丙戊酸联合骨髓间充质干细胞移植对大鼠脊髓损伤的影响  

Effect of valproate combined with bone marrow mesenchymal stem cell transplantation on spinal cord injury in rats

作　　者：张振梁[1] 杨新明[2] 孟宪勇[2] 胡长波[2] 成垚昱 康聪[1] ZHANG Zhen - liang YANG Xin - ming MENG Xian - yong HU Chang - bo CHENG Yao -yu KANG Cong(Graduate School of Hebei North University, Zhangjiakou 075000, Hebei Province, China Department of Orthopedics, First Affiliated Hospital of Hebei North University, Zhangjiakou 075000, Hebei Province, China)

机构地区：[1]河北北方学院研究生院,河北张家口075000 [2]河北北方学院附属第一医院骨科,河北张家口075000

出　　处：《中国临床药理学杂志》2017年第8期714-717,共4页The Chinese Journal of Clinical Pharmacology

基　　金：河北省卫生厅医学科学研究重点项目计划基金资助项目(20110176);河北北方学院创新人才培育基金资助项目(2013-CXRC1322)

摘　　要：目的研究腹腔注射丙戊酸(VPA)辅佐骨髓间充质干细胞(BMSCs)对大鼠脊髓损伤(SCI)后神经功能恢复的作用。方法选择雄性3~4周龄SD幼鼠,体外分离培养BMSCs,收集第3代BMSCs。动物脊髓损伤模型选用改良的Allen’s法制作。假手术组,只切除椎板,不打击脊髓。按照体重将8周龄雄性SD大鼠随机分为5组(每组均12只):除了假手术组以外,模型组、VPA组、移植组及联合组均进行SCI;然后,模型组与假手术组均腹腔注射0.9%氯化钠,并一次性在损伤节段脊髓(或相当于损伤节段的脊髓)注入0.9%氯化钠1 mL;移植组在损伤节段脊髓注入1×10~6mL^(-1)BMSCs 1 mL;VPA组腹腔注射VPA,按300 mg·kg^(-1)·d^(-1)每12 h给药1次;联合组是移植组+VPA组。在SCI后7,14 d,先进行后肢运动功能评分(BBB)。用免疫组化法检定半胱氨酸天冬氨酸蛋白酶3(Caspase-3)蛋白表达情况,通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)染色观察脊髓组织切片神经细胞凋亡情况。结果在SCI后14 d,假手术组、模型组、丙戊酸组、移植组和联合组的BBB评分分别为21,(4.51±0.41),(7.25±0.95),(7.32±0.55),(9.76±0.95)分。3个治疗组的BBB评分较模型组均明显增高,差异均有统计学意义(均P<0.05)。在SCI后14 d,假手术组、模型组、丙戊酸组、移植组和联合组的Caspase-3的表达分别为(1.64±0.41)%,(26.22±4.81)%,(21.21±4.04)%,(21.47±3.32)%,(16.15±3.09)%,3个治疗组的Caspase-3表达明显低于模型组,差异均有统计学意义(均P<0.05)。假手术组、模型组、丙戊酸组、移植组和联合组的细胞凋亡率分别为(4.18±0.39)%,(40.51±3.81)%,(19.95±2.04)%,(20.87±2.32)%,(12.83±1.09)%,与模型组相比,3个治疗组的凋亡指数较低,差异均有统计学意义(均P<0.05)。结论腹腔注射VPA辅佐BMSCs移植对SCI后运动神经功能的恢复效果显著,其机制可能和下调Caspase-3的表达、抑制凋亡有关。Objective To investigate the effects of intraperitoneal injection of valproie acid （VPA） combined with transplantation of bone marrow mesenchymal stem cells （BMSCs） on neurological function recovery after spinal cord injury （SCI） in rats. Methods BMSCs from 3 -4 weeks old healthy male Sprague- Dawley （SD） rats were cultured in vitro. The third generation BMSCs were detected by flow cytometry. Spinal cord injury model was made by modified Allen＇ s technique. Sixty adult male SD rats were randomly divided into five groups（ n = 12 in each group） ： Sham operation group and model group（ received the same dose of normal saline at the same time point）, VPA group（VPA, one time per q12 h with 300 mg·kg^-1·d^-1 ）. BMSCs group （ 1 × 10^6 cell · mL^-1 BMSCs 1 mL injected into the spinal cord of injured segment）and combined group（ VPA ＋ BMSCs）. The BBB scale at the 14 d after injury was evaluated. The expression of Caspase - 3 protein was detected by immunohistochemistry. Then the neuronal apoptosis rate was observed by TdT- mediated dUTP nick- end labeling（TUNEL） staining. Results BBB Rating： On the 14 d after SCI,the rate in sham operation group, model group, VPA group, BMSCs group and combined group were 21, （4.51± 0. 41 ）, （7.25 ± 0.95 ）, （7.32±0.55 ）, （9.76 ± 0. 95 ） point. The BBB score of the three treatment groups was significantly higher than that of model group with significantly （ P 〈 0. 05 ）. On the 14 d after SCI, the caspase -3 expression in sham operation group, model group, VPA group and BMSCs group and combined group were （1.64 ±0.41 ）% ,（26.22±4. 81）% ,（21.21 ±4.04）%, （21.47±3.32）%, （16. 15 ±3.09）%. The expression of Caspase -3 in three treatment groups was significantly lower than that model group with significantly（ P 〈 0. 05 ）. While the expression of caspase -3 in combined group was significantly lower than that in VPA group and BMSCs group with significantly（P 〈0.05）. On the 14 d after SCl,

关 键 词：丙戊酸 骨髓间充质干细胞 脊髓损伤 细胞凋亡 半胱氨酸天冬氨酸蛋白酶3 

分 类 号：R971[医药卫生—药品]