V-i PSCs alkaline phosphorus Acid enzyme staining (AP) was strong positive. Immunofluorescence staining suggested that h-AF-Se Vi PSCs expressed embryonic stem cell specific protein Oct4, Sox2, SSEA-4 and Tra-1-81.3. H-AF-Se V-i PSCs was injected into the groin subcutaneous of male mice of 7 weeks of age. After 10 weeks of injection, the swelling block was removed and fixed, embedded, sliced, stained and immunohistochemical. The results of staining, HE staining and immunohistochemical staining suggested that the endoderm contained the endoderm in the teratoma, the mesoderm and the ectoderm tissue.H-AF-Se V-i PSCs could form a cystic embryoid after suspension culture. After adherent culture, it could also differentiate into three germ layers,.4, h-AF-Se V-i PSCs tenth generation and the 20 generation cell G banding chromosome karyotype analysis. The normal karyotype (46, XY).H-AF-Se V-i PSCs and its original amniotic fluid cell DNA Gap-PCR examination suggested that the genotype of thalassemia was --SEA/--SEA.5, and the embryonic stem cell like clones appeared in amniotic fluid fourth days after Sendai virus transduction. After transduction, the amniotic fluid cells were inoculated on the feeder layer cells, and the clones were visible for ninth days after transduction. There were 30 ES like clones. The alkaline phosphatase staining was positive, the transduction efficiency was about 0.05%. four, and the conclusion was 1. The amniotic fluid cells of the hemoglobin Bart 's fetal edema syndrome could be induced by Sendai virus to I PSCs.2 without exogenous gene integration in vitro, and h-AF-Se V-i PSCs had self renewal and three embryos. H-AF-Se V-i PSCs can be used as an ideal cell model for the study of intrauterine treatment of hemoglobin Bart 's fetal edema syndrome, and amniotic fluid cells can be an ideal cell source for I PSCs, as the ability of amniotic fluid cells to maintain their genetic material stability after reprogramming and in long-term subculture in vitro.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.5

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