【作者】 袁健东；

【导师】 赵杰； 侯铁胜； 傅强；

【作者基本信息】 第二军医大学 ， 外科学， 2009， 博士

【摘要】 一、背景脊髓损伤(spinal cord injury, SCI )是骨科临床工作中最常见的创伤之一。随着现代建筑业、交通运输业的发展,SCI的发生率也逐年呈上升趋势。据报道,SCI在我国的年发生率约为60/100万。脊髓损伤常导致损伤节段平面以下感觉、运动功能不同程度的丧失,给病人带来终生痛苦,给社会带来巨大的损失和沉重的负担,已成为世纪医学挑战之一。目前临床上对脊髓损伤的治疗主要有防治脊髓损伤后继发性损害、保护残存神经元功能,但是疗效都不理想。这是由于损伤局部星形胶质细胞增生形成胶质疤痕,再生的神经纤维无法穿越损伤区域,神经再生困难,并且受损神经元轴突由于缺少神经营养因子的趋化作用,失去正常的迁移能力从而阻碍轴突再生。目前国内外脊髓损伤修复的研究主要策略为促进轴突再生、克服再生屏障。1981年Aguayo等通过大量实验证实脊髓的神经轴突在合适的微环境中可以再生,是脊髓损伤修复研究的重要里程碑。随着组织工程学的兴起,脊髓损伤修复又取得了新的进展。组织工程技术治疗SCI的基本模式为“种子细胞+生物分子+生物支架”。实验中发现经过基因修饰的雪旺氏细胞(Schwann cells,SCs),能分泌大量神经生长因子,诱导脊髓神经元的再生,将生物可降解支架制成圆柱状种植雪旺氏细胞一起移植修复脊髓损伤也取得了一定疗效。二、目的1、建立体外原代培养雪旺氏细胞的方法,实现短时间内获得大量高纯度雪旺氏细胞这一要求。2、制作具有三维结构的新型生物可吸收支架。3、构建携带人神经营养素3(homo sapiens neurotrophin 3,hNT3)基因的慢病毒载体,将hNT3基因导入体外培养的SCs中,使其高效稳定地表达hNT3。4、通过将转染hNT3基因的SCs种植到三维支架上,一起移植至脊髓半横断损伤处,观察脊髓轴突再生及脊髓传导功能恢复情况,探讨新型三维支架搭载SCs移植对半横断脊髓损伤的修复作用。三、研究方法1、应用植块法与消化法相结合的方法进行原代雪旺氏细胞的培养。以新生SD大鼠的双侧坐骨神经为材料,剥去神经外膜,剪成1mm3大小的碎块,用0.25%的胰蛋白酶和0.03%胶原酶消化,然后种植培养,培养基为含10%胎牛血清的DMEM,24小时后用阿糖胞苷去除成纤维细胞,共24小时,以后每2-3天更换培养液。2、以PLGA为原料,通过熔融纺丝、拉升、编织等步骤制作新型三维可吸收支架,并进行支架的体外、体内生物相容性初步测试。3、由Genebank中查得hNT3片段的基因序列,合成引物后体外扩增,构建重组质粒,接着进行慢病毒载体的包装。慢病毒包装系统由pGC-E1-EGFP载体、pHelper 1.0载体,pHelper 2.0载体三质粒组成,以293T细胞为包装细胞,在病毒携带的增强型绿色荧光蛋白的帮助下,检测重组慢病毒的产生。4、LV-hNT3转染雪旺氏细胞,检测慢病毒的转染效率。然后进行体内实验。以雌性SD成鼠为动物模型,将支架和转染有hNT3的SCs植入大鼠半横断脊髓损伤处,实验动物随机分为A组:hNT3-SCs+三维支架,B组:SCs+三维支架,C组:三维支架。于术后2、4、8、12周,应用BBB评分评价大鼠脊髓功能的恢复情况,使用HE、免疫组织化学染色、透射电镜观察等方法观察雪旺氏细胞的存活、hNT3的表达、损伤处轴突的再生情况。四、结果1、经S-100荧光染色鉴定雪旺氏细胞,结合Hoechst核染色后可计算出原代培养的细胞纯度达95.2%,与文献结果一致。2、通过编织工艺成功研制了新型三维支架,支架外径3mm,微管道内径100μm,PLGA细丝直径25μm,横切面显示中空微管内径均匀一致,呈螺旋上升排列。支架孔隙率为68%,支架具有具有良好的生物相容性,降解时间为3个月。3、应用本方法成功地获得了高滴度的携带hNT3基因的重组慢病毒,病毒滴度达5×107 TU/ml。4、LV-hNT3对雪旺是细胞进行转染,结果显示当MOI值为10时转染效率最高,可达85%。经RT-PCR证实转染的SCs中hNT3 mRNA的表达明显高于对照组。5、移植治疗脊髓损伤后,术后4、8、12周,A组的BBB评分较其他组明显提高(P<0.05) ,其中,A组好于B组,B组好于C组,HE染色观察8周时脊髓材料交界处可见巨噬细胞和炎性细胞聚集,支架的表面有小炎性细胞浸润,但较四周时少。12周时脊髓支架交界处巨噬细胞很少见,可见小的炎性细胞聚集,支架已经完全吸收。移植术后3月的脊髓冰冻切面,可见脊髓移植物处有较多的雪旺氏细胞存活,术后4周的脊髓切片在荧光显微镜下观察到在脊髓损伤处有EGFP荧光表达, 8周时,EGFP荧光表达数量较前减少,支架已开始吸收。12周时,仍有EGFP荧光表达,证明LV-hNT3转染后在雪旺氏细胞内持续稳定的表达。12周时顺标和逆标证实有新生的轴突穿越脊髓损伤区域。12周时经Western Blotting检测到hNT3得到了良好的表达。透射电镜观察:术后8周,有新的突触形成,至术后12周,脊髓损伤处可见较多成簇的髓鞘,新生的无髓及有髓神经纤维明显增多。五、结论1.采用消化法与植块法联合应用的方法,进行SD新生鼠雪旺氏细胞的培养。能够在短期内快速获得大量高纯度的雪旺氏细胞,可作为种子细胞进行SCI修复。2.利用编织工艺制作的新型三维支架,具有良好的生物相容性和恰当的生物降解时间,适合修复SCI。3.根据Genebank中hNT3序列体外扩增了hNT3 cDNA,利用慢病毒三质粒系统包装生产了慢病毒LV-hNT3,病毒滴度达5×107TU/ml。4.LV-hNT3在体外对SCs的转染效率可达85%以上,植入体内的SCs保持良好的生物活性并可存活3个月以上,hNT3基因得到持续表达,三维支架3个月后吸收,脊髓损伤处发现新生的轴突和突触,脊髓的神经功能得到部分恢复。更多还原

【Abstract】 BackgroundSpinal cord injury(SCI) is a frequently encountered trauma in clinic of Orthopedic department. With the developing of modern architecture and transporting trade, the incidence of SCI presents a increasing trend every year. It is reported that the incidence of SCI is 60/1,000,000 every year in china. SCI often results in long-lasting deficits, involving partial or complete paralysis and loss of sensation below the level of the injury site, for the reason that SCI leads to huge damage for society and economy, and SCI becomes one of the challenges of medicine in 21th century. Presently, prevention the secondly injury of spinal cord and protection of remaining Neurons are still the treatment strategies for SCI clinically. However, the prognosis is ugly because of limited regenerative ability of spinal cord, the glial scar barrier and lack of neurotrophic factors blocking the axons regeneration.Treatment strategies for spinal cord injury are two aspects at present, involving promoting axonal regeneration and overcoming regeneration barrier. In 1981, Aguayo had proven that axons have the pontentail of regeneration in some appropriate conditions. It is an important landmark in Repairing spinal cord injury. With the emergence of tissue engineering, repair of spinal cord injury made new progress. The basic model of treatment of SCI was "seed cells + biological molecules + biological scaffolds ." Research found that genetically modified Schwann cells can secrete a large number of nerve growth factors to induce the regeneration of neurons, cylindrical biodegradable scaffolds seeded with Schwann cell transplantation for spinal cord injury repair has also been made a certain effect .Obiective1. Primary culture method for Schwann cells is established in vitro to obtain a large number of high-purity Schwann cells within a short period of time.2. Producing a new type of three-dimensional bioabsorbable scaffolds.3. SCs are transduced with lentiviral vectors encoding homo sapiens neurotrophin (LV-hNT3) to expresse transgenes lasting in high level.4. Three-dimensional scaffords seeded with SCs is transplanted to Spinal cord hemisection site , promoting axonal regeneration and restoring spinal cord function. We aim to explore the role of novel 3D scaffold seeded with LV-NT3-transfected Schwann cells for spinal cord injury repair.Materials and Methods1. We introduced a modified protocol combining the explanting and assimilation to culture primary SCs. the sciatic nerve of the rats were harvested and the epineurium was separated, then the nerve was cut in 1mm3 segments, dissociated with 0.03% collagenase and 0.25% trypsin and incubated at 37°C for 12 min. The segments were placed in culture dishes with DMED+10% fetal bovine serum. 24 hours later arabinosylsytoxin was added to eliminate the fibrablasts. Then, the growth medium was changed every 2-3 days.2. The novel three-dimensional scaffolds were made by means of melt spinning, extension and weaving., then biocompatibility and biodegradability were tested preliminarily in vivo and in vitro.3. From genebank we got the serial of homo sapiens neurotrophin 3 gene, and PCR was used to amplify the hNT3 cDNA, and LV-NT3 stocks were produced by cotransfection of the vector, packaging, and envelope plasmids into 293T cells. the number of transducing particles was defined by infecting 293T cells and counting the number of EGFP-expressing cells after 48h. Lentiviral vectors consisted of pGC-E1-EGFP, pHelper 1.0 (gag/pol ),Helper 2.0 (VSVG).4.We measured the transfection efficiency of the LV-NT3 transduction to SCs. scaffold seeded with LV-NT3-transfected SCs were transplanted to the spinal cord hemisection site , the female SD rats with 200g weight were chosed to study. SD rats were divided into three groups randomly: hNT3-SCs + scaffold, SCs + scaffold and scaffold. BBB locomotion scores are examined to learn functional recovery at 2,4, 8 and 12 weeks after injury. Tissues in SCI sites were observed with H&E, immunohistochemistry staining and electronmicroscopy to identify the survival of SCs, the regeneration of fibres and scaffolds degradation, hNT3 expression was identified by Western Blotting test.Results1. SCs was identified by S-100 staining. The purity of the cultured SCs was determined by comparing the number of Hoechst-labelled nuclei with the number of S-100 immunoreactive cells under a microscope. The purity reached 95.2% for the primary cells , it was Consistent with the literature.2. The novel 3D scaffold was produced by way of weaving, We prepared new type of scaffolds whose outer diameter was 3 mm, and the diameters of the micro-catheters and the fine wires were 100μm and 25μm, respectively. The inner diameters of the micro-catheters were uniform in transverse sections and showed a spiral increasing arrangement in axial sections. The porosity of scaffolds was 68%, and the 3D scaffold possessed features with good biocompatibi1ity , it’s degradation time was 3 months.3. Titers of LV-NT3 were expressed as transducing units (TU) per milliliter and concentrated stocks ranged on the order of 5×107 TU/ml.4.The results showed that transfection efficiency was high reaching 85% when the MOI value was 10. hNT3 expression was identified by RT-PCR test, it was significantly higher compared with the control groups5. From 2 to 12 weeks,The BBB locomotion scores of hNT3-SCs + scaffold group was the best in all (P<0.05), especially group A > group B, group B > group C (P<0.05). H&E staining revea1d the number of cells in the scalford decreases at 8 weeks compared with 4 weeks , and less cells could be seen and the novel 3D scaffolds were completely absorbed at 12weeks. The results showed that 12 weeks after transplantation, the SCs were still alive. Under fluorescence microscope, the slices were observed that there was the expression of EGFP fluorescence after 4 weeks, less after 8 weeks and even after 12 weeks. Anterogradely labeled and retrogradely labeled axons were observed going through the region of spinal cord injury after 12 weeks. hNT3 expression was identified by Western Blotting test. New synapse formation were observed in the injury site after 8 weeks with electronmicroscopy, the region of SCI showed that more clusters of myelin, the new-grown myelinated and non-myelinated nerve fibers increased significantly after 12 weeks.Conclusions1.With the modified protocol combining the explanting and assimilation , the purity reached 95.2% for the primary cells , the cultured SCs could be used for tissue engineering technique for SCI repair.2.The novel 3D scaffold was suitable for SCI repair by tissue engilneering technique with good biocompatibi1ity and biodegradability。3. The hNT3 cDNA were amplified , and LV-hNT3 were constructed Successfully, Titers of LV-hNT3 were concentrated , stocks ranged on the order 5×107 TU/ml.4. The transfection efficiency of SCs was up to more than 85% in vitro, RT-PCR tests confirmed that hNT3 gene expression increased significantly. SCs maintained a good biological activity living for 12 weeks. hNT3 sustainable and efficient expression in SCs was identified, the axonal and synaptic regeneration were found in SCI sites. Spinal cord function got partially restored.更多还原