Effect Of BMSCs On CD4 ~+ CD25~+ Regulatory Cells In Rats With Sap With Combined Treatment Of Traditional Chinese And Western Medicine

Study Background and Objective: Acute pancreatitis(AP)is one of acute abdomen, with high incidence rate and complicated pathophysiologic change.When SIRS and MSOF are complicated by SAP have high mortality and rapid progression.The pathological features of SAP is pancreatic tissue hemorrhagic necrosis on a large scale, and involving surrounding tissue rapidly. The treatment involves a short fast, decompression, supplement the blood volume, improving the microcirculation, inhibiting pancreas secretion, maintain water, electrolyte and acid-base balance, hemodialysis, peritoneal dialysis, antibiotics administration, sedatives, antispasmodics, surgery, nutritional therapy,and so on. The rate of mortality is decrease in recent years, but the treatments still lack sufficient sensitivity and specificity, and cannot get satisfactory curative effect on it, it is difficult to reverse complete the progress of SAP disease. Bone marrow-derived mesenchymal stem cells (BMSCs)is easy to elevate, less invasive, low immunogenicity and can greatly proliferate in vitro. The research on this topic suggests that BMSCs can releases a large number of cytokines and regulatory protein to block apoptosis, postpone the progress of tissular fibrosis and decrease inflammatory injury, The immunoregulation effect of BMSCs can be embodied by The up-regulation of expression of CD4~+CD25~+ T cells which can suppressive immunocyte, to restore and preserve damaged tissue and organs. This study is about the influence of peripheral blood CD4~+CD25~+T cells and inflammation factors were observed in severe acute pancreatitis (SAP) rats receiving BMSCs allogeneic transplant. It confirms that BMSCs can decrease inflammatory injury, restore and preserve damaged pancreatic tissue on SAP, and to provide scientific basis for clinical practice. To study the traditional Chinese and western medicine treatment of SAP in rats, maybe provide the new thoughts for the treatment to delaying the progression and reducing mortality rate of SAP.Method:The first part, separate and cultivate BMSCs through percoll density gradient centrifugation and stick wall separating, the third generation of BMSCs were identified through Flow cytometry to detect the surface markers of CD45 and CD90 and confirms the potency of multi -directional differentiation by osteoblast and lipoblast formation induction. Rats model of SAP was made by retrograde infusion of 1.5% sodium deoxycholate into biliopancreatic duct of rats. Sixty SD rats were randomly divided into five groups: Sham-operated group (SO), model control group (SAP), BMSCs treatment group (BMSCs), Dachengqi Decoction treatment group(ZY) and the group of united medication(ZXY). SAP group were administered with Dachengqi Decoction and injected Labeled BMSCs via tail vein, at the sametime, the BMSCs was labeled by DAPI. The rats blood serum of abdominal aorta was collect to detect the IL-6 and IL-10 contents with IL-6 or IL-10 Elisa kit, the level of the serum amylase, lipase levels.The rats'gross anatomy and pathological histology of pancreatic tissue, flow cytometry for the expression pattern of CD4~+CD25~+T cells from peripheral blood.Results:1.After several subcultures, BMSCs form a more homogeneous, and show spindly, triangle or polygon. Cells growth trend to form BMSCs colony-like cell groups which proliferated quickly, these components purifying gradually. The positive rate of CD90 that sit on the surface of BMSCs was 91.39%,and the negative rate of CD45 was 99.72%by flow cytometry (FCM). BMSCs demonstrated positive staining for alizarin red after induce osteoblastic differentiation. Oil Red"O"staining of the BMSCs after 10 days of culture demonstrated numerous intracellular lipid droplets.2. After operation 24 hours, SAP group rats displayed the pancreas become swell, hemorrhagic necrosis, a lot of intra-abdominal hemorrhagic ascites, the omentum were found saponification spot. The tissue sections in histology and pathology displayed large areas of bleeding and coagulation necrosis in the pancreatic tissue, and there is no discernable l pancreas lobular. There may be profuse neutrophil and mononuclear cells infiltration of the pancreatic tissue.Gross anatomy and the pancreatic pathological section observation of the treated groups, changes in various indicators of disease extent of change were significantly lighter than the model group, the severity of the lightest of which ZXY group.3. (1)DAPI labeled BMSCs in vivo tracer experiments: under fluorescence microscope frozen sections of the lumen tissue BMSCs group could seen bright blue fluorescence in the cells, SAP group were not observed the bright blue fluorescence of the cells. BMSCs in the SAP model rats proved able to locate the damaged pancreatic tissue. (2)The level of the serum amylase and lipase, SAP group were significantly higher than the SO group(P﹤0.01); compared with SAP group, the treatment groups were significantly decreased, and the decreased level of ZXY group were the most significant (P﹤0.01).(3)Compared with SAP group,SO group and the untreated groups IL-6 contents decreased significantly(P﹤0.01), especially ZXY group decreased significantly(P﹤0.01). compared with SO group, SAP group and the untreated groups IL-10 contents increased significantly(P﹤0.01), BMSCs group IL-10 contents increased(P>0.05). ZY group showing no significant difference compared with SAP group (P>0.05). (4)The rates of CD4~+CD25~+T cells of SAP group were obviously lower(P﹤0.01), ZY group were increasing, but have no statistics significance (P>0. 05). the rates of CD4~+CD25~+T cells of BMSCs group and ZXY group were obvious increasing compared with SAP group(P﹤0.05).Conclusion:1. Established good separation of BMSCs, culture and identification methods and obtained BMSCs after 3rd generation into uniform spindle, more than 90% purity.Cell surface markers by flow cytometry and Induced differentiation of osteoblast cells and adipocyte cells were obtained confirmed features consistent with BMSCs.2. By BMSCs transplantation and medicine Dachengqitang treatment, reduced the inflammation, ascites, hemorrhage necrosis and other lesions of SAP rats, and reduced serum amylase, lipase and the inflammatory mediators IL-6 levels, anti-inflammatory cytokine IL-10 of the treatment groups increased with varying degrees, the mortality in each treatment group were lowered than the SAP group. DAPI labeled BMSCs in vivo by tracer experiments show that through the blood circulation BMSCs migrate to the damaged parts of pancreatitis, to repair damaged tissue and inhibit the inflammatory response.Peripheral blood CD4~+CD25~+ T cells of SAP were significantly decreased compared with SO group. Afte BMSCs transplantation, peripheral blood CD4~+CD25~+ T cells was increased, prompting CD4~+CD25~+T cells may be involved in the inflammatory reaction against the process of BMSCs.Among the treatment groups compared, ZXY group improved the most obvious pathological, and the indicators to be detected were optimal. So that BMSCs in combination with traditional Chinese medicine maybe by increasing the peripheral blood CD4~+CD25~+ T cells to against the inflammatory response, curb the development of the disease SAP,improve prognosis,this treatment is an effective method for SAP.