【作者】 何俊山；

【导师】 许家军；

【作者基本信息】 第二军医大学 ， 人体解剖与组织胚胎学， 2011， 硕士

【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。更多还原

【Abstract】 Objective: Survival cells of retinal ganglion decrease significantly after optic nerve injured. Formation of glial scar and various molecules inhibit regeneration by releasing molecules that hinder the axons regenerating through the damaged area. Nerve regeneration is difficult. In perspective of neural tissue engineering, amniotic epithelial cells were seeded into collagen sponge to build amniotic epithelial cells / collagen sponge complex in vitro. Then it was implanted into the damaged parts of the optic nerve in rats to preliminarily observe and discuss repair effects of complex on the injured optic nerve and related mechanisms.Methods: (1)Amniotic tissue was taken from late pregnancy SD rats. After sequential digestion, amniotic epithelial cells were culture primarily. Subcultured cells were stained by immunofluorescence and quantified by PCR. (2)Amniotic epithelial cells were seeded into the collagen sponge, in vitro cultured for one week. Using immunofluorescence staining, quantitative PCR to reassure cells in the collagen sponge. (3)Using H&E staining, scanning electron microscopy, fluorescent dye to label cells, identifying cell growth, and CCK-8 to detect cell proliferation in vitro. (4)The amniotic epithelial cells / collagen sponge composition adult male SD rats were transplanted into the optic nerve injury model. The animals were divided into 4 groups: normal control group, injured group. Rats in the injuried group got complete optic nerve transected by using1ml syringe with a vertical incision of epineurium at 2 mm retrobulbar, cutting off about 0.5 mm of the nerve. But epineurium and vessels intact should be kept complete. Injecting 10μl serum-free culture medium into the injured part. Stent control group was based on the injured group, collagen sponge which was cultured for 1 week was implanted into injured part of the nerve. Experimental group was based on the injured group. 10μl The amniotic epithelial cells / collagen sponge composition (5×10~6 /ml) were injected into the injured part of the nerve. (5) For some of the animal models, utilizing CM-Dil as a membrane marker before the cell implantation. Rats were fixed by cardiac perfusion on 4weeks and 8 weeks after surgery. The optic nerve were harvested for frozen sectioning to observe distribution and numbers of the labeled cells. (6)CTB was injected into vitreous body to label RGCs in some of the animals 48 h before being sacrificed. 2 days later, rats were fixed by cardiac perfusion, and eyeballs were taken for retina flattening to count CTB labeled cells. Some of the animals were sacrificed 4 weeks after surgery ,whose eyeballs were taken for retina flattening and Nissl staining to count RGCs. Some of the animals were sacrificed 4,8 weeks after surgery, the optic nerves were collected for HE staining to observe the changes of tissue structure and cell concentration, and for SABC immunohistochemistry staining to show the expression of GAP-43.Results:(1)Rat AECs were successfully cultured. Immunocytochemical staining showed the rat AECs could express epithelial-specific markers CK-19, neural stem cell marker Nestin, as well as pluripotent cells marker Oct-4, Nanog, and so on. (2)Quantitative RT-PCR showed the same genes were expressed by AECs after grew on the collagen sponge scaffold, and upregulated the mRNA of Nestin. (3)By using H&E staining, scanning electron microscopy, immunofluorescence staining and cck-8, amniotic epithelial cells grew well on the collagen sponge scaffold, collagen sponge can promote the proliferation of amniotic epithelial cells.(4)After CM-Dil labeled cell complex were transplanted into the model in which the optic nerve was cut, frozen section of optic nerve showed great amount of labeled cells survived in the injury area 4 and 8 week after surgery. And these cells could spread to the proximal segment and distal segment of damaged optic nerve, and migration along the nerve membrane.(5)At 4 weeks after injury, CTB and NISSL staining of retinal ganglion cells counts showed that ganglion cell density in each group was significantly lower than normal. In the cell complex group, cell density of ganglion was higher than that in injured group, stent control group; there was no significant difference between the injured group and stent control group. (6)HE staining of optic nerve showed the Collagen sponge transplanted into body can integrated with the injured nerve tissue. it degraded after 8 weeks. There were great amount of cells, forming irregular streak and spreading towards both directions. Shape of the nuclear in the distal part of the optic nerve became smaller, but seemed much similar with normal ones in the cell transplanted groups. By counting the cells within the distal part of the optic nerve in 4 and 8 weeks, we learned that the number of stent control group higher than injury group,the number of complex cells was significantly higher than injury group and stent control group.(7)Immunohistochemistry staining of GAP-43 in the injured group and control stent group, only small amount of GAP-43 expressed in the injury area, and none in the distal part. The expression of GAP-43 in amniotic epithelial cell / collagen sponge graft injury area significantly increased, showing a small amount of GAP-43 positive, similar to the regeneration of axon-like structure stretching from the injured area to the distal segment. The structure transplanted 8 weeks reach farther than to it was transplanted 4 weeks .Conclusions: (1)Amniotic epithelial cells and collagen sponge have good histocompatibility. Collagen cultured in vitro can promote the proliferation of amniotic epithelial cells and enhanced amniotic epithelial cells differentiating towards neural stem cells, the complex have better vitality.(2)After transplanting the complex, part of cells in collagen sponge can survive at least 8 weeks in damaged optic nerve and could migrate to both sides of the damaged zone(.3)After transplanting the complex, it could protect retinal ganglion cells and optic nerve glial cells and improve the growth ability of the regenerating axons, and could facilitate some of the axons to pass injury area and grow into the distal part of the optic nerve. These results indicate that the complex could improve the optic nerve regeneration microenvironment and promote axon regeneration.更多还原