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Objective@#To investigate the role of bone marrow mesenchymal stem cells (BMSCs) with CTLA4Ig and CD40LIg gene modification in rejection reaction after liver transplantation in rats and possible mechanisms.@*Methods@#The modified Kamada’s two-cuff technique was used to establish a Lewis-BN rat model of orthotopic liver transplantation, and a total of 75 rats were randomly divided into groups A, B, C, D, and E, with 15 rats in each group. The rats in group A (control group) were given infusion of isotonic saline via the portal vein during liver transplantation, those in group B (BMSC group) were given infusion of BMSCs via the portal vein during liver transplantation, those in group C (BMSCs with CTLA4Ig gene modification) were given infusion of BMSCs carrying the CTLA4Ig gene via the portal vein during liver transplantation, those in group D (BMSCs with CD40LIg gene modification) were given infusion of BMSCs carrying the CD40LIg gene via the portal vein during liver transplantation, and those in group E (BMSCs with CTLA4Ig and CD40LIg gene modification) were given infusion of BMSCs carrying CTLA4Ig and CD40LIg gene modification via the portal vein during liver transplantation. Postoperative survival and change in liver function were observed. HE staining was used to observe the pathomorphological changes of the graft liver, and ELISA was used to measure the levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon-γ (IFN-γ) in peripheral blood. A one-way analysis of variance was used for comparison of means of multiple samples, and the Kaplan-Meier survival curve analysis was used for comparison of survival rates between multiple groups.@*Results@#Group E had a significantly longer survival time after surgery than groups A, B, C, and D (P < 0.05), groups C and D had a significantly longer survival time than groups A and B (P < 0.05), and there was no significant difference between groups C and D (P > 0.05). On day 10 after surgery, group A had significantly higher levels of alanine aminotransferase and total bilirubin than the other four groups (P < 0.05). HE staining showed severe rejection reaction in group A, moderate rejection reaction in group B, and mild rejection reaction in groups C and D; pathological examination showed no marked rejection reaction in group E. Group A had significant increases in the levels of IL-2 and IFN-γ and significant reductions in the levels of IL-4 and IL-10 after surgery compared with the other four groups (all P < 0.05).@*Conclusion@#Infusion of BMSCs with modification of both CTLA4Ig and CD40LIg genes can significantly inhibit acute rejection reaction after liver transplantation in rats and effectively prolong the survival time of the graft liver, with a better effect than infusion of BMSCs alone or BMSCs with modification of CTLA4Ig or CD40LIg gene.
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Objective To explore the mechanism of microRNA (miRNA, miR)-155 in the rejection after liver transplantation in rats. Methods The rats were divided into two groups. In the xenograft model group (rejection group, n=10),the donors were male Lewis rats and the recipients were male BN rats.In the allograft model group(control group, n=10),both the donors and recipients were male Lewis rats.The rat models with orthotopic liver transplantation were established by two-cuff technique in two groups. At postoperative 7 d, the animals were sacrificed for the collection of blood and liver tissue samples. The serum levels of alanine aminotransferase (ALT), total bilirubin (TB), and cytokines of interleukin (IL)-2, IL-4, interferon (IFN)-γ were quantitatively measured. The pathological changes of liver tissues were observed under light microscope. In each group, three liver tissue samples were prepared and subject to high-throughput sequencing. The miRNAs related to rejection were identified for bioinformatics analysis to predict and analyze relevant signaling pathways and genes. Results In the rejection group, the serum levels of ALT and TB were significantly higher than those in the control group (both P<0.01). Compared with the control group, the levels of IL-2 and IFN-γ were considerably up-regulated (both P<0.01), whereas the level of IL-4 was dramatically down-regulated (P<0.01). Pathological examination demonstrated that more evident rejections were observed in the rejection group than the control group. High-throughput sequencing revealed that the expression level of miR-155 was significantly up-regulated in the rejection group, which was 5.89 times of that in the control group. Bioinformatics analysis demonstrated that up-regulation of miR-155 was associated with the mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK) and T cell receptor signaling pathways. The genes which were probably responsible for regulation included the yeast autophagy related gene 1(ATG1) and its homologous gene ULK2, insulin-like growth factor-1 (Igf-1) and G protein-coupled receptor regulatory gene(Arrb1),etc.Conclusions miR-155 might promote the incidence and progression of rejection after liver transplantation in rats. The involved signaling pathways probably include the mTOR, MAPK signaling pathway and T cell receptor signaling pathway.ATG1,ULK2,Igf-1,and Arrb1 genes may participate in this process.
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Objective To explore the effects of the Chinese medicine, modified Erchen decoction, on the serum lipid spectrum of ApoE-/-mice, and to explore its possible anti-atherosclerotic mechanism.Methods Forty-four male 7-8-week old ApoE-/-mice were used in this experiment.ApoE-/-mouse models of atherosclerosis were generated by high-cholesterol diet for 4 weeks.And then, they were given simvastatin or modified Erchen decoction by gavage.The body weight of mice was recorded every week, The mice were sacrificed after treated with the drugs for 8 weeks continuously, and the plasma lipid was determined by enzymatic method.The aortic valves and arches were stained with oil red O to depict atherosclerotic plaques and liver structural changes of the mice were examined by pathology.Results Modified Erchen decoction lowered plasma lipid ( including TCHOL and LDL-C ) significantly ( P<0.01 ) .The body weight was increased in the mice of all groups, but it was more pronounced in the mice of model group than in the blank and modified Erchen decoction groups.The serum CHOL and LDL-C levels were significantly lowered in the modified Erchen decoction group (P<0.01).The area of atherosclerotic plaques in the aortic wall was significantly reduced in the mice of modified Erchen decoction group as shown by oil red O staining.The pathological changes of hepatocytes were less severe and the structure of hepatic lobules was better preserved in the mice of modified Erchen decoction group.Conclusions The Chinese medicin modified Erchen decoction can effectively reduce serum lipids, regulate lipid metabolism, and ameliorate the process of atherosclerosis in ApoE-/-mice.
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To investigate the effects and mechanism of bone marrow mesenchymal stem cell (MSC)modified by cytotoxicity T lymphocyte-associated antigen 4-immunoglobulin (CTLA4-Ig)gene on the rejection of orthotopic liver transplantation (OLT)in rats. Methods MSC was infected with recombinant adenoviruses (Ad)5 containing CTLA4-Ig gene. After recombinant Ad-5 containing CTLA4-Ig infected MSC for 72 h,the total proteins were extracted. The protein expression of CTLA4-Ig was assessed by Western-blot.The suppression to lymphocyte proliferation by MSC and transgenic MSC were tested by cell counting kit (CCK)-8 analysis. Forty models of acute rejection after OLT in rats were established by modified Kamada’s two-cuff technique,with male Lewis and BN rats serving as liver donors and recipients respectively. Forty recipient rats were randomly divided into 4 groups with 10 rats in each group including control group (group A, only saline solution was injected into portal venous during transplantation),MSC group (group B,MSC was injected into portal venous during transplantation),transgenic MSC group (group C,transgenic MSC was injected into portal venous during transplantation),immunosuppressant group [group D,saline solution was injected into portal venous during transplantation,and ciclosporin (CsA)was administered intramuscularly at a dose of 1.5 mg /(kg·d) for 8 days]. On the 9 th day after operation,5 rats were killed randomly in every group,then the levels of interleukin (IL)-2,interferon (IFN)-γ,IL-4 in peripheral blood were measured and the pathological changes and rejection expression of liver tissues were observed by light microscope. The survival condition of other 5 rats in 4 groups was observed. Results After recombinant Ad-5 containing CTLA4-Ig infected MSC for 72 h,the protein expression of CTLA4-Ig gene in MSC infected with Ad5-CTLA4-Ig could be detected by Western-blot.When the ratios of MSC∶peripheral blood monouclear cell (PBMC)were 1∶10 and 1∶20,the rates of suppression to lymphocyte proliferation were 85.60% and 76.69% respectively.When the ratios of transgenic MSC∶PBMC were 1∶10 and 1∶20,the rates of suppression to lymphocyte proliferation were 90.50% and 84.20% respectively. Compared with MSC,MSC infected with Ad5-CTLA4-Ig had stronger effect on suppression to lymphocyte proliferation (P <0.05 ). The survival time after liver transplantation of rats in group A,B,C,D was (13 ±3),(41 ±6),(90 ±15),(102 ±18)d respectively.There were significant differences among group A,B,C (P<0.05 )and there was no significant difference between group C and D (P>0.05 ). Compared with group A,the serum levels of IL-4 in group B and C were significantly higher (P<0.05 ). The serum levels of IL-4 in group C were significantly higher than that in group B (P<0.05 ). There was no significant difference in the serum levels of IL-4 between group C and D (P>0.05 ). Compared with group A,the serum levels of IL-2 and IFN-γin group B and C were lower significantly (P<0.05 ). The serum levels of IL-2 and IFN-γin group C were significantly lower than those in group B (both in P<0.05 ). There were no significant differences in the serum levels of IL-2 and IFN-γbetween group C and D (P>0.05 ). The pathological result of liver tissues of rats showed that the grafts of group A developed severe rejection,and the grafts of group B developed moderate rejection. And the grafts of group C and D developed slight rejection. Conclusions MSC infected with recombinant Ad5-CTLA4-Ig can inhabit the rejection in liver transplantation,and the effect is superior to MSC alone.
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Objective To evaluate the protective effect of adiponectin in early atherosclerosis and the diagnostic value of adiponectin in metabolic syndrome in obese children. Methods Total 176 obese children and 88 normal weight children aged 9-12 years were included in the present study. All participants underwent hematologic and biochemical tests including serum adiponectin, high sensitivity C-reactive protein (hsCRP),fasting blood glucose, insulin, and plasma lipids. Homeostasis model assessment of insulin resistance (HOMA-IR)was calculated. Noninvasive ultrasound measurement including intima-media thickness of the common carotid artery(IMT), brachial flow-mediated dilatation (FMD), carotid artery compliance (CAC), and the maximum fatthickness ahead of peritoneum (Pmax) were obtained to investigate arterial mechanical properties and endothelial function. Results (1) The level of adiponectin was negatively correlated with obese index, blood pressure,fasting insulin, hsCRP, HOMA-IR, and IMT(P<0.05 or P<0. 01 ); but not with triglyceride, fasting blood glucose, CAC, high-density lipoprotein-cholesterol (HDL-C), and FMD. (2) The risk of metabolic syndrome increased 3.43 times in children with adiponectin level <7. 060 mg/L compared with ＞7. 060 mg/L. (3)Receiver operating characteristic( ROC ) curve was used to choose the optimal cutpoint of adiponectin to identify obese children with the metabolic syndrome. The area under the curve (AUC) for adiponectin to discriminate the sensitivity of metabolic syndrome was 0. 769 (95% CI0. 714-0.816, P< 0. 0 1 ). (4) The obese children were divided into three groups according to the cut-off value for adiponectin (high, middle, low groups). There were significant differences in the prevalences of severe obesity, visceral fat accumulation, hypertension, insulinemia,low HDL-C, metabolic syndrome among three groups (P<0.05). Conclusions High levels of serum adiponectin could prevent early stage of atherosclerosis. The lower the adiponectin level, the higher the incidence of metabolic syndrome.
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ObjectiveTo investigate the effect of pravastatin on serum levels of interleukin-6(IL-6) and tu- mor necrosis faetor-α(TNF-α) in patients with acute coronary syndromes(ACS). Methods50 patients with ACSwere randomly divided into pravastatin group( n = 25) and routine therapy group( n = 25). Serum TNF-α and IL-6levels were measured before and four weeks after the two treatment options respectively. ResultsThe level of TNF- α and IL-6 were higher than routine therapy group before therapy(P < 0.01 ). The level of TNF-α and IL-6 weredecreased significantly after pravastatin therapy,and higher thancontrols(P < 0.05). The levels of TNF-α and IL-6changed only slightly after the routine therapy(P > 0.05). ConclusionThe level of TNF-α and IL-6 becominghigher in ACS patients may be related to the pathogenesis of ACS. Pravastatin can reduce serum level of TNF-α and IL-6 contributing to treatment of ACS.