【作者】 苗巍巍；

【导师】 杨有庚；

【作者基本信息】 吉林大学 ， 外科学， 2009， 博士

【摘要】 TNF-α是各种炎症和免疫反应中由多种细胞分泌的一类多向性细胞因子。作为一种炎症细胞因子,在脊髓损伤的急性期,TNF-α有促进细胞凋亡作用,在慢性期还具有诱导小胶质细胞及星形细胞增殖及聚集的作用。本实验旨在通过应用Allen’s改良法复制大鼠SCI模型,同时局部应用抗TNF-α单克隆抗体(Anti - TNF–αmAb)治疗,从形态学角度、细胞因子表达以及凋亡信号转导通路角度观察抗TNF-α单克隆抗体治疗对大鼠SCI发生及愈后的影响,并对其机制作深入探讨,得到如下结论:1.细胞凋亡是SCI早期主要的生物学事件;2.细胞凋亡的调控基因家族bax/bcl-2通过调节细胞凋亡参与SCI的发生发展;3. TNF-α在SCI发生发展中起重要作用,尤其是在调节细胞凋亡信号通路中起重要作用;4.在SCI损伤部位局部应用抗TNF-α特异性抗体,可延缓或抑制SCI的发生发展,其机制主要通过调节bcl-2、bax基因及蛋白表达,使二者表达量比值改变,并可调节IL-6、IL-8等多种炎症相关因子含量,从而实现对SCI的治疗作用。本研究结果可为临床急性脊髓损伤的治疗提供新的思路和方法。更多还原

【Abstract】 It is a worldwide subject for therapy of spinal cord injury (SCI). Although there have been taken amazing outcome on gene therapy of SCI in experiment study,for the complex effect factor and injury mechanism,there is still a lot of key point unknown for people to study incontinuous.TNF-αis a cytokine of inflammation, which can be released from neuron and others.In this research, we will detect the protection role of monoclone TNF-αAntibody in spinal cord injury.In this research, the BBB achievement of the SCI group and TNF-αtreated group were lower than that of the sham group at after 12 h, 1 d after SCI.the BBB achievement of the SCI group and TNF-αtreated group were lower than that of the sham group at 3 d, 7 d, and 14 d after SCI,While at 7 d after SCI, compared with sham group, the BBB of TNF-αtreated group was higher significantly than that of SCI group( P < 0.05).While at 14 d after SCI, compared with SCI group, the BBB of TNF-αtreated group was higher significantly than that of SCI group( P < 0.05).Histology detection found that morphorlogy of spinal cord nerve cell of the sham group was normal. spinal cord nerve cell edema can be seen, but no apoptosis can be seen at 12h after SCI,inflamatory cell infiltration can be seen in spinal cord tissue at 3d after SCI,repair change can be seen in spinal cord tissue at 7d after SCI,scar formation can be seen in injured spinal cord tissue at 14d after SCI,while at 7d after anti- TNF-αtreatment,nerve cell morphology begin to repair, and function of spinal cord. at 14d after anti- TNF-αtreatment, spinal cord tissue structure arrange regular, inflammatory cell disappear.The immunochemistry results of Image analysis of bcl-2 and bax showed that the index of bcl-2/bax of SCI group decresed significantly at 12 h after SCI, compared with the shamI group, P<0.05. which increase gradually.While at 12h、1d、3d、7d after anti-TNF-αtreatment, the index of bcl-2/bax of TNF-αtreated group increased significantly, compared with SCI group, P<0.05.The RT-PCR results show that bcl-2 mRNA expression level of SCI group increase at 12h after SCI, reach the peak walue at 24h after SCI, compared with sham group, P<0.05. then which decrease gradually, but which is higher compared with sham group, P<0.05. After treated with anti-TNF-αantibody, the bcl-2 mRNA expression of TNF-αtreated group was higher significantly than that of SCI group(P<0.05)at12 h、1d、3d、7d after SCI.the bax mRNA expression was increasing and reach the peak value at 12h after SCI, compared with sham group, P<0.05.and then decreased.the bax mRNA expression of SCI group was higher significantly than that of sham group(P<0.05)at 1d、3d、7d after SCI.while anti-TNF-αtreated was applied, bax mRNA expression of TNF-αtreated group was no significantly difference from that of SCI group at each time point. (P﹥0.05)Western blot results show that,the bcl-2 protein expression was increasing at 12 h after SCI and reach the peak value at 24h after SCI, and then decreased. compared with sham group, P<0.05.While anti-TNF-αtreated was applied, bcl-2 protein expression of TNF-αtreated group was significantly higher than that of SCI group, compared with SCI group at each time point., P<0.05.bax protein expression of SCI group was at the peak value at 12h after SCI, and decreased furtherly at 1d、3d and 7d after SCI, compared with sham group, P<0.05.After treated with anti-TNF-αantibody, the bax protein expression of TNF-αtreated group was no significantly difference from that of SCI group at each time point. (P﹥0.05)ELISA analysis found thatat 12 h after SCI, amount of IL-6 and IL-8 expression was at the peak value, compared with the sham group, P<0.05.with times go on, the amount of IL-6 and IL-8 expression were lower grandually than that of SCI group, and at 14 d after SCI, the amount of IL-6 and IL-8 expression were access to the normal level.While after anti-TNF-αtreatment was applied, IL-6 and IL-8 expression of TNF-αtreated group decreased significantly, compared with SCI group, P<0.05. TUNEL detection was applied to evaluate the apoptosis in the SCI rats. It was found that the apotosis cell can be seen clearly 3 days after SCI, which can be prevented after treatment of anti-TNF-α.From our research, we can get these conclusions:1. apoptosis is an important event of SCI; 2. Bax/bcl-2 family take part in the development of SCI through control the processof apoptosis; 3. TNF-αplay an important role in SCI, especially in regulating signal transduction pathway of apoptosis; 4. Anti-TNF-αtreatment by monoclone antibody can regulate the expression of IL-6、IL-8 and bcl-2,inhibit the process of apotosis after SCI in rats.更多还原