交通医学

目的 ：研究S100钙结合蛋白B(S100 calcium-binding protein B,S100B)在成年大鼠急性脊髓损伤（spinal cord injury,SCI）后对星形胶质细胞增殖的调控作用。方法：将70只成年雄性SD大鼠随机分为假手术组14只和脊髓损伤组56只，采用NYU撞击器建立脊髓损伤模型。大鼠脊髓损伤后采用Basso-Beattie-Bresnahan(BBB)评定量表评估大鼠后肢运动功能，采用蛋白质印迹法测定脊髓组织中S100B蛋白表达水平，免疫组织化学法和双重免疫荧光染色技术检测S100B在大鼠脊髓组织中的表达与定位。体外培养原代星形胶质细胞，脂多糖（lipopolysaccharide,LPS）诱导星形胶质细胞后采用蛋白质印迹法分别测定S100B蛋白表达水平；将S100B siRNA转染至原代星形胶质细胞中，采用CCK8法测定细胞增殖能力，流式细胞术测定细胞周期。结果：BBB运动功能评分显示，大鼠脊髓损伤后6 h均丧失后肢运动功能，但从损伤后12 h开始运动功能自发性恢复。蛋白质印迹结果显示，S100B蛋白表达水平在脊髓损伤后6 h开始升高，于第3天达到峰值。免疫组织化学结果显示，S100B蛋白主要表达于脊髓白质，仅少量表达于灰质。脊髓损伤后3天白质中S100B表达明显增加。双重免疫荧光染色结果显示，S100B在星形胶质细胞和小胶质细胞中表达，且与增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）共定位。LPS诱导原代星形胶质细胞后S100B蛋白表达逐渐升高，敲低S100B蛋白表达后，原代星形胶质细胞增殖能力显著低于Control组和NC siRNA组。流式细胞术检测结果显示，与Control组和NC siRNA组比较，S100B siRNA组S期细胞比例降低，G0/G1期细胞比例升高。结论：大鼠脊髓损伤后白质中S100B蛋白表达显著上调，从而促进星形胶质细胞增殖，并通过调控细胞周期参与SCI后的病理生理过程，为脊髓损伤的临床干预提供了新的理论基础。

Objective: To investigate the regulatory effect of S100 calcium-binding protein B(S100B) on astrocyte proliferation in adult rats after acute spinal cord injury(SCI). Methods: Seventy adult male SD rats were randomly divided into the sham operation group(14 rats) and the spinal cord injury group(56 rats). The spinal cord injury group was modeled using the NYU impactor. The Basso-Beattie-Bresnahan(BBB) scale was used to evaluate the motor function of the rats after spinal cord injury. The expression level of S100B protein in the spinal cord tissue of rats was determined by Western blotting. The expression and localization of S100B in rat spinal cord tissue were detected by immunohistochemistry and dual immunofluorescence staining techniques. Further, primary astrocyte cultures were conducted in vitro. The expression level of S100B protein was determined by Western blotting after lipopolysaccharide(LPS) was used to induce astrocytes. S100B siRNA was transfected into primary astrocytes, then the cell proliferative ability was determined by CCK8 assay. The cell cycle was determined by flow cytometry. Results: The BBB motor function score indicated that the rats lost their hind limb motor function 6 hours after spinal cord injury. However, starting from 12 hours after the injury, the motor function of the rats spontaneously recovered. Western blotting results showed that the expression level of S100B protein began to increase 6 hours after spinal cord injury and reached its peak on the third day. Immunohistochemical results indicated that S100B protein was mainly expressed in the white matter and was slightly expressed in the gray matter. The expression of S100B protein in the white matter of the rats increased 3 days after injury. The results of double immunofluorescence staining showed that S100B was expressed in astrocytes and microglia, and S100B was co-localized with proliferating cell nuclear antigen(PCNA). The expression of S100B protein gradually increased after LPS induced primary astrocytes. After the expression of S100B protein was knocked down, the proliferative ability of primary astrocytes was significantly lower than that of the Control group and the NC siRNA group. The flow cytometry test results showed that compared with the Control group and the NC siRNA group, the proportion of S phase cells in the S100B siRNA group decreased, while the proportion of G0/G1 phase cells increased. Conclusion: The expression of S100B protein in the white matter significantly increased after spinal cord injury in rats, thereby promoting the proliferation of astrocytes and participating in the pathological and physiological processes after SCI by regulating the cell cycle. This provides a new theoretical basis for clinical intervention in spinal cord injury.