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目的研究胰腺干细胞与胰岛混合培养对增加胎鼠胰腺干细胞向β前体细胞转化率的作用及机制。方法实验分为胰腺导管干细胞单独培养组(Ⅰ组),胰岛单独培养组(Ⅱ组)及干细胞-胰岛混合培养组(Ⅲ组)。V型胶原酶消化大鼠胰腺获得胰岛后,使用Ficoll-400梯度离心法纯化胰岛;采用胶原酶消化法获得胎鼠胰腺干细胞进行培养,通过RT-PCR及免疫组织化学染色的方法 ,检测细胞角蛋白-19(CK-19)、巢蛋白(Nestin)、胰高血糖素和胰岛素等干细胞相关标志物。干细胞诱导培养基中加入纯化的胰岛进行混合培养,通过观察干细胞表达胰岛素的阳性率评价其诱导转化率。结果 V型胶原酶逆行胰管灌注继而Ficoll-400梯度离心可获得生物活性良好的胰岛;所培养的干细胞表达CK-19、Nestin、胰高血糖素,诱导后部分表达胰岛素。干细胞-胰岛混合培养组及干细胞单独培养组诱导后,细胞胰岛素阳性率分别为38.2%和23.9%,差异有统计学意义(P<0.05);CK-19阳性率分别为89.3%和81.6%,其差异亦有统计学意义(P<0.05)。结论胰腺导管干细胞与胰岛混合培养可提高胰腺导管干细胞向β前体细胞的转化率,其作用与CK-19的表达密切相关。
Objective To study the effect and mechanism of pancreatic stem cells and islet mixed culture on increasing the conversion of fetal rat pancreatic stem cells to β precursor cells. Methods The experiment was divided into group of pancreatic ductal stem cells alone (group Ⅰ), group of islets alone (group Ⅱ) and stem cell - islet mixed culture group (group Ⅲ). The pancreatic islets were isolated from rat pancreas by digestion with collagenase type V, and then the islets were purified by Ficoll-400 gradient centrifugation. The fetal rat pancreatic stem cells were obtained by collagenase digestion and cultured. RT-PCR and immunohistochemical staining Protein-19 (CK-19), Nestin, glucagon and insulin-related markers. Stem cell induction medium was added to the purified islets mixed culture, by observing the positive rate of insulin expression of stem cells to evaluate the induction of conversion rate. RESULTS: Reverse transcatheter perfusion of type V collagenase followed by Ficoll-400 gradient centrifugation to obtain islets with good bioactivity. The cultured stem cells expressed CK-19, Nestin, and glucagon, and induced partial expression of insulin. The positive rate of insulin was 38.2% and 23.9%, respectively, and the difference was statistically significant (P <0.05). The positive rate of CK-19 was 89.3% and 81.6% respectively after induced by stem cell-islet mixed culture group and stem cell alone group, The difference was also statistically significant (P <0.05). Conclusions Incubation of pancreatic duct stem cells and islets can increase the conversion rate of pancreatic duct stem cells to β-precursor cells, and its role is closely related to the expression of CK-19.