Objective To investigate the effect of cyclosporine A (CsA) on autophagylysosomal pathway in tubular epithelial cells.Methods Human renal tubular epithelial cell line (HK-2 cell) was treated with different concentrations (3,5 and 10 μmol/L) of CsA for 24 h.Then the viability and apoptosis of cells were measured by MTT assay or AnnexinV-PI staining followed by flow cytometry analysis,respectively.Autophagy-related protein LC3-Ⅱ and p62 were detected by immunofluorescence assay.Autophagic flux was analyzed in HK-2 cells transfected with a tandem mRFP-GFP fluorescent-tagged LC3 (ffLC3) plasmid by laser confocal microscope.The lysosomal degradation was evaluated by DQ-ovalbumin staining followed by flow cytometry analysis.Results The viability of HK-2 cells was significantly decreased with CsA stimulation when compared with control group (P ＜ 0.01),but the number of apoptotic cells was markedly increased by CsA treatment (P ＜ 0.05).Compared with the control group,different doses of CsA dramatically increased the expressions of LC3-Ⅱ (P ＜ 0.01) and p62 (P ＜ 0.05) in HK-2 cells.Moreover,HK-2 cells treated with CsA displayed a significant increase in autophagosomes but a marked decrease in autolysosomes.In HK-2 cells,exposured to CsA caused a decrease in lysosomal degradation by DQ-ovalbumin staining when compared with control group (P ＜ 0.01).Conclusion Blockade of autophagy via disrupting lysosome degradation may represent a novel mechanism of CsA-induced tubular epithelial cells injury.

Objective To investigate the role of basophils in the imbalance of Th 1/Th2 response in mouse models of collagen-induced arthritis(CIA).Methods 4-6-weeks old C57/BL6 mice were immunized with collagen at multiple points on the back and foot twice (0 and 3 weeks) to establish a mouse model of collagen-induced arthritis.Blood samples were collected before the first immunization and 1, 3, 6 and 9 weeks after immunization , and cells from lymph nodes were collected.Flow cytometry and ELISA were employed to detect the levels of basophils and IL-4, and the joint swelling was scored.Results Mouse model of CIA was successful established .The ratio of IL-4/IFN-γof the CIA group was significantly lower than that in the mice before CIA modeling and the control group , indicating a Th2-dominant response .At the same time, the peripheral basophils counting and percentage of IL-4 positive basophils of the CIA group were significantly higher than those of the control group .While, the IL-4/IFN-γratio of the CIA group was significantly higher than that of the control group , indicating a Th1-dominant response .The peripheral basophils counting of the CIA group was slightly lower than that of the control group .Conclusion Basophils may participate in the development of CIA in mouse models through affecting the imbalance of Th 1/Th2 response.

ObjectiveTo study the role of middle hepatic vein (MHV) on the early function and regeneration of the donor remnant liver in living donor liver transplantation (LDLT).Methods Between August 2007 and August 2008,66 LDLT were performed,36 without MHV (group A),and 30 with MHV (group B) in the donor liver.The donor operation time,intraoperative blood loss,postoperative hospital stay,serum bilirubin,international normalized ratio (INR),alanine aminotransferase (ALT) and albumin were analyzed.We measured the volume of remnant liver with CT scan at 2 weeks after operation,and compared the function and regeneration of the remnant liver between the two groups. Results At 2 weeks after operation,there was no significant difference (P=0.16) in the volume of remnant liver between group A (959.3±195.2 ml) and group B (883.7±155.5 ml).There was also no difference (P=0.62) in the regeneration rate of segment IV between group A (78.2 ％ ± 29.1 ％) and group B (82.7 ％ ± 40.4％).The serum bilirubin,INR and ALT in group B was significantly higher than group A immediately after liver transplantation,but there was no difference at 1 week after transplantation.ConclusionExtended right hepatectomy with MHV was safe,and did not significantly impact early liver function and regeneration in the donor.

ObjectiveTo investigate the effect of living donor right liver graft transplantation (LDLT) with middle hepatic vein (MHV) on the early congestion and regeneration of the donor remnant liver.MethodsBetween August 2008 and August 2009,28 LDLT were performed with 11 LDLT without MHV (group A) and 17 LDLT with MHV (group B).The donor operative time,intraoperative blood loss,postoperative hospital stay,bilirubin,INR,and ALT level were recorded in detail.We measured the volume of remnant liver by means of CT scan 2 weeks after operation and compare the degree of congestion and regeneration of the remnant liver between the two groups.ResultsThere were 10 cases in group B and 0 cases in group A suffering from congestion at segment Ⅳ,and the difference was significant(P =0.006).In group B,6 cases in type Ⅰ and 4 cases in type Ⅱ developed congestion at segment Ⅳ,and the difference was significant(P=0.035).Two weeks post operation,the volume of segment Ⅳ in group B was smaller than in group A(P=0.005).The regeneration rate of segment Ⅳ in group B was smaller than in group A (P =0.007),on the contrary,the regeneration rate of segment Ⅰ - Ⅲ in group B was larger than in group A( P =0.008 ).But the regeneration rate of remnant liver was the same in both groups (P =0.63 ).ConclusionsThe right lobe hemihepatectomy with MHV does not damage the early liver function of the donor significantly.The segment Ⅳ of the remnant liver suffered from congestion and impeded the regeneration,but was compensated by the regeneration of segments Ⅰ - Ⅲ.

Objective To explore the role of VEGF positive expression in tumor tissue in the prognosis of liver transplantation for hepatocellular carcinoma (HCC).Method Fifty cases of liver transplant recipients with HCC confirmed immunohistochemically were enrolled in this study.The MaxVisionTM two-step method was applied to detect the expression of vascular endothelial growth factor(VEGF),and the microvessel density (MVD) was measured in para-cancerous tissues by using DAB staining.The correlation of the VEGF tumor tissue in tumor tissue with Child-Pugh,MELD,tumor diameter and number,differentiation,MVD,Milan criteria and UCSF criteria for HCC liver transplantation was analyzed.Result In the HCC tissue,the VEGF positive expression rate was 52％(26/50).The one-year survival of recipients positive and negative for VEGF was 78％ and 100％,respectively,and one-year recurrence rate was 32％ and 12％,respectively,with the difference being significant (P =0.043 and P =0.048 respectively).The expression of VEGF was associated with Child-Pugh,tumor diameter,MVD,Milan criteria and UCSF criteria (P＜0.05 for all).Logistic regression analysis showed that low differentiation and VEGF positive expression were independent prognostic factors for HCC recurrence after liver transplantation.Conclusion VEGF has a certain reference value to judge HCC invasiveness and prognosis of liver transplantation.

Objective To evaluate the influence of right ventricular outflow tract septal ( RVS) pacing with right ventricular apical ( RVA) pacing on left ventricular remodeling and brain natriuretic peptide ( BNP). Methods Sixty patients with indication of pacemaker implantation were randomized into two groups, RVA group and RVS group. BNP was measured with ELISA, and echocardiography was performed to measure the left ventricular end diastolic volume ( LVEDD), left ventricular end systolic volume ( LVEDV) and left ventricular ejection fraction (LVEF) at pre-operation,and after 6,12,24 months pacing. The difference of cardiac remodeling and BNP in the two groups was observed. Results Compared to BNP at pre-operation (( 60. 2 ± 15. 7 ) ng/L) , BNP increased significantly in the RVA group at the 6th,12th and 24th month after operation( ( 108. 2 ±29. 8) , ( 190. 3 ±46. 7) ,(308. 2 ±56. 5)ng/L,respectively) (P <0. 05). In the RVS group,BNP increased only at 24 months after pacing ( (75. 2 ± 15. 8) ng/L vs. (63. 9 ± 15. 1 ) ng/L) (P < 0. 05). There was significant difference on BNP between the two groups. LVEDD,LVEDV increased,LVEF declined at 12 months after pacing in the RVA group,which were not observed in the RVS group. There was significant difference on LVEDD,LVEDV and LVEF in the RVA group (P< 0. 05) between the 12th month and pre-operation,and there were no significant difference in the RVS group (P > 0. 05). Conclusions Compared to RVA pacing,RVS pacing was more beneficial to improve heart function,prevent cardiac remodeling and decline the activation of nerve-endocrine.

[ ABSTRACT] AIM:To investigate the effects of pathological products, urinary proteins and advanced glycosyla-tion end products ( AGE) produced in the progression of chronic kidney disease ( CKD) , on the structure and function of lysosomes in renal tubular epithelial cells ( TECs ) , and try to find a novel approach for preventing or delaying CKD. METHODS:The renal specimens of the untreated patients with minimal change nephrotic syndrome (MCNS), diabetic nephropathy (DN) or normal kidney were collected.The expression of lysosomal-associated membrane protein 1 (LAMP1) and cathepsin B ( CB) was studied in TECs by indirect immunofluorescent staining.Human renal tubular epithelial cell line HK-2 was incubated with 8 g/L urinary proteins or 100 mg/L AGE.The expression of LAMP1 and CB was investigated by indirect immunofluorescence and the activity of CB and cathepsin L ( CL) was measured by biochemical and enzymatic as-says.The degradation of DQ-ovalbumin was also determined.RESULTS: The lysosomal membrane permeabilization oc-curred in the TECs of MCNS and DN patients.After treatment with urinary proteins or AGE-BSA, the lysosomal membrane permeabilization of the HK-2 cells was increased.The activity of CB and CL and degradation of DQ-ovalbumin were de-creased as compared with normal control group.CONCLUSION:The digestive function of lysosome was decreased and ly-sosomal membrane permeabilization occurred in the TECs exposed to urinary proteins and AGE, which might be a key factor to induce the tubulointerstitial fibrosis.

Objective:To investigate the activation of peripheral basophils from patients with rheumatoid arthritis ( RA) and its mechanisms. Methods:The activation markers including CD203c and the proportion of IL-4 positive rate of peripheral basophils from RA patients and healthy controls were detected by flow cytometry. Serum levels of IgE in RA patients and healthy controls were detected by electrochemilu minescence immunoassay. Basophils were negatively isolated and co-cultured with or without purified IgE,anti-IgE as positive control,then,the expression of CD203c and propotion of IL-4 positive basophils were detected by flow cytometry. Results:The expression of CD203c and IL-4 positive rate of basophils from RA patients were higher than that of healthy controls (P<0. 05). Serum levels of IgE in RA were higher than that of healthy controls (P<0. 05). After co-cultured with isolated IgE from RA patients,basophils negatively isolated from healthy controls were activated,and higher expression of CD203c and proportion of IL-4 (P<0.05). Conclusion:Basophil activation is related with development of RA,and its activation is mainly mediated by IgE. Targeting basophil and its activation pathway would be expected to provide new strategies for the treatment of RA.

Objective:To investigate levels of autophagy in T cells and B cell of patients with systemic lupus erythematosus ( SLE) and its clinical significance.Methods: 68 SLE patients without treatment within 4 weeks were enrolled in this study.We accessed the levels of autophagy in T cells and B cells of 23 healthy controls and 68 patients before and after treatment by flow cytometry,and analyzed their correlations with serum levels of C3 and anti-dsDNA antibodies,SLEDAI score,et al.Results: Before treatment,a significantly increased levels of LC3-Ⅱ was observed in SLE patients than healthy controls, the active group ( SLEDAI score≥10) was significantly higher than the stable group(SLEDAI score<10),and the newly diagnosed group was significantly higher than the recurrent group(all P<0.05).While the levels of LC3-Ⅱ was decreased in B lymphocytes in SLE patients,the active group was significantly lower than stable group,and the active group was significantly lower than the newly diagnosed group(all P<0.05). Correlation analysis found that,a positively correlation was observed for the levels of LC3-Ⅱwith SLEDAI score in T lymphocyte( rs=0.289,P<0.05),and the levels of C3 were positively correlated the levels of LC3-Ⅱ in B lymphocyte(rs=0.371,P<0.01).After treatment for five days, levels of autophagy in T lymphocytes of SLE patients with good prognosis ( SLEDAI score decreased ≥4 ) significantly decreased(P<0.05).Also,three days after treatment,levels of autophagy in B lymphocytes of SLE patients with good prognosis were increased significantly ( P<0.05 ) .However, SLE patients with poor prognosis had no such difference ( P>0.05 ) . Conclusion:Levels of autophagy in T and B lymphocytes of SLE patients are abnormal compared to healthy controls,and these changes are associated with disease activity.Also,these changes are expected to be the indicators of disease activity and potential therapeutic targets in SLE.

Objective To observe the changes of renal tubular injury and the extent of interstitial fibrosis in the C57BL/6 mouse models of chronic kidney disease(CKD),and provide experimental animal evidence for study of the pro-gression of acute kidney injury(AKI)to chronic kidney disease as well as its mechanisms. Methods Twenty-four 8-week-old male C57BL/6 mice were randomly and equally divided into control group, low-dose, medium-dose, and high-dose cisplatin groups,6 mice in each group. Mice in the cisplatin groups were administrated with 5,7 or 10 mg/kg cispla-tin by intraperitoneal injection once a week for 4 weeks. Plasma creatinine and 24-hour urinary protein were detected to as-sess the renal function. The mice were sacrificed, and plasma and kidney samples were collected for subsequent tests. Pathological changes were observed using periodic acid-Schiff(PAS)staining. To evaluate renal tubules injury, the ex-pression of kidney injury molecule 1(KIM-1)was examined by immunohistochemistry and the level of urinary N-Acetyl-β-D-glucosaminidase was detected with a commercial kit. The infiltration of CD3-positive T cells and F4/80-positive macro-phages was observed by immunohistochemistry(IHC)and immunofluorescence. The expression of collagen I and α-smooth muscle actin(α-SMA)were tested by immunohistochemistry to assess the renal fibrosis, while total kidney collagen was detected by Picrosirius red staining. Results In contrast to the normal control group,the kidney injury became more seri-ous in the cisplatin-treated mice as cisplatin concentration increased. Particularly,significant kidney damage was observed in the high-dose cisplatin group. Compared with the control group,the plasma creatinine and 24-hour urinary protein were significantly increased in the high-dose cisplatin group(P<0.05 and P<0.001)indicating impaired renal function. Mor-phologically,numerous clear vacuoles and necrosis were present in renal tubule epithelial cells in the high-dose cisplatin group. The expression of KIM-1 was markedly up-regulated and the level of urinary NAG was elevated. Infiltration of CD3-positive T cells and F4/80-positive macrophages was enhanced in the mice of high-dose cisplatin group. Data from immuno-histochemistry and picrosirius red staining showed that mice of the high-dose cisplatin group developed renal fibrosis evi-denced by markedly up-regulated expression of collagen I and α-SMA. Conclusions Repeated administration of 10 mg /kg cisplatin for 4 weeks can induce chronic renal insufficiency in mice,which may serve as a novel model for the research on underlying mechanisms of progression from acute kidney injury to chronic kidney disease.