BDNF基因修饰施万细胞结合组织工程技术治疗周围神经损伤的研究
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摘要
目的: (1)研究体外高纯度Wistar乳鼠施万细胞(SCs)的培养方法;(2)研究SCs与壳聚糖支架的组织相容性;(3)将BDNF基因转染的SCs注入预制壳聚糖导管,构建组织工程人工神经桥接Wistar大鼠10mm坐骨神经缺损,研究此人工神经桥接体的修复效果及这些促神经再生因素之间的相互作用,为组织工程神经的进一步研究和临床应用提供实验依据。
方法: (1)取3-5d的Wistar乳鼠双侧坐骨神经、臂丛神经,用酶消化后组织块种植法培养SCs。用0.25%的胰蛋白酶常规消化、2次25分钟差速贴壁法纯化SCs,经过1周培养,长满瓶底后,加入0. 25%的胰蛋白酶常规消化分瓶传代, S-100免疫组化染色计算SCs纯度。(2)取上述SCs制成悬液,密度为1×105/ml。24孔培养板2个,分为正常对照组和实验组,壳聚糖膜平铺于孔底。SCs接种到培养板内,在37℃和5%CO2的条件下培养。培养24h后,培养液中加入DAPI(终浓度50μg/ml)孵育过夜后液,PBS洗涤培养瓶至少3次,以去除未与细胞结合的DAPI。加培养液继续培养。观察SCs和壳聚糖组织相容性。(3)将1×105/ml BDNF基因转染的SCs接种到壳聚糖导管内,构建组织工程人工神经桥接体,桥接SD大鼠10mm坐骨神经缺损(A组),研究其修复效果。另设空白壳聚糖导管组(B组)、假手术组(C组)、自体神经移植组(D组)作为对照。在术后6, 12周进行实验动物的经电生理检测、组织学切片,光镜观察,评价其修复效果。
结果: (1)用酶消化后组织块贴壁培养的的方法,SCs“爬出”速度较直接组织贴壁培养方法快,进一步纯化后做S-100细胞免疫组织化学鉴定为SCs,纯度达到95%以上。(2)SCs与壳聚糖支架的组织相容性:正常对照组和实验组各时点凋亡细胞的百分率经差异无显著性意义(P> 0.05)。(3)大体观察:饲养过程中无大鼠死亡,大鼠患肢屈曲挛缩情况及拖曳步态在6周以后有所改善。12周后大鼠处死,未见导管残留及桥接段上下神经萎缩,周围有血管增生、粘连现象。(4)电生理检测:四组NCV相互比较,A组与B组、A组与C组均有显著性差异(P<0.05),A组与D组无显著性差异(P>0.05)。组织学观察组织工程神经体内长期、稳定表达目的基因-BDNF和报告基因-GFP、FLAG。
结论: (1)先用酶消化,后组织块贴壁培养SCs和差速贴壁综合运用的纯化方法所“生产”的SCs数量较大,纯度较高,且细胞的理化性质及生物学特性受外界环境影响较小。同时又节约了神经组织的材料来源,避免了反复多次取材的繁琐操作过程,节省人力和物力,并且保证了实验所需的SCs尽可能来源于同一只或同一批动物。(2)使用冷冻干燥技术研制的壳聚糖导管表面光滑,透明状,质地均匀。厚度0.15 mm,与盖玻片的厚度相仿。实验表明具有良好生物相容性,SCs可以在其表面生长增殖。可以作为神经桥接管。(3)用壳聚糖制作三维支架,以BDNF基因转染的SCs为种子细胞,构建成的组织工程化人工神经,修复神经缺损,其修复效果接近于自体神经移植,相对于单纯应用壳聚糖神经导管修复神经缺损可进一步提高神经修复的效果。具有广阔的应用前景,值的进一步研究。
Objectine: (1) Researching external high-purity“Wistar”cultural method of suckling rat cells. (2) Inserting BDNF schwann cell into presetting Chitosan tubes, constructing tissue engineering artificial nervous bridge connect rat 10mm SCs sciatic nerve damnify, researching the repairing effect of artificial nervous bridge and these interaction between nerve regeneration factor, providing experiment evidence for tissue engineering further studies and clinical application.
Method: (1) Take 3-5 days suckling rat two sides sciatic nerve and brachial plexus, after digesting by enzyme, explants planting insolating cultivate SCs. After a week’s cultivation, full of bottom, adding 0.25% trypsase general digested to each bottles to generation, using S-100 immunohistochemistry dyeing and calculating SCs purity. (2)Get such schwann cell and make them into suspension, density is 1×105/ml a 24 hole costar, divided into normal group and experimental group, Chitosan tiles on the bottom of holes, ultra-clean platform drying. SCs vaccinates into costar, cultivating is in the condition of 37 oC and 5% CO2 After 24h,adding DAPI (final concentration 50μg/ml) into incubation , After 24h,washing bottle using PBS at least 3 times, to be rid of DAPI which is not combined with cells. Adding inoculums to continue cultivate. Observing SCs and Chitosan histocompatibility. (3)Using lentivirus taking BDNF gene infected schwann cell, and infuse its into Chitosan tubes, construct Nerve tissue engineering bridge, connected Wistar rat sciatic nerve (A-group),researching the renovation effection,observe Chitosan tubes (B-group), sham operation (C-group)、autogenous nerve graft(D group) as the comparison. After 6、12 weeks, taking the experiment by electrical physiological test、histology section, microscope and estimate its therapeutic efficacy.
Result: (1) After enzyme digested, using explants organisms cultivate method, the creeping speed of SCs is faster than direct explants organisms cultivate method, take furthering purification and then identified SCs by S-100 immunohistochemistry, and the purity reached more than 95%. (2)dealed normal group and experimental group, and percentage of apoptosis cells each time point were calculated, the percentage of apoptosis cells at each time points were no significant(P<.0.05). (3)General observation: no rat death in the process of feeding, the condition of rats which suffer from contracture and drag-to gait would improve in four weeks. There were no Chitosan tubes remaining and no neuratrophy in the explantation nerve place, Vascular proliferation was discovered transpl surrounding. (4)Electrophysiology observation: comparisons of three group NCV, group A and B, B and C had significant differences (P<.0.05), group A and group D had no significant differences.
Conclusion: (1) Digesting enzyme, the purity method which composed by backer explants organisms cultivate method and differential adherence method "produce" large number of SCs, high purity and low attached by outer atmosphere of cell physicochemical property and biological characteristics. At the same time, saving nerve organization material sources, avoiding repeated draw material processes, saving manpower and material resources sparingly and guaranteeing SCs derive from the same animal which was used in experiment.(2) The Chitosan tubes made by Freeze-drying technology had smooth surface, transparency, even texture. 0.15mm thickness, the same as Cover slip. Experiments show that both have good biocompatibility and SCs proliferation in the surface. Could serve as a nerve bridge over. (3) Using Chitosan tubes to act as supporting frame, Using lentivirus taking BDNF gene infected SCs as seeds cells, Construct tissue engineered artificial nerves, repairing nerve defection, the effect is closed to autogenous nerve graft, relative to single application Chitosan tubes, it could further raise nerve repairing effects. This study had broad application prospects and value of further investigation.
方法: (1)取3-5d的Wistar乳鼠双侧坐骨神经、臂丛神经,用酶消化后组织块种植法培养SCs。用0.25%的胰蛋白酶常规消化、2次25分钟差速贴壁法纯化SCs,经过1周培养,长满瓶底后,加入0. 25%的胰蛋白酶常规消化分瓶传代, S-100免疫组化染色计算SCs纯度。(2)取上述SCs制成悬液,密度为1×105/ml。24孔培养板2个,分为正常对照组和实验组,壳聚糖膜平铺于孔底。SCs接种到培养板内,在37℃和5%CO2的条件下培养。培养24h后,培养液中加入DAPI(终浓度50μg/ml)孵育过夜后液,PBS洗涤培养瓶至少3次,以去除未与细胞结合的DAPI。加培养液继续培养。观察SCs和壳聚糖组织相容性。(3)将1×105/ml BDNF基因转染的SCs接种到壳聚糖导管内,构建组织工程人工神经桥接体,桥接SD大鼠10mm坐骨神经缺损(A组),研究其修复效果。另设空白壳聚糖导管组(B组)、假手术组(C组)、自体神经移植组(D组)作为对照。在术后6, 12周进行实验动物的经电生理检测、组织学切片,光镜观察,评价其修复效果。
结果: (1)用酶消化后组织块贴壁培养的的方法,SCs“爬出”速度较直接组织贴壁培养方法快,进一步纯化后做S-100细胞免疫组织化学鉴定为SCs,纯度达到95%以上。(2)SCs与壳聚糖支架的组织相容性:正常对照组和实验组各时点凋亡细胞的百分率经差异无显著性意义(P> 0.05)。(3)大体观察:饲养过程中无大鼠死亡,大鼠患肢屈曲挛缩情况及拖曳步态在6周以后有所改善。12周后大鼠处死,未见导管残留及桥接段上下神经萎缩,周围有血管增生、粘连现象。(4)电生理检测:四组NCV相互比较,A组与B组、A组与C组均有显著性差异(P<0.05),A组与D组无显著性差异(P>0.05)。组织学观察组织工程神经体内长期、稳定表达目的基因-BDNF和报告基因-GFP、FLAG。
结论: (1)先用酶消化,后组织块贴壁培养SCs和差速贴壁综合运用的纯化方法所“生产”的SCs数量较大,纯度较高,且细胞的理化性质及生物学特性受外界环境影响较小。同时又节约了神经组织的材料来源,避免了反复多次取材的繁琐操作过程,节省人力和物力,并且保证了实验所需的SCs尽可能来源于同一只或同一批动物。(2)使用冷冻干燥技术研制的壳聚糖导管表面光滑,透明状,质地均匀。厚度0.15 mm,与盖玻片的厚度相仿。实验表明具有良好生物相容性,SCs可以在其表面生长增殖。可以作为神经桥接管。(3)用壳聚糖制作三维支架,以BDNF基因转染的SCs为种子细胞,构建成的组织工程化人工神经,修复神经缺损,其修复效果接近于自体神经移植,相对于单纯应用壳聚糖神经导管修复神经缺损可进一步提高神经修复的效果。具有广阔的应用前景,值的进一步研究。
Objectine: (1) Researching external high-purity“Wistar”cultural method of suckling rat cells. (2) Inserting BDNF schwann cell into presetting Chitosan tubes, constructing tissue engineering artificial nervous bridge connect rat 10mm SCs sciatic nerve damnify, researching the repairing effect of artificial nervous bridge and these interaction between nerve regeneration factor, providing experiment evidence for tissue engineering further studies and clinical application.
Method: (1) Take 3-5 days suckling rat two sides sciatic nerve and brachial plexus, after digesting by enzyme, explants planting insolating cultivate SCs. After a week’s cultivation, full of bottom, adding 0.25% trypsase general digested to each bottles to generation, using S-100 immunohistochemistry dyeing and calculating SCs purity. (2)Get such schwann cell and make them into suspension, density is 1×105/ml a 24 hole costar, divided into normal group and experimental group, Chitosan tiles on the bottom of holes, ultra-clean platform drying. SCs vaccinates into costar, cultivating is in the condition of 37 oC and 5% CO2 After 24h,adding DAPI (final concentration 50μg/ml) into incubation , After 24h,washing bottle using PBS at least 3 times, to be rid of DAPI which is not combined with cells. Adding inoculums to continue cultivate. Observing SCs and Chitosan histocompatibility. (3)Using lentivirus taking BDNF gene infected schwann cell, and infuse its into Chitosan tubes, construct Nerve tissue engineering bridge, connected Wistar rat sciatic nerve (A-group),researching the renovation effection,observe Chitosan tubes (B-group), sham operation (C-group)、autogenous nerve graft(D group) as the comparison. After 6、12 weeks, taking the experiment by electrical physiological test、histology section, microscope and estimate its therapeutic efficacy.
Result: (1) After enzyme digested, using explants organisms cultivate method, the creeping speed of SCs is faster than direct explants organisms cultivate method, take furthering purification and then identified SCs by S-100 immunohistochemistry, and the purity reached more than 95%. (2)dealed normal group and experimental group, and percentage of apoptosis cells each time point were calculated, the percentage of apoptosis cells at each time points were no significant(P<.0.05). (3)General observation: no rat death in the process of feeding, the condition of rats which suffer from contracture and drag-to gait would improve in four weeks. There were no Chitosan tubes remaining and no neuratrophy in the explantation nerve place, Vascular proliferation was discovered transpl surrounding. (4)Electrophysiology observation: comparisons of three group NCV, group A and B, B and C had significant differences (P<.0.05), group A and group D had no significant differences.
Conclusion: (1) Digesting enzyme, the purity method which composed by backer explants organisms cultivate method and differential adherence method "produce" large number of SCs, high purity and low attached by outer atmosphere of cell physicochemical property and biological characteristics. At the same time, saving nerve organization material sources, avoiding repeated draw material processes, saving manpower and material resources sparingly and guaranteeing SCs derive from the same animal which was used in experiment.(2) The Chitosan tubes made by Freeze-drying technology had smooth surface, transparency, even texture. 0.15mm thickness, the same as Cover slip. Experiments show that both have good biocompatibility and SCs proliferation in the surface. Could serve as a nerve bridge over. (3) Using Chitosan tubes to act as supporting frame, Using lentivirus taking BDNF gene infected SCs as seeds cells, Construct tissue engineered artificial nerves, repairing nerve defection, the effect is closed to autogenous nerve graft, relative to single application Chitosan tubes, it could further raise nerve repairing effects. This study had broad application prospects and value of further investigation.
引文
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[1]顾立强,秦煌,叶震海.雪旺细胞与周围神经组织工程[J].中国创伤骨科杂志,2000. 2: 323-5.
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[19]张黎,于炎冰,徐晓利.壳聚糖材料在神经导引管桥接周围神经缺损中的应用[J],生物医学工程研究,2005,03:51-54.
[20]蒋良福,劳杰,何继银,等.几丁糖胶原复合膜及激活态施万细胞修复周围神经缺损的实验研究[J].中华手外科杂志,2005;03:46-48.
[21]张文捷,李兵仓,王建民,等.组织工程人工神经诱导管的制备及其在体外与大鼠体内降解性能研究[J].第三军医大学学报,2004,26(23):1165-1168.
[22] Godard CW, de Rutter,Marti JN, et al.Desighning ideal conduits forperipheralnerve repair[J]. Neurosurg Focus, 2009, 26(2):1.
[23] Keilhoff G,Goihl A, Stang F, et al.Peripheralnerve tissue engineering: autologous schwann cells VS transdifferentiated mesenchymal stem cells[J].Tissue Eng, 2006,12(6):1451.
[24]刘文静,高英茂,李振华,等.应用胶原-壳聚糖桥接管引导大鼠坐骨神经再生.解剖学杂志[J]2008;31(3):42-44.
[25] Maquet V, Martin D, Malgrange B, et al. Peripheral nerve regeneration using bioresorbable macroporous polylactide scaffolds [J]. J Biomed Mater, 2000, 52 (4): 639-51.
[26]戴传昌,商庆新,王炜,等.用组织工程方法桥接周围神经缺损的实验研究[J].中华外科杂志,2000,38(5):388-90.
[27] Rutkowski GE, Heath CA. Development of a bioartificial nerve graft [J]. I Design based on a reaction-difusion model. Biltechnol Prog, 2002, 18:362-72.
[28]陈明,夏仁云.构建生物人工神经修复周围神经缺损的实验研究[J].中国矫形外科杂志,2003, 11(8): 536-8.
[29]张勇杰,金岩,聂鑫,等.组织工程周围神经修复坐骨神经缺损应用研究[J].中华神经外科疾病研究杂志,2004,3:141-4.
[30]杜怀栋,周梁,田宏斌,等.GDNF转染施万氏细胞构建神经导管复合体[J],中华耳科杂志, 2007,5(4):88-92.
[31]王晓芹,李晓辉,王秀芹.壳聚糖胶原复合敷料生物安全性研究[J].四川生理科学杂志,2001, 23(3):160.
[32]卢凤琦,曹晶,林志勇,等.防术后粘连膜的组织病理学观察[J].山东医科大学学报,2000, 38 (2):216-217.
[33] Wang A, Ao Q, Cao W,et al. Porous chitosan tubular scaffolds with knitted outer wall and controllable inner structure for nerve tissue engineering. J Biomed Mater Res A. 2006;79(1):36-46 .