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目的:观察大鼠胰腺导管上皮经四步法诱导分化为胰岛样细胞团后移植治疗糖尿病的效果。方法:采用Ⅴ型胶原酶胆管内灌注消化法,经Ficoll不连续密度梯度离心后分别获得导管上皮细胞和新鲜胰岛。导管上皮细胞经原代培养及传代后取2～6代细胞开始进行四步18天的诱导,分化为胰岛样细胞团,行免疫荧光鉴定。将链脲佐菌素造模成功的18只SD大鼠采用完全随机法均分成3组,空白对照组、新鲜胰岛组、胰岛样细胞团组进行左肾包膜下移植。分别给予RMPI1640、新鲜胰岛和胰岛样细胞团,移植后隔日固定时间监测血糖。结果:经免疫荧光鉴定,分离纯化的胰腺导管细胞具有干细胞特性,诱导后的胰岛样细胞团胰岛素、胰高血糖素染色均为阳性。移植后空白对照组血糖维持在高血糖范围。新鲜胰岛组移植后血糖逐步下降至正常水平,并在2周之内基本稳定在正常范围内但略有上升。胰岛样细胞团组移植后前2天血糖不降反升,之后有所下降但始终未能降至正常范围,然后又逐渐上升。结论:胰腺导管上皮细胞经该方案诱导后分化为胰岛样细胞团,使糖尿病大鼠血糖有一定程度的下降,但下降幅度及维持时间不能令人满意,可能与移植量不够及诱导的胰岛样细胞团功能不良有关。
OBJECTIVE: To observe the effect of transplanted pancreatic ductal epithelium on the differentiation of rat pancreatic ductal epithelium into islet-like cells by four-step method. Methods: The ductal epithelial cells and fresh islets were obtained by collagenase type Ⅴ biliary instillation and digestion, respectively, after Ficoll discontinuous density gradient centrifugation. Catheterial epithelial cells after primary culture and passage of 2 to 6 generations of cells to take four 18-day induction, differentiation into pancreatic islet-like cell mass, immunofluorescence identification. Eighteen Sprague-Dawley rats successfully treated with streptozotocin were divided into three groups randomly. Random control group, fresh islet group and islet-like cell group were transplanted into the left renal capsule. RMPI1640, fresh islet and islet-like cell mass were given respectively, and the blood glucose was monitored every other day after transplantation. Results: The results of immunofluorescence showed that the isolated and purified pancreatic ductal cells had stem cell characteristics. The induced islet-like cell mass was positive for insulin and glucagon staining. After transplantation, the blood glucose of the blank control group remained in the hyperglycemic range. Fresh islet transplantation group blood glucose gradually decreased to normal levels, and within 2 weeks of basic stability in the normal range but slightly increased. In the first two days after transplantation, the islet-like cell mass group did not fall but rise or fall in blood sugar, then declined but never fell to the normal range. CONCLUSION: The pancreatic ductal epithelial cells differentiate into islet-like cell clusters induced by this protocol. The blood glucose of diabetic rats is decreased to a certain extent, but the decrease and maintenance time are not satisfactory, which may be related to the insufficient transplantation and induced islet-like Cell dysfunction related.