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目的探讨缺血再灌注小肠中DNA甲基化对小肠细胞凋亡和增殖是否具有调控作用。方法将35只健康雄性Wistar大鼠随机分为正常组(n=5)、假手术组(n=5)及缺血再灌注(0、3、6、12、24 h,n=5)组。利用脱氧核糖核苷酸末端转移酶介导的缺口末端标记测定法、透射电镜和免疫组织化学方法分别检测细胞凋亡和细胞增殖的变化,最后通过DNA甲基化位点组织末端酶链接检测法检测DNA甲基化。结果①与正常组和假手术组比较,缺血再灌注组缺血再灌注后3、6及12 h时在小肠绒毛上皮、黏膜固有层及隐窝上皮中凋亡细胞均明显增加(P<0.01),且透射电镜检测证实,黏膜固有层的凋亡细胞主要是淋巴细胞和少量吞噬细胞。②与正常组和假手术组比较,缺血再灌注后3、6、12及24 h时在小肠绒毛上皮中细胞增殖明显(P<0.01),缺血再灌注后6 h和12 h时在小肠黏膜固有层和隐窝上皮中细胞增殖明显(P<0.01)。③正常组和假手术组DNA甲基化在小肠绒毛上皮和隐窝上皮部分有弱表达;在缺血再灌注组中,小肠隐窝上皮部分DNA甲基化在近小肠干细胞部位表达最强,从隐窝上皮到绒毛上皮是以从强到弱的趋势发生变化的。结论本研究的初步研究结果提示,缺血再灌注小肠中DNA甲基化可能对小肠细胞凋亡和增殖具有调控作用。
Objective To investigate whether DNA methylation plays an important role in the regulation of apoptosis and proliferation of small intestine after ischemia-reperfusion. Methods Thirty - five healthy male Wistar rats were randomly divided into normal group (n = 5), sham operation group (n = 5) and ischemia - reperfusion group (0, 3, 6, 12, 24 h, n = 5) . The changes of apoptosis and cell proliferation were detected by DNA nick end labeling (TdT), nick end labeling (TdT), transmission electron microscopy (TEM) and immunohistochemistry respectively. Finally, Detection of DNA methylation. Results ① Compared with normal group and sham operation group, apoptotic cells in intestinal villus epithelium, lamina propria and crypt epithelium were significantly increased at 3, 6 and 12 h after ischemia / reperfusion (P < 0.01), and transmission electron microscopy confirmed that the lamina propria apoptotic cells are mainly lymphocytes and a small amount of phagocytes. ②Compared with normal group and sham-operated group, cell proliferation in intestinal villi was significantly increased at 3, 6, 12 and 24 h after ischemia-reperfusion (P <0.01), and at 6 h and 12 h after ischemia-reperfusion Small intestinal mucosa lamina propria and crypt epithelial cell proliferation significantly (P <0.01). DNA methylation was weakly expressed in the epithelial and crypt epithelial portions of the small intestine in the normal and sham-operated groups. In the ischemic-reperfusion group, DNA methylation in the intestinal crypt epithelial was the strongest in the proximal small intestine stem cells, From the crypt epithelium to the villi epithelial changes from strong to weak trend. Conclusion The preliminary results of this study suggest that DNA methylation may play a regulatory role in apoptosis and proliferation of small intestine during ischemia-reperfusion injury.