【作者】 蒋良福；

【导师】 劳杰； 赵新； 虞聪； 张丽银；

【作者基本信息】 复旦大学 ， 外科学， 2006， 博士

【摘要】 引言：周围神经缺损的修复仍是外科领域的一大难题，临床上主要采用自体神经移植的方法修复，但有明显的缺陷。组织工程为修复周围神经缺损提供了新的方法，本研究的目的就是利用激活态雪旺细胞和生物可吸收材料几丁糖—胶原膜构建人工神经，探索一种修复周围神经缺损的有效方法。本研究共分五部分： 第一部分 改良植块法培养成年大鼠雪旺细胞 目的：通过改良植块培养，探索快速获得高纯度成年大鼠雪旺细胞的培养方法。 方法：成年SD大鼠20只，分二组，每组10只大鼠，A组改良植块法，B组传统植块法。取大鼠坐骨双侧坐骨神经剥除外膜后，剪成0.5mm~3小块。A组：植块用0.03％胶原酶消化20分钟后培养；B组：植块直接培养。植块隔5天反复培养，传代纯化，共3次。通过细胞计数比较每次植块所得细胞量，雪旺细胞S—100染色测定纯度。 结果：改良植块法在细胞数量和纯度上均高于传统植块法，改良植块法第一次植块培养所得主要为成纤维细胞，起到分离成纤维细胞的目的，第二次雪旺细胞纯度较高85％，经纯化后可以达到95％，第三次植块雪旺细胞纯度95％。经3次反复植块就可以完全利用神经植块。 结论：改良植块法可以快速获得高纯度的雪旺细胞，方法简单，廉价。 第二部分 激活态雪旺细胞培养及其增殖规律研究 目的：1．利用预变性神经和激活液培养激活态雪旺细胞 2．比较激活态雪旺细胞和正常雪旺细胞的增殖能力 方法：SD大鼠10只，200g～250g。右侧坐骨神经切断一周后取材，剥除神经外膜，称重，用0.03％胶原酶加激活液(0.1ml/ml消化液)，按照改良植块法培养激活态雪旺细胞。左侧坐骨神经取材剥除外膜后称重，称重，用0.03％胶原酶消化，按照改良植块法培养正常雪旺细胞。比较单位神经所得的细胞量。取1×10~5的第二代细胞分别接种35个培养皿，通过各时间点(2d、4d、6d、8d、10d、12d、14d)计数，绘制生长曲线，计算倍增时间，比较激活态雪旺细胞和正常雪旺细胞增殖能力。培养第8天细胞计数后提取蛋白，western blot法比较两者CDK1的含量。取接种前及培养第14天的雪旺细胞作S—100染色。 结果：改良植块第二次培养7天后，每毫克预变性神经可得到7.5×10~4个激活态雪旺细胞正常神经为1.5×10~4个正常雪旺细胞。传代培养后，激活态雪旺细胞在培养2天后计数均高于正常雪旺细胞，具有统计学差异(p＜0.01)，激活态雪旺细胞倍增时间5天，正常雪旺细胞为7天，激活态雪旺细胞增殖快于正常雪旺细胞。而且western blot测定激活态雪旺细胞CDK1含量高于正常雪旺细胞。 结论：神经预变性刺激雪旺细胞增殖，利用预变性7天的神经培养可以获得大量激活态SC，而且激活态SC增殖能力均高于正常SC，可能与激活了CDK1有关。 第三部分： 激活态雪旺细胞GAP43、BDNF、C-JUN、P21 mRNA表达研究 目的：比较激活态雪旺细胞和正常雪旺细胞GAP43、BDNF、C-JUN、P21基因表达区别，寻找激活态雪旺的marker，分析激活态雪旺细胞增殖及促进神经再生的机制。 方法：选取10只SD大鼠，将大鼠右侧坐骨神经切断，1周后取右预变性坐骨神经改良植块法培养激活态雪旺细胞；取左侧正常坐骨神经培养正常雪旺细胞作为对照。自细胞提取mRNA，应用rt-PCR的方法扩增GAP43、BDNF、C-JUN、P21基因，取PCR产物行1％琼脂糖凝胶电泳，拍照，图像分析计算目标基因和内标基因(GAPDH)PCR产物的吸光度比值，以配对t检验比较。 结果 激活态雪旺细胞GAP43、c-Jun、P21PCR产物积分吸光度比值较正常雪旺细胞明显增高，差异有统计学意义(P＜0.001)。表明激活态雪旺细胞较对照组正常雪旺细胞GAP43 mRNA表达明显增多。正常雪旺细胞培养未检测到BDNFmRNA，而激活态雪旺细胞BDNFmRNA表达明显，两者有质的区别。 结论 1．BDNF可以作为激活态雪旺细胞的marker区别于正常雪旺细胞 2．激活态雪旺细胞GAP43、BDNF、c-Jun、P21基因表达增强，可能是其促进神经再生和加速细胞增殖的原因之一。第四部分 激活态雪旺细胞在几丁糖—胶原膜上生长规律的研究 目的：研究激活态雪旺细胞在几丁糖—胶原膜上的生长规律和亲和力，探索两者构建人工神经的可能性。 方法：成年SD大鼠的坐骨神经切断预变性7d，改良植块法培养激活态雪旺细胞。以1×10~5数量接种于几丁糖—胶原膜上和培养皿。通过相差显微镜观察细胞生长情况，培养2d、4d、6d、8d、10d、12d、14d各取5皿细胞计数，绘制生长曲线，计算倍增时间。培养3、7、10天几丁糖—胶原膜行扫描电镜观测细胞情况。 结果：相差显微镜和扫描电镜下观察显示激活态雪旺细胞在几丁糖—胶原膜上贴壁生长情况良好。2周后细胞量在几丁糖—胶原膜和培养皿上分别达到10.26×10~5和7.22×10~5个，体外倍增时间分别为4天和5天。激活态雪旺细胞在几丁糖胶原膜上的增殖速度快于培养皿。 结论：几丁糖—胶原膜具有促进激活态雪旺细胞增殖的作用，两者具有良好的亲和性。以几丁糖—胶原膜为载体可以进行激活态雪旺细胞移植是可行的。 第五部分 激活态雪旺细胞和几丁糖—胶原膜修复周围神经缺损的实验研究 目的 1．研究几丁糖—胶原膜和激活态雪旺细胞促进周围神经再生的作用，探索修复神经缺损的方法 2．比较激活态雪旺细胞和正常雪旺细胞修复神经缺损的能力，为激活态雪旺细胞应用提供实验依据 方法 自体激活态雪旺细胞和自体正常雪旺细胞培养于几丁糖—胶原膜，将膜缝制成导管修复大鼠坐骨神经10mm缺损，以单纯几丁糖胶原复合膜管及自体神经移植做对照，术后4、8、12周，行大体观察，测量肌电图动作电位波幅和传导速度。术后12周取材，通过小腿肌肉测量、组织学切片观测与计算机图像分析测量有髓神经密度和髓鞘厚度进行效果评价。 结果 几丁糖—胶原膜加自体激活态雪旺细胞在肌电图、肌肉湿重和神经再生质量和数量方面与自体神经移植无明显差异，激活态雪旺细胞组比正常雪旺细胞组效果好，而单纯的几丁糖胶原膜修复效果较差。 结论 1．几丁糖胶原复合膜加激活态雪旺细胞能有效促进周围神经再生 2．激活态雪旺细胞比正常雪旺细胞促进神经再生的能力强更多还原

【Abstract】 Introduction:The effective repair of the peripheral nerve gap following injury continues to be a considerable clinical challenge. Peripheral nerve autograft is nearly clinically sole selection of repairing the gap. That has inevitable disadvantages, such as limited supply of available nerve graft material and permanent loss of the donor nerve function. An attractive to autografts seems to be artificial nerve with tissue engineering. The aim to this study was to develop a novel effective substitute composed of activated Schwann cells and chitosan-collagen film to repair peripheral nerve gap. This study includes five major parts.Part one Modified Schwann cell culture from adult ratsObjective: To introduce a modified technique of explants culture to obtain highly purified Schwann cells.Methods: 20 adult SD rats were randomly divided into 2 groups, group A for modified explants culture and group B for tranditional explants culture. The sciatic nerve was dissected bilaterally through a dorsal incision and collected in DMEM. The epineurium was gently stripped and nerve fascicles were subsequently cut in 0.5mm~3 segments. Group A: nerve segments were digested in 0.03% collagenase for 20min before translation to Petri dish. Grou B: nerve segments were transplanted to Petri dish directly. The individual nerve explants were transplanted to a new dish every 5 days, totally 3times. The cell number were counted separately and compared. Schwann cell purity was determined by immunostainning the cells for S-100.Results Cell population in group A was several folds to that in group B. Accordingly Schwann cell purity in group A was also higher than group B. In modified culture, the cell obtained from the first time explants culture were mainly fibrocytes. It contributed to remove fibrocytes from explants. Schwanncell purity reached 85% in the second time explants culture by modified methods. And Schwann cell purity was 95% after third replantation with modified method, while explants were disappeared and fully used.Conclusion: Modified explants culture is a quick way to obtain highly purified Schwann cells.Part two Activated Schwann cell culture and growth ruleObjective: To culture activated SC from predegenerated nerve by addition of activated liquid and explore the growing rule of the activated SC in culture.Methods: 10 male SD rats, weight 200g～250g. Activated SC culture:The right sciatic nerve of SD rats were transected for predegeneration. 1 week later, the distal segment of the transected right sciatic nerve was harvest. The epineurium was gently stripped and each nerve was weighted. Nerve segments were digested in 0.03% collagenase and active liquid (0.1ml/ml) for 20 min according to modified explant culture. Normal SC culture: The untreated left sciatic served as control. The epineurium was gently stripped and each nerve was weighted. Nerve segments were digested in 0.03% collagenase without of active liquid for 20 min according to modified explant culture. Cell number per mg was compared. 1×10~5 cells of second generation were planted on 35 petri dish. The numbers of activated SCs and normal SCs were counted at various growing point (2d、4d、6d、8d、10d、12d、14d), then the growing curve was drawn. Doubling time was also calculated. At the 8 day, protein was extracted after cell number counting. CDK1 was measured by western blot. Schwann cell purity was determined by immunostainning the cells for S-100 at 1 and 14 days.Results: Acivated SC was 7. 5×10~4/mg from pregenerated nerve after modified explant culture. Normal SC was 1.5×10~4/mg from normal nerve. After subcultivation, activated SC amounts were more than normal SC in various point except first 2 days. The doubling period of activated SC was 5 days, while normal SC was 7days. Activated SC proliferates faster than normal SC. The CDK1 expression was also higher in activated SC than in normal SC. At 14 days onculture, purity of activated SC was 95% and normal SC was 92%.Conclusion: A large mount of activated SC can be obtained from predegenerated nerve. Activated SC proliferate faster than normal SC. it may be partly explained by CDK1 over-expression in activated SC.Part three Gene expression of BDNF, GAP43, c-Jun and p21 in activated Schwann cellObjective: To compare activated Schwann cell and normal Schwann cell in BDNF, GAP43, c-Jun and p21 gene expression.Methods: 10 male SD rats, weight 200g～250g. The right sciatic nerves of SD rats were transected for predegeneration. 1 week later, the distal segment of the transected right sciatic nerve was harvest. The untreated left sciatic nerve was harvested as control. Activated schwann cell were cultured from predegenerated nerve and normal Schwann cells were harvest from contralateral norm sciatci nerve. rt-PCR was employed for gene enlargement. mRNA was distilled from activated Schwann cells and Schwann cells respectively. Then the mRNA was reverse transcript to cDNA with SuperScriptTM, and cDNA worked as template for PCR enlargement. The product of PCR was separated with 1% agarose gel electrophoresis for 40min～50min. PCR products of GAP43、BDNF、 C-JUN、P21 was measured and then compared between the experiment group and control group.Results:GAP43、 C-JUN、P21 mRNA of activate Schwann cells are much more than that of the normal Schwann cells. BDNF mRNA was manifest in activated Schwann cells, however it was not showed in normal schwann cells.Conclusion1. BDNF may act as a marker for activated schwann cell.2. GAP43、BDNF、C-JUN、P21 gene expression is up regulated in Activated Schwann in contrast to normal Schwann cell. It may partly elucidate why activated Schwann cells proliferate quickly and promote nerve regeneration.Part four Growth rule of activated Schwann cells on Chitosan-collagen filmObjective: To explore the growth rule of activated SC cultured on the surface of Chitosan-collagen film.Methods: Adult SD rat sciatic nerves were transected and predegenerated for 7 days. Activated SCs were harvested by modified piece replant way. 1×10~5 cells were planted on the surface of Chitosan-collagen film. 1×10~5 cells were planted on petri dish severed as control. Activated SCs were observed through the phase contrast microscope. The numbers of activated Schwann cells were counted at various growing point (2d、4d、6d、8d、10d、12d、14d) , then the growing curve of activated Schwann cells was drawn. Doubling time was also calculated. Scanning electron microscope was employed to observe cell growth on chitosan-collagen film at 3d,7d,10d.Results: Activated SCs grow well on the surface of the Chitosan-collagen film under observation of phase contrast microscope and scanning electron microscope. Cell amount reached were up to 10×10~5 on Chitosan- collagen film and 7×10~5 petri dish separately after 2 weeks compared to the primary 1× 10~5. The doubling period of activated SCs was 4 days on chitosan-collagen film and 5 days on Petri dish. Activated proliferated faster on chitosan-collagen film than on Petri dish.Conclusions: Chitosan-collagen film posses an ability to promote activated SCs proliferation. The fine affinity made it possible to transplant activated SCs by chitosan-collagen film.Part five An experimental study on repair of peripheral nerve defects with Chitosan-collagen film and activated Schwann cellObjective: To study the efficiency of artificial nerve made of Chitosan-collagen and activated Schwann cells on promoting nerveregeneration.Methods: Activiated Schwann cells and normal Schwann cells from left sciaticnerve were cultured on Chitosan-collagen film. Then the films were sutured intoconduits to bridge 10mm defect of right sciatic nerve. Autograft and complainChitosan-collagen film were served as control. General observation, amplitudeof active potential (AMP) and nerve conduction velocity (NCV) were measuredat the end of 4, 8, 12 weeks. Evaluation included triceps surae measurementand histological examination was made at the end of 12 weeks. The myelinatednerve fibers density, axon area percentage and thickness of mylin sheath weremeasured by computerized image analysis.Results: There was no differenc between the group of (Chitosan-collagen withactivated Schwann cells) and the group of autograft in electromyogrphicexamination, triceps surae measurement and axon regeneration. Whereas thegroup of activated Schwann cells got better results than the normal Schwanncells. The group of Chitosan-collagen film without Schwann cell showed a poorresult of axon regeneration.Conclusion: Chitosan-collagen and activated Schwann cells can effectivelypromote nerve regeneration. It provides a novel method for repairing theperipheral nerve defects. Additionally, activated Schwann cell is better thannormal Schwann cells in encouraging nerve regeneration.更多还原