半定量分析方法用于评估脊髓损伤后细胞凋亡程度的可行性研究

Feasibility study on the method of semiquantitative analysis for the evaluation of apoptosis after spinal cord injury

目的:探讨免疫组化半定量分析方法用于评估大鼠脊髓损伤后细胞凋亡程度的可行性,为进一步实验研究提供基础依据。方法:成年SD大鼠6只,采用改良Allen法致T9-T10脊髓损伤,伤后1周处死,取脊髓损伤节段及其上、下节段以及膀胱,另处死2只同批健康SD大鼠2只,取T9-10脊髓节段以及膀胱作正常对照。HE染色观察脊髓和膀胱壁的病理学变化,Nissal染色观察神经元数目的变化,并通过免疫组织化学技术标记caspase-3蛋白阳性细胞,用半定量分析方法评估脊髓损伤后脊髓内和膀胱内膜中细胞凋亡的程度。结果:HE染色发现脊髓损伤后脊髓内空洞形成、炎性细胞浸润等病理学变化,膀胱壁中可见到肌束断裂、肌细胞肥大增生、胶原增生、炎性细胞浸润等病理学改变;Nissal染色显示脊髓损伤后损伤节段及其上、下节段均出现神经元数量的减少;免疫组化结果表明3组均有不同程度的caspase-3表达,损伤组脊髓和膀胱内膜caspase-3表达较正常组显著增加(P<0.01),损伤上段及下段caspase-3表达较损伤节段为少(P<0.01),但均高于正常对照组(P<0.01)。损伤上段和下段caspase-3表达差异无显著性意义(P>0.05)。结论:免疫组化半定量分析方法可以用于评估脊髓损伤大鼠脊髓及膀胱内细胞凋亡的程度。

Objective： To observe the feasibility of semiquantitative analysis for the evaluation of apoptosis after spinal cord injury. Method： Six mature adult SD rats were crashed at T9-T10 （modified Allen＇s method） which caused spinal cord injuries. The injure segments, upper and lower segments of the spinal cord and bladder were taken for analysis, which contrasted with T9-10 spinal cord and bladder of the control rats. HE dyeing was used to observe the pathological changes of spinal cord and wall of bladder, and Nissal dyeing for the amount change of neurons. Caspase-3 positive cells were marked with immunohistochemistry method, and the results were evaluated with semiquantitative analysis. Result： Porosis and inflammatory cell infiltration in the spinal cord, rupture of muscle bundles, hypertrophia and over growth of muscle cells and collagens, and inflammatory cell infiltration in the bladder wall could been seen under HE dyeing. Nissal dyeing showed the decrease of neurons at the injured segment, upper and lower segments of the spinal cord. Result of immunohistochemistry showed there were caspase- 3 positive expression in all the segments. The injured spinal cord and bladder expressed significantly more caspase-3 positive results （P〈0.01）, upper and lower segments were lower than the injured segments （P〈0.01）, but higher than the control team （P〈0.01）. There were no differences between upper and lower segments （P=0.869）. Conclusion： Immunohistochemistry semiquantitative analysis can be used for the evaluation of the extent of apoptosis after spinal cord injury.