本文選題：骨髓間充質(zhì)干細(xì)胞　切入點(diǎn)：感音神經(jīng)性耳聾　出處：《南昌大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：目的： 1.建立SD大鼠BMSCs分離、培養(yǎng)、鑒定及標(biāo)記的方法；2.觀察標(biāo)記的BMSCs移植到藥物性耳聾的SD大鼠耳蝸內(nèi)存活、分布和1分化情況,為利用BMSCs移植治療感音神經(jīng)性耳聾提供實(shí)驗(yàn)依據(jù)。 方法： 1. BMSCs的分離、培養(yǎng)、純化和鑒定：取大鼠股骨、脛骨骨髓,采用貼壁培養(yǎng)法培養(yǎng)SD大鼠的骨髓間充質(zhì)干細(xì)胞,進(jìn)行傳代擴(kuò)增,對(duì)細(xì)胞的形態(tài)學(xué)和生長(zhǎng)特性進(jìn)行觀察,并應(yīng)用免疫細(xì)胞熒光染色法對(duì)細(xì)胞表面抗原CD29、CD90、CD117、CD34 CD45進(jìn)行表型鑒定。 2.耳毒鼠模型的建立與鑒定：應(yīng)用新霉素連續(xù)皮下注射12天,通過(guò)測(cè)定大鼠聽(tīng)性腦干反應(yīng)進(jìn)行鑒定。 3. BMSCs的標(biāo)記：取第四代的BMSCs用CM-Dil進(jìn)行熒光標(biāo)記。 4. BMSCs耳蝸移植：經(jīng)圓窗徑路將用CM-Dil標(biāo)記的骨髓間充質(zhì)干細(xì)胞移植到藥物性聾的SD大鼠耳蝸內(nèi),移植兩周后處死SD大鼠,通過(guò)免疫熒光染色觀察植入細(xì)胞在耳蝸的存活、分布和分化情況。 結(jié)果： 1.貼壁培養(yǎng)法培養(yǎng)的SD大鼠骨髓間充質(zhì)干細(xì)胞表達(dá)CD29(+)、CD90(+)、CD117 (+), CD34 (-) CD45 (-),符合大鼠BMSCs的特征。 2.應(yīng)用新霉素連續(xù)皮下注射12天后,SD大鼠聽(tīng)閩達(dá)80dB以上 3. BMSCs經(jīng)圓窗植入藥物性耳聾鼠耳蝸后,存活2周以上,主要分布于耳蝸底回,中回和頂回較少。 4.用CM-Dil標(biāo)記的骨髓間充質(zhì)干細(xì)胞移植到藥物性耳聾的SD大鼠耳蝸內(nèi),部分分化細(xì)胞可表達(dá)內(nèi)耳毛細(xì)胞的特異性標(biāo)志物Myosin7a和Mathl。 結(jié)論： 1.貼壁培養(yǎng)法可以分離、傳代培養(yǎng)出較純化的BMSCS。 2.應(yīng)用新霉素后,SD大鼠呈現(xiàn)感音神經(jīng)性耳聾,用該方法作耳聾模型結(jié)果穩(wěn)定、可靠。 3. CM-Dil可用于BMSCs體內(nèi)示蹤標(biāo)記。 4. BMSCs移植到藥物性聾的SD大鼠耳蝸內(nèi)可以存活兩周以上并可分化為具有內(nèi)耳毛細(xì)胞標(biāo)志物的類毛細(xì)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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R764

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本文編號(hào)：1561678