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Effect of small intestinal submucosa on islet recovery and function in vitro culture

Hepatobiliary & Pancreatic Diseases International 2005年第4期4卷 524-529页

作者： Xiao-Hui tian Wu-Jun Xue Xin-Lu Pang Yan Teng pu-xun tian Xin-Shun Feng Department of Renal Transplantation First Affiliated Hospital College of Medicine Xi'an Jiaotong University Xi'an 710061 China.

BACKGROUND: The ability to maintain isolated human islet preparation in tissue culture has recently been adopted by most islet transplant centers to improve the safety and practicality of islet transplantation. However, maintaining islet viability and recovery remains a challenge in clinical setting. Extracellular matrix (ECM) is one of the most important components of islet microenvironment. The reconstruction of the cell-matrix relationship seems to be effective in improving the loss of differentiated islet structure and function. Small intestinal submucosa ( SIS ) , a naturally occurring ECM, has been investigated to be able to promote wound healing, tissue remodeling, and cell growth. The purpose of this study was to evaluate the recovery and function of isolated rat pancreatic islets after in vitro culture with SIS. METHODS: Pancreatic islets were isolated from Wistar rats by using standard surgical procurement followed by intra-ductal collagenase distension, mechanical dissociation, and EuroFicoll purification. Groups of purified islets were cultured in plates which were coated with multilayer SIS (SIS-treated group) or without ( standard cultured group) for 7 days and 14 days in standard islet culture conditions of RP-MI 1640 tissue culture media in humidified atmosphere containing 95% air and 5% CO2 at 37 ℃. The mean recovery of islets after the culture period was determined by sizing duplicate counts of a known volume and their viability was assessed by static incubation with low glucose (2.7 mmol) , high glucose (16.7 mmol) and high glucose solution supplemented with 50 μm 3-isobutyl-1-methylxanthine (IB-MX) solution. RESULTS: After 7 days and 14 days of in vitro tissue culture, the SIS-treated group showed a significantly higher recovery compared with those cultured under standard conditions. The recovery in the SIS-treated group was about two times of the control group cultured in standard conditions after 14 days culture. In the SIS-treated group, there

关键词： islet culture islet recovery islet function small intestinal submucosa

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Allograft rejection-related gene expression in the endothelial cells of renal transplantation recipients after cytomegalovirus infection

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2009年第11期10卷 820-828页

作者： Yang LI Hang YAN Wu-jun XUE pu-xun tian Xiao-ming DING Xiao-ming PAN Xin-shun FENG Xiao-hui tian He-li XIANG Jun HOU Department of Renal Transplantation Center of Nephropathy the First Affiliated Hospital School of Medicine Xi 'an Jiaotong University Xi 'an 710061 China The two authors contributed equally to this work

Objective： To explore the effects of cytomegalovirus （CMV） infection on rejection-related gene expression in the endothelial cells of renal transplantation recipients. Methods： Endothelial cells （ECs） were cultured and stimulated by a variety of factors： A, normal control group; B, inactivated human cytomegalovirus （HCMV） infection group; C, HCMV infection group; D, HCMV supematant infection group; and E, ganciclovir HCMV group. Expression of intercellular adhesion molecule-1 （ICAM-1） and major histocompability complex （MHC） class I and class II antigens was detected by flow cytometry （FCM） and immuno- histochemistry. Results： We found characteristic CMV-infected ECs in this study. There were no significant differences among groups A, B and D （P〉0.05）. Although the expression levels of ICAM-1 were not significantly different between groups C and E （P〉0.05）, the ICAM-1 expression in these two groups was significantly higher than that in group A （P〈0.05）. ICAM-1 expression was detected in groups C and E, while there was no expression in groups A, B and D. Furthermore, there was no significant difference of ICAM-1 mRNA expression between groups C and E （P〉0.05）. Human leucocyte antigen （HLA）-ABC expression was detected in all the groups, while HLA-DR expression was only detected in groups C and E. There were no significant dif- ferences of HLA-ABC and HLA-DR expression among groups A, B and D （P〉0.05）. However, the HLA-ABC and HLA-DR expression levels in groups C and D were higher than those of the remaining groups previously reported （P〈0.05）. Meanwhile, the HLA-ABC and HLA-DR expression levels in group E were lower than those of group C （P〈0.05）. Conclusion： CMV could up-regulate the expression levels of ICAM-1 and MHC antigens, which was closely related to allograft rejection.

关键词： Renal transplantation Cytomegalovirus (CMV) Rejection Intercellular adhesion molecule-1 (ICAM-1) Major histocompability complex (MHC)

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Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD^4+ CD25^+ Treg cells

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2009年第12期10卷 928-932页

作者： Hang YAN Chen-guang DING pu-xun tian Guan-qun GE Zhan-kui JIN Li-ning JIA Xiao-ming DING Xiao-ming PAN Wu-jun XUE Department of Renal Transplantation Center of Nephropathy the First Affiliated Hospital Medicine College Xi'an Jiaotong University Xi'an 710061 China

Objective: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4+ CD25+ regulatory T (Treg) cells, in order to inform further studies in Treg cell function. Methods: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4+ CD25+ Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4+ CD25+ Treg cells, trypan blue staining to detect cell viability, and propidium iodide (PI) staining to assess the cell viability. We detected the immune inhibition of CD4+ CD25+ Treg cells in the in vitro proliferation experiments. Results: The results showed that compared to flow cytometry sorting, magnetic cell sorting took more time and effort, but fewer live cells were obtained than with flow cytometry sorting. The CD4+ CD25+ Treg cells, however, obtained with both methods have similar immunosuppressive capacities. Conclusion: The result suggests that both methods can be used in isolating CD4+ CD25+ Treg cells, and one can select the best method according to specific needs and availability of the methodologies.

关键词： CD4+ CD25+ Treg cells Flow cytometry sorting Magnetic cell sorting

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The clinical value of enzyme-multiplied immunoassay technique monitoring the plasma concentrations of cyclosporine A after renal transplantation

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Journal of Pharmaceutical Analysis 2011年第2期1卷 139-142页

作者： Xiao-Hui Luo wu-Jun Xue pu-xun tian Xiao-Ming Ding Hang Yan He-Li Xiang Yang Li Department of Renal Transplant Center of Nephropathy the First Affiliated Hospital Medical School of Xi'an Jiaotong University Xi'an 710061 China.

The feasibility and the clinical value of the enzyme-multiplied immunoassay technique （EMIT） monitoring of blood concentrations of cyclosporine A （CsA） in patients treated with CsA were investigated after kidney transplantation. The validation method was performed to the EMIT determination of CsA blood concentration, the CsA whole blood trough concentrations （Co） of patients in different time periods after renal transplantation were monitored, and combined with the clinical complications, the statistical results were analyzed and compared. EMIT was precise, accurate and stable, also with a high quality control. The mean postoperative blood concentration of CsA was as follows：〈1 month, （281.4± 57.9）ng/mL; 2 - 3 months, （264.5 ± 41.2） ng/mL; 4 - 5 months, （236.4 ± 38.9） ng/mL; 6 - 12 months, （206.5± 32.6）ng/mL; 〉12 months, （185.6± 28.1）ng/mL. The toxic reaction rate of CsA blood concentration within the recommended therapeutic concentration was 14.1%, significantly lower than that of the none-recommended dose group （37.2%）（P〈0.05）; the transplantation rejection rate was 4.4%, significantly lower than that of the none- recommended dose group （22.5%）（P〈0.05）. Using EMIT to monitor the blood concentration of CsA as the routine laboratory method is feasible, and is able to reduce the CsA toxicity and rejection significantly, leading to achieving the desired therapeutic effect.

关键词： enzyme-multiplied immunoassay technique renal transplantation cyclosporin A blood concentration monitoring

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Sinomenine promotes differentiation of induced pluripotent stem cells into immature dendritic cells with high induction of immune tolerance

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World Journal of Stem Cells 2022年第8期14卷 599-615页

作者： Xiao-Yan Huang Zhan-Kui Jin Meng Dou Bing-Xuan Zheng Xiang-Rong Zhao Qing Feng Yang-Meng Feng Xiang-Long Duan pu-xun tian Cui-Xiang Xu Shaanxi Provincial Key Laboratory of Infection and Immune Diseases Shaanxi Provincial People’s HospitalXi'an 710068Shaanxi ProvinceChina Department of Orthopedics Shaanxi Provincial People’s HospitalXi'an 710068Shaanxi ProvinceChina Department of Kidney Transplantation The First Affiliated Hospital of Xi’an Jiaotong UniversityXi'an 710061Shaanxi ProvinceChina Second Department of General Surgery Shaanxi Provincial People's HospitalXi'an 710068Shaanxi ProvinceChina

BACKGROUND Immature dendritic cells(imDCs)play an important role in the induction of donor-specific transplant ***,these cells have limitations,such as rapid maturation and a short lifespan in *** previous studies,induced pluripotent stem cells(iPSCs)differentiated into imDCs,and sinomenine(SN)was used to inhibit the maturation of *** To study the capacity of SN to maintain iPSC-derived imDCs(SN-iPSCs-imDCs)in an immature state and the mechanism by which SN-iPSCs-imDCs induce *** In this study,mouse iPSCs were induced to differentiate into imDCs in culture medium without or with SN(iPSCs-imDCs and SN-iPSCs-imDCs).The imDCrelated surface markers,endocytotic capacity of fluorescein isothiocyanate Dextran and apoptosis were analyzed by flow *** effects of iPSCs-imDCs and SNiPSCs-imDCs on T-cell stimulatory function,and regulatory T(Treg)cell proliferative function in vitro were analyzed by mixed lymphocyte *** expression was detected by *** apoptosis-related proteins of iPSCs-DCs and SN-iPSCs-DCs were analyzed by western *** induced immunotolerance of SN-iPSCs-DCs was evaluated by treating recipient Balb/c skin graft *** evaluation of graft survival was performed using Kaplan–Meier *** Both iPSCs-imDCs and SN-iPSCs-imDCs were successfully obtained,and their biological characteristics and ability to induce immunotolerance were ***-iPSCs-imDCs exhibited higher CD11c levels and lower CD80 and CD86 levels compared with *** major histocompatibility complex II expression,worse T-cell stimulatory function,higher Treg cell proliferative function and stronger endocytotic capacity were observed with SN-iPSCs-imDCs(P<0.05).The levels of interleukin(IL)-2,IL-12,interferon-γin SN-iPSCs-imDCs were lower than those in iPSCs-imDCs,whereas IL-10 and transforming growth factor-βlevels were higher(P<0.05).The apoptosis rate of these cells was significan

关键词： Immature dendritic cells Induced pluripotent stem cells Sinomenine Immune tolerance Organ transplantation

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Small intestinal submucosa improves islet survival and function during in vitro culture

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World Journal of Gastroenterology 2005年第46期11卷 7378-7383页

作者： Xiao-Hui tian Wu-Jun Xue Xiao-Ming Ding Xin-Lu Pang Yan Teng pu-xun tian Xin-Shun Feng Center of Renal Transplantation The First Hospital of Xi'an Jiaotong UniversityXi'an 710061 Shaanxi Province China

AIM： To evaluate the recovery and function of isolated rat pancreatic islets during in vitro culture with small intestinal submucosa （SIS）. METHODS： Pancreatic islets were isolated from Wistar rats by standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and Euroficoll purification. purified islets were cultured in plates coated with multilayer SIS （SIS-treated group） or without multilayer SIS （standard cultured group） for 7 and 14 d in standard islet culture media of RPMI 1640. After isolation and culture, islets from both experimental groups were stained with dithizone and counted. Recovery of islets was determined by the ratio of counts after the culture to the yield of islets immediately following islet isolation. Viability of islets after the culture was assessed by the glucose challenge best with low （2.7 mmol/L） and high glucose （16.7 mmol/L） solution supplemented with 50 mmol/L 3-isobutyl-1- methylxanthine （IBMX） solution. Apoptosis of islet cells after the culture was measured by relative quantification of histone-complexed DNA fragments using ELISA. RESULTS： After 7 or 14 d of in vitro tissue culture, the recovery of islets in SIS-treated group was significantly higher than that cultured in plates without SIS coating. The recovery of islets in SIS-treated group was about twice more than that of in the control group. In SIS treated group, there was no significant difference in the recovery of islets between short- and long-term periods of culture （95.8±1.0% vs 90.8±1.5%, P〉0.05）. When incubated with high glucose （16.7 mmol/L） solution, insulin secretion in SIS-treated group showed a higher increase than that in control group after 14 d of culture （20.7±1.1 mU/L vs 11.8±1.1 mU/L, P〈0.05）. When islets were placed in high glucose solution containing IBMX, stimulated insulin secretion was higher in SlS-treated group than in control group. Calculated stimulation index of SlS-treated group was about 23 times of

关键词： Islet culture Islet survival Islet function Small intestinal submucosa

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Predictive Score Model for Delayed Graft Function Based on Hypothermic Machine Perfusion Variables in Kidney Transplantation

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Chinese Medical Journal 2018年第22期131卷 2651-2657页

作者： Chen-Guang Ding Yang Li Xiao-Hui tian Xiao-Jun Hu pu-xun tian Xiao-Ming Ding He-Li Xiang Jin Zheng Wu-Jun Xue Department of Kidney Transplantation Nephropathy HospitalThe First Affiliated Hospital of Xi'an Jiaotong UniversityXi'anShaanxi 710061China Institute of Organ Transplantation Xi'an Jiaotong UniversityXi'anShaanxi 710061China

Background: Hypothermic machine perfusion(HMP) is being used more often in cardiac death kidney transplantation; however, the significance of assessing organ quality and predicting delayed graft function(DGF) by HMP parameters is still controversial. Therefore,we used a readily available HMP variable to design a scoring model that can identify the highest risk of DGF and provide the guidance and advice for organ allocation and DCD kidney ***: From September 1, 2012 to August 31, 2016, 366 qualified kidneys were randomly assigned to the development and validation cohorts in a 2:1 distribution. The HMP variables of the development cohort served as candidate univariate predictors for DGF. The independent predictors of DGF were identified by multivariate logistic regression analysis with a P < 0.05. According to the odds ratios(ORs) value, each HMP variable was assigned a weighted integer, and the sum of the integers indicated the total risk score for each kidney. The validation cohort was used to verify the accuracy and reliability of the scoring ***: HMP duration(OR = 1.165, 95% confidence interval [CI ]: 1.008–1.360, P = 0.043), resistance(OR = 2.190, 95%CI: 1.032–10.20, P < 0.001), and flow rate(OR = 0.931, 95% CI: 0.894–0.967, P = 0.011) were the independent predictors of identified DGF. The HMP predictive score ranged from 0 to 14, and there was a clear increase in the incidence of DGF, from the low predictive score group to the very high predictive score group. We formed four increasingly serious risk categories(scores 0–3, 4–7, 8–11, and 12–14)according to the frequency associated with the different risk scores of DGF. The HMP predictive score indicates good discriminative power with a c?statistic of 0.706 in the validation cohort, and it had significantly better prediction value for DGF compared to both terminal flow(P = 0.012) and resistance(P = 0.006).Conclusion: The HMP predictive score is a good noninvasive tool for assessing the qu

关键词： Delayed Graft Function Donation after Cardiac Death Hypothermic Machine Perfusion Kidney Transplantation

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Comparison of the characteristics of macrophages derived from murine spleen, peritoneal cavity, and bone marrow

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2017年第12期18卷 1055-1063页

作者： Yan-long ZHAO pu-xun tian Feng HAN Jin ZHENG Xin-xin XIA Wu-jun XUE Xiao-ming DING Chen-guang DING Department of Kidney Transplantation Hospital of Nephropathythe First Affiliated Hospital of Xi'an Jiaotong UniversityXi'an 710061China Institute of Organ Transplantation Xi'an Jiaotong UniversityXi'an 710061China Department of Traditional Chinese Medicine the First Affiliated Hospital of Xi'an Jiaotong UniversityXi'an 710061China

Macrophages have a diverse set of functions based upon their activation states. The activation states, including resting（M0） and polarizing（M1 and M2） states, of macrophages derived from the mouse bone marrow, spleen, and peritoneal cavity（BMs, SPMs, and PCMs, respectively） were compared. We evaluated the macrophage yield per mouse and compared the surface markers major histocompatibility complex（MHC） Ⅱ and CD86 by flow cytometry. The relative mRNA levels of tumor necrosis factor-α（TNF-α）, interleukin（IL）-1β, mannose receptor（MR）, and Ym1 in the M0, M1, and M2 states were also compared using real-time polymerase chain reaction（PCR） analysis. Bone marrow yielded the most macrophages with the best homogeneity, but they were polarized toward the M2 phenotype. All three types of macrophages had the capacity to polarize into the M1 and M2 states, but SPMs had a stronger capacity to polarize into M1. The three types of macrophages showed no differences in their capacity to polarize into the M2 state. Therefore, the three types of macrophages have distinct characteristics regardless of their resting or polarizing states. Although bone marrow can get large amounts of homogeneous macrophages, the macrophages cannot replace tissue-derived macrophages.

关键词： Macrophage Murine splenocytes Murine bone marrow Peritoneal cavity

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Polyglycolic Acid Fibrous Scaffold Improving Endothelial Cell Coating and Vascularization of Islet

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Chinese Medical Journal 2017年第7期130卷 832-839页

作者： Yang Li Ping Fan Xiao-Ming Ding Xiao-Hui tian Xin-Shun Feng Hang Yan Xiao-Ming Pan pu-xun tian Jin Zheng Chen-Guang Ding Wu-Jun Xue Department of Renal Transplantation Center of Nephrology The First Affiliated Hospital of Xi'an Jiaotong University School of Medicine Xi'an Shaanxi 710061 China Department of Rheumatism and Immunology The First Affiliated Hospital of Xi'an Jiaotong University School of Medicine Xi'an Shaanxi 710061 China

Background：Improving islet graft revascularization has become a crucial task for prolonging islet graft *** cells （ECs） are the basis of new microvessels in an isolated islet,and EC coating has been demonstrated to improve the vascularization and survival of an ***,the traditional method of EC coating of islets has low efficiency in *** study was conducted to evaluate the effect of a polyglycolic acid （PGA） scaffold on the efficiency of islet coating by ECs and the angiogenesis in the coated islet ***：A PGA fibrous scaffold was used for EC coating of islet culture and was evaluated for its efficiency of EC coating on islets and islet graft ***：In in vitro experiments,we found that apoptosis index of ECs-coating islet in PGA group （27% ± 8%） was significantly lower than that in control group （83% ± 20%,P 〈 0.05） after 7 days *** index was significantly greater in the PGA group than in the control group at day 7 after ECs-coating （2.07 ± 0.31 vs.1.80 ± 0.23,P 〈 0.05）.vascular endothelial growth factor （VEGF） level in the PGA group was significantly higher than the coating in the control group after 7 days culture （52.10 ± 13.50 ng/ml vs.16.30 ± 8.10 ng/ml,P 〈 0.05）.Because of a tight,circumvallated,adhesive and three-dimensional growth microenvironment,islet cultured in a PGA scaffold had higher coating efficiency showing stronger staining intensity of enzyme than those in the control group after 14 days of culture following *** in vivo study,PGA scaffold significantly prolonged the average survival time of EC-coated islet graft after transplantation compared with control group （15.30 ± 5.60 days vs.8.30 ± 2.45 days,P 〈 0.05）.The angiogenesis and area of survived grafts were more in the PGA group compared with the control group by measuring the mean microvessel density （8.60 ± 1.21/mm2 vs.5.20 ± 0.87/mm2,P 〈 0.05）.In addition,expression of VEGF and tyrosin-protein kinase receptor

关键词： Endothelial Cells Islet Polyglycolic Acid Fibrous Revascularization

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DCD肾移植中DGF高风险受者应用EC-MPS联合低剂量他克莫司治疗的预后分析（英文）

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2018年第6期19卷 481-489页

作者： Li-zi JIAO Chen-guang DING pu-xun tian Xiao-ming DING Xiao-ming PAN He-li XIANG Xiao-hui tian Yang LI Jin ZHENG Wu-jun XUE Department of Kidney Transplantation the First Affiliated Hospital of Xi'an Jiaotong University Institute of Organ Transplantation Xi'an Jiaotong University

目的:分析心脏死亡供体(DCD)肾移植中移植物功能延迟恢复(DGF)高风险受者应用米芙(EC-MPS)联合低剂量他克莫司治疗术后1年的有效性及安全性,指导临床用药。创新点:对比DGF高风险受者术后发生DGF及正常恢复受者的免疫抑制剂药代动力学特... 详细信息

目的:分析心脏死亡供体(DCD)肾移植中移植物功能延迟恢复(DGF)高风险受者应用米芙(EC-MPS)联合低剂量他克莫司治疗术后1年的有效性及安全性,指导临床用药。创新点:对比DGF高风险受者术后发生DGF及正常恢复受者的免疫抑制剂药代动力学特征。方法:将本中心进行肾移植的61例DGF高风险受者按照实际病情纳入DGF组及正常恢复组,均行米芙联合低剂量他克莫司免疫抑制治疗。对比两组免疫抑制剂血药浓度及预后各项指标。结论:DGF组及正常恢复组间米芙及他克莫司剂量无显著差异。术后1周DGF组EC-MPS血药浓度曲线下面积小于30(mg·h)/L的比率显著高于正常恢复组,同时他克莫司谷浓度显著低于正常恢复组,DGF组经活检证实的急排反应的发生率显著高于正常恢复组,术后1周的EC-MPS血药浓度曲线下面积低于30(mg·h)/L的受者中经活检证实的急排反应的发生率显著高于其他受者。1年移植物存活率、移植物功能及常见不良反应在两组间未见明显差异。

关键词：米芙(EC-MPS) 他克莫司移植物功能延迟恢复(DGF) 心脏死亡供体(DCD) 肾移植

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