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  本文選題：人類　切入點：臍帶間充質(zhì)干細(xì)胞　出處：《中國人民解放軍醫(yī)學(xué)院》2016年博士論文　論文類型：學(xué)位論文

【摘要】：[目的]培養(yǎng)人臍帶間充質(zhì)干細(xì)胞及人誘導(dǎo)多能干細(xì)胞,對細(xì)胞進(jìn)行鑒定。對兩種細(xì)胞進(jìn)行比較,探討適用于大量培養(yǎng)以滿足體內(nèi)移植治療的細(xì)胞類型。[方法]使用組織塊貼壁法從臍帶組織中分離人臍帶間充質(zhì)干細(xì)胞。培養(yǎng)、鑒定人臍帶間充質(zhì)干細(xì)胞及人誘導(dǎo)多能干細(xì)胞,繪制兩種細(xì)胞的生長曲線,并比較細(xì)胞形態(tài)。[結(jié)果]組織塊貼壁法成功分離間充質(zhì)干細(xì)胞,經(jīng)鑒定其表型CD73(+),CD105 (+),CD 31(一),CD34(-),符合臍帶間充質(zhì)干細(xì)胞特點。誘導(dǎo)多能干細(xì)胞NANOG (+)、OCT-4(+)、SOX-2(+)、SSEA-4(+)、TRA-60(+)和TRA-81(+),符合誘導(dǎo)多能干細(xì)胞特點。臍帶間充質(zhì)干細(xì)胞呈梭形,以漩渦樣排列,其倍增時間約42小時。誘導(dǎo)多能干細(xì)胞呈圓形或橢圓形,呈集落生長,其倍增時間約18小時。[結(jié)論]人臍帶間充質(zhì)干細(xì)胞及誘導(dǎo)多能干細(xì)胞增殖速度快,狀態(tài)穩(wěn)定,適合用于以移植治療為目的的大量繁殖。[目的]摸索建立一種在人類感音神經(jīng)性耳聾大型哺乳動物模型上可行的內(nèi)耳干細(xì)胞移植方法,檢測經(jīng)腰椎穿刺蛛網(wǎng)膜下腔注射移植的人誘導(dǎo)多能干細(xì)胞在Mitf基因突變耳聾豬的內(nèi)耳及全身的分布情況,檢測對聽性腦干反應(yīng)閾值的影響。[方法]培養(yǎng)及鑒定人誘導(dǎo)多能干細(xì)胞。選擇Mitf基因突變的榮昌豬作為神經(jīng)性耳聾模型的實驗對象,移植干細(xì)胞的榮昌豬為實驗組,單純注射生理鹽水的榮昌豬為對照。將人誘導(dǎo)多能干細(xì)胞經(jīng)蛛網(wǎng)膜下腔注射的途徑移植到實驗組榮昌豬體內(nèi)。設(shè)置觀察期為移植后1天、移植后3天和移植后7天,在移植前和處死前行全身麻醉后測定聽性腦干反應(yīng)閾值。按觀察期的時間點處死動物取耳蝸及其他組織。制作冰凍組織切片,行免疫組織熒光染色,用人特異性抗體檢測供體細(xì)胞。提取蛋白質(zhì),使用蛋白印跡法檢測移植細(xì)胞蛋白的表達(dá)。提取RNA,使用RT-PCR法檢測供體細(xì)胞基因在實驗對象體內(nèi)分布。[結(jié)果]經(jīng)蛛網(wǎng)膜下腔穿刺注射的人誘導(dǎo)多能干細(xì)胞在移植后可以在實驗對象內(nèi)耳檢出；同過蛋白質(zhì)印跡法在移植后1天、3天和7天可以在內(nèi)耳、腰椎段脊髓、胸椎段脊髓、頸椎段脊髓、延髓、小腦、大腦、心、肝、脾、肺、腎中檢出移植細(xì)胞的蛋白表達(dá)。通過RT-PCR方法移植后1天、3天和7天可以在腰椎段脊髓、胸椎段脊髓、頸椎段脊髓、延髓、小腦、大腦、心、肝、脾、肺、腎中檢出移植細(xì)胞的基因表達(dá)。在移植前后,Mitf基因突變榮昌豬的聽性腦干反應(yīng)的波形未能引出。[結(jié)論]數(shù)據(jù)表明,可以在內(nèi)耳及其余臟器中檢測出移植細(xì)胞的蛋白及基因的表達(dá),移植后Mitf突變耳聾豬的聽性腦干反應(yīng)測聽在120 dB SPL未能引出波形,移植后螺旋神經(jīng)節(jié)細(xì)胞病變未見繼續(xù)進(jìn)展。經(jīng)腰椎穿刺注射是內(nèi)耳干細(xì)胞移植的新途徑,但對耳聾治療效果有待進(jìn)一步探索。
[Abstract]:[objective] to identify human umbilical cord mesenchymal stem cells and human induced pluripotent stem cells. Methods Human umbilical cord mesenchymal stem cells were isolated from umbilical cord tissue by tissue mass adherence method. Culture, identification of human umbilical cord mesenchymal stem cells and human induced pluripotent stem cells, The growth curves of the two kinds of cells were drawn and the morphology of the cells was compared. [results] the mesenchymal stem cells were successfully isolated by tissue mass adherence method. Its phenotype CD73 was identified as CD105 (CD31), which was consistent with the characteristics of umbilical cord mesenchymal stem cells (NANOG). Induced pluripotent stem cells (NANOG) (OCT-4 (OCT-4) (OCT-4) (OCT-4)) and TRA-81 (TRA-81) were similar to the characteristics of induced pluripotent stem cells. The mesenchymal stem cells were spindle-shaped and arranged in whirlpool shape. The doubling time was about 42 hours. The pluripotent stem cells were induced to be round or oval, and the multiplication time was about 18 hours. [conclusion] the proliferation of human umbilical cord mesenchymal stem cells and induced pluripotent stem cells is fast and stable. Suitable for mass reproduction for transplantation therapy. [objective] to explore a feasible method for transplantation of inner ear stem cells in large mammal models of human sensorineural hearing loss. The distribution of human induced pluripotent stem cells in the inner ear and whole body of Mitf gene mutant deafness pigs was detected by subarachnoid injection through lumbar vertebrae. The effect of the threshold on auditory brainstem response was detected. [methods] pluripotent stem cells were cultured and identified. Rongchang pig with Mitf gene mutation was selected as the experimental object of neurodeafness model and Rongchang pig transplanted with stem cells as experimental group. Rong Chang pigs were injected with normal saline as control. The human induced pluripotent stem cells were transplanted into the experimental group by subarachnoid injection. The observation period was 1 day after transplantation, 3 days after transplantation and 7 days after transplantation. The threshold of auditory brainstem response was measured before and after general anesthesia. Cochlea and other tissues were taken from animals according to the time point of observation period. Frozen tissue sections were made and immunofluorescence staining was performed. Human specific antibodies are used to detect donor cells. Protein expression of transplanted cells was detected by Western blotting. RNAs were extracted and donor cell genes were detected by RT-PCR method. [results] Human pluripotent stem cells were induced by subarachnoid puncture after transplantation. It can be detected in the inner ear of the experimental object. The same Western blotting method can be used in the inner ear, lumbar spinal cord, thoracic spinal cord, cervical spinal cord, medulla oblongata, cerebellum, brain, heart, liver, spleen, lung at 1 and 7 days after transplantation. The protein expression of transplanted cells was detected in the kidney by RT-PCR method. The expression of protein in the lumbar spinal cord, thoracic spinal cord, cervical spinal cord, medulla oblongata, cerebellum, brain, heart, liver, spleen, lung, heart, liver, spleen and lung were detected 1 day and 7 days after transplantation by RT-PCR method. The gene expression of transplanted cells was detected in kidney. The waveform of auditory brainstem response of Rongchang pig with Mitf gene mutation before and after transplantation could not be induced. [conclusion] data showed that, The expression of protein and gene in the transplanted cells could be detected in the inner ear and other organs. After transplantation, the auditory brainstem response audiometry of Mitf mutant deafness pigs could not elicit the waveform at 120dB SPL. There was no further progress in spiral ganglion cytopathic changes after transplantation. Lumbar puncture injection is a new approach for transplantation of inner ear stem cells, but the therapeutic effect for deafness needs to be further explored.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R764.9

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