胰岛分离纯化过程中细胞凋亡发生的分子基础与解析

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。

Study on the Molecular Mechanisms of Islet Cell Apoptosis in Isolation and Purification of Islet

Objective To observe the changes of islet cell apoptosis, oxidation- antioxidation system and cytokines in the process of islet isolation and purification before transplantation, so as to elucidate the molecular mechanisms of islet cell apoptosis. Methods Fifteen human pancreases were perfused with Hank＇ s solution containing collagenase and then were subjected to digestion and gradient eentrifugation to isolate islets. Samples were collected during this process for detection of islet cell apoptosis by TUNEL method, TNF -α IL - 1, IL - 8 by ELISA and SOD, MDA by colorimetric method. The morphologic alterations of islets and islet cells were observed after H- E and dithimne staining. Results In the process of pancreases perfusion and digestion, the tissues around the islets were loose and structures of some islets were damaged. Apoptotie islet cells with positive staining by TUNEL and high levels of TNF-α, IL - 8, IL - 1 and MDA occurred, which were all significantly higher before perfusion, after perfusion and during digestion than those after islet isolation. Conclusions During the process of islet isola- tion and purification, collagenase perusion and digestion can induce islet cell apoptosis, which may be related with elevation of cell cytokines and oxidation. It suggests that inhibition of cytokines secretion and anti - oxidation can be the starting point for islet protection.