胰腺脱细胞支架内胰岛素分泌细胞循环灌注培养

Circulation perfusion culture of insulin-producing cells using rat decellularized pancreatic scaffolds

目的观测小鼠骨髓间充质干细胞（mBMSCs）诱导的胰岛素分泌细胞（IPCs）在大鼠胰腺脱细胞支架上的生长及功能发挥。方法经脾动脉持续灌注洗脱剂制备胰腺脱细胞支架，对其进行组织染色，DNA、糖胺多糖（GAG）含量，生物相容性检测。含小鼠肌腱膜纤维肉瘤癌基因同系物A（MafA）、胰腺十二指肠同源异型盒-1（PDX-1）和神经源性分化因子-1（NeuroD1）的腺病毒联合导入mBMSCs，免疫荧光、实时荧光定量聚合酶链反应（FQ-PCR）检测胰岛素（Insulin）表达；酶联免疫吸附测定（ELISA）检测不同浓度葡萄糖刺激后胰岛素分泌情况。IPCs再种植于脱细胞支架内，培养后行苏木素-伊红（HE）染色、免疫荧光、FQ-PCR检测细胞生长及功能发挥。结果经灌注后未见细胞残留，可见细胞外基质（ECM）保留，DNA定量提示细胞含量为（44.92±3.89） ng/mg（t=15.160，P=0.001），GAG含量为（30.27±2.70） ng/mg，（t=2.862，P=0.046），免疫组织化学显示胶原Ⅰ、纤连蛋白保留。三基因修饰的mBMSCs Insulin表达阳性，葡萄糖刺激试验显示其对不同浓度葡萄糖有反应。将IPCs在胰腺脱细胞支架内循环灌注培养，HE染色显示细胞在支架内生长良好，免疫荧光和FQ-PCR显示Insulin表达阳性，与二维培养比较，Insulin表达提高（t=15.030，P=0.004）。结论制备的大鼠胰腺脱细胞支架保存了基本的脉管结构及ECM，生物相容性良好。PDX-1、NeuroD1和MafA协同促进mBMSCs分化并获得Insulin合成和分泌能力。经三维循环灌注培养，IPCs能够在支架内生长及发挥功能，且细胞功能得到促进。

ObjectiveTo cultivate mouse bone marrow mesenchymal stem cells （mBMSCs） drived insulin-producing cells （IPCs） in rat decellularized pancreatic scaffolds and evaluate the growth and function of cells.MethodsDecellularized pancreatic scaffolds were obtained by continuous perfusion of detergent by the splenic artery. HE staining, Masson staining, DNA content, glycosaminoglycan （GAG） content, extracellular matrix （ECM）, biological compatibility were adopted to evaluate the result. mBMSCs were differentiated into IPCs via infected by pancreatic and duodenal homeobox-1 （PDX-1）, neurogenic differentiation-1 （NeuroD1） and V-mar musculoaponeurotic fibrosarcoma oncogene homologue A （MafA）. Immunofluorescence and real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） were detected of insulin expression of the IPCs. The IPCs were exposed to a glucose gradient and insulin release was measured by enzyme linked immunosorbent assay （ELISA） assay. The IPCs were cultivated in the decellularized pancreatic scaffolds in the circulation perfusion device. Hematoxylin-eosin staining （HE） and immunofluorescence were implemented to evaluate the growth and function of cells. The insulin gene expression was compared with traditional culture by FQ-PCR.ResultsAfter perfused of detergent, HE staining and Masson staining demonstrated no residual cells remained and most of the collagen fiber components were well retained. DNA content of decellularized scaffolds was （44.92±3.89） ng/mg （t=15.160, P=0.001）and GAG content was （30.27±2.70） ng/mg, （t=2.862, P=0.046）. Immunohistochemical staining showed collagen I and fibronectin were retained. Scaffolds were desirable biocompatibility. Immunofluorescence and FQ-PCR showed gene and protein expression of insulin after infected by PDX-1, NeuroD1 and MafA. The IPCs were glucose responsiveness to different glucose concentration according to ELISA assay. After cultivated in the decellularized pancreatic scaffolds, HE and immunofluorescence demostrated that the IPCs grew well in the scaffolds. FQ-PCR showed that insulin gene expression was increased compared with the traditional two-dimensional culture （t=15.030, P=0.004）.ConclusionThe rat decellularized pancreatic scaffolds not only preserved the ECM and structure of vascular, but also good biocompatibility. The IPCs differentiated from mBMSCs had the gene expression of insulin expression. The decellularized pancreatic scaffolds not only supported the growth and function of IPCs, but provided a favourable platform compared with the two-dimensional culture.