角脘蛋白生长因子与胰腺导管上皮细胞的体外增殖

Effect of keratinocyte growth factor on proliferation of rat pancreatic ductal epithelium cells in vitro

背景:胰腺导管上皮细胞在体内外均能分化为胰岛素分泌细胞,但胰腺导管上皮细胞体外增殖潜能较差,限制了胰岛细胞移植治疗糖尿病的发展。目的:探讨不同质量浓度角脘蛋白生长因子对SD大鼠胰腺导管上皮细胞体外增殖活力的影响,并寻找最佳浓度,从而得到大量增殖的胰腺导管上皮细胞。方法:运用胶原酶逆行灌注法分离大鼠胰腺导管上皮细胞,密度梯度离心法纯化细胞,而后体外扩增,分别通过免疫细胞化学染色及RT-PCR进行鉴定。在培养基中加入不同质量浓度的角脘蛋白生长因子刺激胰腺导管上皮细胞增殖,通过细胞计数及MTT法测定细胞增殖能力。结果与结论:胰腺导管上皮细胞表达CK19蛋白;不同浓度的角脘蛋白生长因子培养基中胰腺导管上皮细胞形态正常,5μg/L角脘蛋白生长因子组比其他浓度组细胞数量少(P<0.01),而10,15与20μg/L角脘蛋白生长因子组的细胞数量相近(P>0.05)。提示不同浓度的角脘蛋白生长因子均可促进大鼠胰腺导管上皮细胞的增殖,10μg/L角脘蛋白生长因子可能是促进大鼠胰腺导管上皮细胞增殖的最佳培养剂量。

BACKGROUND：Pancreatic ductal epithelium cells（PDECs） can differentiate into insulin-producing cells in vitro or in vivo,but the cells have poor proliferation in vitro.Thus,the cell transplantation therapy for diabetes is limited.OBJECTIVE：To investigate the effect of keratinocyte growth factor（KGF） at different concentrations on the proliferation of PDECs in order to seek the optimal serum dose.METHODS：The PDECs of rats were isolated by injecting collagenase Ⅴ solution and purified by removing the islets with Ficoll density gradient centrifugation.Then the PDECs were identified by immunocytochemical staining and RT-PCR.Different concentrations of KGF were added into medium.The proliferation ability of PDECs was determined by cells counting and MTT assay.RESULTS AND CONCLUSION：Immunocytochemical staining results showed positive expression of CK19 in PDECs;Morphology of the PDECs in the different groups were similar.The PDECs numbers of the 5 μg/L KGF group were significantly lower than that of the other KGF groups（P 0.01）,and the proliferation of the groups cultured with 10,15 and 20 μg/L KGF were similar（P 0.05）.The KGF at different concentrations can significantly stimulate the proliferation of PDECs,the optimized concentration is 10 μg/L.