骨髓间充质干细胞体外培养及耳蜗移植的实验研究

发布时间：2018-03-03 15:49

本文选题：骨髓间充质干细胞　切入点：感音神经性耳聋　出处：《南昌大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的： 1.建立SD大鼠BMSCs分离、培养、鉴定及标记的方法；2.观察标记的BMSCs移植到药物性耳聋的SD大鼠耳蜗内存活、分布和1分化情况,为利用BMSCs移植治疗感音神经性耳聋提供实验依据。方法： 1. BMSCs的分离、培养、纯化和鉴定：取大鼠股骨、胫骨骨髓,采用贴壁培养法培养SD大鼠的骨髓间充质干细胞,进行传代扩增,对细胞的形态学和生长特性进行观察,并应用免疫细胞荧光染色法对细胞表面抗原CD29、CD90、CD117、CD34 CD45进行表型鉴定。 2.耳毒鼠模型的建立与鉴定：应用新霉素连续皮下注射12天,通过测定大鼠听性脑干反应进行鉴定。 3. BMSCs的标记：取第四代的BMSCs用CM-Dil进行荧光标记。 4. BMSCs耳蜗移植：经圆窗径路将用CM-Dil标记的骨髓间充质干细胞移植到药物性聋的SD大鼠耳蜗内,移植两周后处死SD大鼠,通过免疫荧光染色观察植入细胞在耳蜗的存活、分布和分化情况。结果： 1.贴壁培养法培养的SD大鼠骨髓间充质干细胞表达CD29(+)、CD90(+)、CD117 (+), CD34 (-) CD45 (-),符合大鼠BMSCs的特征。 2.应用新霉素连续皮下注射12天后,SD大鼠听闽达80dB以上 3. BMSCs经圆窗植入药物性耳聋鼠耳蜗后,存活2周以上,主要分布于耳蜗底回,中回和顶回较少。 4.用CM-Dil标记的骨髓间充质干细胞移植到药物性耳聋的SD大鼠耳蜗内,部分分化细胞可表达内耳毛细胞的特异性标志物Myosin7a和Mathl。结论： 1.贴壁培养法可以分离、传代培养出较纯化的BMSCS。 2.应用新霉素后,SD大鼠呈现感音神经性耳聋,用该方法作耳聋模型结果稳定、可靠。 3. CM-Dil可用于BMSCs体内示踪标记。 4. BMSCs移植到药物性聋的SD大鼠耳蜗内可以存活两周以上并可分化为具有内耳毛细胞标志物的类毛细胞。
[Abstract]:Objective:. 1. To establish the method of isolation, culture, identification and labeling of BMSCs in SD rats. 2. To observe the survival, distribution and differentiation of labeled BMSCs in cochlea of SD rats with drug-induced deafness, and to provide experimental evidence for the treatment of sensorineural hearing loss by BMSCs transplantation. Methods:. 1. Isolation, culture, purification and identification of BMSCs: bone marrow from femur and tibia of rats was isolated and cultured with adherent culture method. The phenotypes of CD29, CD90, CD117 and CD34 CD45 were identified by immunofluorescence staining. 2. Establishment and identification of ototoxic rat model: neomycin was injected subcutaneously for 12 days, and the auditory brainstem response was determined. 3. BMSCs labeling: 4th generations of BMSCs were labeled with CM-Dil. 4. BMSCs cochlear transplantation: bone marrow mesenchymal stem cells labeled with CM-Dil were transplanted into cochlea of SD rats with drug-induced deafness through round window pathway. SD rats were killed two weeks after transplantation. The survival of implanted cells in cochlea was observed by immunofluorescence staining. Distribution and differentiation. Results:. 1. Bone marrow mesenchymal stem cells (BMSCs) of SD rats cultured by adherent culture method expressed CD29 (CD45), which was consistent with the characteristics of rat BMSCs. 2.After 12 days of continuous subcutaneous injection of neomycin, the auditory threshold of SD rats was more than 80 dB. 3. The cochlea of drug-induced deafness rats implanted with BMSCs through round window survived for more than 2 weeks, mainly distributed in the basal gyrus of the cochlea, but less in the middle gyrus and parietal gyrus. 4. Bone marrow mesenchymal stem cells labeled with CM-Dil were transplanted into cochlea of SD rats with drug-induced deafness. Some differentiated cells could express Myosin7a and Mathl, the specific markers of inner ear hair cells. Conclusion:. 1. The method of adherent culture can be used to isolate and subculture BMSCs. 2. SD rats presented sensorineural deafness after neomycin, and the results were stable and reliable. 3. CM-Dil can be used for BMSCs in vivo tracer labeling. 4. BMSCs transplantation into the cochlea of drug-induced deafness SD rats could survive for more than two weeks and differentiate into hair-like cells with markers of inner ear hair cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R764

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