| Objective: The experiment by measuring endothelial progenitor celltransplant to rat acute pancreatitis model induced by sodium taurocholateof CD133and CD34immunohistochemical expression and HE stainingthe change of the density of blood vessels,research on endothelialprogenitor cells to the improvement of microcirculation in rats with acutepancreatitis.Methods:1. The isolation and identification method ofEPCs: choose adult healthy SD rat isolate rat femur and tibia, PBS rinsemarrow cavity, collect all bone marrow cells,and make from cellsuspension.Join in the lymphoid cell separation fluid, by gradient densitycentrifugation, collect the middle white membrane layer of nucleatedcells, put in PBS,centrifuge and wash three times. By the differentialadherence method，collect not adherent cells after48h，and put incontaining20%fetal bovine serum,5ng/ml bFGF,20ng/ml VEGF M199nutrient solution cultivate EPCs in6orifice plate. Culture cells after10days, identify of cells by immunohistochemical and double stainingfluorescence method. Cell grow to the needful quantity, collect cells,wash twice in serum free medium, make cells suspension in serum free medium, and prepare for transplant.2. Establishment of rat model ofacute pancreatitis and transplantation of EPCs:72healthy adult male ratsare randomly divided into normal control group (group A, n=24), acutepancreatitis model group (group B, n=24), and acute pancreatitisendothelial progenitor cell transplantation group (group C, n=24). SDrats with1%sodium pentobarbital intraperitoneal injection of0.6ml/100g, after anesthesia,laparotomize rat, find the pancreas,and inject1%sodium taurocholatel1ml under the pancreas envelope (normalcontrol group inject normal saline), and carry out needle catheterjejunostomy,after surgery parenteral nutrition.Endothelial progenitor cellswitch cultured in vitro inject into external jugular vein in rats.Every groupwould put to dead6rats and collet specimen of pancreatic after C groupwas transplantated, the specimen fixed by formaldehyde, pancreatic tissueafter HE staining pathology examination, observation of pancreaticpathological morphological changes, recording density of blood vessels;Immune histochemical method (SP method) detection of CD133andCD34in pancreatic tissue accumulation of optical density (IOD)values.Data adopt mean±standard deviation (x±s) to show, usingSPSS17.0statistical software for statistical processing;the sample meancompared use the single factor analysis of variance; The correlation ofboth use linear correlation and linear regression analysis, P <0.05, thedifference is statistically significant. Results：(1) Pathological score of group B（model group）and group C(transplantation group) higher thangroup A(control group) at various time points(the3th day、the7th day andthe14th day)(P<0.05), pathological score of group B higher than groupC at the7th day and the14th day (P<0.05).(2)In the pancreas HE dyeingof micro vessel at the7th day and the14th day, group C more than Agroups and B group(P<0.05).(3)At various time points,group C in thepancreas of CD133IOD values were higher than in A and B group(P<0.05).(4)At various time points,group C in the pancreas of CD34IODvalues is higher than A and B group (P<0.05).(5) Histopathologicalgrading of the pancreas and CD133IOD values is rectilinear correlation(r=0.814，P=0.000). Conclusion:(1) By density gradient centrifugationcombined with differential sticking wall method,EPCs can be isolatedfrom rat bone marrow, and can be used for celltransplantation.(2)Endothelial progenitor cells witch cultivated in vitrocan be successfully transplanted into the rat pancreatic tissue of acutepancreatitis.(3)Endothelial progenitor cells witch had transplanted intothe acute pancreatitis rat can increase the number of pancreascapillaries,and had make the pancreas microcirculation improvement. |
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