| 1.The establishment of animal models of severe acute pancreatitis(SAP)and evaluation of capillary leakage in rats with SAP.Objective:To establish reliable animal models ofsevere acute pancreatitis(SAP)and evaluate the functional impairment of renal in SAP with renal capillary leakage.Method:SD rat were randomly divided into SAP and control group.The SAgroups were disposed by retrograde injection of 5% sodium taurocholate into biliopancreatic duct to induce SAP.Meanwhile,the pancreas of control group were only flipped and the abdomen were closed.The serum of two groups were collected and the concentration of serum amylase(AMY),ascitic fluid,albumin,creatinine(Cr)and urea nitrogen(BUN)were detected.The level of TNF-ɑ,IL-6,IL-10 were detected by using ELISA.The pancreatic and renal tissue disposed with H&E straining were observed by microscopy to acquire pathological score.The ratio of wet and dry weight(w/d)of kidney and pancreas,and the concentration of Evan’s Blue(EB)in tissue,and the degree of capillary leakage were evaluated by change of renal capillary endothelial barrier observed by transmission electron microscope,All data at the same time point were compared between SAP and control groups.Results:(1).At the same time point,the serum AMY,Cr,BUN,TNF-ɑ,IL-6,IL-10 in SAP group were significantly higher than control group(P< 0.05).Besides the IL-10,the level of all these factors were positively correlated with time.The level of serum albumin in SAP group were significantly lower than control group(P< 0.05),the ratio of w/d and EB of kidney and pancreas in SAP group were significantly higher than control group(P<0.05).(2).No obvious pathological change were existed in control group.The pathological score of kidney and pancreas in SAP group were significantly higher than control group(P< 0.05)and aggravated along with time.The renal capillary endothelial barrier in mesenchymedemonstrated a time-dependent damage,and contrarily,no obvious damage in control groups.Thus,the SAP with capillary leakage and renal impairment were successfully established.Conclusion:The SD rat modelof SAP could be established successfully by using retrograde injection of 5% sodium taurocholate,which demonstrated excessive inflammatory reaction,typicalcapillary leakage and functional impairment.This models could served as the platform for further investigation to the pathogenesis of renal capillary leakage in SAP.2.The role of RhoA/Rock signal pathway in renal capillary leakage in SAPObjective:To investigate the regulatory functional of RhoA/Rock signal pathway to the renal capillary leakage in SAPMethod:SD rat were randomly divided into SAP and control group.The SAgroups were disposed by retrograde injection of 5% sodium taurocholate into biliopancreatic duct to induce SAP.The renal tissue were collected to be experimental object.The distribution of RhoA、ROCK-1、ZO-1、VE-cadherin were visualized by immumohistochemical straning.The expression of mRNA and protein including RhoA、Rock-1、ZO-1、VE-cadherin were detected by RT-PCR and Western-blot,respectively.All data of 6h,12 h and 24 h from two group were compared.Results: According to microscopic observation,RhoA 、 ROCK-1,VE-Cadherinand ZO-1 distributed in the epithelial cell of blood capillaryaround kidney tubules.At the same time point,the expression of mRNA and protein of RhoA、ROCK-1 in SAP groups were significantly higher than control group(P< 0.05)and elevated over time.At the same time point,the expression of mRNA and protein of VE-Cadherinand ZO-1 were significantly lower htan control group(P< 0.05)and reduced over time.Conclusion: Combined with the results of Part I,the RhoA/Rock signal pathway may be activated and sequentially suppress the expression of VE-Cadherin and ZO-1 which cause endothelial cell skeleton actin remodeling and lead to the damage of endothelial connection of vessel endotheliumand increase of permeability.These could trigger capillary leakage around whole body and renal impairment,providing theoretical foundation for further treatment of renal capillary leakage 3.The impact of bone marrow stem cell on renal capillary leakage of SAP in rats and the study of regulatory function of RhoA/ROCK signal pathways on SAP.Objective : To investigate the function and regulatory mechanism of RhoA/Rock signal pathway in the treatment of bone marrow stem cells(BMSCs)for renalcapillary leakage of SAP,providing a new approach for treatment of SAP..Method:(1)Collected and cultured the primary BMSCs to the third generation and identified the cells with morphological examination,survivorship curve,flow cytometry identification of surface biomarkers.(2)Depending the method of intervene,the SD rats were randomly divided in to BMSC-treated(BMSCs-SAP),ROCK-I inhibitor-treated(Y-27632-SAP)and Control SAP groups.ALL groups were disposed with retrograde injection of 5% sodium taurocholate into biliopancreatic duct to induce SAP.The serum AMY,ascitic fluid,Cr,BUN,albumin,and W/D weight of kidney and renal EB were detected in 6h,12 h,24h.The level ofTNF-ɑ,IL-6 andIL-10 were detected with ELISA.The migration of BMSCs were observed by fluorescence microscope.The histological sectionswith H&E stainingwere observed withmicroscope and theultrastructureof renal were observed by transmission electron microscope.The distribution and expression of RhoA,Rock-1,ZO-1 and VE-cadherin were detected with immunohistochemistry.The RT-PCRand Western-blotwere used to detect the expression of RhoA、Rock-1、ZO-1、VE-cadherin in renal tissue.All indicators from BMSCs-SAP、Y27632-SAP were pair-compared with SAP,respectively.Results:(1)After passage,the BMSCs arranged like fibroblast-like and arrange orderly.(2)The BMSCs were highly expressed the CD90(99.8%),CD90(96.7%)and lowly expressed CD34(1.9%),CD45(0.8%),which were consistent with the characteristics of BMSCs.(3)The comparison of renal pathomorphism:The pathological damage of renal tissue from BMSC-treated group was less than that in Control-SAP group at each time point.The pathological damage of renal tissue inY-27632-SAP group was less than that in Control-SAP group at point of 6h,but insignificantly at point of 12 h and 24 h.(4)Thecomparison of renal ultrastructure: The damage ofrenal capillary endothelial barriers in BMSCs-SAP group were less than Control-SAP groups but were similar betweenY-27632-SAP and Control-SAP group.(5)The comparison of serum biochemistry,renal EB and W/D weight of kidney: The serum AMY,ascitic fluid,Cr,BUN,IL-6,TNF-ɑ,W/Dweight of kidney and renal EB in BMSCs-SAP were significantly lower than that in Control-SAP group.The serum albumin and IL-10 were significantly higher than that in Control-SAP group(p<0.01or0.05).The serum AMY,ascitic fluid,Cr,BUN,renal EB and the ratio of W/D of kidney in Y27632-SAP groupwere significantly lower than that in SAP groups at the point of 6h(p<0.05),but not significantly at the point of 12 and 24 h.The serumIL-6,TNF-ɑandIL-10 were not different between Y27632-SAP and SAP group.(6)The mRNA and protein expression of RhoA,ROCK-1,VE-cadherin and ZO-1: The expression of RhoA and ROCK-1in BMSCs-SAP group were significantly lower than Control-SAP and increased along with time(P<0.05).The expression of VE-cadherinand ZO-1 in BMSCs-SAP group decreased along with time were significantly higher than Control-SAP group(P<0.05).The expression of RhoA and Rock-1 in Y27632-SAP group were significantly lower than SAP group while the expression of VE-cadherin and ZO-1 were significantly higher than Control-SAP group at the point of 6h(P<0.05).The expression of RhoA 、 Rock-1 、VE-cadherinand ZO-1 in were not different between Y27632-SAP and Control-SAP group at the point of 12 h and 24h(P>0.05).Conclusion:BMSCs could migrate to impaired kidney due to SAP and colonized.Consequently,BMSCs could persistently suppress the expression ofinflammatory factors TNF-αandIL-6,and activate the release of anti-inflammatoryfactors IL-10,sequentially suppress the over activation of RhoA/ROCK pathwayand up-regulate the expression of the ZO-1 and VE-Cadherin in epithelial cells,thus to relieve th erenal capillary leakage and function.Y-27632 could not suppress the release of inflammatory factors and ameliorate SAP through directly inhibiting the RhoA/ROCK pathway for a short time. |
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