本文選題：血紅蛋白Bart’s胎兒水腫綜合征 + 羊水細胞��； 參考：《廣州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：α-地中海貧血(α-地貧)是一組由于α-珠蛋白基因缺失或突變使α-珠蛋白鏈合成不足或缺乏而引起的遺傳性溶血性貧血,是人類最常見的單基因疾病之一。臨床上將α-地中海貧血分為靜止型攜帶者、α-地中海貧血特性、Hb H病和血紅蛋白Bart’s胎兒水腫綜合征。血紅蛋白Bart’s胎兒水腫綜合征又稱重型α-地中海貧血,主要由于缺少4個α-基因引起。因α-珠蛋白鏈合成完全缺乏,從而導(dǎo)致胎兒嚴重貧血。若不進行宮內(nèi)治療,胎兒多在孕晚期死亡或出生后數(shù)小時死亡。重型α-地中海貧血是一個重要的全球性健康問題。誘導(dǎo)多能干細胞(i PSCs)是可以通過在已分化的體細胞中同時表達在胚胎干細胞(ESC)中通常以高水平表達的幾種轉(zhuǎn)錄因子而產(chǎn)生的一種多能干細胞。該干細胞與胚胎干細胞相似,具有自我更新和向三個胚層分化的能力。因此,誘導(dǎo)多能干細胞的產(chǎn)生可以避免使用胚胎干細胞帶來的倫理問題,同時建立個體化疾病特異性i PSCs來治療遺傳性和退行性疾病可以避免免疫排斥的風(fēng)險。一、目的建立羊水細胞來源、無外源基因整合的血紅蛋白Bart’s胎兒水腫綜合征患兒特異性誘導(dǎo)多能干細胞,為基因治療研究及實現(xiàn)個性化宮內(nèi)治療提供模型。二、方法1、獲得知情同意后,胎兒被診斷為血紅蛋白Bart’s胎兒水腫綜合征(--SEA/--SEA)的孕婦于孕16+周在廣州市婦女兒童醫(yī)療中心產(chǎn)前診斷中心行超聲引導(dǎo)下的羊膜腔穿刺術(shù),抽取10毫升羊水用于本研究。2、利用非整合型仙臺病毒介導(dǎo)Klf4、Oct3/4、Sox2及c-Myc 4種轉(zhuǎn)錄因子將血紅蛋白Bart’s胎兒水腫綜合征胎兒的羊水細胞重編程為誘導(dǎo)多能干細胞。標記為h-AF-Se V-i PSCs。3、通過堿性磷酸酶試驗,免疫熒光標記等對h-AF-Se V-i PSCs進行多能性鑒定。通過擬胚體形成和自發(fā)分化實驗、畸胎瘤形成實驗對h-AF-Se V-i PSCs在體外及體內(nèi)向3個胚層細胞分化的能力進行鑒定。4、將h-AF-Se V-i PSCs第10代及第20代的細胞進行G顯帶染色體核型分析,檢測細胞經(jīng)重編程及傳代培養(yǎng)后遺傳物質(zhì)的穩(wěn)定性。將h-AF-Se V-i PSCs及原代羊水細胞提取DNA,進行缺失型α-地貧基因檢測,對突變基因型進行驗證,同時排除細胞污染。5、6×104個仙臺病毒轉(zhuǎn)導(dǎo)后的羊水細胞接種在飼養(yǎng)層細胞上,觀察出現(xiàn)克隆的時間,并計算ES樣克隆(堿性磷酸酶染色陽性)的個數(shù)。三、結(jié)果1、成功將血紅蛋白Bart’s胎兒水腫綜合征胎兒的羊水細胞利用仙臺病毒重編程為非整合型誘導(dǎo)性多能干細胞(h-AF-Se V-i PSCs)。2、h-AF-Se V-i PSCs堿性磷酸酶染色(AP)強陽性。免疫熒光染色提示h-AF-Se Vi PSCs表達胚胎干細胞特異性蛋白Oct4、Sox2、SSEA-4和Tra-1-81。3、將h-AF-Se V-i PSCs注射到7周齡SPF級NOD/SCID雄性小鼠腹股溝皮下,i PS細胞注射10周后摘除腫塊,并進行固定、包埋、切片、HE染色及免疫組化染色,HE染色和免疫組化染色結(jié)果均提示畸胎瘤內(nèi)含有內(nèi)胚層、中胚層及外胚層組織。h-AF-Se V-i PSCs懸浮培養(yǎng)后可形成囊性擬胚體,貼壁培養(yǎng)后同樣能向三個胚層分化。4、h-AF-Se V-i PSCs第10代及第20代的細胞G顯帶染色體核型分析提示均為正常核型(46,XY)。h-AF-Se V-i PSCs及其原代羊水細胞提DNA行Gap-PCR檢查提示其地中海貧血基因型均為--SEA/--SEA。5、仙臺病毒轉(zhuǎn)導(dǎo)后第4天,羊水細胞中出現(xiàn)胚胎干細胞樣的克隆。轉(zhuǎn)導(dǎo)后第7天,將羊水細胞接種到飼養(yǎng)層細胞上,轉(zhuǎn)導(dǎo)后第9天,可見克隆樣生長的細胞。共出現(xiàn)ES樣克隆30個,堿性磷酸酶染色均陽性,其轉(zhuǎn)導(dǎo)效率大約為0.05%。四、結(jié)論1、血紅蛋白Bart’s胎兒水腫綜合征胎兒的羊水細胞在體外可被仙臺病毒誘導(dǎo)為無外源基因整合的i PSCs。2、h-AF-Se V-i PSCs具有自我更新和向三個胚層分化的能力,且羊水細胞經(jīng)過重編程及在體外長期傳代培養(yǎng)仍能維持其遺傳物質(zhì)的穩(wěn)定,因此,h-AF-Se V-i PSCs可作為研究血紅蛋白Bart’s胎兒水腫綜合征宮內(nèi)治療的理想細胞模型。3、羊水細胞可作為是i PSCs的理想細胞來源。
[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.
【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】：R714.5

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1 蔣滿波;曾敏慧;章鈞;文艷飛;張濱;蔡柳洪;;地中海貧血“integration-free”誘導(dǎo)多能干細胞的建立及造血分化的研究[J];中國病理生理雜志;2015年02期

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