| BackgroundChronic pancreatitis (CP) was a recognized clinical refractory disease. Chronic pancreatic persistent inflammation damage and fibrosis were the main pathological features, but its precise mechanism had not yet been clarified. Current clinical treatment results were not satisfactory. Even after the removal of pathogenic factors of pancreatic fibrosis, development process remained irreversible, and the persistence of fibrosis was a potential carcinogen. Therefore, the search for safe and effective treatment for blocking or reversing the fibrosis of the pancreas with chronic pancreatitis And improve pancreatic function were important to improve survival in patients with chronic pancreatitis and quality of life.Represented by stem cell transplantation and tissue engineering in the modern GM as the representative of modern genetic engineering technology, both provided a new exploration area for a variety difficult of diseases. Mesenchymal stem cells (MSCs) were a class of pluripotency and high self-renewal of adult stem cells, with sources rich, easy isolation and culture and expanded long-term survival, gene transfection efficiency, transfection and stable expression of low, which were other stem cell immunogenicity unparalleled advantages.MSCs transplantation for treatment of diseases have become a hot research. The results show that, MSCs to the heart, liver, lung, kidney, nervous system and other organs are protected and the pathological injury repair. However, current MSCs research in pancreatic diseases was less. Research of domestic Professor Li Zhaoshen showed that bone marrow mesenchymal stem cells may be involved in acute tissue injury and repair.Through the transplantation of bone marrow mesenchymal stem cells or injection of granulocyte colony stimulating factor (GCSF), Cui Huifei et al treated severe acute pancreatitis (SAP) induced by L-arginine, which suggested bone marrow mesenchymal stem cells helped to alleviate the condition of SAP.PurposeDiscussed the mechanism and its value of MSCs in treatment of CP.Methods1. the SD rats, purchased from the Animal Center of Zhengzhou University,80 of them were randomly divided into control group, model group, sham treatment group and treatment group,each 20 rats.2. BMSCs isolation, culture, identification:Reference to the literature, used the improved adherence screening method. When the 80%-90% of the cells covered the culture bottom, used with 0.25% trypsin to subculture. When the cells spread to the 5th generation, digested MSCS with 0.25% trypsin. Made cell suspension with the concentration of 2 x 109/L. Repackaged the cell suspension to two 5ml test tube, which were added to mouse anti rat CD29, CD44, CD45 monoclonal antibody and its doing the same type of control identified by flow cytometry.rAAV2-EGFP-mediated cell transfection. When the fourth generation of culture BMSCs90% covered bottom, used 0.25% trypsin digestion, and then adjusted the cell concentration of 5 x 104/L.Seeded them in 6-well plates and placed them in 5% CO2, 37℃incubator. When 90% MSCs reached confluence, Subcultured at 1:2 ratio. 3.80 healthy male SD rats,about 250g, were randomly divided into control group, model group, sham treatment group and treatment group, each 20rats.8 weeks after producing rat chronic pancreatitis model, treatment group rats were injected a concentration of 3×106/L, green fluorescent protein BMSCs (GFP-BMSCs) 1ml at 20d,40d,60d.The control group and the sham treatment group were both injected equivalent sterile distilled water, while the model group had no treatment. After 80d, took rat pancreas, part of the production sections, HE staining, MASSON staining and immunohistochemical staining to detect PSC activation, proliferation and differentiation.Some pancreatic tissues of rats ware took to detect expression ofⅠ,Ⅲcollagen content, L-1, IL-6, IL-8, IL-10, TNF-α, TGF-β.4. Detection indicatorEvaluation of pancreatic lesions:①the degree of inflammatory cell infiltration②necrosis:③structural damage④myxedema⑤atrophy Evaluation of fibrosis; expression of inflammatory cytokines in pancreatic tissue.5. Statistical MethodsMeant the difference between the application of one-way analysis of variance, data as mean±standard deviation (χ±s), and analysis using statistical software SPSS 16.0, P <0.05 as significant difference.Results1. In model group, pancreatic tissue got edema after 1d, with neutrophils infiltration. After 7d, the inflammation in pancreatic tissue gradually increased and extensive infiltration of inflammatory cells was showed. A wide range of inflammatory cell infiltration and mild fibrosis occurred in pancreatic tissue after 14d.28d later progression of pancreatic fibrosis increased, showing that a large number of collagen deposition. After 60d, pathology showed damage of pancreatic lobular structure and a wide range of inflammatory cell infiltration, at the same time interstitial fibrosis got more severe.Sham treatment group and model group, roughly showed the same pathology.Treatment group status to reduce inflammatory cell infiltration, collagen deposition area and interstitial fibrosis were reduced.Control group did not change during the period. 2. In the treatment group, expression of desmin, GFAP,α-SMA,Ⅰ,Ⅲcollagen content, IL-1, IL-6, IL-8, TGF-βwere all lower than the sham treatment group and model group (P<0.05),but IL-10, TNF-a were higher than that in the sham treatment group and model group (P<0.05). There was no significant difference between model group and sham treatment groups, about the expression of desmin, GFAP,α-SMA,Ⅰ,Ⅲcollagen content, IL-1, IL-6, IL-8, IL-10, TNF-α, TGF-β(P> 0.05)3. the apoptotic pancreatic cells could be observed around MSCs in the pancreas.Conclusions1. mesenchymal stem cells could reduce inflammation in pancreatitis, the activation, proliferation, differentiation of pancreatic stellate cells, the deposition of collagen fibers and block, even reverse pancreatic fibrosis.2. Mesenchymal stem cells which were injected into the body of the individual rat model of chronic pancreatitis, colonization in the damaged pancreas, could repair damaged pancreatic tissue. The research provided a basis for clinical research. |
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