Yu Fu-Xiang, Su Long-Feng, Dai Chun-Lei, Wang Yang, Teng Yin-Yan, Fu Jun-Hui, Zhang Qi-Yu, Tang Yin-He
Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, Wenzhou Medical College, Wenzhou 325000, China.
Hepatobiliary Pancreat Dis Int. 2015 Apr;14(2):215-21. doi: 10.1016/s1499-3872(14)60283-6.
Pancreatic stellate cells (PSCs) play a critical role in the development of pancreatic fibrosis. In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory effects of adipose-derived stem cells (ADSCs) on activation and proliferation of PSCs.
Pancreatic tissue was obtained from Sprague-Dawley rats for PSCs isolation. Transwell cell cultures were adopted for co-culture of ADSCs and PSCs. PSCs proliferation and apoptosis were determined using CCK-8 and flow cytometry, respectively. alpha-SMA expressions were analyzed using Western blotting. The levels of cytokines [nerve growth factor (NGF), interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1)] in conditioned medium were detected by ELISA. Gene expression (MMP-2, MMP-9 and TIMP-1) was analyzed using qRT-PCR.
This method produced 17.6+/-6.5X10(3) cells per gram of the body weight with a purity of 90%-95% and a viability of 92%-97%. Co-culture of PSCs with ADSCs significantly inhibited PSCs proliferation and induced PSCs apoptosis. Moreover, alpha-SMA expression was significantly reduced in PSCs+ADSCs compared with that in PSC-only cultures, while expression of fibrinolytic proteins (e.g., MMP-2 and MMP-9) was up-regulated and anti-fibrinolytic protein (TIMP-1) was down-regulated. In addition, NGF expression was up-regulated, but IL-10 and TGF-beta1 expressions were down-regulated in the co-culture conditioned medium compared with those in the PSC-only culture medium.
This study provided an easy and reliable technique to isolate PSCs. The data demonstrated the inhibitory effects of ADSCs on the activation and proliferation of PSCs in vitro.
胰腺星状细胞（PSCs）在胰腺纤维化的发展中起关键作用。在本研究中，我们使用一种新方法分离和培养大鼠PSCs，然后研究脂肪来源干细胞（ADSCs）对PSCs激活和增殖的抑制作用。
从Sprague-Dawley大鼠获取胰腺组织用于分离PSCs。采用Transwell细胞培养法对ADSCs和PSCs进行共培养。分别使用CCK-8和流式细胞术测定PSCs的增殖和凋亡。使用蛋白质免疫印迹法分析α-SMA的表达。通过酶联免疫吸附测定法检测条件培养基中细胞因子[神经生长因子（NGF）、白细胞介素-10（IL-10）和转化生长因子-β1（TGF-β1）]的水平。使用定量逆转录聚合酶链反应（qRT-PCR）分析基因表达（基质金属蛋白酶-2、基质金属蛋白酶-9和金属蛋白酶组织抑制因子-1）。
该方法每克体重产生17.6±6.5×10³个细胞，纯度为90%-95%，活力为92%-97%。PSCs与ADSCs共培养显著抑制PSCs增殖并诱导PSCs凋亡。此外，与仅培养PSCs相比，PSCs+ADSCs中α-SMA表达显著降低，而纤溶蛋白（如基质金属蛋白酶-2和基质金属蛋白酶-9）表达上调，抗纤溶蛋白（金属蛋白酶组织抑制因子-1）表达下调。另外，与仅培养PSCs的培养基相比，共培养条件培养基中NGF表达上调，但IL-10和TGF-β1表达下调。
本研究提供了一种简便可靠的分离PSCs的技术。数据表明ADSCs在体外对PSCs的激活和增殖具有抑制作用。
