器官移植

Abstract:
Objective To explore the value of pediatric end-stage liver disease (PELD) score system in predicting prognosis after pediatric living donor liver transplantation(LDLT). Methods Clinical data of 101 infants undergoing living-donor liver transplantation from October 2006 to December 2012 in Department of Liver Surgery in Affiliated Renji Hospital of School of Medicine of Shanghai Jiaotong University, were analyzed retrospectively. All infants were diagnosed as biliary atresia. PELD scores before LDLT were graded. According to PELD scores, all the patients were divided into two groups: low score group(PELD score<16,n=62)and high score group(PELD≥16,n=39).The basic data during perioperative period and incidence of postoperative complications were compared between two groups. Results There were significant differences in age and body weight between two groups(both in P<0.05). But there was no significant difference between the two groups in gender,graft to recipient weight ratio(GRWR),cold isehemia time and intraoperative blood loss(all in P>0.05). The incidence of lung infection and biliary complications in high score group were significantly higher than those in low score group. Conclusions Preoperative PELD score can be used in predicting prognosis after pediatric LDLT and provide a reference for the treatment, caring and nursing during perioperative period of pediatric LDLT. For infants with high PELD score before operation, the care of perioperative complications should be enhanced.

Variation and significance of T helper 17 cells in peripheral blood in patients after liver transplantation

Li Ping, Fan Hua, Kou Jiantao, Han Dongdong, Zhu Jiqiao, He Qiang

2014, 5(4): 217-221,241. doi: 10.3969/j.issn.1674-7445.2014.04.005

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Abstract:
Objective To study the relationship between T helper (Th)17 cell [CD4⁺ interleukin (IL)-17⁺T lymphocytes] in peripheral blood and acute rejection in patients after liver transplantation. Methods A total of 76 patients receiving orthotopic liver transplantation (OLT) for benign end-stage liver diseases in Department of Hepatobiliary and Pancreas-Spleen Surgery, Affiliated Beijing Chaoyang Hospital of Capital Medical University from June 2008 to December 2012 were included in this study. According to whether the acute rejection occurred after operation, the patients were divided into rejection group (n=17) and non-rejection group (n=59). All the patients were followed up regularly by routine. The incidence of rejection and the treatment of patients were recorded. Patients in the rejection group received liver biopsy when suffered acute rejection to decide the severity. The percentage of CD4⁺IL-17⁺T lymphocytes to CD4⁺T lymphocytes (CD4⁺IL-17⁺T%) in peripheral blood in all patients was measured at different time points: pre-OLT, at regular intervals (3-6 months) within 1 year after hospital discharge or before the treatment of acute rejection and after the remission (3-6 months). The CD4⁺IL-17⁺T% of every time point were compared between two groups. The correlations of CD4⁺IL-17⁺T% with the rejection activity index (RAI), blood concentration of immunosuppressor were analyzed. Results The acute rejection occurred in 0.7-12.0 (median: 2.5)months after OLT. The CD4⁺IL-17⁺T% in the rejection group increased significantly compared with that in the non-rejection group after OLT [(2.56±0.43)% vs.(1.79±0.44)%, P<0.001]. In the rejection group, the CD4⁺IL-17⁺T% increased significantly when acute rejection occurred compared with that when acute rejection had not occurred [(2.56±0.43)% vs.(1.50±0.25)%,P<0.001)]. The variation of CD4⁺IL-17⁺T% was not obvious at different time points in non-rejection group(P>0.05). The CD4⁺IL-17⁺T% was positively correlated with RAI when acute rejection occurred in the rejection group (r=0.72,P=0.001). The blood concentration of tacrolimus, cyclosporin in rejection and non-rejection group were not correlated with CD4⁺IL-17⁺T% (r=0.21,-0.13; both in P>0.05). Conclusions CD4⁺IL-17⁺T%in peripheral blood can be used as a monitoring index for deciding and assessing severity of acute rejection after OLT. The increase of CD4⁺IL-17⁺T% in peripheral blood indicates a severe acute rejection.

Prognostic analysis of liver transplantation in treating patients with end-stage autoimmune liver disease

Zou You, Zhang Lei, Li Ruidong, Guo Wenyuan, Ding Guoshan, Fu Zhiren, Wang Zhengxin

2014, 5(4): 222-226. doi: 10.3969/j.issn.1674-7445.2014.04.006

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Abstract:
Objective To investigate the prognosis of patients underwent liver transplantation (LT) for end-stage autoimmune liver disease (AILD). Methods Clinical data of 48 patients with end-stage AILD undergoing LT from May 1996 to April 2013 in Affiliated Changzheng Hospital of the Second Military Medical University were analyzed retrospectively. The postoperative cumulative survival rates of the recipients were calculated, and the cause of death was analyzed. The postoperative rejections, new-onset viral hepatitis and AILD recurrence were analyzed. Results In 48 AILD recipients, 38 cases survived and the postoperative 5-year cumulative survival rate was 76%. Causes of death for the 10 dead cases were multiple organ failure, liver graft failure, sepsis, pulmonary infection, hemorrhage, hepatic artery embolization and renal failure. In 48 AILD recipients, 9 cases (19%) suffered acute rejection after operation, 3 cases suffered new-onset hepatitis B infection in 1-2 years after operation, 2 recipients suffered primary disease (primary biliary cirrhosis) recurrence 2 years after operation and all survived for a long term after positive treatments. Conclusions Most liver transplant recipients with end-stage AILD can obtain a long-term survival. Attentions should be paid on the immunosuppressive regimens in early period after LT, prevention of infection, rejection and postoperative new-onset viral hepatitis, timely diagnosis of primary disease recurrence.

Relation between donor-recipient HLA mismatching and combined malignant tumor after renal transplantation

Jia Baoxiang, Lin Jun, Wu Junjie, Ma Weiran

2014, 5(4): 227-230. doi: 10.3969/j.issn.1674-7445.2014.04.007

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Abstract:
Objective To study the relation between donor-recipient human leukocyte antigen (HLA) mismatching and combined malignant tumors after renal transplantation. Methods Clinical data of 1 021 patients who received renal transplantation from 1993 to 2009 in Department of Urology of Beijing Friendship Hospital of Affiliated Capital University of Medical Sciences over 5 years and had complete HLA typing were analyzed. In the 1 021 patients after renal transplantation, 928 cases were non-tumor patients and 93 cases were malignant tumor patients. The mismatching data of 3 locus (HLA-A, B and DR) with a total of 6 antigens of the donors and recipients were collected. The relation between donor-recipient HLA mismatching number and postoperative combined malignant tumors was analyzed. And that between genders was also analyzed. Results Malignant tumors occurred in 9.11%(93/1 021)of the patients. The malignant tumor incidences of patients with HLA mismatch of 0-1, 2, 3, 4, 5 and 6 antigens were 14%, 13%, 14%, 6%, 3% and 4% respectively. Patients with HLA over half-matched (0-3 antigens mismatch) had higher incidence of malignant tumors compared with that in patients with HLA less than half matched (4-6 antigens mismatch) (14% vs. 5%;χ²=24.11,P<0.005). In the malignant tumor patients of 0-3 antigens mismatch, 12 cases were males and 54 cases were females. In the patients of 4-6 antigens mismatch, 12 cases were males and 15 cases were females. The proportion of female patients of 0-3 antigens mismatch was higher than that of male patients(χ²=5.60,P<0.025). Conclusions For the renal transplant patients, especially female patients, the lower the HLA mismatching number is, the higher the malignant tumor incidence is.

Experimental Researche

Bone marrow mesenchymal stem cell modified by CTLA4-Ig gene can inhibit the rejection of liver transplantation in rats

Yin Dongliang, Sun Chong, Zhu Huanbin, Li Kun, Li Haohui, Zhang Jian

2014, 5(4): 231-236. doi: 10.3969/j.issn.1674-7445.2014.04.008

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Abstract:
Objective 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-γ in group B and C were lower significantly (P<0.05). The serum levels of IL-2 and IFN-γ in 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-γ between 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.

Dendritic cells phagocytized apoptotic lymphocytes induce the development of regulatory B cells with high secretion of interleukin-10

Wei Yuxiang, Wang Xiaogang, Xiao Li, Zhou Wenqiang, Zheng Dehua, Yu Tao, Shi Bingyi

2014, 5(4): 237-241. doi: 10.3969/j.issn.1674-7445.2014.04.009

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Abstract:
Objective To investigate the feasibility of immature dendritic cells (imDC) phagocytized psoralen ultraviolet A (PUVA)-treated splenic lymphocytes (PUVA-SP DC) in mice inducing B lymphocytes to be regulatory B cells(Breg) with high secretion of interleukin(IL)-10(IL-10⁺Breg). Methods Bone marrow-derived DC of mice was cultured. Spleen lymphocytes of mice were isolated and treated by PUVA, and turned to be PUVA-SP. The bone marrow-derived imDC was co-cultured with PUVA-SP in vitro to obtain PUVA-SP DC. Splenic B lymphocytes of mice were separated by anti-CD19 magnetic beads and co-cultured with different kinds of DC for 48 hours. The levels of interferon(IFN)-γ, transforming growth factor(TGF)-β, IL-12p70, and IL-10 in the culture supernatant of B lymphocytes, imDC, imDC+B lymphocytes, PUVA-SP DC and PUVA-SP DC+B lymphocytes were measured by enzyme-linked immune absorbent assay(ELISA). The accounts of IL-10⁺Breg in B lymphocytes, imDC+B lymphocytes, mDC+B lymphocytes and PUVA-SP DC+B lymphocytes were detected by flow cytometry. Results Compared with the other 4 groups, the level of IL-10 in cell culture supernatant of PUVA-SP DC+B lymphocytes was significantly higher(all in P<0.05). Compared with the other groups, the account of IL-10⁺Breg in PUVA-SP DC+B lymphocytes was significantly higher. Conclusions PUVA-SP DC can induce splenic B lymphocytes to differentiate into IL-10⁺Breg.

Establishing a model of oxidative damage of L02 hepatocytes in vitro

Zhao Hui, Meng Wei, Yi Shuhong, Chen Guihua

2014, 5(4): 242-246,250. doi: 10.3969/j.issn.1674-7445.2014.04.010

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Abstract:
Objective To investigate the method of establishing a model of oxidative damage of L02 hepatocytes. Methods L02 hepatocytes were cultured divided into 3 h damage group, 6 h group and 12 h group. Each group was divided into 6 subgroups according to different concentrations of hydrogen peroxide (H₂O₂) added: 100, 200, 300, 500, 750, 1 000 μmol/L. The control group was treated without H₂O₂. All groups were tested with cell counting kit (CCK)-8 after incubating for 3 h, 6 h or 12 h respectively. The other L02 hepatocytes were cultured and divided into 3 h damage group, 6 h group and 12 h group. The 3 h group was divided into 5 subgroups according to different concentrations of H₂O₂ added: 100, 200, 300, 500 and 750 μmol/L, the 6 h group was divided into 4 subgroups according to different concentrations of H₂O₂ added: 100, 200, 300 and 500 μmol/L, and the 12 h group was divided into 3 subgroups according to different concentrations of H₂O₂ added: 100, 200 and 300 μmol/L. The control group was treated without H₂O₂. All groups were tested by flow cytometry with Annexin V-fluorescein isothiocyanate(FITC)/propidium iodide(PI)double staining after incubating for 3 h, 6 h or 12 h respectively. The model of oxidative damage of L02 hepatocytes was established and identified. The L02 hepatocytes in damage group were treated with 200 μmol/L H₂O₂ after culture and L02 hepatocytes in control group were treated without H₂O₂ after culture. Indicators such as mitochondrial membrane potential, malondialdehyde (MDA), oxygen free radical (ROS), superoxide dismutase (SOD), alanine aminotransferase (ALT), aspartate aminotransferase (AST) were tested after L02 hepatocytes were incubated for 6 h. Results Compared with control group, significant differences were observed in cell survival rates of 200, 300, 500, 750 and 1 000 μmol/L subgroup in 3 h damage group (all in P<0.01). So were in 6 h damage group and 12 h damage group (all in P<0.01). The correlation coefficient between H₂O₂ concentration and cell viability was -0.993 in 3 h group, -0.955 in 6 h group, and -0.819 in 12 h group. Flow cytometry with Annexin V-FITC/PI double staining was used to detect the apoptosis/necrosis of L02 hepatocytes when treated with different concentrations and actuation duration of H₂O₂. In 3 h, 6 h and 12 h damage group, significant differences were observed between damage subgroups and control groups(all in P<0.01). The correlation coefficients between H₂O₂ concentration and cell apoptosis/necrosis in 3 h, 6 h and 12 h group were 0.971, 0.992 and 0.986 respectively. Compared with control group, significant differences were observed in mitochondrial membrane potential, MDA, ROS, SOD, ALT, and AST in damage group after treated with 200 μmol/L H₂O₂ for 6 h(all in P<0.01). Conclusions L02 hepatocytes treating with 200 μmol/L H₂O₂ for 6 h is an appropriate model simulating the ischemia-reperfusion or oxidative damage in vitro.