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目的观察移植前胰岛提取过程中胰岛细胞凋亡情况,以及细胞因子与氧化-抗氧化因素变化,以阐明胰岛细胞凋亡的分子生物学机制,为胰岛保护策略提供实验基础。方法选取15例人胰腺,进行胶原酶灌注、消化以及梯度离心分离胰岛,在此过程中取材,采用TUNEL法进行胰岛细胞凋亡检测,胰腺或胰岛组织中TNF-α、IL-8、IL-1等细胞因子采用ELISA法检测,而组织中SOD与MDA水平采用比色法检测,同时进行HE染色、双硫腙染色观察胰岛及胰岛细胞形态改变。结果在人胰腺灌注与消化过程,形态学观察发现胰岛周围组织疏松以及部分胰岛结构的损害,有TUNEL染色阳性凋亡细胞出现,伴随较高水平的TNF-α、IL-8、IL-1以及MDA出现,在灌注前、灌注后与消化中均显著高于胰岛分离后的水平。结论移植前胰岛提取过程中胶原酶灌注以及消化可引起胰岛细胞凋亡,可能与细胞因子与氧自由基水平升高有关,提示抑制细胞因子的释放与抗氧化可以作为胰岛保护策略切入点。
Objective To observe the apoptosis of pancreatic islet cells during the process of pancreatic islet transplantation and the changes of cytokines and oxidative-antioxidative factors in order to clarify the molecular mechanism of islet cell apoptosis and provide the experimental basis for islet protection strategy. Methods Fifteen human pancreas were selected for collagenase perfusion, digestion and gradient centrifugation. The pancreatic islets were isolated from the pancreatic islets by TUNEL method. The levels of TNF-α, IL-8 and IL- 1 and other cytokines were detected by ELISA, while the levels of SOD and MDA in tissues were detected by colorimetric method. At the same time, HE staining and dithizone staining were used to observe the morphological changes of islet and islet cells. Results In the course of human pancreas perfusion and digestion, TUNEL-positive apoptotic cells appeared with morphological observation of loose tissue around islets and some islet structure accompanied by higher levels of TNF-α, IL-8, IL-1 and MDA, before perfusion, after perfusion and digestion were significantly higher than the level after islet isolation. Conclusions Collagenase perfusion and digestion during pretransplantation of pancreatic islets may cause islet cell apoptosis, which may be related to the increase of cytokines and oxygen free radicals. It suggests that inhibiting the release of cytokines and antioxidation may be the entry point for islet protection strategies.