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

发布时间：2018-06-10 05:46

本文选题：丙戊酸 + 骨髓间充质干细胞　；参考：《河北北方学院》2017年硕士论文

【摘要】：为了探讨腹腔注射丙戊酸(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。本实验选用3-4周龄健康雄性SD幼鼠,体外分离培养BMSCs,对第3代BMSCs进行流式细胞仪检测,收集备用。把60只成年雄性SD大鼠随机分为5组:假手术组(A组)、模型组(B组)、丙戊酸组(C组)、移植组(D组)和联合组(E组)。动物脊髓损伤模型选用改良的Allen’s法制作。丙戊酸组伤后给予腹腔注射VPA(300 mgkg-1),每12 h给一次药直至取材;移植组在损伤段脊髓注入BMSCs l mL浓度为1×106 mL-1;联合组同时注入VPA和BMSCs,假手术组和模型组注射等体积的0.9%氯化钠。大鼠在伤后7 d、14 d先行后肢(BBB)运动功能评分,随后处死取材。用苏木精-伊红染色法(HE)染色观察脊髓空洞面积,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。实验结果显示,BBB评分:假手术组、模型组、丙戊酸组、移植组和联合组在SCI后7 d分别为(21),(2.22±0.33),(3.80±0.53),(3.81±0.83),(4.02±0.83);在SCI后14 d分别为(21),(4.51±0.41),(7.25±0.95),(7.32±0.55),(9.76±0.95)。假手术组运动功能正常,3个治疗组的BBB评分明显比模型组高(P0.05)。在SCI后第14 d联合组的评分明显高于丙戊酸组和移植组(P0.05)。HE染色结果:模型组、丙戊酸组、移植组和联合组损伤区域的面积分别是(4.57±0.26)mm2,(3.34±0.21)mm2,(3.51±0.18)mm2,(2.43±0.35)mm2。联合组损伤空洞及瘢痕组织面积明显比其余的3个组低(P0.05)。免疫组化染色:假手术组、模型组、丙戊酸组、移植组和联合组在SCI后7 d的Caspase-3免疫组化阳性目标面密度分别为(1.51±0.23)%,(28.51±5.23)%,(24.39±4.65)%,(24.51±3.81)%,(20.73±3.43)%。在SCI后14 d的Caspase-3免疫组化阳性目标面密度分别为(1.64±0.41)%,(26.22±4.81)%,(21.21±4..04)%,(21.47±3.32)%,(16.15±3.09)%。假手术组少量caspase-3阳性表达,3个治疗组的caspase-3表达明显低于模型组(P0.05)。而联合组的caspase-3阳性表达明显低于丙戊酸组和移植组(P0.05)。细胞凋亡结果:假手术组、模型组、丙戊酸组、移植组和联合组在SCI后14 d的凋亡指数分别为(4.18±0.39)%,(40.51±3.81)%,(19.95±2..04)%,(20.87±2.32)%,(12.83±1.09)%。伤后14 d假手术组存在少量凋亡阳性细胞。与模型组相比,3个治疗组的凋亡指数较低(P0.05),而3个治疗组之间,联合组凋亡指数明显比丙戊酸组和移植组低(P0.05)。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。
[Abstract]:To investigate the effect of intraperitoneal injection of valproic acid (VPA) combined with bone marrow mesenchymal stem cells (BMSCs) on the recovery of neural function after spinal cord injury (SCI) in rats. BMSCs were isolated and cultured in vitro from 3-4 week old male SD rats. The third generation BMSCs were detected by flow cytometry and collected. Sixty adult male Sprague-Dawley rats were randomly divided into 5 groups: sham operation group (group A), model group (group B), valproic acid group (group C), transplantation group (group D) and combined group (group E). The animal spinal cord injury model was made by modified Allen's method. The valproic acid group was intraperitoneally injected with VPA-300 mg / kg ~ (-1) of BMSCs every 12 hours, the transplantation group was injected with BMSCs (1 脳 10 ~ 6 mL / mL) in the injured spinal cord, and the combined group was injected with VPA and BMSCs, 0.9% sodium chloride was injected into the sham operation group and the model group. The motor function of hind limbs was scored 14 days after injury, and then killed. The area of syringomyelia was observed by hematoxylin-eosin staining and the expression of Caspase-3 protein was detected by immunohistochemistry. Apoptosis of neurons in spinal cord sections was observed by dNT-mediated dUTP Nick end labeling (Tunel) staining. The results showed that the scores of BBB in sham operation group, model group, valproic acid group, transplantation group and combined group were 2.22 卤0.33, 3.81 卤0.83C, 4.02 卤0.83N, 7.32 卤0.555D, 7.32 卤0.555L, 4.02 卤0.83g / d after sci, respectively. The motor function of sham-operation group was normal, and BBB score of three treatment groups was significantly higher than that of model group (P 0.05). On the 14th day after sci, the score of the combined group was significantly higher than that of the valproic acid group and the transplantation group. The result of HE staining: the area of the injured area in the model group, valproic acid group, transplantation group and combined group was 4.57 卤0.26 mm ~ 2 + 3.34 卤0.21 卤0.21 卤0.18 mm ~ (2) and 2.43 卤0.35 mm ~ (2) ~ 2 respectively. The area of injury cavity and scar tissue in the combined group was significantly lower than that in the other three groups (P 0.05). Immunohistochemical staining: the positive surface density of Caspase-3 in sham-operation group, model group, valproic acid group, transplantation group and combined group were 1.51 卤0.23 and 28.51 卤5.23 respectively on the 7th day after sci. The density of Caspase-3 immunocytochemical positive target was 24.39 卤4.65 + 24.51 卤3.81 and 20.73 卤3.43 respectively in sham operation group, model group, valproic acid group, transplantation group and combined group on the 7th day after sci. On the 14th day after sci, the positive surface density of Caspase-3 was 1.64 卤0.41 and 21.21 卤4.81, respectively. The density of Caspase-3 was 21.47 卤3.32 and 16.15 卤3.09. A small amount of caspase-3 positive expression was found in the sham-operation group, and the caspase-3 expression in the three treatment groups was significantly lower than that in the model group (P 0.05). The positive expression of caspase-3 in combined group was significantly lower than that in valproic acid group and transplantation group (P 0.05). Results: the apoptotic index of sham operation group, model group, valproic acid group, transplantation group and combined group were 4.18 卤0.39 on the 14th day after sci. The apoptotic index was 19.95 卤2.81 and 20.87 卤1.09 in the sham operation group, model group, valproic acid group, transplantation group and combined group respectively on the 14th day after sci. There were a few apoptotic positive cells in sham-operation group 14 days after injury. Compared with the model group, the apoptotic index of the three treatment groups was lower than that of the valproic acid group and the transplantation group, but the apoptotic index of the combined group was significantly lower than that of the valproic acid group and the transplantation group. The effect of intraperitoneal injection of VPA-assisted BMSCs on the recovery of motor nerve function after sci may be related to the down-regulation of caspase-3 expression and inhibition of apoptosis.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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