以Matrigel作为神经干细胞移植支架治疗急性脊髓损伤的实验研究

发布时间：2018-07-26 09:03

【摘要】：目的本研究旨在探讨Matrigel作为急性脊髓损伤后细胞移植支架材料的可行性。主要方法1.取新生24 h的SD大鼠大脑海马组织,在体外用无血清的神经干细胞培养液[成分为DMEM/F12(1∶1)、20 ng/ml EGF、20 ng/ml b FGF、1%PS]进行扩增培养。加入胎牛血清使其自然分化,应用细胞免疫荧光技术对细胞进行鉴定。体外分别采用Matrigel、可吸收明胶海绵(absorbale sponge)、TCP/POC作为支架材料培养感染Ad-G FP腺病毒的NSCs,于培养后2 h、1 d、3 d、7 d、14 d在荧光显微镜下观察细胞在不同支架材料中的存活情况。将Matrigel/NSCs混合物接种至裸鼠皮下成瘤,对瘤体进行组织学分析观察移植的NSCs在M atrigel中的生长和分化情况。2.制备SD大鼠脊髓的Allen’s打击损伤模型。将动物分为三组:PBS对照组(A组,于损伤位点注射PBS),单纯Matrigel组(B组,于损伤位点单纯注射Matrigel),Matrigel/NSCs组(C组,于损伤位点植入Matrigel与NSCs的复合物)。暴露T10节段脊髓后予10×2.5g·cm的能量打击。各组在SCI后7 d进行移植处理,C组将Matrigel与用PKH67绿色荧光染料标记的第三代NSCs(细胞密度为5×107/ml)在体外混匀后植入大鼠SCI后7 d的脊髓损伤位点,分别于移植后1d,7d,14d,28d在激光共聚焦扫描显微镜下观察脊髓损伤部位移植NSCs的存活情况。术后通过BBB评分评估大鼠后肢运动功能,观察8周;移植处理后4,8周取损伤段脊髓行HE染色和免疫组织学检测,观察和评估Matrigel对损伤脊髓以及移植细胞的作用。结果1.新生鼠海马组织中分离出的NSCs具有较强的克隆能力,可形成细胞团,呈不规则球形,免疫荧光结果提示早期形成的神经球Nestin抗原呈阳性;加入血清促使其分化2周,细胞免疫荧光结果提示分化后的细胞GFAP抗原、MAP2抗原呈阳性。2.与absorbale sponge和TCP/POC两种支架材料相比较,NSCs在Matrigel中的存活率以及存活时间优于另外两组,NSCs可在Matrigel中存活较长时间;免疫荧光结果提示NSCs可在Matrigel中分化为β-tubulin 3阳性的神经元;裸鼠皮下成瘤瘤体病理学检测结果提示Matrigel中神经细胞生长良好,并可见少许新生血管;组织免疫学检测结果提示移植的细胞可分化为β-tubulin 3和MAP2阳性的神经元。3.PKH67示踪NSCs结果示NSCs在脊髓损伤部位可存活较长时间。4.C组各时相点BBB评分最高,与其他两组比较差异有统计学意义(p0.05);A组与B组各时相点无统计学差异(p0.05)。植入处理4周时,脊髓组织HE染色结果显示A组损伤部位神经组织液化坏死形成体积不等的空洞;B组损伤部位空洞被植入的Matrigel填补,Matrigel中可见细胞生长;C组损伤部位空洞被植入的Matrigel填补,可见大量神经细胞在植入的Matrigel中生长,且有少许血管生成。免疫组织学结果显示:A组损伤部位无neun、NF-200、MAP2阳性的神经元,损伤部位边缘可见较多Nestin阳性的神经干细胞和GFAP阳性的星形胶质细胞,损伤中心则无Nestin表达;在B、C组损伤部位可见Nestin、neun、NF-200、MAP2、GFAP阳性的神经细胞,C组表达多于B组。植入处理8周时,支架材料有一定程度降解,上述神经细胞标记物表达降低。结论神经干细胞在Matrigel中可以存活和分化;以Matrigel作为急性脊髓损伤后神经干细胞移植支架材料,具有使用方便,不易造成二次损伤的特点。
[Abstract]:Objective the purpose of this study was to explore the feasibility of Matrigel as a scaffold for transplantation of cells after acute spinal cord injury. 1. the main method was to take the brain hippocampus of SD rats with new 24 h, and to expand the culture in vitro using serum free neural stem cell culture medium (DMEM/F12 (1: 1), 20 ng/ml EGF, 20 ng/ml B FGF, 1%PS], and to add fetal bovine blood. The cells were identified by cell immunofluorescence technology. Matrigel was used in vitro to absorb gelatin sponge (absorbale sponge) and TCP/POC as scaffold material to cultivate NSCs of Ad-G FP adenovirus. After culture, 2 h, 1 D, 3 D, 7 d, and 14 d in different scaffold materials under fluorescence microscope. The Matrigel/NSCs mixture was inoculated into the subcutaneous tumor of nude mice, and the tumor body was histologically analyzed to observe the growth and differentiation of the transplanted NSCs in M atrigel.2. to prepare the Allen 's damage model of the spinal cord of SD rats. The animals were divided into three groups: the PBS control group (A group, PBS injecting site injection), and the simple Matrigel group. The injury site was simply injected with Matrigel), group Matrigel/NSCs (group C, the complex of Matrigel and NSCs at the site of damage). After exposure to the T10 segment spinal cord, the energy shock was given to 10 * 2.5G. Cm. Each group was transplanted after SCI 7 d. The C group was in vitro (the cell density of 5 x) marked with the green fluorescent dye. The spinal cord injury site of 7 d after SCI was implanted, and the survival of NSCs was observed in 1D, 7d, 14d and 28d after the laser confocal scanning microscope after the transplantation. The hind limb movement function was evaluated by BBB score for 8 weeks, and the spinal cord of the injured segment of the transplanted spinal cord was stained with HE and immuno tissue after the transplantation. The effects of Matrigel on the injured spinal cord and the transplanted cells were observed and evaluated. Results the isolated NSCs in the hippocampus of 1. neonatal rats had a strong clone ability, which could form a cell group, which showed irregular sphere. The immunofluorescence results suggested that the early formation of the neural ball Nestin was positive, and the serum was added to promote its differentiation for 2 weeks. The immunofluorescence results suggest that the differentiated cell GFAP antigen, MAP2 antigen positive.2. and absorbale sponge and TCP/POC two scaffolds are compared, the survival rate and survival time of NSCs in Matrigel are better than the other two groups, NSCs can survive for a long time in Matrigel, and the immunofluorescence results suggest that NSCs can be divided into beta in Matrigel. Ulin 3 positive neurons; the pathological examination results of subcutaneous tumor tumor in nude mice suggest that the nerve cells in Matrigel grow well, and a few neovascularization is visible; the tissue immunological detection results suggest that the transplanted cells can differentiate into the.3.PKH67 NSCs of the neurons of beta -tubulin 3 and MAP2 positive neurons indicating that NSCs can survive in the injured part of the spinal cord. The BBB score of each time point in the.4.C group was the highest for a long time, and there was significant difference with the other two groups (P0.05). There was no statistical difference between the A group and the B group (P0.05). At the time of implantation, the spinal tissue HE staining showed that the nerve tissue of the injured part of the group A was liquefied and necrotic to form a void in the group A, and the cavity in the lesion site was planted in the group B. In the Matrigel filling, the cell growth was seen in Matrigel, and the injured site in group C was filled with the implanted Matrigel, and a large number of nerve cells were grown in the implanted Matrigel, and a little angiogenesis was found. The immunohistochemical results showed that there were no NeuN, NF-200, MAP2 positive neurons in the A group and more Nestin in the edge of the injury site. Positive neural stem cells and GFAP positive astrocytes had no Nestin expression in the damage center; in B, Nestin, NeuN, NF-200, MAP2, GFAP positive neurons were seen in group C, and the expression of C group was more than that in B group. The scaffold material was degraded to a certain extent and the expression of nerve cell markers decreased at 8 weeks. Conclusion nerve stem was found. Conclusion nerve stem Cells can survive and differentiate in Matrigel, and Matrigel is used as a scaffold for neural stem cell transplantation after acute spinal cord injury. It is easy to use and is not easy to cause two damage.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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