锌离子抑制气管移植后上皮细胞凋亡的实验研究

The inhibition of zinc ion on epithelial apoptosis after tracheal transplantation

目的:利用小鼠异体异位气管移植模型,研究锌离子(Zn2+)对气管移植后上皮细胞凋亡及其促发的闭塞性细支气管炎综合征的抑制作用,为临床实践提供理论依据。方法:采用成年近交系雄性小鼠,平均体质量(21±0.5)g。BALB/c小鼠为供体,C57BL/6小鼠为受体,建立气管异位移植模型,每组16只。对照组:移植术后生理盐水灌胃。锌离子(Zn2+)组:移植术后3.5%葡萄糖酸锌0.15mL(Zn2+2.5mg/kg)灌胃,1次/12h;环孢菌素A(CsA)组:移植术后口服环孢菌素A10mg·kg-1·d-1;联合用药组:移植术后葡萄糖酸锌0.15mL(Zn2+2.5mg/kg)灌胃,1次/12h并口服环孢菌素A10mg·kg-1·d-1。术后7d和14d每组随机处死8只小鼠。对每组受体鼠移植气管上皮细胞脱落和管腔闭塞进行评估。使用Tunel法检测上皮细胞的凋亡情况并计算凋亡指数。采用免疫组织染色方法检测上皮组织中Caspase-3的表达情况。结果:Zn2+组、CsA组及联合用药组的上皮细胞脱落和管腔闭塞程度较对照组明显减轻(P<0.01),联合用药组与Zn2+组、CsA组间差异有统计学意义(P<0.01),同时发现各组上皮细胞脱落和管腔闭塞程度存在正相关性(r=0.445,P<0.01)。通过计算凋亡指数(apop-tosis index,AI)发现,Zn2+组、CsA组、联合用药组的移植气管上皮细胞凋亡程度低于对照组(P<0.05),联合用药组与Zn2+组、CsA组间差异有统计学意义(P<0.05)。通过计算Caspase-3表达指数(caspase-3 express index,CEI)发现,Zn2+组、CsA组、联合用药组的Caspase-3阳性表达的气管上皮细胞数目明显低于对照组(P<0.01),联合用药组与Zn2+组、CsA组间差异有统计学意义(P<0.01)。同时发现各组上皮细胞AI和Caspase-3表达指数(CEI)之间有正相关性(r=0.394,P<0.01)。结论:气管移植后管腔闭塞程度随上皮细胞的脱落增多而加重,说明气管上皮细胞的破坏丢失是导致管腔闭塞的因素之一。气管移植后上皮细胞凋亡数目随Caspase-3表达增多而增加,说明Caspase-3与气管移植后上皮细胞凋亡之间关系密切。锌离子(Zn2+)可以通过抑制Caspase-3的表达起到抑制气管上皮细胞凋亡的作用,并可以与CsA起到协同作用。

Objective：With the method of heterotopic tracheal allograft model of mouse,we investigated the effect of zinc ion on the epithelial apoptosis in bronchiolitis obliterans syndrome after transplantation,which could provide theoretical basis for clinical practice.Methods：The experimental animals were inbred male mice.Mouse heterotopic tracheal allograft model was made from BALB/c to C57BL/6（n = 16）,and average weight was（21 ± 0.5） g.Control group was received NaCl po after transplantation.Zn2 ＋ group was received 3.5% Zn-G 0.15mL（Zn2 ＋ 2.5 mg/kg） po q12h after transplantation.CsA group was received CsA 10 mg·kg-1·d-1 po after transplantation.Combined group was received 3.5% Zn-G 0.15mL（Zn2＋ 2.5 mg/kg） po q12h and CsA 10 mg·kg-1·d-1 po after transplantation.The mice were sacrificed serially on postoperative days 7th and 14th.The samples were obtained for assessment of epithelial loss and luminal occlusion.The apoptosis of epithelial was detected by the Tunel method and the apoptosis index was calculated.The expression of Caspase-3 in the epithelium was detected by immunohistochemical method and the Caspase-3 express index was calculated.Results：The epithelial loss and luminal occlusion of intervention groups were lowest than control group（P 0.01）.Furthermore,there was a positive correlation between epithelial loss and luminal occlusion（r = 0.445,P 0.01）.By using the method of Tunel,the apoptosis index was calculated.The apoptosis index of intervention groups were lower than control group（P 0.05）,and the combined group was lower than Zn2 ＋ group and CsA group（P 0.05）.By using immunohistochemical method,the Caspase-3 express index was calculated.The Caspase-3 express index of intervention groups were lowest than control group（P 0.01）,and the combined group was lowest than Zn2 ＋ group and CsA group（P 0.01）.Furthermore,there was a positive correlation between apoptosis index and Caspase-3 express index（r = 0.394,P 0.01）.Conclusion：Through the experiment,first we proved that the degree of luminal occlusion in heterotopic tracheal allograft progressed with the increasing number of epithelial loss,which indicated that epithelial loss is one cause of luminal occlusion.Second we proved that the number of epithelial apoptotic cell in heterotopic tracheal allograft increased with the rising of Caspase-3 expression,which prompted a relationship between the Caspase-3 expression and airway epithelial apoptosis.Third we proved that Zinc acted anti-apoptosis effect on airway epithelium by reducing the Caspase-3 expression,and had synergistic effect on CsA.