本文關(guān)鍵詞：人羊膜間充質(zhì)干細(xì)胞移植對(duì)大鼠脊髓損傷后神經(jīng)功能的影響　出處：《遵義醫(yī)學(xué)院》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人羊膜間充質(zhì)干細(xì)胞 脊髓損傷 移植 大鼠

【摘要】： 目的人羊膜間充質(zhì)干細(xì)胞(hAMSCs)具有表型可塑性特征,作為再生醫(yī)學(xué)的細(xì)胞資源已頗受重視。但目前尚未見有關(guān)hAMSCs體內(nèi)移植治療脊髓損傷的報(bào)道。本研究旨在探討hAMSCs移植對(duì)大鼠脊髓損傷后神經(jīng)功能恢復(fù)的影響,以評(píng)價(jià)hAMSCs在脊髓損傷修復(fù)中的應(yīng)用價(jià)值。 方法①采用胰酶-膠原酶兩步消化法從足月分娩的人羊膜組織中分離hAMSCs,用流式細(xì)胞儀(FCM)和免疫熒光染色對(duì)其細(xì)胞表型進(jìn)行鑒定。②雌性SD大鼠40只,制備脊髓全橫斷損傷模型,隨機(jī)分為hAMSCs移植組和PBS對(duì)照組,每組20只,脊髓橫斷后立即以明膠海綿吸附10μl約2×10~5個(gè)hAMSCs植入脊髓兩斷端之間,對(duì)照組用明膠海綿吸附10μl PBS液同法植入。③術(shù)后每周按Basso Beatlie Bresnahan(BBB)評(píng)分標(biāo)準(zhǔn)測(cè)定各組大鼠神經(jīng)功能,連續(xù)8周。④移植后第14 d各組隨機(jī)處死3只大鼠,采用免疫熒光染色檢測(cè)細(xì)胞移植區(qū)脊髓組織人細(xì)胞核抗原及神經(jīng)細(xì)胞特異性蛋白MAP-2和GFAP的表達(dá),以分析hAMSCs植活及分化情況。⑤術(shù)后第8周,采用免疫組化染色檢測(cè)各組動(dòng)物脊髓橫斷遠(yuǎn)端組織中神經(jīng)纖絲(NF-200)的表達(dá)。 結(jié)果①FCM分析結(jié)果顯示,采用二酶法分離的hAMSCs不同程度表達(dá)CD29、CD44、CD71,其表達(dá)率分別為98.16%、50.96%和12.30%,而不表達(dá)造血細(xì)胞的表面標(biāo)志CD34和CD45,HLA-DR亦為陰性;免疫熒光染色結(jié)果顯示,hAMSCs表達(dá)間充質(zhì)細(xì)胞標(biāo)志波形蛋白,而不表達(dá)上皮細(xì)胞標(biāo)志CK19。②免疫熒光染色顯示,hAMSCs移植后第2周,大鼠脊髓移植區(qū)可見人細(xì)胞核抗原陽性細(xì)胞,但未檢測(cè)到神經(jīng)細(xì)胞特異性蛋白MAP-2和GFAP表達(dá)。③BBB評(píng)分結(jié)果表明,木后第2周開始直至第8周,hAMSCs移植組大鼠BBB分值從3.95±0.78升至7.67±0.90,均高于對(duì)照組(均P＜0.05)。④hAMSCs移植后8周,損傷脊髓遠(yuǎn)端組織中NF-200的表達(dá)強(qiáng)于對(duì)照組。 結(jié)論hAMSCs移植能有助于大鼠脊髓損傷后的神經(jīng)功能恢復(fù),其機(jī)制可能與hAMSCs促進(jìn)受損脊髓遠(yuǎn)端組織表達(dá)NF-200有關(guān)。
[Abstract]:Objective to study the phenotypic plasticity of human amniotic mesenchymal stem cells (hAMSCs). As a cell resource of regenerative medicine, however, there is no report on the treatment of spinal cord injury by in vivo transplantation of hAMSCs. The purpose of this study was to investigate the effect of hAMSCs transplantation on the nerve after spinal cord injury in rats. Effects of functional recovery. To evaluate the value of hAMSCs in spinal cord injury repair. Methods 1 hAMSCs was isolated from human amniotic membrane by trypsin collagenase two-step digestion. 40 female Spinal Cord Sprague-Dawley (SD) rats were identified by flow cytometry (FCM) and immunofluorescence staining. 20 rats in each group were randomly divided into hAMSCs transplantation group and PBS control group. After spinal cord transection, 10 渭 l of gelatin sponge was adsorbed into the spinal cord between the two ends of the spinal cord with about 2 脳 10 ~ 5 hAMSCs. In the control group, 10 渭 l PBS solution was absorbed by gelfoam and implanted weekly with Basso Beatlie Bresnahan. The neurological function of rats in each group was measured by scoring standard. Three rats in each group were randomly killed on the 14th day after transplantation for 8 weeks. The expression of human nuclear antigen (HNA) and neuron-specific proteins (MAP-2 and GFAP) in the spinal cord of the transplanted area were detected by immunofluorescence staining. The expression of NF-200 was detected by immunohistochemical staining at 8 weeks after transplantation and differentiation of hAMSCs. Results 1 the results of FCM analysis showed that hAMSCs isolated by two-enzyme method expressed CD29, CD4, CD41 and CD71 in different degrees, and the expression rates were 98.16%, respectively. 50.96% and 12.30%, while the surface markers of CD34 and CD45 were also negative. Immunofluorescence staining showed that hAMSCs expressed mesenchymal cell marker vimentin, but no epithelial cell marker CK19.2 immunofluorescence staining showed that hAMSCs were transplanted 2 weeks after transplantation. The positive cells of human nuclear antigen were found in the transplanted area of spinal cord of rats, but the expression of neuronal specific protein MAP-2 and GFAP were not detected. The BBB score of rats in the hAMSCs transplantation group increased from 3.95 鹵0.78 to 7.67 鹵0.90 from the 2nd week after wood transplantation to the 8th week. The expression of NF-200 in the injured distal spinal cord tissue was higher than that in the control group at 8 weeks after transplantation (P < 0.05). Conclusion hAMSCs transplantation can contribute to the recovery of neural function after spinal cord injury in rats, and the mechanism may be related to the promotion of NF-200 expression in the distal spinal cord by hAMSCs.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329.28

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