GDNF修饰的脂肪间充质干细胞对外周神经电损伤后修复作用的研究

发布时间：2018-05-29 11:57

本文选题：慢病毒 + 胶质细胞源性神经营养因子　；参考：《第四军医大学》2015年硕士论文

【摘要】：研究背景随着现代化社会发展,工业用电及生活用电的使用日益频繁,人类在日常生产及生活中因意外接触造成的电损伤常有发生。人体作为导电体,接触电流后,因电流通过组织产生高热而造成损伤,故临床上也称为电烧伤。电流通过同一横截面时,电阻越大的部位,产热越高,对人体造成损伤也越明显,因此皮肤骨骼等部位损伤严重,神经损伤较轻。同时根据电流传导的方向最初接触皮肤进入人体的部位为电流入口,经组织传导流出体外的部位为电流出口。电流接触或流出皮肤的瞬间均可能产生放电现象,对人体造成损伤,损伤程度甚至高于热损伤,电流出口放电现象更明显,因而组织损伤更严重。由于接触放电造成损伤创面的修复目前临床上手段多样,效果良好。然而电流通过时产热或引起组织其他病理改变而造成的血管栓塞、肌肉坏死、神经变性等问题目前还无法得到有效的解决。据统计,电损伤多缘于四肢接触,周围神经损伤多发,目前临床上缺乏有效的对神经损伤的修复手段,损伤后预后较差,肢体功能减退、丧失甚至截肢发生率高,严重影响患者生活质量,因此如何有效促进周围神经电损伤修复是目前研究的热点及难点。胶质细胞源性神经营养因子(Glial cell line-derived neurotrophic factor,GDNF)是目前发现的生物活性最强的靶源性神经营养因子,在损伤后可以减少神经元死亡,促进神经元存活、生长和分化。脂肪间充质干细胞(adipose-derived stem cells,ADSCs)是目前研究的热点,其多向分化潜能及旁分泌功能对于促进神经修复有着巨大的潜力。本实验通过慢病毒转染基因的方法使得ADSCs持续过表达GDNF,观察其对于大鼠坐骨神经电损伤后肢体运功功能恢复的作用。方法1.采用SD雄性大鼠5只,提取腹股沟脂肪,采用胰酶消化法及差速贴壁法分离并纯化大鼠来源脂肪间充质干细胞,连续传代培养至第3代,进行分化鉴定及表面标记流式细胞鉴定。2.构建过表达GDNF的慢病毒,以适当滴度感染ADSCs,观察感染效率。3.采用SD雄性大鼠33只,建立大鼠坐骨神经220V电损伤模型,其中3只用于电损伤病理验证,余30只按随机数字表法分为一个正常组(6只,常规饲养,不做任何处理),四个不同处理组(每组6只大鼠):生理盐水对照组(NS组),脂肪间充质干细胞组(ADSCs组),胶质细胞源性神经营养因子组(GDNF组),胶质细胞源性神经营养因子基因修饰的脂肪间充质干细胞组(GDNF-ADSCs组)。将蛋白溶液或细胞悬液进行损伤神经表面注射,等量盐水对照处理。观察时间8周,每周同一时间进行垂绳积分及后肢平均步幅测量,记录结果。并于第8周最后一天处死,收集伤侧及正常组同侧坐骨神经,进行形态学观察。4.采用SD雄性大鼠120只,每组24只,分组及处理同上。伤后4周处理组取伤侧坐骨神经,正常组收取同侧神经提取组织蛋白,通过蛋白印迹法对GDNF蛋白进行定量测定。结果培养并鉴定了SD大鼠腹股沟脂肪来源的ADSCs;成功构建过表达GDNF的慢病毒载体;慢病毒感染ADSCs成功,感染效率高达95%。垂绳积分平均分显示GDNF-ADSC是组恢复趋势较好。根据统计分析,伤后第8周,GDNF-ADSCs组垂绳积分明显优于NS组,ADSCs组、GDNF组,μ值分别为6.02、2.54、2.67,p0.05或p0.01,余组间比较无统计学差异。NS组、ADSCs组、GDNF组、GDNF-ADSCs组在伤后各时间点后肢步幅评分均明显低于正常组(p值均小于0.05);伤后第3、5、7周,ADSCs组及GDNF组后肢步幅均明显优于NS组(p值均小于0.05),第8周GDNF组后肢步幅优于NS组(p值小于0.05);伤后4~8周,GDNF-ADSCs组后肢步幅评分明显优于其他伤后组(p值均小于0.05)。伤后8周,NS组与其他各组比较有髓纤维数明显减少(p值均小于0.05);ADSCs组、GDNF组GDNF-ADSCs组与正常组比较,有髓纤维数明显升高,轴突直径明显减小,髓鞘厚度明显增加(p值均小于0.05);GDNF-ADSCs组有髓神经纤维数及髓鞘厚度明显高于ADSCs组及GDNF组(p值均小于0.05)。伤后4周4个伤后组GDNF蛋白表达明显高于正常组(p值均小于0.05);GDNF-ADSCs组蛋白表达量明显高于对照组、ADSCs组、GDNF组(p值均小于0.05)。结论1.慢病毒转染脂肪间充质干细胞效率较高,且稳定,对干细胞正常生长无明显影响。2.过表达GDNF的慢病毒转染的ADSCs对于大鼠坐骨神经电损伤后神经修复及肢体运动功能的恢复具有更明显的促进作用。3.采用慢病毒转染的手段可以有效改变ADSCs对GDNF的表达;在伤后4周,经GDNF慢病毒载体修饰的ADSCs处理组大鼠损伤神经内依然稳定持续过表达GDNF,其表达量与最终恢复效果呈正相关。
[Abstract]:With the development of modern society, the use of industrial electricity and living electricity is increasingly frequent, and the electrical damage caused by accidental contact in daily production and life is often occurring. As a conductive body, the human body is also called electrical burns. In the same cross section, the higher the resistance part, the higher the heat production, the more obvious the damage to the human body. Therefore, the skin and bone are damaged seriously and the nerve damage is lighter. At the same time, the current entrance of the skin into the body is first contact with the skin in the direction of current conduction, and the part of the outflow and out of the body is the current outlet. The current contact is in contact with the current. At the moment of or out of the skin, the discharge phenomenon may be produced, causing damage to the human body, and the damage degree is even higher than the heat damage. The discharge phenomenon of the current outlet is more obvious, thus the tissue damage is more serious. Other pathological changes, such as vascular embolism, muscle necrosis and neurodegeneration, are still unable to be effectively solved. According to statistics, electrical injury is mainly caused by contact of limbs and many injuries of peripheral nerve. There is no effective repair method for nerve injury in clinic, poor prognosis after injury, loss of limb function, loss or even intercepting. The incidence of limb is high, which seriously affects the quality of life of the patients. Therefore, how to effectively promote the repair of peripheral nerve damage is a hot and difficult point of study. Glial cell line-derived neurotrophic factor (GDNF) is the most bioactive target derived neurotrophic factor, which is found at present, after injury. Adipose-derived stem cells (ADSCs) is a hot topic at present. The multidirectional differentiation potential and paracrine function have great potential for the promotion of nerve repair. In this experiment, the method of gene transfection by lentivirus caused ADSCs to continue. GDNF was used to observe the function of the limb function recovery after the electrical injury of the sciatic nerve in rats. Method 1. 5 male rats of SD were used to extract the fat of the groin. The adipose mesenchymal stem cells were isolated and purified by trypsin digestion and differential adherence, and cultured to third generations for differentiation and surface labeling. Flow cytometry was used to identify the lentivirus expressing GDNF in.2., to infect ADSCs with appropriate titer, and to observe the infection efficiency of.3. in 33 male SD rats, and to establish the 220V electrical injury model of the sciatic nerve of the rat, of which 3 were used for the pathological examination of electrical injury, and the remaining 30 were divided into a normal group by random number table method (6 rats were reared routinely without any treatment). Four different treatment groups (6 rats in each group): normal saline control group (group NS), adipose mesenchymal stem cell group (group ADSCs), glial derived neurotrophic factor group (group GDNF), glial derived neurotrophic factor gene modified adipose mesenchymal stem cell group (group GDNF-ADSCs). Protein solution or cell suspension was used to damage nerve. Surface injection, equal amount of saline control treatment. Observation time 8 weeks, the same time per week to carry out the vertical rope integral and the average hind leg measure, record the results. And on the last day of eighth weeks to death, collect the injured side and the same side of the normal group of the sciatic nerve, observe the morphological observation of.4. SD male rats 120, each group of 24, group and treatment of the same. 4 after injury. 4 The peripheral sciatic nerve was injured in the week treatment group, and the normal group collected the protein of the ipsilateral nerve extract, and the GDNF protein was measured by Western blot. The ADSCs of the fat source of the groin in SD rats was cultured and identified, and the lentivirus vector expressing GDNF was successfully constructed. The slow virus infection ADSCs was successful and the infection efficiency was as high as 95%. vertical rope product. According to statistical analysis, eighth weeks after injury, the vertical rope score of group GDNF-ADSCs was obviously better than that of group NS, ADSCs group, GDNF group, P0.05 or P0.01, respectively, and there was no statistical difference between the other groups.NS group, ADSCs group, GDNF group, and the hind limb stride score at each time point after injury. It was significantly lower than the normal group (P value was less than 0.05), and the hind limb stride of group ADSCs and GDNF group was significantly better than that of group NS (P value was less than 0.05) in group ADSCs and GDNF, and the hind limb stride of GDNF group was better than that of NS group (p less than 0.05) in eighth weeks, and the hind limb stride of the GDNF-ADSCs group was better than that of other groups after injury (P values were less than 0.05). 8 weeks after injury, the group was more than 8 weeks after injury. The number of myelinated fibers in each group was significantly reduced (P value was less than 0.05). In group ADSCs, the number of myelinated fibers increased significantly, the diameter of axon decreased significantly and the thickness of myelin sheath was significantly increased (P value was less than 0.05) compared with the normal group, and the myelinated nerve fiber dimension and the thickness of myelin sheath in group GDNF-ADSCs were significantly higher than that of the ADSCs group and GDNF group (P values were small). The expression of GDNF protein was significantly higher than that of the normal group (P value was less than 0.05) after 4 injuries at 4 weeks after injury. The expression of GDNF-ADSCs protein in group GDNF-ADSCs was significantly higher than that of control group, ADSCs group and GDNF group (P value was less than 0.05). Conclusion 1. lentivirus transfected adipose mesenchymal stem cells were more efficient and stable, and there was no significant effect of.2. over expression GDN on the normal growth of stem cells. The ADSCs transfected with lentivirus of F has a more obvious promoting effect on the nerve repair and the recovery of motor function after the electrical injury of the sciatic nerve in rats..3. can effectively change the expression of ADSCs to GDNF by the means of lentivirus transfection; 4 weeks after the injury, the ADSCs treated group of the GDNF lentivirus carrier is still stable in the injured nerve. The expression level of GDNF was positively correlated with the final recovery.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R647

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