大鼠羊膜上皮细胞与胶原海绵复合体修复视神经损伤的初步研究

发布时间：2019-07-05 17:05

【摘要】：目的:视神经损伤后存活视网膜节细胞数量明显减少,胶质瘢痕形成及胶质细胞释放的抑制再生的各种分子,阻碍了再生轴突通过损伤区域,神经再生困难。从组织工程的角度,我们将羊膜上皮细胞接种于胶原海绵上,体外构建羊膜上皮细胞/胶原海绵复合体,移植入大鼠视神经受损部位,初步观察和探讨复合体对损伤后视神经修复的作用及相关机制。方法:(1)取妊娠晚期SD大鼠的羊膜组织,贯序消化后进行羊膜上皮细胞原代培养,传代细胞进行免疫荧光细胞化学和定量PCR鉴定。(2)将传代羊膜上皮细胞接种于胶原海绵上,体外共培养1周,行免疫荧光细胞化学、定量PCR鉴定。(3)用荧光染料标记细胞、扫描电镜、HE染色、CCK-8法等方法,检测复合体上羊膜上皮细胞生长及增殖情况。(4)将构建的羊膜上皮细胞/胶原海绵复合体移植入成年雄性SD大鼠视神经切断伤模型中,设以下4组:正常对照组;损伤组,于眼球后约2 mm处用1ml注射器针尖将神经外膜纵向切开,完全切除一段长约0.5 mm的神经,致视神经完全断开,但保持视神经外膜和血管完整,于视神经缺损处注入10μl无血清培养基;空支架组是在损伤组的基础上,于视神经缺损处植入预培养1周的胶原海绵;实验组是在损伤组的基础上,于视神经缺损处移植入羊膜上皮细胞/胶原海绵复合体并注入10μl羊膜上皮细胞悬液(5×10~6个/ml)。(5)在部分羊膜上皮细胞/胶原海绵复合体移植组,用CM-Dil标记支架上细胞的细胞膜,术后4周、8周经心灌注固定,取视神经作冰冻切片,观察标记细胞的存活和分布。(6)部分动物取材前48 h,玻璃体注射CTB标记视网膜节细胞,术后4周经心灌注固定,取眼球视网膜铺片,计数CTB标记细胞;部分动物术后4周经心灌注固定,取眼球视网膜铺片,尼氏染色计数视网膜节细胞;部分动物术后4周、8周取视神经眶内段HE染色观察组织结构和细胞密度变化,免疫组织化学染色法显示GAP-43表达。结果: (1)体外成功培养获得大鼠羊膜上皮细胞,接种于胶原海绵,免疫荧光细胞化学染色鉴定后显示,接种胶原海绵前后,羊膜上皮细胞均能表达上皮细胞特异性标志物CK-19、神经干细胞标志物Nestin、以及细胞多能性标志分子Oct-4、Nanog等。(2)定量PCR检测,结果显示羊膜上皮细胞接种胶原海绵前后均有CK-19、Nestin、Oct-4、Nanog、bFGF的mRNA表达,且细胞接种胶原海绵后Nestin的mRNA表达显著上调。(3)经荧光标记、扫描电镜、HE染色、CKK-8法检测,结果显示羊膜上皮细胞能较好地粘附于胶原海绵上生长,胶原海绵能促进羊膜上皮细胞增殖。(4)将用CM-Dil标记细胞的复合体移植入大鼠视神经切断伤模型后,术后4周、8周取视神经行冰冻切片,可观察到损伤区有标记的细胞存活,能向受损视神经近侧段和远侧段迁移,并可沿神经外膜向两侧迁移。(5)损伤后4周,CTB及尼氏染色视网膜节细胞的计数结果显示,各组节细胞密度较正常组明显降低,细胞复合体组节细胞密度较损伤组、空支架组增加;空支架组的节细胞密度较损伤组无明显差异。(6)HE染色视神经显示,胶原海绵移植体内后,能与断端神经组织相融合,术后8周基本降解。视神经损伤区有大量细胞存在,呈不规则条索状分布并向两端延伸。视神经损伤后远侧段细胞核明显变小,而复合体移植组的胞核与正常对照组大小相似。伤后4周、8周计数远侧段细胞结果显示,各组细胞数量均高于正常组,空支架组较损伤组细胞数量无增加,细胞复合体组细胞数量增加明显高于损伤组及空支架组。(7)免疫组织化学法对视神经GAP-43染色后显示,损伤组、空支架组中GAP-43在损伤区仅有少量表达,远侧段未见表达;复合体移植后损伤区GAP-43表达明显增多,可见少量GAP-43阳性、类似再生轴突样结构,由伤区伸入至远侧段,8周时能较4周达到更远处。结论:(1)羊膜上皮细胞与胶原海绵组织相容性好,胶原海绵能促进羊膜上皮细胞的增殖,促进羊膜上皮细胞向神经干细胞方向分化,复合体活性较好;(2)复合体移植受损视神经后,部分复合体细胞能在损伤部位存活至少8周并向损伤区两侧迁移,能在一定程度上保护视网膜节细胞和视神经的神经胶质细胞,增强视神经再生轴突的生长活力,特别是能促使少量再生轴突通过损伤部位进入到远侧段神经,表明所构建的复合体能改善视神经再生微环境、促进轴突再生。
[Abstract]:Objective: The number of viable retinal ganglion cells after optic nerve injury was significantly reduced, and the formation of glial scar and the release of glial cells inhibited the regeneration of various molecules, which prevented the regeneration of axons from the area of injury and the difficulty of nerve regeneration. From the angle of tissue engineering, the amniotic epithelial cells were inoculated on the collagen sponge, the amniotic epithelial cells/ collagen sponge complex was constructed in vitro, the damaged part of the optic nerve of the rat was transplanted, and the effect of the complex on the optic nerve repair after injury and the related mechanism were observed and discussed. Methods: (1) The amniotic epithelial cells of SD rats were cultured in the late stage of pregnancy, and the primary culture and passage of the cells were carried out by immunofluorescence cell chemistry and quantitative PCR. and (2) inoculating the subcultured amniotic epithelial cells on the collagen sponge, co-culturing for 1 week in vitro, carrying out fluorescent cell chemistry and quantitative PCR in vitro, (3) detecting the growth and proliferation of amniotic epithelial cells on the complex by means of fluorescent dye-labeled cells, scanning electron microscopy (SEM), HE staining, and CCK-8 method. Cases. (4) The constructed amniotic epithelial cell/ collagen sponge complex was transplanted into the optic nerve cut-out model of adult male SD rats, and the following four groups were set: normal control group; injury group;1 ml of syringe needle tip was used to machine the outer membrane of the nerve in the longitudinal direction at about 2 mm after the eyeball. Incision, complete removal of a length of nerve with a length of about 0.5 mm, complete disconnection of the optic nerve, but keep the outer membrane of the optic nerve and the blood vessel intact, inject 10. m u.l of serum-free medium at the optic nerve defect; the empty stent group is on the basis of the damaged group, and the pre-cultured 1-week collagen is implanted in the optic nerve defect. Sponge; the experimental group was transplanted into the amniotic epithelial cell/ collagen sponge complex at the optic nerve defect and injected with 10 & mu; l of amniotic epithelial cell suspension (5-10-6/ m) on the basis of the injury group. (1). (5) In the transplantation group of part of the amniotic epithelial cell/ collagen sponge complex, the cell membrane of the cell was marked with CM-Dil, the cell membrane of the cell was labeled with CM-Dil, the cell membrane of the cell was fixed at 4 and 8 weeks after operation, the optic nerve was taken as a frozen section, and the survival of the labeled cells was observed. Distribution. (6) The vitreoretinal ganglion cells were labeled with CTB in vitreoretinal injection for 48 hours before and after 4 weeks of operation, and the retina of the eye was set to count the CTB-labeled cells. The 4-week post-operation of some animals was fixed by heart-perfusion, and the retina of the eye was taken. The changes of tissue structure and cell density were observed at 4 weeks and 8 weeks after operation, and the changes of tissue structure and cell density were observed in 8 weeks after operation, and GAP-43 was shown by immunohistochemical staining. Results: (1) In vitro, the amniotic epithelial cells of the rat were successfully cultured and inoculated in the collagen sponge. After the chemical staining and identification of the immunofluorescence cells, the epithelial cell-specific markers CK-19 and the neural stem cell marker N could be expressed in the amniotic epithelial cells before and after the inoculation of the collagen sponge. estin, and cell multi-functional marker molecule Oct-4, N The expression of CK-19, Nestin, Oct-4, Nanog, and bFGF in the amniotic epithelial cells was detected by quantitative PCR. The results showed that the amniotic epithelial cells could be better adhered to the collagen sponge and the collagen sponge could promote the amniotic membrane. (4) After transplantation of the complex of the CM-Dil-labeled cells into the rat optic nerve-cutting injury model, the optic nerve line frozen section was taken at 4 and 8 weeks after the operation, and the labeled cells in the damaged area were observed to survive, and the injured optic nerve proximal section and the damaged optic nerve were observed. The distal segment is migrated and can be along the outer membrane of the nerve The results showed that the cell density of the cell complex was lower than that in the normal group, the cell density of the cell complex group was lower than that of the group, the empty stent group was increased, and the cell density in the empty stent group was lower than that in the group. There was no significant difference. (6) HE staining the optic nerve showed that after the collagen sponge was transplanted into the body, it could be fused with the end-end nerve tissue, and the operation was 8. The optic nerve injury area has a large number of cells, which are distributed in the form of irregular strips. The nucleus of the distal segment was significantly smaller after the optic nerve injury, and the nucleus of the complex transplantation group and the normal control. The results showed that the number of cells in the group was higher than that in the normal group, and the number of cells in the cell complex group was significantly higher than that of the injured group. (7) The GAP-43 expression of GAP-43 was only expressed in the injured area after GAP-43 was stained by immunocytochemical method. The expression of GAP-43 in the damaged area was not seen in the distal segment. The expression of GAP-43 in the damaged area after the complex transplantation was significantly increased, and a small amount of GAP-43 was found to be positive and similar to the regeneration. The axon-like structure, which extends into the distal section from the wound area, can be more than 4 weeks at 8 weeks Conclusion: (1) The collagen sponge has good compatibility with the collagen sponge, and the collagen sponge can promote the proliferation of the amniotic epithelial cells and promote the differentiation of the amniotic epithelial cells in the direction of the neural stem cells, and the activity of the complex is better; (2) the complex after the damaged optic nerve is transplanted, the part of the complex cells can survive for at least 8 weeks at the damaged site and migrate to the two sides of the damaged area, so that the retinal ganglion cells and the glial cells of the optic nerve can be protected to a certain extent, the optic nerve is enhanced, The growth vigor of the axons, in particular, can cause a small amount of regenerated axons to enter the nerve of the distal section through the lesion site, indicating that the constructed composite can improve the micro-ring of the optic nerve regeneration.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.6

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