经腰椎穿刺注射干细胞内耳移植新途径的探索研究

发布时间：2018-03-08 16:04

本文选题：人类　切入点：脐带间充质干细胞　出处：《中国人民解放军医学院》2016年博士论文　论文类型：学位论文

【摘要】：[目的]培养人脐带间充质干细胞及人诱导多能干细胞,对细胞进行鉴定。对两种细胞进行比较,探讨适用于大量培养以满足体内移植治疗的细胞类型。[方法]使用组织块贴壁法从脐带组织中分离人脐带间充质干细胞。培养、鉴定人脐带间充质干细胞及人诱导多能干细胞,绘制两种细胞的生长曲线,并比较细胞形态。[结果]组织块贴壁法成功分离间充质干细胞,经鉴定其表型CD73(+),CD105 (+),CD 31(一),CD34(-),符合脐带间充质干细胞特点。诱导多能干细胞NANOG (+)、OCT-4(+)、SOX-2(+)、SSEA-4(+)、TRA-60(+)和TRA-81(+),符合诱导多能干细胞特点。脐带间充质干细胞呈梭形,以漩涡样排列,其倍增时间约42小时。诱导多能干细胞呈圆形或椭圆形,呈集落生长,其倍增时间约18小时。[结论]人脐带间充质干细胞及诱导多能干细胞增殖速度快,状态稳定,适合用于以移植治疗为目的的大量繁殖。[目的]摸索建立一种在人类感音神经性耳聋大型哺乳动物模型上可行的内耳干细胞移植方法,检测经腰椎穿刺蛛网膜下腔注射移植的人诱导多能干细胞在Mitf基因突变耳聋猪的内耳及全身的分布情况,检测对听性脑干反应阈值的影响。[方法]培养及鉴定人诱导多能干细胞。选择Mitf基因突变的荣昌猪作为神经性耳聋模型的实验对象,移植干细胞的荣昌猪为实验组,单纯注射生理盐水的荣昌猪为对照。将人诱导多能干细胞经蛛网膜下腔注射的途径移植到实验组荣昌猪体内。设置观察期为移植后1天、移植后3天和移植后7天,在移植前和处死前行全身麻醉后测定听性脑干反应阈值。按观察期的时间点处死动物取耳蜗及其他组织。制作冰冻组织切片,行免疫组织荧光染色,用人特异性抗体检测供体细胞。提取蛋白质,使用蛋白印迹法检测移植细胞蛋白的表达。提取RNA,使用RT-PCR法检测供体细胞基因在实验对象体内分布。[结果]经蛛网膜下腔穿刺注射的人诱导多能干细胞在移植后可以在实验对象内耳检出；同过蛋白质印迹法在移植后1天、3天和7天可以在内耳、腰椎段脊髓、胸椎段脊髓、颈椎段脊髓、延髓、小脑、大脑、心、肝、脾、肺、肾中检出移植细胞的蛋白表达。通过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、颈椎段脊髓、延髓、小脑、大脑、心、肝、脾、肺、肾中检出移植细胞的基因表达。在移植前后,Mitf基因突变荣昌猪的听性脑干反应的波形未能引出。[结论]数据表明,可以在内耳及其余脏器中检测出移植细胞的蛋白及基因的表达,移植后Mitf突变耳聋猪的听性脑干反应测听在120 dB SPL未能引出波形,移植后螺旋神经节细胞病变未见继续进展。经腰椎穿刺注射是内耳干细胞移植的新途径,但对耳聋治疗效果有待进一步探索。
[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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R764.9

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