汉防已甲素对内源性神经干细胞增殖分化影响的前瞻性研究

摘要

目的：探讨汉防己甲素(tetrandrine，Tet)对大鼠急性脊髓损伤(acute spinal cord injury，ASCI)后脊髓损伤段内源性神经干细胞(endogenous neural stem cells，ENSCs)增殖分化的影响。方法选用78只大鼠，随机分为3组：损伤对照组(n=36只)，Tet组(n=36只)，假手术组(n=6)。损伤组、Tet组采用Allen’s打击法造模，Tet组于脊髓损伤后30 min、术后24 h、术后48 h给予Tet 22.5 mg/kg体重治疗，损伤组注射同等剂量的生理盐水。于脊髓损伤后1 d、3d、1w、2w、3w、4w取损伤段脊髓组织，HE染色观察脊髓组织的形态结构变化，免疫荧光组织化学方法检测各组大鼠脊髓组织在不同时间段巢蛋白（Nestin）及BrdU的表达变化情况。结果 ASCI后HE染色显示Tet组脊髓组织损伤较损伤组轻；ASCI后1d损伤对照组及Tet组均可见少量Nestin表达阳性细胞及BrdU表达阳性细胞，ASCI后1 w对照组及Tet组可见大量阳性细胞，阳性细胞表达达高峰，持续至2 w后逐渐开始下降，ASCI后4 w时，Tet组、损伤对照组中BrdU及Nestin阳性细胞表达数明显减少，较假手术组高，但无统计学差异。Tet组BrdU阳性细胞数量和Nestin阳性细胞数量在各时间点均高于损伤对照组，其中术后1 d、3 d、1 w、2 w、3 w差异具有统计学意义（P<0.05），术后4 w 2者阳性细胞数比较无统计学差异。损伤对照组及Tet组在ASCI后1 d、3 d、1 w、2 w、3 w时，与假手术组相比，BrdU、Nestin阳性细胞数量差异有统计学意义（P<0.05）。损伤对照组及Tet组在ASCI后1 d、3 d、1 w、2 w、3 w时BrdU、Nestin阳性细胞数量差异有统计学意义（P<0.05）。结论 Tet可通过改善微环境增加BrdU、Nestin阳性细胞，增加ENSCs的增殖，有利于神经功能的恢复。%Objective To discuss the effect of tetrandrine on endogenous neural stem cell proliferation and differentiation after spinal cord injury in rats. Methods 78 rats were randomly divided into 3 groups: control group(n=36), Tet-treated group(n=36), sham-operated group(n=6). Control group and Tet-treated group were adapted with Allen's combat modeling method. Rats in Tet group were injected Ted with a dosage 22.5 mg/kg in 30 minutes, 24 hours and 48 hours after ASCI, and the same dose of saline was injected into injured group as control .Samples were dissected from the spinal cord injury sites at 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks after ASCI, and tested by HE staining for morphology and by immunolfuorescence staining for the expression of BrdU and nestin. Results A little Nestin positive cells and BrdU positive cells were found in control group and Tet-treated group at 1 day after injury. A large number of positive cells were found in both groups at 1 week after injury and reached the peak which lasted for 2 weeks and then decreased gradually. The expression of Nestin positive cells and BrdU positive cells in control group and Tet-treated group were decreased significantly at 4 weeks after injury, but were still more than that in sham operation group. The number of Nestin positive cells and BrdU positive cells in Tet-treated group were more than that in control group at each time point after injury. The expression was higher in Tet-treated group than control group at 1 day, 3 days, 1 week, 2 weeks, 3 weeks after injury and had no difference at 4 weeks after injury. Conclusions Tetrandrine could increase the number of Nestin positive cells, BrdU positive cells and endogenous neural stem cells though improving the microenvironment, and it is beneficial for the recovery of spinal cord injury in rats.