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Role of Bone Marrow Mesenchymal Stem Cells in the Development of Pancreatic Injury and Chronic Pancreatitis and Expression of Micrornas in the Cp-pdac Rat Model

Author: HanFei
Tutor: WangChunYou
School: Huazhong University of Science and Technology
Course: Surgery
Keywords: Bone mafrow mesenchymal stem cells Pancreatic injury Chronicpancreatitis Pancreatic ductal adenocarcinoma MicroRNA MMPs
CLC: R-332
Type: PhD thesis
Year: 2012
Downloads: 124
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Abstract

Purpose1. To investigate the role of BMSCs in repair process of injured pancreas2. To investigate the role of BMSCs in the resolution process of chronic pancreatitis3. To investigate the whether microRNA expression was a potential biomarker to distinguish chronic pancreatitis (CP) from PanTNs and PDAC in a CP-PDAC rat modelMethods1. In this study, we intravenously infused GFP+ BMSCs into mice received 65%-70% partial pancreatectomy via the tail vein, while mice received partial pancreatectomy only and mice with BMSCs injected only to be used as control. Immunophenotyping for surface markers on BMSCs was detected by FACS to characterize this cell population. To assess pluripotency of the BMSCs, BMSCs were induced to differentiate into adipocyte- and osteoblast-like cells in vitro. GFP+ cells were recognized by immunohistochemistry and immunoflurescent using anti-GFP antibody. The differentiation of BMSCs in pancreatic sections was assessed by double fluorescent staining for CK19, Desmin,α-SMA, CD31, Insulin, Nestin and GFP. Four weeks after partial pancreatectomy and BMSCs implantation, the whole body and total pancreas of mice was weighted with auto-electronic balance. We performed qRT-PCR to assess the mean relative levels of four candidate miRNAs (miR-9, miR-204, miR-34a and miR-375) in engrafted GFP+BMSCs which were sorted out of the residual pancreas, compared to wild type and GFP+BMSCs.2. Mice received repeated intra-abdominal cerulein injections for eight weeks to induce chronic pancreatitis. Four weeks after initial cerulein treatment, GFP-positive murine BMSCs were injected into these mice through the tail vein. The distribution and characteristics of GFP-positive cells present in fibrotic pancreas tissue were examined at different time points after injection of cerulein. The differentiation of BMSCs in pancreatic sections was assessed by double fluorescent staining for Desmin,α-SMA and GFP. The expression of MMP-2 and MMP-9 in engrafted BMSCs was detected by double fluorescent. The percent area of pancreatic fibrosis was analyzed by sirius red staining and Image Pro Plus. The expression of MMP-2 and MMP-9 in pancreatic sections was detected by western blotting.3. In this study, four miRNAs (miR-34a, miR-204, miR-107 and miR-21) related to pancreatic ductal adenocarcinoma (PDAC) were selected in this study. Pancreatic samples were obtained from a CP-PDAC rat model, which was induced by partial ligation of biliary-pancreatic ducts and implantation of 7,12-dimethylbenzanthracene (DMBA) in rats. Expression of miRNAs was investigated in these samples by using quantitative real-time polymerase chain reaction (qRT-PCR) technique.Results1. Four weeks later, injected GFP+ BMSCs were diffusely engrafted in pancreatic parenchyma and mesenchyma of recipient mice with pancreatic injury, differentiated into pancreatic ductal epithelial cells (accounted for 1.7±0.3%), vascular endothelial cells (accounted for 3.2±0.6%) and pancreatic stellate cells (PSCs) (accounted for 5.2±1.6%), but not any pancreatic beta cells or neural cells. Significantly more engrafted and differentiated GFP+BMSCs were observed in regenerating pancreas than in normal pancreas. For the mice received partial pancreatectomy, the pancreatic weight/body weight of the mice with BMSCs treated ((0.225±0.042)%) was more than that of the mice without BMSCs treated ((0.183±0.021)%) (p<0.05). In addition, real time RT-PCR showed that the expression levels of miR-9 and miR-204 in the engrafted BMSCs (5.2-fold and 2.6-fold, p<0.05, respectively) were increased compared to wild type BMSCs. We also observed significant downexpression of miR-375 (0.71-fold, p<0.05) in engrafted GFP+BMSCs compared to wild type BMSCs.2. Double fluorescent immunohistochemistry showed that a large number of GFP-positive BMSCs, as well as GFP-negative pancreatic resident cells, expressed matrix metalloproteinase (MMP)-2, but only a small number of GFP+ cells expressed MMP-9. Furthermore, our data showed increased MMP-2 and MMP-9 expression in the pancreas with BMSCs treatment compared with the pancreas treated with cerulein alone. Eight weeks after BMSCs injection, mice had significantly reduced pancreatic fibrosis, as assessed by sirius red staining of the pancreas (4.56%±0.63%), compared to that of mice treated with cerulein alone (7.37%±0.92%). As demonstrated by the coexpression of GFP and a-SMA, a subset of donor-derived activated PSCs were observed in periacinar fibrotic regions within the pancreas.3. Relative to chronic pancreatitis, significant overexpression of miR-34a was observed in PanIN-1, PanIN-2, PanIN-3 and PDAC (p<0.05), while significantly low expression of miR-204 were observed in PanIN-2, PanIN-3 and PDAC (p<0.05). In contrast, significant overexpression of miR-21 (p<0.05) was observed in PanIN-3 and PDAC. miR-107 was upregulated in PDAC only.Conclusion1. Here we demonstrate that BMSCs can be a potential cell bank for treating pancreatic injury by contributing to a variety of cell types, which might be related with the expression of miR-9, miR-204 and miR-375.2. BMSCs contribute to the spontaneous regression of pancreatic fibrosis through expressing MMP-2 and MMP-9.In addition, BMSCs can differentiate into PSCs in chronic pancreatitis mice.3. Aberrant miR-34a and miR-204 production was an early event in the multistep progression of PDAC.

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