舒芬太尼丙泊酚静脉复合麻醉对骨髓间充质干细胞移植治疗创伤性脑损伤大鼠的影响

doi:10.3969/j.issn.1007-3205.2021.04.018

摘要/Abstract

摘要： 目的观察舒芬太尼丙泊酚静脉复合麻醉对骨髓间充质干细胞移植治疗创伤性脑损伤大鼠的影响。
方法取3周龄幼鼠若干，体外分离、培养骨髓间充质干细胞（bone marrow mesenchymal stem cells）稳定传代3次备用。将45只受试大鼠随机分为对照组、干细胞治疗组及联合治疗组，每组15只。采用电子颅脑损伤仪（electric cortical contusion impactor,eCCI）建立颅脑损伤（traumatic brain injury,TBI）大鼠模型，造模完成后尾静脉给予干细胞治疗组干细胞悬液0.5 mL（1×105/mL），联合治疗组给予干细胞的同时尾静脉注射舒芬太尼0.5 g/kg和丙泊酚2.0 mg/kg，对照组给予等量生理盐水。神经功能评分用于判定治疗过程中大鼠神经损伤程度；HE染色观察大鼠脑组织病理学变化；干湿比重法、光谱扫描仪及酶联免疫吸附法（enzyme linked immunosorbene assay,ELISA）用于检测脑水含量、总钙含量及血清S100β含量变化；TUNEL法检测脑组织神经细胞凋亡情况，Western blot用于检测凋亡相关蛋白表达；qRT-PCR与免疫组化检测脑部炎性因子表达水平及分布。
结果 1：2传代后细胞呈放射状或旋涡状集落生长趋势，细胞形态随传代次数增加逐渐稳定均一，最终稳定生长为梭形成纤维细胞样。TBI造模结束后治疗3 d内，3组mNSS评分差异无统计学意义（P＞0.05）；治疗7 d后，联合治疗组mNSS评分显著低于对照组；治疗14 d、21 d后干细胞治疗组及联合治疗组评分均显著低于对照组，联合治疗组低于干细胞治疗组(P＜0.05)。干细胞治疗组与联合治疗组海马组织较对照组病理学变化显著改善，且联合治疗组脑组织神经细胞密度显著恢复，海马区细胞排列整齐，细胞核完整且着色较浅，较干细胞治疗组治疗效果更佳。干细胞治疗组及联合治疗组脑组织水含量、总钙含量及血清S100β含量均低于对照组，联合治疗组低于干细胞治疗组（P＜0.05）。干细胞治疗组、联合治疗组脑组织细胞凋亡数目及Caspase-3、Bax、Cytc蛋白表达量低于对照组，联合治疗组低于干细胞治疗组，干细胞治疗组、联合治疗组Bcl-2蛋白表达高于对照组，联合治疗组高于干细胞治疗组（P＜0.05）。干细胞治疗组、联合治疗组IL-6、IL-1β、TNF-α mRNA表达水平均低于对照组，联合治疗组低于干细胞治疗组（P＜0.05）。
结论舒芬太尼丙泊酚静脉复合麻醉联合骨髓间充质干细胞可减少大鼠脑水含量、总钙含量及血清S100β积聚，抑制神经细胞凋亡及炎性因子表达，加快创伤性脑损伤大鼠神经功能恢复。

关键词: 脑损伤, 舒芬太尼, 丙泊酚, 骨髓间充质干细胞

Abstract: Objective To investigate the effect of intravenous anesthesia with propofol and sufentanil in bone marrow mesenchymal stem cell transplantation on the treatment of traumatic brain injury(TBI) in rats.
Methods Bone marrow mesenchymal stem cells were isolated from young rats aged 3 weeks and cultured in vitro to 3 generations. A total of 45 healthy female SD rats were randomly divided into control group, stem cell treatment group and combination treatment group, with 15 rats in each group. TBI models were established by electric cortical contusion impactor(eCCI). After establishment of TBI modeling, the rats in stem cell treatment group were given 0.5 mL stem cell suspension(1×105/mL), the rats in combination treatment group were given stem cell suspension plus injection of 0.5 g/kg sufentanil and 2.0 mg/kg propofol, while the control group rats were given an equal volume of saline. Modified neurological function scores(mNSS) was performed to determine the degree of nerve damage in rats during treatment; morphological changes of brain tissue were observed by HE staining. The water content of brain, total calcium content and serum level of S100β were measured by dry-and-wet gravity method, spectral scanner and enzyme linked immunosorbene assay. Neuronal apoptosis in hippocampal structural region was observed by TUNEL staining, western blot was used to detected the expression of apoptosis-related proteins, and quantitative real-time(qRT-PCR) and immunohistochemistry were used to detect the expression and distribution of inflammatory factors in brain tissues.

Results After passage, the cells showed the trend of radial or vortex-like colony growth, and the cell morphology gradually became stable and uniform with the increase of passage times. Finally, the cells grew stably into spindle-like fibroblasts. Within 3 days after TBI modeling, there was no significant difference in mNSS score among the three groups(P＞0.05); At 7 days after treatment, the mNSS score of the combination treatment group was significantly lower than that of the control group; At 14 and 21 days after treatment, the scores of the stem cell treatment group and the combination treatment group were significantly lower than that of the control group, and the scores were lower in combination treatment group than in the stem cell treatment group(P＜0.05). Compared with the control group, the pathological changes of the hippocampus in the stem cell treatment group and the combination treatment group were significantly improved, and the density of nerve cells in the brain tissue of the combination treatment group was significantly restored. In addition, the cells in the hippocampus were orderly arranged, the nucleus was complete and the staining was lighter, which was better than the stem cell treatment group. The brain water content, total calcium content and serum S100β content in stem cell treatment group and combination treatment group were lower than those in control group, and the indicators were lower in combination treatment group than in stem cell treatment group(P＜0.05). The number of apoptotic cells and the expression of Caspase-3, Bax and cytc protein in brain tissue of stem cell treatment group and combination treatment group were lower than those of control group, while the indicators were lower in combination treatment group than in the stem cell treatment group. The expression of Bcl-2 protein in stem cell treatment group and combination treatment group was higher than that of control group, and the expression was higher in the combination treatment group than in the stem cell treatment group(P＜0.05). The mRNA expression levels of IL-6, IL-1 β and TNF-α in the stem cell treatment group and the combination treatment group were lower than those in the control group, which were lower in the combination treatment group than in the stem cell treatment group(P＜0.05).
Conclusion Intravenous anesthesia with sufentanil and propofol in bone marrow mesenchymal stem cell transplantation can promote the recovery of neurological function after TBI, by reducing brain water content, total calcium content and serum S100β accumulation in rats, and inhibiting the neuronal apoptosis and expression of inflammatory factors.

Key words: brain injuries, sufentanil, propofol, bone marrow mesenchymal stem cell

武婕, 陈燕, 杜晓宣. 舒芬太尼丙泊酚静脉复合麻醉对骨髓间充质干细胞移植治疗创伤性脑损伤大鼠的影响[J]. 河北医科大学学报, 2021, 42(4): 454-460,465.

WU Jie, CHEN Yan, DU Xiao-xuan. Effects of intravenous anesthesia with sufentanil combined with propofolin bone marrow mesenchymal stem cell transplantation on rats with traumatic brain injury[J]. Journal of Hebei Medical University, 2021, 42(4): 454-460,465.

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舒芬太尼丙泊酚静脉复合麻醉对骨髓间充质干细胞移植治疗创伤性脑损伤大鼠的影响

Effects of intravenous anesthesia with sufentanil combined with propofolin bone marrow mesenchymal stem cell transplantation on rats with traumatic brain injury

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