【作者】 李长文；

【导师】 郑启新；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分移植骨髓间充质干细胞治疗大鼠脊髓损伤的实验研究目的：研究体外培养的骨髓间充质干细胞（MSCs）神经营养因子的表达，以及移植后对大鼠脊髓损伤的治疗作用。方法：取大鼠骨髓，用全骨髓贴壁法分离MSCs，流式细胞仪检测CD34、CD90、CD44和CD45等抗原的表达。逆转录-聚合酶链反应（RT-PCR）检测MSCs中脑源性神经营养因子（BDNF）和神经生长因子（NGF）mRNA的表达，酶联免疫吸附（ELISA）检测其BDNF和NGF蛋白的表达。用改良Allen重物打击法造大鼠脊髓损伤模型，1周后将MSCs注射到损伤部位，术后4周每周进行BBB评分，术后第4周取损伤部位脊髓组织，ELISA检测组织中BDNF和NGF的表达：另取损伤部位脊髓组织进行NF200和GFAP免疫荧光染色。结果：MSCs贴壁生长，呈纺锤形及梭形，流式细胞术检测表达CD90、CD44，不表达CD34和CD45，RT-PCR发现其表达BDNF和NGF mRNA，且ELISA检测到了BDNF和NGF蛋白的表达，移植MSCs的大鼠运动功能改善，BBB评分高于对照组；移植MSCs的脊髓组织中BDNF和NGF蛋白含量高于对照组；免疫荧光发现移植MSCs的大鼠脊髓囊腔较小，且周围有较多轴突再生。结论：MSCs表达神经营养因子BDNF和NGF，移植后改善了局部微环境，能够促进大鼠脊髓损伤后运动功能恢复。第二部分多肽原位植入对大鼠脊髓损伤治疗作用的实验研究目的：设计合成两亲性多肽C₁₆H₃₁O-A₃G₄D₂IKVAV，研究其自组装形成的三维凝胶结构，并研究其植入后对大鼠脊髓损伤的治疗作用。方法：将两亲性多肽C₁₆H₃₁O-A₃G₄D₂IKVAV用仿生体液促发自组装，用透射电镜（TEM）检测。改良Allen植物打击法造大鼠脊髓损伤模型，将多肽溶液注入损伤部位，术后6周每周进行BBB评分；术后3d、1w、2w、3w、4w、6w分别取损伤部位脊髓组织，实时荧光定量PCR检测胶质纤维酸性蛋白（GFAP）的表达；术后第6周取损伤部位脊髓组织，进行NF200和GFAP免疫荧光染色。结果：多肽溶液在仿生体液促发下自组装为三维凝胶，TEM显示为纳米纤维，直径7-8nm，纤维空隙较大；术后第5周、第6周多肽注入组BBB评分高于对照组；实时定量荧光PCR显示多肽组损伤部位GFAP表达低于对照组；免疫荧光染色发现植入多肽组局部GFAP表达较少，且有较多轴突再生。结论：两亲性多肽C₁₆H₃₁O-A₃G₄D₂IKVAV能自组装为三维凝胶，植入脊髓损伤大鼠能够抑制GFAP形成，并能促进轴突再生。第三部分组织工程化神经移植物的构建目的：使用两亲性多肽自组装三维凝胶作为支架材料，结合骨髓间充质干细胞，构建组织工程化神经移植物。方法：将多肽溶液用MSCs细胞悬液促发自组装，形成复合细胞的三维凝胶，光镜观察细胞的分布；培养1d、7d及21d，用钙黄绿素和PI进行活死细胞染色，观察细胞在凝胶内的存活情况；细胞凝胶复合物进行培养，用CCK-8法检测细胞增殖；ELISA法检测细胞培养液中BDNF和NGF的表达。结果：细胞悬液促发了多肽溶液自组装为三维凝胶，且细胞以三维形式存在于凝胶内；活死细胞染色发现培养至21d大部分MSCs仍存活，细胞增殖测试发现MSCs在凝胶内增殖良好，与三维培养无显著性差异，ELISA发现凝胶中的MSCs产生并分泌了BDNF和NGF。结论：用细胞悬液成功促发了多肽溶液自组装，构建了神经组织工程构件，体外测试其活性良好。更多还原

【Abstract】 Part 1Experimental research of transplant mesenchymal stem cells to treat spinal cordinjuryObjective: To investigate the expressions of neurotrophins in MSCs in vitro, and thetherapeutic effects of MSCs transplantation for SCI.Methods: MSCs were isolated from marrow of rats, and purified by continuous culture.The cells were characterized by the expression of CD34, CD90, CD44 and CD45 by flowcytometry. The expression of BDNF and NGF were detected in MSCs using RT -PCR andELISA. SCI model was established by modified Allen’s method. MSCs were injected intothe epicenter of injured spinal cord in one week. Locomotive function of SCI rats wasgraded with BBB score weekly, and in four weeks injured spinal cord was taken, theprotein level of BDNF and NGF was detected by ELISA, and NF200, GFAPimmunofluorescent staining were performed in frozen sections of injured spinal cord.Results: Isolated cells were adherent to culture flask and were spindle-shaped, and werepositive for CD90 and CD44, negative for CD34 and CD45 by flow cytometry. MSCsexpressed BDNF and NGF mRNA, and expressed the protein. The rats treated with MSCstransplantation showed improvement in functional outcome, the BBB scorces were higherthan the control, the protein levels of BDNF and NGF in spinal cord were higher than thecontrol. And immunofluorescent staining show smaller cysts and more regenerated axonsin spinal cord tn transplantation rats.Conclusions: MSCs could expressed BDNF and NGF, and could improve functionaloutcome in SCI rats. neurotrophic factor, nerve growth factorPart 2Experimental study of Peptide implantation in situ of spinal cord injuried rats for SCItreatmentObjective: To design Peptide-Amphiphile C₁₆H₃₁O-A₃G₄D₂IKVAV, investigate 3-Dconstruction of self-assembly gel and study the therapeutic effects of its implantation forSCI treatment in rats.Methods: Self-assembly of PA C₁₆H₃₁O-A₃G₄D₂IKVAV was improved by simulated bodyfluid and detected under TEM. SCI rats models were established by modified Allen’smethod, peptide solution was injected into injuried site. BBB test was performed everyweek postoperatively in 6 weeks, spinal cord samples were collected 3d, 1w, 2w, 3w, 4wand 6w after SCI. the expression of GFAP was detected by Real-time PCR. Spinal cordsample of injuried sites were collected again and observed after immunofluorescentstaining with NF200 and GFAP.Results: Self-assembly of peptide solution into 3-D gel could be trigged by SBF, andpresented as nanofiber under TEM, with 7-8nm in diameter and big gaps between the fibers.BBB scores in peptide implantation group were higher than controls in the 5th and 6thweeks after surgery, Real-time quantitative fluorescent PCR demonstrated the expression ofGFAP in the injured sites was weaker than controls. Immunofluorescent staining showedthe low expression of GFAP and many axon regenerations after peptide transplantation.Conclusion: PA C₁₆H₃₁O-A₃G₄D₂IKVAV could self-assembly into 3-D gel, GFAPformation could be inhibited and axon regeneration could be improved after itstransplantation into rats. Objective: To construct tissue-engineered neural grafts with Self-assembled PA 3-D gel asscaffold material and MSC.Methods: Self-assembly of peptide solution was trigged by MSCs suspension, 3-D gelcombined with MSCs was formed, cells distribution was observed under microscope.Live/dead cell staining was performed with Calcein-AM PI after 1d, 7d and 21d culture.Living condition of cells in the gel was observed. The gel combined with cells was culturedand detected by CCK-8 method to determine the cell proliferation. The expressions ofBDNF and NGF in the medium were detected by ELISA.Results: Cell suspension trigged peptide solution to self-assembly into 3-D gel, the cellswere kept alive in the gel. Live/dead cell staining presented most MSCs were still aliveafter 21 days culture. Cell proliferation test demonstrated MSCs proliferation was prettywell, and there’s no significant difference with 2-D culture. ELISA showed BDNF andNGF were generated and secreted by MSCs in the gal.Conclusion: peptide solution self-assembly was trigged by MSCs suspension successfully;tissue-engineered material was constructed and presented good activity in vitro experiment.更多还原