NSCs与PLGA复合体移植对大鼠脊髓半横断损伤的修复作用

发布时间：2017-12-28 08:15

  本文关键词：NSCs与PLGA复合体移植对大鼠脊髓半横断损伤的修复作用　出处：《石河子大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 神经干细胞 脊髓半横断损伤 PLGA组织工程支架 运动诱发电位(MEPs) 移植

【摘要】：背景:目前脊髓损伤(spinal cord injury,SCI)的预防、治疗和康复是目前临床工作种难以攻克的难题。而生物细胞学技术和组织工程技术的发展为治疗脊髓损伤带来新的治疗方案。目的:研究乳鼠脑组织源神经干细胞(neural stem cells,NSCs),联合聚乳酸-乙醇酸工程支架(Poly-Lactic-co-glycolicacid,PLGA)移植治疗SD大鼠脊髓半横断损伤的效果。方法:(1)乳鼠NSCs的分离和培养的优化:取出生3天的SD乳鼠大脑颞叶皮质,分别用机械吹打法、胰蛋白酶消化法分离培养,通过免疫荧光法对NSCs进行鉴定和诱导分化;分别于培养的第2、5、7天计数细胞球直径及数目;通过MTT法比较原代及传代后第3亚代NSCs克隆增值速率。(2)NSC与PLGA复合体移植治疗SD大鼠脊髓半横断损伤修复作用:将72只雌性SD大鼠分成6组:正常组、假手术组、手术组、NSCs移植组、PLGA移植组、NSCs+PLGA复合体共移植组。制作脊髓半横断模型,正常组不做处理,假手术组仅摘除椎板;其他各组行脊髓右侧半横断术;术后第7天,再次打开脊髓,手术组于损伤处移入20ul培养液,NSCs移植组移入含NSCs培养液;PLGA移植组将PLGA组织工程支架植入脊髓损伤处;NSCs+PLGA复合体共移植组将NSCs+PLGA复合体植入脊髓损伤处。每组大鼠于术前1d、术后7d、移植后第1、2、4、8周行BBB评分、IP评分等行为学,于移植后第2、4、8周,行电生理MEPs检查、病理HE染色。结果:(1)NSCs的分离和培养的优化:机械吹打组与胰酶消化组获得的NSCs,行免疫荧光发现,Nestin、NF-200、GFAP染色均阳性,培养第5、7天时,机械吹打组获得的细胞球数量和直径均高于相同培养时间点的胰酶消化组,差异有统计学意义(p0.05)。机械吹打组原代和第3亚代细胞第2、5天细胞克隆增殖能力均优于同时间节点的胰酶消化组,差异有统计学意义(p0.05)。(2)行为学评价:移植后第4、8周时NSCs移植组、PLGA移植组、NSCs+PLGA复合体移植组BBB评分、IP评分高于手术组,差异有统计学意义(p0.05);NSCs移植组、PLGA移植两组比较,差异无统计学意义(p0.05)。移植后第4、8周,NSCs+PLGA复合体移植组BBB评分、IP评分高于NSCs移植组、PLGA移植组,差异具有统计学意义(p0.05)。(3)电生理:运动诱发电位(MEPs)潜伏期比较显示:移植后第2、4、8周,NSCs移植组、PLGA移植组、NSCs+PLGA复合体移植组优于手术组(p0.05);移植后第2、4、8周NSCs+PLGA复合体移植组优于NSCs移植组、PLGA移植组(p0.05)。同时间点各组健侧MEPs的潜伏期轻度优于手术侧,但是在统计学上差异无意义(p0.05)。(4)病理HE染色:各实验组脊髓组织行HE染色后,发现移植后第8周时,NSCs移植组、PLGA移植组、NSCs+PLGA移植组损伤处脊髓组织结构较前有所恢复,空洞、瘢痕组织少于手术组。NSCs+PLGA复合体移植组周围组织结构恢复更好,空洞、瘢痕组织少于NSCs移植组、PLGA移植组。结论:1.机械吹打法、胰酶消化法均可成功分离培养、诱导分化NSCs,但是机械吹打法分离培养的NSCs的克隆增殖能力优于胰酶消化法。2.通过行为学、电生理及病理学评估发现NSCs+PLGA复合体移植对SD大鼠脊髓半横断损伤有一定的修复作用,尤其对后肢运动能力的恢复比较明显。但是对脊髓损伤的解剖结构的重塑和功能的恢复作用有限。3.NSCs、PLGA单独移植时行为学、电生理、病理学评价较手术组有一定的改善,但改善程度弱于两者复合体移植治疗。
[Abstract]:Background: the prevention, treatment and rehabilitation of spinal cord injury (SCI) is a difficult problem to be tackled in the current clinical work. The development of biological cytology and tissue engineering will bring new treatment for the treatment of spinal cord injury. Objective: To study the effect of neural stem cells (NSCs) combined with poly (lactic acid glycolic acid) engineering scaffold (Poly-Lactic-co-glycolicacid, PLGA) transplantation on the treatment of spinal cord hemisection injury in SD rats. Methods: (1) NSCs rats were isolated and cultured for optimization: 3 day old SD rat cerebral cortex respectively by mechanical trituration and trypsin digestion method for isolation and cultivation, identification and differentiation of NSCs by immunofluorescence; on the second, fifth, seventh day count and number of training ball diameter the MTT method; through comparison of primary and passage third sub NSCs clone VAT rate. (2) transplantation of NSC and PLGA complex to treat spinal cord hemisection injury in SD rats: 72 female SD rats were divided into 6 groups: normal group, sham operation group, operation group, NSCs transplantation group, PLGA transplantation group and NSCs+PLGA complex transplantation group. Production of spinal cord hemisection model, normal group was not treated, the sham operation group only with each other on the right side of the lamina; spinal cord hemisection; seventh days after surgery, open surgery group in spinal cord injury. Again, in 20ul medium, NSCs medium containing NSCs in the transplantation group; PLGA transplantation group PLGA scaffolds for tissue engineering implantation of spinal cord injury; NSCs+PLGA complex were transplanted NSCs+PLGA complex was implanted into spinal cord injury. Rats in each group were subjected to 1D, 7d, first, second, fourth and 8 weeks after operation, BBB score, IP score and other behaviors. Second, fourth, eighth weeks after transplantation, electrophysiological MEPs examination and pathological HE staining were performed. Results: (1) NSCs isolate and culture: the optimization of the mechanical percussion group and group NSCs obtained by trypsin digestion, immunofluorescence, Nestin, NF-200 and GFAP staining were positive when cultured for fifth, seventh days, cell number and diameter of mechanical percussion group were higher than those obtained by trypsin digestion group culture of the same point in time, the difference was statistically significant (P0.05). Mechanical percussion group primary and third sub cells second, fifth days of cell clone proliferation were better than trypsin digestion group at the same time node, the difference was statistically significant (P0.05). (2) behavioral evaluation: at fourth, eighth weeks after transplantation, BBB score and IP score in NSCs transplantation group, PLGA transplantation group and NSCs+PLGA complex transplantation group were higher than those in operation group, the difference was statistically significant (P0.05), NSCs group and PLGA transplantation group had no significant difference between two groups (P0.05). Fourth, eighth weeks after transplantation, the BBB score and IP score of the NSCs+PLGA complex group were higher than those in the NSCs transplantation group and the PLGA transplantation group, and the difference was statistically significant (P0.05). (3) electrophysiological: motor evoked potentials (MEPs) were compared: second, fourth, eighth weeks after transplantation, NSCs transplantation group, PLGA transplantation group, NSCs+PLGA transplantation group than the complex operation group (P0.05); second, fourth, eighth weeks after transplantation NSCs+PLGA complex transplantation group was better than NSCs transplantation group, PLGA transplantation group (P0.05). At the same time, the latency of MEPs in each group was slightly better than that in the operation side, but there was no significant difference in Statistics (P0.05). (4) pathological HE staining: after the HE staining of spinal cord tissue in each experimental group, it was found that at eighth weeks after transplantation, the spinal cord structure of NSCs transplantation group, PLGA transplantation group and NSCs+PLGA transplantation group recovered earlier than that of the operation group, and the cavity and scar tissue were less than those of the operation group. The surrounding tissue structure of the NSCs+PLGA complex transplanted group was better, and the cavity and scar tissue were less than that of the NSCs transplantation group and the PLGA transplantation group. Conclusion: 1., mechanical blow and trypsin digestion methods can successfully isolate, culture and induce NSCs differentiation, but the NSCs cloning ability of mechanical blow up culture is better than that of trypsin digestion. 2. through behavioral, electrophysiological and pathological evaluation, we found that NSCs+PLGA complex transplantation has certain repair effect on spinal cord hemisection in SD rats, especially for the recovery of hind limb movement ability. However, the reshaping and functional recovery of the anatomical structure of spinal cord injury are limited. The evaluation of behavior, electrophysiology and pathology of 3.NSCs and PLGA was better than that of the operation group, but the degree of improvement was weaker than that of the two groups.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R651.2
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本文编号：1345246