【作者】 崔颜宏；

【导师】 张志坚； 龚爱华；

【作者基本信息】 江苏大学 ， 人体解剖与组织胚胎学， 2009， 硕士

【摘要】 目的:探讨纤维蛋白支架对神经干细胞和星形胶质细胞分化及增殖的影响;评价纤维蛋白支架在神经组织内的生物相容性,观察支架在体内的改建过程。为纤维蛋白用于构建组织工程支架移植修复脊髓损伤提供可行性研究资料。方法:1.用培养基将纤维蛋白原制成胶状溶液,在新鲜鼠血浆中凝血酶的促凝作用下,使其凝固成多孔的具有三维网状结构的纤维蛋白支架。显微镜观察支架的表面结构,并用电子显微镜观察支架内部的三维结构。2.分别培养胚胎大鼠脊髓来源的神经干细胞和新生鼠脊髓神经胶质细胞,接种于纤维蛋白支架上,同时用多聚赖氨酸修饰的玻片作为对照。于体外培养不同时间(3天、7天、14天)后,用神经丝蛋白(NF200)对神经细胞进行免疫荧光染色,测量各复孔(n=4)内NF阳性细胞的突起长度,计算其平均值;培养14天后,用胶质纤维酸性蛋白(GFAP)对胶质细胞进行染色,各复孔(n=4)内统计5个不同视野的胶质细胞总数和GFAP阳性细胞数,计算GFAP阳性细胞相对数量的平均值。用免疫印迹技术对荧光染色结果进行验证。比较在纤维蛋白支架和玻片上神经干细胞分化、神经纤维延伸及神经胶质细胞增殖的差异,分析支架对神经干细胞和星形胶质细胞分化及增殖影响。3.将纤维蛋白支架植入大鼠脊髓完全横断缺损部位,同时以脊髓完全横断缺损大鼠作为对照组。动物存活不同时间后,取出支架植入部位的组织,应用透射电镜观察支架移植部位的组织学结构及支架在神经组织内的降解和改建过程,评价支架的组织相容性。4.将纤维蛋白支架植入大鼠脊髓完全横断缺损部位,同时以脊髓完全横断缺损大鼠作为对照组。动物存活不同时间后,取出支架/脊髓组织,用免疫荧光双标和免疫印迹技术分析损伤部位/支架移植区的神经纤维再生和胶质纤维增生情况。评价纤维蛋白支架移植对大鼠脊髓损伤后神经再生及胶质瘢痕形成的影响。5.将纤维蛋白支架植入大鼠脊髓完全横断缺损部位,同时以脊髓完全横断缺损大鼠作为对照组。分别于术后4周,8周,12周对各组动物下肢运动功能进行BBB评分。比较各组动物后肢运动功能的恢复程度。评价支架移植用于修复脊髓损伤的效果和临床应用的可行性。结果:1.纤维蛋白支架呈半透明多孔海绵状,质地柔软有弹性。支架纵切面经伊红染色后用光镜观察,支架内部呈多孔的网状结构。用扫描及透射电镜观察支架内部纤维蛋白纤维的纵、横切面,具有三维多孔网状结构。2.于体外培养的各观察时间点,纤维蛋白支架组的NF阳性纤维明显长于对照组(3天和7天组,P＜0.05;14天组,P＜0.01);而各时间点的GFAP的表达水平明显低于对照组(P＜0.05)。体外培养14天后,支架组GFAP细胞阳性率为23.5%,对照组为69.1%,支架组的GFAP阳性星形胶质细胞相对数量明显少于对照组。3.实验组术后12周,仔细分离移植部位粘连的周围组织取出整体脊髓,可见脊髓缺损部位移植支架后已完全愈合,局部组织增生,脊髓外观饱满完整;对照组术后12周,缺损部位有致密的瘢痕增生并与背侧结缔组织粘连,分离周围组织,取出整体脊髓,可见缺损部位大部分已修复,局部有暗红色塌陷区,整体脊髓外观欠饱满。透射电镜观察实验组脊髓支架移植部位和对照组脊髓缺损瘢痕修复部位的超微结构,可见实验组支架内有毛细血管和有髓神经纤维分布;对照组局部组织内存在空洞,空洞内有组织碎片和浆细胞,洞壁细胞排列紧密。4.术后4～12周,支架移植组可见支架内有少量神经纤维,此后神经纤维逐渐增多,而胶质细胞增生不明显;对照组脊髓损伤局部可见坏死空洞,空洞周边胶质细胞增生明显。免疫印迹检测结果显示:术后各时间点,支架移植组神经丝蛋白(NF200)相对含量高于对照组;对照组胶质纤维酸性蛋白(GFAP)的相对含量高于支架移植组。结果经统计学处理,均具有显著性差异(P＜0.05)。5.术后两组动物的运动功能均有不同程度的恢复,但对照组动物恢复较慢。各观察时间点BBB评分,支架移植组均高于对照组,统计学有显著性差异(P＜0.05)。结论:1.纤维蛋白支架外观半透明具有较好的韧性,可促进神经干细胞向神经细胞分化并有利于神经纤维的延伸而抑制星形胶质细胞的增殖成熟。2.纤维蛋白支架具有三维多孔网状结构。移植入神经组织内则具有可降解性和良好的组织相容性,移植后促进大鼠脊髓损伤后神经再生和神经纤维的延伸而抑制胶质瘢痕形成。3.纤维蛋白支架移植对脊髓损伤动物后肢运动功能的恢复具有一定的促进作用。纤维蛋白作为生物材料用于构建修复脊髓损伤的组织工程支架具有良好的应用前景。更多还原

【Abstract】 Objective:To investigate the effects of the fibrin scaffold on the differentiation and the proliferation of neural stem cells and astrocytes and evaluate the biocompatibility of the scaffold with the spinal cord tissue.Methods:1.Fibrinogen was mixed with medium,then added the fresh rat blood serum. The reticular structured fibrin scaffolds were prepared.The surface and internal structures of the scaffolds were observed by electron microscopy.2.The spinal cord derived neural stem cells and the gliocytes were cultured in vitro respectively.The purified neural stem cells or gliocytes were seeded separately onto the fibrin scaffolds as experimental group and the glass slides modified with poly-L-lysine(PLL) as control group.At different time in culture(3d,7d and 14d) the neural stem cells were immunofluorescence stained with antibodies against the marker of neurons ie.neurofilament (NF).The length of NF-positive neuritis was masured and the average value was calculated in the culture well(n=4).The gliocytes were immunofluorescence stained with antibodies against the marker of astrocytes ie.glial fibrillary acidic protein(GFAP) at 14 days in vitro.The total cells and the GFAP-positive cells were counted from 5 different fields of vision in the culture well(n=4),then the average ratio of GFAP-positive cells was calculated.The differentiation of neural stem cells,the extension of neurites and the proliferation of astrocytes on the fibrin scaffolds were compared with those on the slides.The protein of GFAP was detected by Western blotting to analyse the mature degree of astrocytes.3.The rats were divided into two groups at random,the scaffold transplantation group and the spinal cord injury control group.The rats in two groups were suffered from spinal cord injury.In the scaffold transplantation group,the cylindrical fibrin scaffolds were implanted into the injured spinal cord.The rats of each group were sacrificed respectively at different times after operation,and the spinal segments or the scaffolds were removed out.The tissue reconstruction of the scaffold in the injured spinal cord was observed with electron microscope, in order to evaluate the scaffold histocompatibility.The regeneration of the nerve fibers and the proliferation of the gliacytes were analysed with immunofluorescence and immunoblotting methods,in order to invesgate the effect of fibrin scaffold transplantation on the regeneration of nerve fiber and the formation of glial scar after spinal cord injury in rats.4.The rats of each group were respectively estimated by system of the BBB locomotion score in 4 weeks,8 weeks and 12 weeks after operation.Comparison of hindlimb movements recovery in rats of different groups,in order to appraise the effect and feasibility of this scaffold to repair the injured spinal cord.Results:1.The profile of fibrin scaffold appears translucent and posseses soft like spongy. The light microscope image of the longitudinal section of the cylindrical scaffold stained with Eosin,showed the reticular structure.SEM and TEM images showed that the scaffold formed a three-dimension(3-D) porous network structure.2.The comparison of average length of the NF-positive neurites at different time points in vitro.lmmunofluorescence staining showed that the NF-positive neurites in the fibrin scaffold group were longer than those in the control group,(at 3 days and at 7 days,P＜0.05.at 14 days, P＜0.01).whereas GFAP-positive cells were fewer than those in the control group(P＜0.05).The comparison of percentage of GFAP-positive cells cultured after 14 days.The percentage of cells was 23.5%in the scaffold group,and 69.1%in the control group,respectively.The expression of GFAP in the cells on the scaffold was lower than that in the control group.3.The adhesions around the transplanted segment were separated carefully,then,we removed out the whole spinal cord from the experimental group 12 weeks after operation.The implanted scaffold was fused with the spinal cord and the appearance of spinal cord was plump. A large number of glial scars were formed and adhered to the dorsal connective tissue in the control group 12 weeks after operation.Majority of the injured spinal cord was healed,and there were collapses in the injured segment.The spinal cord was thinner.The spinal cord specimens and TEM images of the cross-sections of the implanted scaffold / injured spinal cord 12 weeks after operation.The TEM image of the cross-section of the implanted scaffold/spinal cord of experimental group,there were myelinated nerve fibers and the capillary in the area.The TEM image of the cross-section of the injuredsegment of the spinal cord in the control group,there were necrotic cavities with the thick wall,Asterisks showed the plasma cells in the cavities.4.From 4 weeks to 12 weeks after operation,the regenerating nerve fibers could grow into the scaffold then become more and more in case of scaffold transplantation,but there were fewer gliocytes in the scaffold.In the control group,the necrotic cavities were formed after spinal cord injury.There were a lot of gliacytes around the cavity.The results of Western blotting were that the contents of the neurofilament(NF) were higher than the glial fibrilliary acidic protein(GFAP) in the scaffold transplantation group.However,the results of the control group were on the contrary.There were significant differences between the scaffold group and the control group(P＜0.05).5.The control group rats of hindlimb movements recovery were lower than scaffold transplantation group after spinal cord injury.From 4 weeks to 12 weeks,the BBB locomotion scores of scaffold transplantation group were improved significantly contrasting to that of control group(P＜0.05).Conclusion:1.The profile of fibrin scaffold appears translucent and posseses flexibility. It could promote differentiation of the neural stem cells to neurones and extension of the neurites.Meanwhile,the scaffold could inhibit proliferation and mature of the astrocytes.2.The fibrin scaffold possessed a 3-D porous network structure.It has exhibit satisfactory biodegradation and histocompatibility in vivo.With regard to the biological functions,this scaffold could not only promote the regeneration and extension of nerve fiber,but also inhibit the formation of the glial scar.3.The fibrin scaffolds implanted into rats’ spinal cords could promote the recovery of hindlimb movements.Fibrinogen as a biomaterial has a bright perspective in construction of the tissue engineering scaffold to repair the injured spinal cord.更多还原