间充质干细胞治疗感音神经性耳聋的实验研究

发布时间：2018-05-27 09:32

本文选题：脂肪间充质干细胞 + 脐带间充质干细胞　；参考：《南开大学》2014年博士论文

【摘要】：第一部分：目的：寻找获得用于移植的间充质干细胞的适宜方法；培养猪脂肪间充质干细胞(ADMSC).猪骨髓间充质干细胞(BMSC).人脐带间充质干细胞(UMSC).人脂肪间充质干细胞；对培养的间充质干细胞进行鉴定；筛选适合对荣昌猪进行移植的间充质干细胞类型。方法：胶原酶消化法原代分离培养猪脂肪间充质干细胞,用WST-1法比较猪ADMSC在37度和38度条件下的生长曲线,向成骨、成脂和成软骨方向诱导分化ADMSC,鉴定其多能性。密度梯度离心法原代分离培养猪骨髓间充质干细胞,用细胞计数法描绘其生长曲线,向成骨和成脂方向诱导分化鉴定其多能性。组织块贴壁法原代培养人脐带间充质干细胞,用流式法鉴定干细胞表面抗原。扩增培养成人脂肪间充质干细胞,用WST-1法比较在5%和15%血清浓度下培养人ADMSC的生长曲线。结果：用胶原酶消化法可以从猪颈部脂肪组织中分离培养出脂肪间充质干细胞,贴壁生长,P2代以后形态均一,稳定增殖,约5天可传代,在一定条件诱导下可以向成骨、成脂或成软骨方向分化,适宜在5%CO2,38℃,饱和湿度条件下培养。用密度梯度离心法可以从猪股骨骨髓中提取分离出骨髓间充质干细胞,贴壁生长,P3代以后形态均一,P4代以内增殖稳定,约10天可传代,在一定条件诱导下可以向成骨及成脂方向分化。用组织块贴壁法可以从人新鲜脐带组织中分离出脐带间充质干细胞,组织贴壁约20天后可见UMSC爬出,细胞形态均一,呈漩涡样排列,P1-P10代生长迅速,约4天即可传代,经流式细胞仪检测其表型为CD73(+), CD105(+),CD31(-),CD34(-),符合间充质干细胞特点。结论：原代培养的人脐带间充质干细胞,分离方便、增殖速度快,状态稳定,适合用于移植治疗。第二部分：目的：熟悉小型猪脊柱的解剖结构,模拟脑脊液循环途径,探讨经鞘内注射细胞移植途径的可行性。方法：对小型猪腰、骶椎脊柱区进行局部解剖,了解其结构特点,测量腰椎棘突大小及椎间隙的宽度。行腰椎穿刺向蛛网膜下腔注射美兰染液,2小时后处死动物观察染料分布情况。腰椎穿刺向蛛网膜下腔注射DAPI染液,24小时后处死动物,取听神经及耳蜗制作组织冰冻切片,激光显微镜下观察。结果：小型猪的腰椎棘突向头侧倾斜,高度1cm,厚度0.2cm,宽度1.2-1.5cm,腰5-骶1椎间隙最宽,约0.5cm。经蛛网膜下腔注射的美兰染料可以将听神经内听道段染为蓝色。经蛛网膜下腔注射DAPI染料,24小时后在激光显微镜下观察可见听神经内听道段有蓝色荧光,而耳蜗脱钙处理1个月后行冰冻组织切片后再观察未见荧光。结论：小型猪脊柱解剖结构与人类相似,在麻醉恰当的情况下,腰椎穿刺手术顺利,可以完成脑脊液的取样和蛛网膜下腔药物注射,经此途径注射的染料可以对脑脊液进行短期示踪,可对听神经内听道段进行活体染色标记。第三部分：目的：检测经腰椎穿刺蛛网膜下腔注射移植的间充质干细胞在动物内耳及全身的分布,以及这种移植方法对听性脑干反应电位的影响。方法：原代分离培养人脐带间充质干细胞。选择Mitf-M突变的白化耳聋荣昌猪作为模型,正常荣昌猪为对照。将P5-P6代的人脐带间充质干细胞经蛛网膜下腔注射的途径移植到正常及耳聋的荣昌猪体内,每只猪移植细胞量为10～6-10～7个。分别在移植前、移植后2小时、3天、1周、2周、3周、4周直至动物处死前检测其听性脑干反应电位。在移植后2小时、3天、1周、2周、4周、8周处死动物取其耳蜗,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。在移植后3天、1周和3周处死动物取其全身器官标本制作冰冻组织切片、提取DNA,用免疫组织荧光以及聚合酶链反应的方法,用人特异性抗体及引物检测供体细胞在动物体内分布。结果：经蛛网膜下腔注射的人脐带间充质干细胞在移植后3天、1周、2周、3周、4周可以在动物内耳的血管纹、螺旋神经节及基底膜等处检出；在移植后3天和1周可以在大脑、小脑、延髓、心、肝和肺中检出。通过PCR方法在3天、1周后可以在神经系统、心、肝、肺组织中检出供体细胞的DNA。在移植后3天和1周,白化荣昌猪ABR出现了与移植前明显不同的波形。结论：经蛛网膜下腔注射移植人脐带间充质干细胞后,8周内不会导致宿主动物死亡,移植细胞可以进入耳蜗及中枢神经系统以及部分内脏,可以对耳聋荣昌猪的听性脑干反应电位造成短期影响。第四部分：目的：总结颞骨巨细胞肉芽肿的临床表现、病理特点、诊断与治疗方法以提高其疗效。方法：回顾性分析我科2001年至2010年十年间发生在颞骨的巨细胞肉芽肿8例,总结其临床和影像学表现、手术进路方法、病理及随访结果。结果：8例中男性4例、女性4例,年龄21-50岁,中位数年龄37岁,左侧4例、右侧4例,病程从5-60个月,中位数病程21个月,其中1例为复发病例,其余均为首次就诊。主诉听力下降5例、耳鸣4例、局部包块2例。影像学检查可见局部肿物、颞骨及颅底骨壁破坏缺损。术前均未明确诊断,全部采用手术治疗,其中颅中窝进路5例,颅-耳颈联合进路3例,术后3例出现不全面瘫,1例脑水肿,经治疗后均恢复。术后病理诊断均为巨细胞肉芽肿。随访7例,12至58个月无一例复发,另有1例失访。结论：颞骨巨细胞无特异性临床和影像学表现,常以听力下降为首发症状,也可表现为耳鸣、眩晕、耳痛以及局部包块等,术前诊断困难,治疗以手术彻底清除病变为为首选,术后复发率较低。
[Abstract]:Part one:
Objective: to find a suitable method for obtaining mesenchymal stem cells for transplantation; to cultivate porcine adipose mesenchymal stem cells (ADMSC), pig bone marrow mesenchymal stem cells (BMSC), human umbilical cord mesenchymal stem cells (UMSC), human adipose mesenchymal stem cells (human adipose mesenchymal stem cells), identification of cultured mesenchymal stem cells (MSCs), and to screen for the transplants suitable for Rongchang pigs. The type of mesenchymal stem cells.
Methods: pig fat mesenchymal stem cells were isolated and cultured by collagenase digestion. WST-1 method was used to compare the growth curve of pig ADMSC at 37 and 38 degrees. It was induced to differentiate into bone, lipid and cartilaginous direction to differentiate ADMSC and identify its pluripotent activity. Density gradient centrifugation was used to separate bone marrow mesenchymal stem cells from pig to be cultured, and the cell count method was used. The growth curve was drawn to identify the pluripotent activity of the osteogenic and fat induced differentiation. The tissue block adherence method was used to culture human umbilical cord mesenchymal stem cells. The stem cell surface antigen was identified by flow method. Adult adipose mesenchymal stem cells were amplified and cultured. The growth curve of human ADMSC was compared with the serum concentration of 5% and 15% by WST-1 method.
Results: the adipose mesenchymal stem cells can be isolated and cultured from porcine neck adipose tissue by collagenase digestion. After P2 generation, the cells can grow evenly and proliferate steadily. It can be passaged for about 5 days, and can be differentiated into osteogenesis, fat or cartilage under certain conditions. It is suitable to be cultivated at 5%CO2,38 C and saturated humidity. The gradient centrifugation method can extract and separate bone marrow mesenchymal stem cells from the bone marrow of pig's femur. After P3 generation, the proliferation of mesenchymal stem cells is uniform, and the proliferation is stable within P4 generation. It can be passaged for about 10 days, and can differentiate into osteogenesis and fat formation under certain conditions. The umbilical cord mesenchymal cells can be separated from human umbilical cord tissue by tissue block wall method. The stem cells were found to climb out of UMSC after 20 days of tissue adherence. The cell morphology was uniform, and the cells were arranged in a whirlpool like arrangement. The growth of the P1-P10 generation was rapid, about 4 days. The phenotype of the cells was CD73 (+), CD105 (+), CD31 (-), CD34 (-), conforming to the characteristics of mesenchymal stem cells.
Conclusion: primary cultured human umbilical cord mesenchymal stem cells are easy to isolate, proliferate fast and stable, and are suitable for transplantation therapy.
The second part:
Objective: to understand the anatomy of the spinal column of the miniature pig and to simulate the circulation of cerebrospinal fluid, and to explore the feasibility of intrathecal injection of cell transplantation.
Methods: the local anatomy of the small pig's waist and the sacral spine area was carried out to understand the structural features of the lumbar spine, and to measure the size of the spine and the width of the intervertebral space. The lumbar puncture was injected into the subarachnoid cavity and the dye was injected into the subarachnoid cavity. After 2 hours, the animals were sacrificed to observe the distribution of the dyestuffs. The lumbar puncture was injected into the arachnoid membrane and the animals were killed after 24 hours. The frozen tissues of the auditory nerve and cochlea were collected and observed under laser microscope.
Results: the lumbar spine of the small pig was inclined to the head side, the height of 1cm, the thickness of 0.2cm, the width 1.2-1.5cm, the width of the lumbosacral 1 vertebra in the lumbar 5-, and the methylene blue dye which was injected by the subarachnoid cavity could dye the auditory canal in the auditory nerve blue. The DAPI dye was injected into the subarachnoid cavity, and the auditory nerve was observed under the laser microscope after the laser microscope. There was blue fluorescence in the passage, but no fluorescence was observed in the frozen tissue after 1 months of cochlear decalcification.
Conclusion: the anatomical structure of the spinal column is similar to that of human. In the case of appropriate anesthesia, the lumbar puncture operation is smooth and the cerebrospinal fluid can be sampled and the subarachnoid drug injection can be completed. The dye injected through this way can trace the cerebrospinal fluid in short term, and can be used to stain the auditory segment of the auditory nerve.
The third part:
Objective: to detect the distribution of mesenchymal stem cells (MSCs) in the inner ear and the whole body of the subarachnoid injection through the lumbar puncture, and the effect of this method on the auditory brainstem response potential.
Methods: human umbilical cord mesenchymal stem cells were isolated and cultured. The Mitf-M mutated albino deafness Rongchang pig was selected as the model and the normal Rongchang pig as the control. The P5-P6 generation human umbilical cord mesenchymal stem cells were transplanted into the normal and deafness Rongchang pigs by subarachnoid injection into 10 to 6-10 to 7 pigs. Before transplantation, 2 hours, 3 days, 1 weeks, 2 weeks, 3 weeks, 4 weeks until the animals were executed, the auditory brainstem response potential was detected before the animals were executed. The cochlea was taken for 2 hours, 3 days, 1 weeks, 2 weeks, 4 weeks and 8 weeks. The frozen tissue sections were stained with immunofluorescence, and the donor cells were detected by human specific antibodies. Frozen tissue sections were taken from the dead animals of Zhou Dynasty to produce frozen tissue sections to extract DNA. The immunofluorescence and polymerase chain reaction (PCR) were used to detect the distribution of donor cells in animals by human specific antibodies and primers.
Results: human umbilical cord mesenchymal stem cells were injected into the subarachnoid space for 3 days, 1 weeks, 2 weeks, 3 weeks, and 4 weeks to detect the vascular lines, spiral ganglion and basal membrane in the inner ear, and can be detected in the brain, cerebellum, medulla, heart, liver and lungs at 3 days and 1 weeks after transplantation. The PCR method can be used in the nerve system for 3 days and 1 weeks after the transplantation. The DNA. of donor cells was detected in the heart, liver and lung tissues. 3 days and 1 weeks after transplantation, the ABR of albino Rongchang pig appeared distinct waveforms before transplantation.
Conclusion: the transplantation of human umbilical cord mesenchymal stem cells by subarachnoid injection can not lead to the death of the host animal within 8 weeks. The transplanted cells can enter the cochlea and the central nervous system as well as some viscera, which can have a short-term effect on the auditory brainstem response potential of the deaf Rongchang pigs.
The fourth part:
Objective: To summarize the clinical manifestations, pathological features, diagnosis and treatment of giant cell granuloma of temporal bone in order to improve its curative effect.
Methods: a retrospective analysis of 8 cases of giant cell granuloma occurring in the temporal bone in our department from 2001 to 2010 was reviewed. The clinical and imaging findings, surgical approach, pathology and follow-up results were summarized.
Results: 4 of the 8 men, 4 women, 21-50 years old, the median age 37, the left 4, the right 4, the course from 5-60 months, the median course 21 months, 1 cases for the first time, the rest were all for the first time. The main complaints were 5, tinnitus 4 cases, and partial lump 2 cases. Imaging examination showed local mass, temporal bone and skull base wall No definite diagnosis was performed before the operation, all of them were treated with surgical treatment, including 5 cases of cranial fossa approach, 3 cases with craniofacial neck combined approach, 3 cases of incomplete facial paralysis after operation, 1 cases of brain edema and recovery after treatment. All the postoperative pathological diagnosis was giant cell granuloma. 7 cases were followed up, 12 to 58 months had no recurrence, and 1 cases were lost.
Conclusion: there is no specific clinical and imaging manifestations of giant cells in the temporal bone, often with hearing loss as the first symptom. It can also show tinnitus, vertigo, ear pain and local mass. It is difficult to diagnose before operation. The first choice is to remove the disease thoroughly by operation, and the recurrence rate is low after operation.
【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.431

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