酸性成纤维细胞生长因子对缺血再灌注小肠绒毛细胞内bax和bcl-2表达的影响

Effects of extrogenous aFGF on bax and bcl-2 expression in intestinal cells after ischemia/reperfusion

目的:探讨外源性酸性成纤维细胞生长因子(aFGF)对大鼠肠缺血再灌注后小肠绒毛细胞凋亡以及凋亡相关基因bax和bcl-2表达的影响. 方法:采用肠系膜上动脉(SMA)夹闭45 min后松夹造成缺血再灌注(I/R)损伤的动物模型,将108只Wistar大鼠随机分成假手术组(C)、肠缺血组(Ⅰ)、肠缺血-再灌注组(R)和aFGR冶疗组(A).根据缺血后再灌注时间的不同又将RN和A组又分成0.25,0.5,1,2,6,12,24和48 h共8组,每组6只动物.A组和R组在松开动脉夹的同时, 分别经尾静脉注入20 μg/kg aFGF和生理盐水0.15 mL.各时相点取小肠组织,通过末端脱氧核糖转移酶介导的生物素化脱氧尿嘧啶缺刻标记技术(TUNEL)检测细胞凋亡率;利用免疫组织化学方法检测bax和bcl-2蛋白表达;用RT- PCR方法测定bax和bcl-2基因的表达水平. 结果:缺血再灌注后2,6,和12 h,A组中大鼠小肠绒毛组织的细胞凋亡率分别为(41.17±3.49%),(42.83±5.23%) 和(53.33±6.92%),显著低于C组中各对应时间点上的细胞凋亡率(P<0.05).I/R后小肠绒毛细胞内bax基因表达逐渐增强,蛋白含量升高,而bcl-2基因转录迅速降低,蛋白含量减少.在再灌注2-12 h,A组中bcl-2基因转录水平和蛋白含量都较C组增加,而bax基因的mRNA含量和蛋白水平均较C组降低. 结论:外源性aFGF能够减轻缺血再灌注对小肠绒毛的损伤,其机制可能与aFGF促进bcl-2基因转录、抑制bax基因表达相关.

AIM: To detect the effects of acid fibroblast growth factor (aFGF) on apoptosis and Bax and bcl-2 expression in rat intestine after I/R injury, and to explore the protective mechanisms of aFGF on intestinal Villus. METHODS: One hundred and eight Wistar rats were randomly divided into 4 groups, namely intestinal ischemia/ reperfusion group (R, n =48), intestinal ischemia group (I, n =6), aFGF treatment group (A, n =48) and sham-operated group (C, n =6). The rats sustained 45 min of arteria mesenterica (SMA) occlusion to establish the ischemia model. At the beginning of reperfusion, rats in group R and-A were treated with normal saline (0.15 mL) and aFGF (20 μg/kg, 0.15 mL) respectively. Then each six rats as a sub-group were reperfused for a duration of 0.25, 0.5, 1, 2, 6, 12, 24, 48 h respectively. Cell apoptotic rates in intestinal villus were determined with terminal deoxynucl eotidy transferase mediated dUTP-biotin nick-end-labeling technique (TUNEL). RT-PCR was used to detect the expressions of bax and bcl-2 gene in intestinal villus. Im munohistochemical methods were adopted to detect bax and bcl-2 protein expressions and distributions. RESULTS: The improvement of intestinal histological structures was observed at 2 h, 6 h and 12 h after the reperfusion in group A, compared with group R. The apoptotic rates were (41.17±3.49 %), (42.83±5.23 %) and (53.33±6.92 %) at 2, 6, 12 h after reperfusion respectively in group A, and these rates were significantly lower than those in group R (P<0.05). The expressions of bax gene and bax protein in intestinal villus were gradually increased after ischemia/ reperfusion, while the transcription of bcl-2 gene and expression of bcl-2 protein were decreased. During the 2-12 h of reperfusion, the transcription of bcl-2 gene and expression of bcl-2 protein were significantly increased in group A compared with those in group R (P<0.05). However, the expressions of bax gene and bax protein were significantly higher than those in group R (P <0.05). CONCLUSION: Intravenous aFGF could alleviate I/R-in-duced injury, in which its effects on the facilitation of bcl-2 transcription and inhibition of bax expression may play an important role.