低氧预处理骨髓间充质干细胞移植治疗大鼠急性脊髓损伤的实验研究

中文关键词:  脊髓损伤  干细胞治疗  骨髓间充质干细胞  低氧  凋亡  迁移，小胶质细胞

英文关键词:Spinal cord injury  Stemcell therapy  Bone mesenchymal stem cells  Hypoxia  Apoptosis  Migration  Microglia

中文摘要:

  【摘要】 目的：研究低氧预处理骨髓间充质干细胞（bone mesenchymal stem cells，BMSCs）移植对大鼠急性脊髓损伤（spinal cordinjury，SCI）治疗效果的影响并探讨其可能的机制。方法：转绿色荧光蛋白（green fluorescent protein, GFP）基因的SD大鼠10只，体重55.6±4.2（50~60）g，通过改良全骨髓贴壁法从大鼠骨髓中分离培养大鼠BMSCs并进行并进行细胞纯度和分化能力鉴定。应用0、10、50、100、200和300μM的二氯化钴（CoCl2）低氧预处理BMSCs后，通过CCK-8法检测低氧对细胞增殖的影响，细胞血清剥夺培养0、6、12和24h后，流式细胞法检测血清剥夺对细胞凋亡的影响，Transwell法检测低氧对BM-MSCs培养6、12、24h后细胞迁移的影响，流式细胞法检测低氧对细胞凋亡的影响，PCR法检测相关分子通路，找出合适的低氧处理条件。体内实验，使用Allen法垂直打击建立SCI模型，实验分为A组（假手术组）、B组（对照组）、C组（BMSCs组）和D组（H-BMSCs组）（n=20）。A组的大鼠进行外科手术，但不锤击脊髓，B、C和D组均进行锤击脊髓行SCI造模和蛛网膜下腔注射注射生理盐水和低氧预处理前后的BMSCs。通过术前，术后1、3、7、10、14、21、28天BBB评分研究大鼠的神经功能恢复，术后72h免疫荧光染色法确定移植细胞的存活情况和小胶质细胞激活情况，术后72h和28d HE染色评估脊髓组织病理损伤情况。结果：CCK-8结果表明CoCl2浓度越高对BMSCs增值能力抑制越大，100μM CoCl2培养24h可以明显降低BMSCs增殖（P<0.05），提高BMSCs的凋亡（P<0.05），但能够显著增强预处理后BMSCs 6和12h后的细胞迁移的数量（P<0.05），显著降低细胞血清剥夺培养24h后细胞凋亡率（P<0.05）。动物实验，与B组相比，C组的治疗可显著提高14d、21d和28d的BBB评分（P<0.05）。D组的BBB评分在21d和28d时明显高于C组（P<0.05）。HE染色结果显示，BMSC移植可以显著减少脊髓损伤部位细胞死亡，出血和炎性细胞浸润，而与C组相比，D组中的脊髓病理学损伤更轻微。细胞移植72h后，在C组和D组中均可见绿色荧光细胞，且绿色荧光的细胞数量D组（254.0±35.5）中明显高于C组（143.2±22.3，P<0.05）。SCI 72h后Iba-1免疫荧光染色显示，与B组（759.0±114.3）相比，BMSC（544.8±37.1）和D组（422.4±56.0）小胶质细胞数明显减少（P<0.05），且D组抑制小胶质细胞激活的能力更强（P<0.05）。结论：低氧预处理BMSCs通过提高移植细胞的存活率，增强抑制小胶质细胞的激活能力，提高了SCI后大鼠神经功能的恢复，机制为低氧预处理能够增强细胞的抗损伤和迁移能力，是一种提高BMSCs移植治疗SCI疗效的有效手段。

英文摘要:

  【Abstract】 Objectives: To investigate the effects of hypoxic preconditioning of bone marrow mesenchymal stem cells (BMSCs) on acute spinal cord injury(SCI) in rats and related mechanisms. Methods: BMSCs were isolated from 10 SD rats(55.6±4.2g) and cultured by modified whole bone marrow adherence methods, and then were transfected with green fluorescent protein(GFP) gene. In vitro, the proliferation ability of BMSCs was tested by using CCK-8 after treated by CoCl2 in different concentrations (0, 10, 50, 100, 200 and 300μM). The anti-apoptosis effect of serum deprivation after 0, 6, 12 and 24h culture migration was tested by using Transwell, and apoptosis was tested by using flow cytometry. In vivo experiment, vertical strike SCI model was used and hypoxic preconditioning BMSCs were injected subcutaneously, then the injected models were divided into sham operation group(group A), control group(group B), normal cell group(group C) and hypoxic preconditioning cell group(group D). The recovered neurological function was studied by BBB score before operation and 1, 3, 7, 10, 14, 21 and 28 days after operation. The pathological damage of spinal cord was evaluated by using HE staining 72h and 28d after SCI. Immunofluorescence staining was used to determine the transplanted cells viability and microglia activation. Results: In vitro experiments, the cck-8 results showed that the higher was the concentration of CoCl2, the greater was the effect on the proliferation capacity of BMSCs, and the 24h culture of 100μM CoCl2 could significantly reduce the proliferation ability of BMSCs(P<0.05) and increase the apoptosis of BMSCs(P<0.05). However, the number of cell migration of BMSCs at 6 and 12h after pretreatment significantly increased(P<0.05), and the apoptosis rate after serum deprivation culture for 24h was significantly reduced(P<0.05). In vivo experiments, compared with group B, the treatment of BMSCs could significantly improve BBB score of 14 days, 21 days and 28 days(P<0.05). BBB score of group D was significantly higher than that of group C at 21 and 28 days(P<0.05). HE staining results showed that BMSCs transplantation could significantly reduce cell death, bleeding and inflammatory cell infiltration at the spinal cord injury site. While compared with group C, the pathological injury of spinal cord in group D was more mild. After 72h of cell transplantation, green fluorescence cells were seen in both group C and group D, and the number of green fluorescence cells in group D(254.0±35.5) was significantly higher than that in group C(143.2±22.3, P<0.05). At 72h after SCI, iba-1 immunofluorescence staining showed that the number of microglia cells in group C(544.8±37.1) and group D(422.4±56.0) decreased significantly(P<0.05), and the ability of group D to inhibit the activation of microglia cells was higher(P<0.05), compared with group B(759.0±114.3). Conclusions: Hypoxic preconditioning manipulation of BMSCs is an effective way to improve the efficacy of cell transplantation in the treatment of SCI.