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Objective To investigate the severity of allogenic rejection after partial liver transplantation (PLTx) with different graft weight to recipient liver weight (GW/RLW).Methods The full-size liver transplantation (group A),GW/RLW ＞33％ PLTx (group B) and GW/RLW ＜ 30％ PLTx (group C) were set up using BN rats and Lewis rats as donors and recipients,respectively.All recipients were observed for 28 days.The Banff RAI grading,survival rate,jaundice and body weight recovery were evaluated to determine the severity of acute allogeneic rejection.Two PLTx groups,group B1 (GW/RLW＞33％) and group C1 (GW/RLW＜30％),were established to assess the mRNA level of IL-2,GranzymeB,Perforin and CD3 48 h and 7 days postoperatively.Additionally,the mRNA level of B7-H1,the ratio of Ki67 + hepatocytes and the liver enzymes were also assessed 7 days postoperatively.Results All recipients in group C died within 22 days postoperatively,presenting with severe lymphocytic infiltration and vascular endothelialitis.All recipients in group A and group B survived until the end of observation time.All recipients in group A survived and presented with a mild lymphocytic infiltration and rare vascular endothelialitis.Group B presented with moderate lymphocytic infiltration and moderate vascular endothelialitis.The Banff RAI grading in group C was significantly higher than that in group A and group B (P ＜ 0.05).In accordance with the result of histology and survival rate,group B and group C presented with earlier jaundice and lower body weight recovery than that of group A (P＜0.05).As compared with group B1,group C1 presented with higher mRNA levels of Perforin,GranzymeB,IL-2 and CD3,higher level of liver enzymes and heavier liver graft weights.Besides,the mRNA level of immunosuppresive molecule B7-H1 in group C1 was lower than that of group B1.However,there was no significant difference in the ratio of Ki67 + hepatocytes between group B1 and group C1.Conclusion The allogenic liver rejection may be enhanced by reducing the GW/RLW.
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Objective:To explore the effect of adipose derived mesenchymal stem cells to regulate the differentiation of macrophage RAW264.7.Methods:First,we used RAW264.7 cells to simulate macrophage and induced them to M 1 macrophage with lipopolysaccharide ( LPS,1 μg/ml) .Then we cultured these RAW264.7 cells in culture mediums which were previously used to culture adipose derived mesenchymal stem cells to imitate the transplantation of ADMSC .Last,the mRNA relative expression of IL-10, IGF-1,Arg-1,TNF-α,FIZZ1,SPHK-1 was detected by real-time PCR.The protein expression of IL-12 p40,IL-27 Rα,IL-10 was detected by Western blot.Results:After been cultured in ADMSCCM and induced by LPS ,M1 markers (TNF-αmRNA,IL-12 p40;P<0.05) of the RAW264.7 cells declined while M2 markers (IGF-1 mRNA,IL-10 mRNA,IL-10;P<0.05) rose.Conclusion: ADMSC can secrete soluble cytokines to induce the RAW264.7 cell,which have been induced to the M1 macrophages,to differentiate towards M2 macrophages.
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Objective To investigate the risk factors of chylous leakage after pancreatioduodenectomy so as to find effective measures to prevent this complication.Methods A retrospective analysis was conducted on 230 patients who underwent pancreatioduodenectomy at the First Affiliated Hospital of Zhejiang University from Jun.2012 to Jun.2014.Patients with chylous leakage were identified and a 1 ∶ 2 patients in the study and the control groups were selected.The parameters for matching included tumor volume,vascular invasion,and extent of lymph node dissection.A logistic analysis was performed to identify independent risk factors of chylous leakage.Results 15 (6.5％) patients developed chylous leakage after pancreatioduodenectomy.The average hospital stay after surgery of the study group was 20.8 days,compared to 13.5 days in the control-group (P =0.004).In the study group,chylous leakage rate increased in patients with 14th and 16th group of lymph nodes dissection (80％ vs 36.7％,P =0.006).Logistic analysis showed that 14th and 16th lymph nodes dissection was an independent risk factor of chylous leakage after pancreatioduodenectomy (P ＜ 0.05,OR =6.909,95％ CI 1.593 ～ 29.958).Conclusions Chylous leakage prolonged hospitalization after pancreatioduodenectomy.Dissection of the 14th and 16th lymph node groups was an independent risk factor of chylous leakage after pancreatioduodenectomy.Careful ligation of the gastrocolic vein near the lymphatic trunk and dissection of 14th and 16th group of lymph nodes were effective interventions to reduce postoperative chylous leakage.
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OBJECTIVE To investigate the effect ofγ-secretase inhibitor N-[N-(3,5-difluorophen?acetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)on phenotypic transformation and matrix accu?mulation induced by aristolochic acid(AA) in renal tubular epithelial cells(NRK-52E)and explore the mechanism. METHODS NRK-52E cells were divided stochastically into normal cell control group,AA 10 mg·L-1 group and AA 10 mg·L-1+DAPT 1 and 10μmol·L-1 group. After 24 h,the mRNA expressions of Notch1,Jagged1,Numb,E-cadherin,transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),bone morphogenic protein 7 (Bmp7),typeⅠ a1 (Col1a1) and Ⅲ collagens a1 (Col3a1)were quantified by quantitative real-time RT-PCR. The protein expressions of Notch1,Jagged1,α-SMA,and Col3a1 in NRK-52E cel s were detected by immunofluorescence staining. RESULTS In NRK-52E cells,AA enhanced the expression of TGF-β1,α-SMA and Col3a1 mRNA(P<0.05),reduced the expression of E-cadherin mRNA(P<0.05),up-regulated the mRNA expression of Notch1 mRNA(P<0.01)and Jagged1(P<0.05),and down-regulated the mRNA expression of Numb mRNA(P<0.05) compared with normal cell control group,indicating that phenotypic transformation and matrix accumu?lation occurred in AA-treated NRK-52E cells,accompanied by activated Notch signaling. Treatment with DAPT inhibited Notch signaling by decreasing the expression of Notch1 and Jagged1 (P<0.05),and increasing the expression of Numb mRNA(P<0.05). Furthermore, DAPT also down-regulated the expression levels of TGF-β1,α-SMA,Col1a1 and Col3a1 mRNA(P<0.05), and up-regulated the expression level of Bmp7 and E-cadherin mRNA(P<0.05) compared with AA group,suggesting that DAPT inhibited phenotypic transformation and matrix accumulation in AA-treated NRK-52E cells. CONCLUSION AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells,which is inhibited by DAPT treatment. The possible mechanism is that DAPT suppresses the activation of Notch signaling,resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.
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<p><b>OBJECTIVE</b>To explore the central mechanism of postoperative fatigue syndrome by detecting the expression of NMDA receptor and tryptophan metabolism.</p><p><b>METHODS</b>After being numbered according to the weight, ninety-six male SD rats were randomly divided into control group (bowel loop was flipped after laparotomy and received intraperitoneal injection of saline at a dose of 1 ml/kg), POFS model(70% of the length of small intestine was resected and received intraperitoneal injection of saline at a dose of 1 ml/kg), and NMDA antagonist groups(70% of the length of small intestine was resected and received intraperitoneal injection of MK801 at a dose of 1 ml/kg). Each group was divided into subgroups by postoperative 1, 3, 5 and 7 d, with 8 rats in each subgroup. The hippocampus was removed at each time point after open field test (OFT) to detect the mRNA expression levels of NMDA receptor 1 and kynurenine aminotransferase III((KATIII() by real-time PCR. Protein level of NMDA receptor 1 was detected by Western blot. High performance liquid chromatography (HPLC) was used to measure the concentrations of tryptophan (TRP), kynurenine (KYN) and kynurenic acid(KYNA). Ultra-structural changes of hippocampal neurons were observed by transmission electron microscopy(TEM).</p><p><b>RESULTS</b>As compared to control group, exercise score decreased(P<0.05), rest time and central panel residence time prolonged, periphery/central panel ratio increased (all P<0.05), mRNA and protein expressions of NMDA receptor 1 increased (P<0.05), mRNA expression of KAT III( decreased (P<0.05), KYN/TRP ratio and KYN/KYNA ratio decreased (all P<0.05) in POFS group on postoperative day 1 and 3. As compared to POFS group, central panel residence time and periphery/central panel ratio decreased on postoperative day 1, and mRNA and protein expressions of NMDA receptor 1 decreased on postoperative day 1 and 3 (all P<0.05) in antagonist group. TEM revealed that degenerated neuron was found in the hippocampus of POFS rats, while such damage was improved in antagonist group.</p><p><b>CONCLUSION</b>The increased expression level of NMDA receptor may play an important role in POFS. NMDA receptor antagonist MK801 may improve the POFS.</p>
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Animals , Humans , Male , Rats , Fatigue , Hippocampus , Injections, Intraperitoneal , Postoperative Period , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate , Signal Transduction , TransaminasesABSTRACT
Objective To investigate the mechanism of islet transplantation lessening renal damage of diabetic nephropathy (DN) rat model.Method Rat DN model was established by intraperitoneal injection of a single-dose streptozotocir.The rats were divided into normal control group, DN group and islet transplant group.Islet transplantation was taken on the right renal capsule in transplantation group at 8th week after the modeling.At week 12 after the modeling, the urinary protein, urinary creatinine and blood glucose in each group were determined.The kidneys were collected, and transplant islet HE staining, immunofluorescence, kidney electron microscopy were done.Synaptopodin and transforming growth factor-β1 (TGFβ1) protein expression was observed in renal tissues of each group by immunohistochemical staining.Result The urine protein and urinary creatinine ratio in islet transplant group was significantly lower than in DN group (P＜0.001).Blood glucose level in islet transplant group had no significant difference (P＞0.05) with the control group, but was significantly lower than in DN group (P ＜ 0.05).Islet cells by HE staining and immunofluorescence staining showed new blood vessels around the islets, and the insulin secretion was exuberant.Under an electron microscope, there was local fusion of podocyte foot processes, and segmental thickening of the basement membrane in DN group;in islet transplant group, the foot processes of podocytes were neat, basement membrane structure was clear and had no thickening.Synaptopodin protein expression was significantly decreased in the glomeruli of DN group and islet transplant group as compared with the control group (P＜0.05), and that in islet transplant group was significantly enhanced (P＜0.05) as compared with DN group.The expression of TGFβ1 in DN group and islet transplant group was significantly increased (P＜0.05) as compared with control group, and that in DN group was significantly higher (P＜0.05) than in islet transplant group.Conclusion Islet transplantation can inhibit TGFβ1 pathway, improve DN podocyte injury in rats, and alleviate or even reverse proteinuria.
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AIM:To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transfor-mation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E.METHODS:NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at con-centrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10μmol/L).After cultured for 24 h, the mRNA expression of Ptch1, Smo,α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR.The protein levels of Shh and TGF-β1 were detected by ELISA .Immunofluorescence staining was used to evaluate the expression of Ptch 1, Smo,α-SMA, E-cadherin and type III collagen in the NRK-52E cells.RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling , and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells.Treatment with cyclopamine inhibited HH signaling by decrea-sing Smo expression and increasing Ptch 1 expression.Moreover, cyclopamine also down-regulated the expression of TGF-β1,α-SMA, type I collagen and III collagen , and up-regulated the expression of BMP-7, ZO-1 and E-cadherin.CON-CLUSION:AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells , which can be inhibited by cyclopamine treatment .The possible mechanism is that cyclopamine suppresses the activation of HH signaling , resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition .
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Objective To investigate the expression of monocyte-macrophage-related factors and interstitial fibrosis in kidney tissues of rats with ureter obstruction and recanalization .Methods Forty-eight male Spragur-Dawley rats were divided randomly into the obstructive group:sham (n=6), unilateral ureteral obstruction(UUO)3 days (n=6), UUO 7 days (n=6), and UUO 14 days (n=6) and recanalization group:bilateral ureteral obstruction(RBUO)0 day (n=6), 3 days after RBUO (n=6), 7 days after RBUO (n=6), and 14 days after RBUO (n=6).The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fibers in kidney was detected with HE and Masson staining . Immunohistochemical analysis was performed to evaluate the protein expressions of monocyte chemoattractant protein -1 (MCP-1), macrophage colony-stimulating factor (M-CSF) and activated-macrophage marker CD68.Real-time PCR was used to detect the mRNA expressions of MCP-1 and M-CSF.TGF-β1 levels were determined by ELISA .Results Fibrosis observed with HE and Masson staining was obviously increased in kidney tissue of UUO rats , and aggravated as time prolonged, but alleviated in rats with recanalization .TGF-β1 levels were increased obviously in the UUO group , but decreased in rats with recanalization compared with those in BUO rats .In UUO rats, mRNA and protein expression levels of MCP-1 and M-CSF were increased .MCP-1 and M-CSF expression was gradually decreased in rats with recanalization compared with those in BUO rats .The dynamic change in expression of MCP-1 and M-CSF in both UUO rats and recanalization rats was consistent with the change in expression of CD 68. Conclusion Dynamic change in expression of MCP-1 and M-CSF in kidney tissues reflects change of activated and accumulated monocyte -macrophages , which may be one of the major mechanisms contributing to fibrosis induced by ureter obstruction .Renal fibrosis is alleviated by down-regulated expression of monocyte-macrophages factors with recanalization operation .
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OBJECTlVE To investigate the association between CYP3A5 genotypes and the early efficacy of tacrolimus ( Tac) and cyclosporin A ( CsA) in renal transplantation recipients, and provide a basis for individualized treatment. METHODS Seventy-four kidney transplantation recipients were en-rolled in this study between August 2012 and April 2013. Thirty-one patients were treated with the combi-nation of CsA, MMF and methylprednisolone while the rest were treated with Tac, MMF and methylpred-nisolone. The genotype CYP3A5 was detected by sequence specific primer-polymerase chain reaction ( SSP-PCR) before transplantation. The levels of Tac and CsA were detected by ELlSA and chemilumi-nescence, respectively, to monitor the blood concentration/dose of drugs ( c/D) at 2 weeks, 1 month, 2 months, 3 months and 6 months after transplantation. Simultaneously, the concentrations of blood glu-cose, creatinine, urea nitrogen and uric acid were determined with hexokinase method, creatininase method, urease method and uricolase method, respectively. RESULTS Among the 74 recipients, 9.5%carried CYP3A5?1/?1, 48.6%carried CYP3A5?1/?3 and 41.9%carried CYP3A5?3/?3. According to the phenotype of CYP3A5, the patients were divided into CYP3A5 expression group ( including CYP3A5?1/?1 and CYP3A5?1/?3) and non-expression group ( including CYP3A5?3/?3) , which accounted for 58.1%and 41.9%of the cases, respectively. Among the patients taking Tac, the median value of c/D at 2 weeks, 1 month, 2 months, 3 months and 6 months was 25.49, 49.64, 53.72, 51.9 and 44.5 in CYP3A5 expression group, and 65.48,100.84,99.54,123.01 and 133.21 in non-expression group. The c/D ratio of CYP3A5 non-expressers was higher than among CYP3A5 expressers at each time point ( P<0.05) . The initial dose of Tac 0.1 mg·kg-1 was high for CYP3A5 non-expressers, and the kidney function recovered more slowly than among CYP3A5 expressers and kidney damage occurred. However, there was no association between CYP3A5 genotype and the early efficacy of CsA. The levels of blood glucose, creatinine, urea nitrogen and uric acid were not significantly different between CYP3A5 expression and non-expression groups. CONCLUSlON CYP3A5 non-expression recipients whose starting amount of Tac was 0.1 mg·kg-1 have drug overdoses. CYP3A5 genotype is one of the factors affecting the efficacy of Tac. CYP3A5 genotype has no association with the efficacy of CsA in renal transplantation recipients.
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[ABSTRACT]AIM:ToinvestigatetheeffectofimmunosuppressantFK506onserumglucoseinratsandtoex-plore its mechanism .METHODS: Sprague-Dawley rats ( n =12 ) were randomly divided into drug group and normal group.The rats in drug group were intraperitoneally injected with FK 506 at dose of 1 mg· kg-1 · d-1 and the rats in nor-mal group received saline (1 mL· kg-1 · d-1 , ip) for 14 d.The fasting weight and fasting glucose were regularly meas-ured every 2 d.Visceral fat was isolated from the rats at the end of experiment .The mRNA expression of adiponectin , lep-tin, visfatin, resistin, retinol-binding protein 4 ( RBP4) and peroxisome proliferator-activated receptors γ( PPAR-γ) was determined by real-time fluorescence quantitative PCR .The protein expression of PPAR-γand adiponectin was measured by Western blotting .RESULTS:Compared with normal group , the concentration of fasting blood glucose in model group was significantly increased from the 10th day (P<0.05).At day 14, the fasting blood glucose of the model group increased from (5.10 ±0.62) mmol/L to (7.73 ±0.73) mmol/L.No significant change of blood glucose in normal group between the 10th day and the 14th day [from (4.66 ±0.32) mmol/L to (5.80 ±0.10) mmol/L] was observed.Compared with normal group , the mRNA expression of PPAR-γ, adiponectin and leptin in the adipose tissue of model group was signifi-cantly decreased ( P <0.01 ) , whereas the expression of visfatin , resistin and RBP4 was significantly increased ( P <0.05).Compared with normal group, the expression of PPAR-γand adiponectin in model group was decreased (P <0.01).CONCLUSION:FK506 may decrease the expression of PPAR-γto change the expression of adipocytokines and induce hyperglycemia in rats .
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OBJECTIVE To investigate the molecular mechanisms of resveratrol( Res)in renal interstitial fibrosis(RlF)in rats with unilateral ureteral obstruction(UUO). METHODS Forty-eight Spra-gur-Dawley rats were randomly divided into UUO( normal saline,n = 16),UUO with Res treatment (Res,20 mg·kg-1 ,n=16),and sham-operation(sham,n=16)models. The kidneys were excised on the 7th and 14th day. The deposition of collagen fiber in the kidney was detected with HE and Masson staining. The levels of sonic hedgehog(SHH,an inducer of SHH pathway)in kidney tissues were deter-mined by ELlSA. lmmunohistochemical analysis was performed to evaluate the protein expression of SHH signaling-related molecules,including SHH,smoothened(Smo),patched-1(Ptch1),and Gli1, proliferating cell nuclear antigen(PCNA)and matrix component typeⅢ collagen. The mRNA expression levels of Smo,Ptch1 and Gli1 were detected by real-time RT-PCR. RESULTS The degree of RlF observed with HE and Masson staining was obviously increased in UUO kidneys,but decreased in Res-treated kidneys. Enhanced expression levels of typeⅢ collagen and PCNA in UUO rats were suppressed by Res treatment(P﹤0.05). Res administration decreased the expression levels of SHH,Smo,and Gli1 (P﹤0.05),but increased the expression of Ptch1(P﹤0.05),suggesting that Res inhibit the obstruction-induced activation of SHH signaling. CONCLUSION Res can attenuate RlF in UUO rats,and the possi-ble mechanism is that Res down-regulates the activity of SHH signaling and inhibits cellular proliferation, resulting in inhibition of matrix accumulation in renal interstitium of UUO rats.
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Proteomic technologies have provided effective approaches to the analysis of the pathogenesis of hepatic fibrosis. A large number of proteins that have been revealed by this technology play a critical role in various aspects of pathological liver fibrosis. Comprehensive evaluations of these proteins have led to the understanding that the mechanisms of hepatic fibrosis can be stratified into several broad classifications. Here, we describe the mechanisms of action that are defined as being related to 1] oxidative stress and mitochondrial damage, 2] inflammatory response and immune injury, 3] abnormal cell proliferation and apoptosis, 4] abnormal metabolism, 5] abnormal cellular signal transduction, and 6] abnormal extracellular matrix metabolism
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Objective To investigate the efficiency of Trichostatin A (TSA) in inducing cell apoptosis and altering the Notch pathway genes expression in PANC-1 cells line.Methods The survival rate and apoptosis of PANC-1 cells were measured by MTT assay and Hoechst 33258 staining,respectively.mRNA expression levels of the genes,numb,gcn512,dll3,hes6,eaf2,cytohesins,in PANC-1 cells were assessed by real-time quantitive PCR.Western blot was used to measure the expression of bcl-2,bax,actived caspase-3 and NICD protein which was the biologically active form of Notch-1.Results After culturing with 0.1,0.2,and 0.4 μmol/L TSA for 24 hours,the cellular survival rate of PANC-1 cells significantly decreased to 72％,58％ and 39％,respectively.The survival rate of PANC-1 was negatively correlated to time length of culture with TSA.Increased apoptosis of PANC-1 cells after 12,24 and 36 h culture with TSA was detected by Hoechst 33258 staining.Western blotting showed that the expression of bax,actived caspase-3 and NICD protein increased while the bcl-2 protein decreased after culture with TSA.In real time quantitive PCR assessment,the mRNA expression of numb and hes6 in PANC-1 cells were upregulated by TSA (P ＜ 0.05),while the mRNA expression of gcn512 and dll3 were down-regulated by TSA (P ＜ 0.05).While mRNA expressions of eaf2 and cytohesin1,2,3,4 were not affected by TSA.Conclusions TSA induces apoptosis of pancreatic cancer cell line PANC-1.The Notch signal pathway may be involved in inducing cellular apoptosis of PANC-1 when cultured with TSA.
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ObjectiveTo investigate the effect of perioperative use of fish oil on post-operative fatigue(POF) of rat.MethodsAfter one week's preoperative behavior training,12 rats presented poor behavior were excluded from 60 healthy adult male SD rats as the normal controls of serum parameters.The remaining 48 rats were randomly divided into model group and fish oil treatment group by random number table.The fish oil treatment group received 10 days' (3 days before surgery and 7 days after surgery) intraperitoneal injection of fish oil [2 ml/( kg · d) ],and the model group with saline.On the 1st,3rd,5th,and 7th post-operative day,rats were assessed by Morris water-maze and tail suspension test.Serum levels of interleukin ( IL)-1β,IL-6,tumor necrosis factor-α (TNF-α),superoxide dismutase (SOD),and glutathione peroxidase (GSH-PX) were measured.ResultsSerum parameters:on the 1st and 3rd post-operative day,the IL-6 level in the fish oil treatment group [ (66.22 ±8.80),(56.03 ± 1.19) pg/ml] was significantly lower than in model group [ (83.30 ± 10.69),(82.72 ± 24.27) pg/ml ] (P =0.034,P =0.038 ) ; on the 1 st,3rd,5th,and 7th post-operative day,the TNF-α level in the fish oil treatment group [ ( 104.36 ±5.02),(84.49 ±7.81 ),(64.47 ±2.89),(39.29 ±2.52)pg/ml ] was significantly lower than in model group [ ( 120.01 ± 14.99 ),( 119.68 ± 8.84),(75.29 ± 2.58 ),(41.96±1.65) pg/ml] (P=0.014,P=0.003,P=0.000,P=0.004); onthe1st,3rd,5th,and 7th postoperative day,the IL-1β level [(155.11 ±9.08),(79.39±5.86),(57.26±16.07),(35.42±1.53) pg/ml]was significantly lower than model group [ (204.87±30.61),(198.82±54.83),(152.12±29.06),(64.35 ± 2.70) pg/ml ] ( P =0.024,P =0.002,P =0.000,P =0.000) ; on the 5th postoperative day,SOD ( 1.08±0.08) μmol/L was significantly higher than model group (0.71±0.06) μmoL/L (P=0.000) ; on the 5th and 7th postoperative day,GSH-PX [ (31.21 ± 1.30), (30.78 ± 1.83) μmol/L] was significantly higher than model group [ (25.03 ±1.74),(27.57±3.57) μ mol/L](P=0.000,P=0.036).Behavior:in tail suspension test,on the 1st and 3rd postoperative day,value of struggle in fish oil treatment group [ (6620 ± 1390),(7011 ± 1472) mv · s] was significantly higher than in model group [ (4739 ± 1040),(4344 ± 1130) mv · s](P=0.048,P=0.043); cumulative fixed time [ (118.42±10.05), (101.02±8.68) s] and single rest time [ (55.39±7.70),(56.60±5.88) s] was lower thanin modelgroup [ (135.08+12.44),(131.02±9.24) s; (65.73±3.78),(64.93±3.25) s] (P=0.042,P=0.012,P=0.043,and P=0.042).In Morris water-maze,on the 3rd and 5th postoperative day,escape latent period of fish oil treatment group [ (48.263 ±1.815),(44.955±2.567) s] was lower than model group [ (51.543±1.990),(49.956±2.888) s] (P=0.035,P=0.035) ; on the 1st,3rd,5th,and 7th postoperative day,the cross platform number (1.04±0.25,1.95±0.49,2.42 ±0.41,3.21 ±0.53) was significantly higher than in model group (0.58 ±0.26,1.20±0.33,1.50±0.39,2.17±0.68) (P=0.002,P=0.003,P=0.018,P=0.035).ConclusionPerioperative use of fish oil can reduce postoperative inflammatory response,enhance antioxidant defense capability,and mitigate post-operative fatigue.
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Objective To investigate the effects and mechanism of Cordyceps sinensis on transplant arteriosclerosis in a rat model Methods Brown Norway aortic allografts were transplanted into Lewis recipient rats,and the recipient rats were randomly divided into four groups:group A,isograft control group,Lewis-Lewis (n =10); group B,allograft control group,BN-Lewis (n =10); groups C and D,allograft experimental groups,BN-Lewis (n =10).Rats were given normal saline via intragastric injection,once every day for 60 days in groups A and group B,and received different doses (1.5 and 3 g·kg-1 ·d 1) of Cordyceps sinensis in groups C and group D respectively.Grafts were harvested on the day 60 after transplantation. Intimal thickness was detected by HE staining.Protein was extracted from the abdominal aortas for Western blotting.Cellular localization was assessed by histology and immunohistochemistry.The serum was analyzed by an enzyme-linked immunosorbent assay (ELISA). Results Transplanted arteries were normal in group A.Transplanted arteries in group A had allograft vasculopathy,and intimal thickness was significantly increased.Transplanted arteries in allograft experimental groups had endometritis changes,and intimal thickness was significantly decreased as compared with that in group B (P ＜ 0.05).Immunohistochemistry and Western blotting revealed that the expression levels of VEGF and PDGF-BB proteins in group A were significantly higher than in group B,and those in groups C and D were significantly reduced as compared with group B (P＜0.05).ELISA showed that serum VEGF and PDGF BB concentrations in group B were significantly increased as compared with group A (P＜0.05).Serum VEGF and PDGF-BB concentrations were significantly reduced in groups C and D as compared with group B (P＜0.05).Conclusion Cordyceps sinensis could significantly inhibit the intimal hyperplasia,and delayed transplant arteriosclerosis caused by chronic rejection,which may be related to the down-regulated expression of VEGF and PDGF-BB.
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Objective To investigate the effect of the polymorphisms of multidrug resistance 1 (MDR1) C3435T and G2677T on Tacrolimus (Tac) individualized treatment and prognosis of grafts in the renal transplantation recipients (RTRs).Methods One hundred and twenty-seven RTRs who treated with Tac regimen and had a stable graft function were enrolled,and were divided into adjuvant treatment group and non-adjuvant treatment group according to whether given adjuvant drugs to raise Tac trough concentrations. MDR1 C3435T and G2677T SNPs were detected by using sequence specific primers PCR.Tac trough concentrations of whole blood were measured by using enzymelabeled immunosorbent assay.Tac concentration-to-dose ratio (C/D) standardized by body weight was compared according to the various genotypes and haplotypes of MDR1 C3435T and G2677TA SNPs.Results Adjuvant treatment group including 36 recipients had a higher frequency of C genotype of C3435T than un-adjuvant treatment group (68.05％ vs 48.35％,P ＜ 0.01 ). The frequency of G2677TA polymorphisms was of no significant difference between the two group recipients (P＞ 0.05).As to non-adjuvant treatment recipients,the mean Tac DD required and C/D were not significantly different among various polymorphisms of MDR1 G2677T/A and C3435T or various haplotypes (P＞0.05).During A follow-up period of 4 years,13 recipients suffered graft dysfunction in which 84.6％ (11/13) carried 3435C genotype (P＞0.05).Conclusion The frequency of MDR1 C3435T polymorphisms in RTRs is high in the recipients given adjuvant treatment to raise Tac concentrations.Recipients with 3435C genotype were prone to graft dysfunction.
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Objective To establish a universal stem loop primer (USLP) based real-time PCR method to scan mature miR profile and quantify it's expression.Methods The common universal stem-loop primer pairs were re-designed; 8 random nucleotides were introduced at 3 ' end for reverse transcription of the mature miR,establishing a miR scanning and quantifying system based on SYBR Green Ⅰ PCR (improved USLP method).10-fold gradient diluted standard miRNA-155 cDNA ( 1 ～ 109 copies/μ1) were utilized to evaluate the sensitivity of this method.The specificity was verified by melting curve assay; the precision was assessed by intra-assay coefficient of variation (ICV) of threshold cycle (Ct value) through 20 repeated detections of the standard miR-155 cDNA (2 × 105,2 × 106,2 × 107 copies/μl) ; cost of the primers and time were evaluated,compared with that of the conventional USLP method.Peripheral blood samples were cultured with phytohaemagglutinin (PHA) for0 h,16 h,24 h,48 h and 72 h,and 87 candidate miR that may be associated with human immunity from PubMed data were scanned and quantified from the cultured T cells.Results The sensitivity of the improved USLP method was 103 copies/μl of standard miR-155 cDNA.Melting curve assay showed a single melting peak at 80 ℃,suggesting the excellent PCR specificity of miR-155.Precision of our method quantifying miR-155 was acceptable (ICV ＜ 2.5％ ).Compared with the traditional stem loop primers,our method saved 75％ cost of primers ( 1 917 bp vs 7 851 bp) and 60％ test time of reverse transcription (85 min vs 205 min).By our method,85 of the 87 miR expression in T cells had no significant difference after the PHA stimulation; the expression of miR-150 (72 h) decreased by 10 times and that of miR-155 (48 h) increased 8 times after culture with PHA (Z =-2.032,P =0.042;Z =- 2.023,P =0.043,respectively ).Conclusions The improved USLP method is fast,precise,sensitive,and cost-effective.It could be used for miR profile scanning and quantifying in T cells.
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Objective To observe the effects of mycophenolate mofetil (MMF) on the differentiation and proliferation of Helper T cells 17 (TH 17),so as to reveal its role and the possible mechanism in inducing immunological suppression.Methods Sixteen Balb/c mice of SPF level aged 8 weeks were randomly divided into two groups:MMF group and control group,with 8 mice in each group.In MMF group,the mice received intragastric administration of MMF (40 mg·kg-1· day-1 ),and those in control group received intragastric administration of identical volumetric saline every day.After three weeks,peripheral blood was collected and spleen cells were prepared.Flow cytometry was used to determine the proportions of CD4+ TH 17 and CD4+ CD25+ Tregs,then the ratio of TH 17/Tregs was calculated,and the concentrations of interleukin-1 7 (IL-1 7) and interleukin-23 (IL-23) in serum were measured by ELISA.Results The proportion of CD4+ TH 17 in the peripheral blood and spleen was (1.95 ± 0.08) and (2.42 ± 0.06) in MMF group,and (3.19 ± 0.07)％ and (4.21 ± 0.25)％ in control group,respectively.There were significant differences between the two groups (P ＜0.05).Meanwhile,the ratio of TH 17/Tregs in MMF group,both in the peripheral blood and spleen,was significantly decreased as compared with the control group (P＜0.05).The concentration of IL-17 in MMF group was lower,but that of IL-23 in MMF group was higher than in the control group (P＜0.05).Conclusion MMF could obviously suppress the differentiation and proliferation of CD4+ TH 17 in vivo,reduce the ratio of TH17/Tregs and the IL-17 secretion,thus facilitate the induction of immune tolerance.
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Objective To develop the hypothesis ‘saturated or non-saturated cytotoxicity model' and explain the various phenomena of antibody mediated immunoresponses in recipients,including rejection and accommodation.Methods The imitating complement dependent cytotoxicity.The threshold set to identify as saturated or non-saturated cytotoxicity depends on antigen-antibody complex(R)whether or not above lethal number(D)in effective time.Feasibility of the hypothesis was examined through explaining various phenomena mediated by anti-donor antibodies,especially some contradictory phenomena.Results Hyperacute rejection,accelerated rejection and acute rejection could be well explained by saturated cytotoxicity.Accommodation of ABO imcompatible transplantion,de novo antibody induced injury,change of protein profile,and C4d deposition in graft could be well elucidated by the hypothesis.Conclusion The hypothesis saturated or nonsaturated cytotoxicity model' help to interpret and interconnect various phenomena of antibodies mediated immune response,such as rejection and accommodation.
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Objective To investigate the expression of survivin in T lymphocytes that were stimulated by Con A and alloantigens in renal grafts in vitro and in vivo. Methods According to the different treatments, the experiment was divided into three parts. (1) The C57BL/6 mice splenocytes stimulated by Con A (10 mg/L) were cultured in the RPMI-1640 medium. Following the proliferation blockade or not, the expression of Survivin in the splenocytes was detected. (2) The GVHR model was established by transfusing the C57BL/6 mice splenocytes into Balb/c× C57BL/6 F1 mice, and the expression of Survivin in the donor splenocytes was detected at the different time points. (3) Seventythree cases of clinical renal allograft biopsy specimens were collected, pathologically diagnosed and classified according to the Banff 97 classification, and then the expression of Survivin was detected.Results Survivin was expressed in the CD3+ splenocytes that received Con A stimulation. The positive cell count reached the peak on the day 3, and subsequently declined. In the GVHR model, the lymphocytes infiltration and Survivin expression were detected around the portal vein and portal area on the post-splenocytes-transfused day (PSTD) 4 to 12. But on the PSTD 14, the Survivin expression could not be detected in the infiltrated lymphocytes. In the renal allograft biopsy specimens,lymphocytes did not express Survivin in 13 specimens of the group without acute cellular rejection.was difference between the two groups (P＜0. 01 ). Conclusion The activated T cells possess the capacity to express Survivin, and the expression is time-dependent. For the characteristics of Survivin expression of T cells, it may be applied as an approach to diagnose the acute cellular rejection and judge its degree and stage in the clinical allograft biopsy specimens.