异体脊髓支架联合环孢素A纳米阳离子脂质体修复脊髓损伤的实验研究

The treatment of spinal cord injury has always been a worldwide problem. According to the research, the formation of glial scar has greatly hindered the regeneration and functional recovery of damaged axons. Therefore, how to reduce the influence of glial scars after spinal cord injury has become a focus of present research. Tissue engineering scaffold implanted could act as the supporting material for the growth of seed cells in the damaged area and at the same time block or reduce the local intrusion of glial scars to promote and guide axon regeneration and penetration. These years, acellular spinal cord scaffold has become the research focus, and this study combined nano-drug-carrying technology with acellular spinal cord transplanting to treat spinal cord injury, in order to solve the problem of high immunogenicity. The freeze thawing and chemical extraction were combined to prepare the acellular spinal cord scaffold, and co-culture of leaching liquid and cells, hemolysis tests and clotting time measuring were used to evaluate its cell toxicity and blood compatibility. The acellular spinal cord was then transplanted to the spinal cord injury area of the rat with CSA nano-cationic liposomes. BBB rating and footprint analysis were given to observe the behavior functional recovery of the rats, HE and GFAP staining were used to observe the change of the glial scars and GAP-43 staining, BDA nerve tracing, nerve electrophysiological examination and electron microscopy observation were used to observe the regeneration of damaged axons to provide new ideas for clinical treatment of spinal cord injury and finally we hope our research will provide a new idea for the clinical treatment of spinal cord injury in the future..

脊髓损伤的治疗一直是世界性难题，课题组之前的研究显示胶质瘢痕的形成极大程度阻碍了损伤轴突的再生和功能恢复，同时阻碍了大分子药物在损伤局部的聚集。组织工程支架植入可作为损伤部位轴突再生的支持基质, 同时减少局部胶质瘢痕侵入, 促进和导向轴突再生并穿越移，课题组之前的研究中成功构建了跨血脊髓屏障纳米载体。本研究将纳米载药技术结合异体脱细胞脊髓移植治疗脊髓损伤，利用环孢素A的免疫抑制作用及神经保护作用，解决异体脊髓支架免疫原性的问题。实验采用冻融+化学萃取法制备异体脊髓支架，通过与细胞共培养，溶血试验评价其细胞毒性与血液相容性。然后将该支架移植到到大鼠脊髓损伤区并联合应用环孢素A纳米阳离子脂质体进行局部靶向给药，用BBB评分，脚印分析观察大鼠行为功能恢复，HE和GFAP染色观察胶质瘢痕变化，GAP-43染色、BDA神经示踪，电镜观察等方法观察损伤轴突再生情况，进而为临床治疗脊髓损伤提供新思路。

【目的】.探讨异硫氰酸荧光素-跨膜肽-聚乙二醇-环孢素-PLGA多功能纳米脂质体的制备、生物相容性和体外药物释放，并观察经尾静脉注射后，跨越大鼠学脊髓屏障聚集情况及对神经细胞的神经保护作用。应用脱细胞技术制备出脱细胞脊髓支架，与大鼠脊髓神经元细胞进行共培养，观察细胞在支架材料上的生长情况。..【方法】.采用反相蒸发法制备兼具跨膜、长循环功能及运载CSA的FITC-TAT-PEG-CSA-PLGA多功能纳米脂质体（PLGA/CsA NPs）；分别通过尾静脉途径注入大鼠体内，观察大鼠一般情况变化，连续记录体重变化情况，评价其安全性；将PLGA/CsA NPs通过尾静脉注入大鼠体内，荧光显微镜观察脂质体跨血脊髓屏障并在脊髓组织分布的情况，并通过GAP-43和GFAP免疫组化染色观察其对于神经细胞的保护作用，通过BBB评分观察脊髓损伤大鼠的功能恢复情况。采用化学萃取法制备脱细胞脊髓支架。采用HE染色、髓鞘染色、扫描电镜观察脱细胞脊髓表面结构，分离培养脊髓神经细胞，共培养，观察其生物相容性。.【结果】.所制备的PLGA/CsA NPs平均有效粒径为251.4±2.2nm，粒径分布范围较窄且少见大粒子，多分散性系数较小，Zeta电位值为25.86±3.33 mV，体外缓释研究显示CSA可缓释释放25h；大鼠在尾静脉注射不同浓度PLGA/CsA NPs后，并未出现呼吸急促、躁动等不适症状，连续观察7d也未出现明显体重变化；荧光显微镜可观察到PLGA/CsA NPs跨过血脊髓屏障分布于脊髓组织中，PLGA/CsA NPs可以提高脊髓损伤阶段神经组织的GAP-43表达，抑制GFAP表达，BBB评分与GAP-43表达一致，GAP-43表达高的组别BBB评分高。大鼠脊髓神经元细胞与大鼠脱细胞脊髓支架共培养后HE染色结果显示大鼠脊髓神经元细胞粘附于大鼠脱细胞脊髓支架生长，细胞生长状态良好。大鼠脱细胞脊髓支架与大鼠脊髓神经元细胞具备良好的生物相容性与适应性。.【结论】.该PLGA/CsA NPs具有良好的生物相容性，作为一种安全药物载体可运载CSA跨过血脊髓屏障，聚集于脊髓损伤处，缓释CSA，保护神经细胞，有望为脊髓损伤治疗提供新的途径。大鼠神经元细胞和大鼠脱细胞脊髓支架具备良好的生物相容性。