肾移植受者移植前免疫状态及其与移植肾功能关系研究

发布时间：2018-04-21 04:29

本文选题：肾移植 + 淋巴细胞　；参考：《复旦大学》2014年博士论文

【摘要】：第一部分肾移植受者移植前外周血白细胞分型与肾移植预后关系目的：肾移植受者免疫状态的监测对评估其罹患排斥反应、肿瘤、感染或免疫抑制剂药物中毒风险的高低十分重要。在肾移植临床工作和科学研究的实践中,对肾移植受者免疫状态尤其是术后相关指标的系统评估方法正逐渐完善。群体反应性抗体的检测已经应用于移植前受者的风险评估,而探寻其他移植前免疫检测指标也逐渐得到了学界的共识。肾移植受者在移植前淋巴细胞功能紊乱,表现为T细胞数显著减少,CD4+/CD8±比例降低,Th1/Th2匕例升高,同时伴有初始T细胞和中央记忆T细胞细胞减少,CD4+CD28-T细胞比例增加,。此种状态导致T细胞免疫的减弱,从而增加术后移植肾功能延迟恢复或急性排斥反应的发生率,影响肾移植预后。本部分旨在研究肾移植受者移植前白细胞及淋巴细胞分型与术后移植物功能恢复情况的关系,并探讨其意义。方法：收集2012年12月至2013年12月于复旦大学附属中山医院泌尿外科肾移植中心行同种异体肾移植术尿毒症患者的相关资料,检测相关临床生化指标,采用流式细胞术检测淋巴细胞分型。根据肾移植受者术后6个月肌酐水平三分位数将患者分为术后6个月肌酐较低组(Group A:Cr≤95 μmol/L)、肌酐中等组(Group B:95Cr≤139μmol/L)、肌酐较高组(Group C:Cr139μmol/L),并采用单因素方差分析方法、单因素线性分析及多因素Logistic回归分析比较移植前白细胞、淋巴细胞、CD3+ T细胞、CD3+CD4+ T细胞、CD3+CD8+ T细胞、B细胞、NK细胞数及CD3+/CD4+比值与术后6个月肌酐的关系。结果：术后6个月肌酐较低组移植前白细胞总数显著低于肌酐较高组(5.36±0.67vs.7.3±1.32,p=0.010),移植前淋巴细胞数显著低于肌酐较高组(0.93±0.46vs.1.86±0.47,p=0.005),移植前CD3+T细胞数显著低于肌酐中等组及肌酐较高组(vs. Group B:61.60±38.35 vs.122.76±61.98,p=0.027; vs. Group C: 61.60±38.35 vs 132.99±43.94, p=0.012),移植前CD3+CD4+ T细胞数显著低于肌酐中等组及肌酐较高组(vs. Group B:36.84±20.86 vs.70.86±29.39, p=0.013; vs. Goup C:36.84±20.86 vs.65.66±20.10, p=0.031),移植前CD3+CD8+ T细胞数显著低于肌酐较高组(22.33±17.53 vs.59.98±25.64,p=0.010)。移植前CD19+B细胞数、NK细胞数及CD3+/CD4+比值在各组间无显著性差异。单因素线性分析显示移植前白细胞总数、淋巴细胞数、CD3+T细胞数、CD3+CD4+ T细胞数、CD3+CD8+ T细胞数与术后6个月肌酐值显著正相关(p均0.05),移植前CD19+B细胞数、NK细胞数及CD3+/CD4+比值与术后6个月肌酐值无显著相关性。进一步术后6个月肌酐较低组、肌酐中等组和肌酐较高组中对年龄、性别、高血压、糖尿病、供肾类型、术前肌酐进行多因素Logistic回归分析,显示移植前白细胞总数在不同分组间不存在显著性差异,而移植前淋巴细胞数、CD3+T细胞数、CD3+CD4+ T细胞数、CD3+CD8+ T细胞数在不同分组间存在显著性差异(p均0.001)。结论：肾移植受者移植前外周血白细胞、淋巴细胞及其亚型水平与术后肾功能水平密切相关。移植前淋巴细胞数、CD3+T细胞数、CD3+CD4+ T细胞数、CD3+CD8+ T细胞数较高的肾移植受者,术后6个月血清肌酐趋于维持在较高水平。因此,肾移植受者移植前免疫功能状态可能影响移植肾预后,该结论尚需进一步的研究证实。第二部分ESRD患者外周血白细胞分类变化及淋巴细胞分型的相关因素分析目的：肾移植受者在移植前ESRD期间因氧化应激、尿毒症毒素潴留、代谢紊乱等,存在免疫激活与免疫抑制并存的状态,对各种免疫细胞功能有广泛的影响,这种免疫功能紊乱可能影响移植肾短期及长期预后。本研究旨在比较ESRD患者与健康志愿者不同白细胞分类水平,分析ESRD患者淋巴细胞亚群水平的相关因素,探讨影响肾移植受者移植前免疫细胞变化的因素。方法：分别收集2012年12月至2013年12月于复旦大学附属中山医院泌尿外科肾移植中心行活体亲属肾移植术之健康供肾者及2012年10月至2013年6月于复旦大学附属中山医院肾脏内科住院之尿毒症患者的相关资料,检测相关临床生化指标,同时采用流式细胞术检测淋巴细胞分型。采用单因素方差分析、独立样本t检验和Kruskal-Wallis检验比较ESRD患者与健康志愿者白细胞分类水平,采用双变量相关分析淋巴细胞亚群与其他临床及实验室指标的相关性,采用多因素线性回归分析T细胞分型与其他临床及实验室指标的相关性。结果：在ESRD组和健康对照组间进行比较发现ESRD组外周血白细胞总数显著高于健康对照组[(6.43±2.19)×109vs.(5.67±1.39)×109,p=0.025],中性粒细胞数显著高于健康对照组[(4.34±1.99)×109vs.(3.28±1.26)×109,p=0.012],淋巴细胞数显著低于健康对照组[(1.45±0.55)×109 vs.(1.88±0.42)×109,p=0.007],中性粒细胞/淋巴细胞比值显著高于健康对照组(3.60±3.08 vs.1.83±0.85,p=0.007)。在ESRD组与健康对照组中对年龄、性别因素回归后,白细胞总数在组间不存在显著性差异,而中性粒细胞数(p=0.015)、淋巴细胞数(p=0.001)、NLR(p0.001)在组间仍有显著性差异。将ESRD组患者根据不同透析方式分为未透析组、血透组和腹透组,发现健康对照组外周血淋巴细胞数分别显著高于未透析组(1.88±0.42 vs.1.49±0.65,p=0.002)、血透组(1.88±0.42 vs.1.45±0.48,p=0.023)和腹透组(1.88±0.42 vs.1.39±0.48,p=0.001)；而不同透析组之间无显著性差异。单因素相关性分析显示ESRD患者外周血中性粒细胞数与糖尿病、CVD、血磷、NT-proBNP、hsCRP降钙素原等呈显著正相关,与白蛋白、转铁蛋白、25OH-vitD等呈显著负相关(p均0.05)；淋巴细胞数与eGFR、血红蛋白、前白蛋白、胆固醇、甘油三酯、LDL-C、二氧化碳、血钙、转铁蛋白等呈显著正相关,与p2-微球蛋白、尿素、肌酐、NT-proBNP、hsCRPiPTH、降钙素原、促红素等呈显著负相关(p均0.05)；B细胞数与收缩压、eGFR、血红蛋白、白蛋白、前白蛋白、胆固醇、甘油三酯、LDL-C、转铁蛋白等呈显著正相关,与年龄、β2-微球蛋白、尿素、肌酐、NT-proBNP、hsCRP、降钙素原、促红素等呈显著负相关(p均0.05)。多因素线性回归分析显示CD3+T细胞数与收缩压、BMI、肌酐、胆固醇、钙呈独立正相关,与吸烟史、舒张压、β2-微球蛋白、白蛋白、尿素呈独立负相关(p均0.05)；CD3+CD4+ T细胞数与收缩压、LDL-C呈独立正相关,与高血压史、舒张压、β2-微球蛋白、尿素、hsCRP呈独立负相关(p均0.05)；CD3+CD8+ T细胞数与肌酐、胆固醇呈独立正相关,与吸烟史、p2-微球蛋白呈独立负相关(p均0.05)。结论：肾移植受者移植前免疫状态和健康对照组不同,这可能与ESRD患者合并脂代谢紊乱、尿毒症毒素潴留等因素有关。第三部分硫酸吲哚酚和硫酸对甲酚对淋巴细胞增殖影响研究目的：硫酸吲哚酚和硫酸对甲酚是新近发现的两种与蛋白质结合的尿毒症毒素,在慢性肾脏病及肾移植受者中,它们与肾功能密切相关。研究表明这两种毒素可以激活固有免疫细胞,但有关其对适应性免疫细胞影响的研究未见报道。本部分拟探讨硫酸吲哚酚和对甲酚对淋巴细胞增殖或凋亡是否存在影响,并初步探讨此种影响是否呈时间或浓度依赖性。方法：收集健康志愿者外周血标本,提取外周血单个核细胞后分离并培养淋巴细胞,在5% CO2、37℃C的环境下用合适的细胞培养液及适宜浓度的PHA培养淋巴细胞,并在培养体系中分别加入不同浓度的硫酸吲哚酚(10μg/ml、 25nμg/ml、50μ/ml)或不同浓度的对甲酚(0.10mmol/L、0.25mmol/L、 0.50mmol/L),与淋巴细胞共同孵育,同时设立对照组,共同孵育12h、24h、48h结束后收集各组细胞,进行细胞计数并用CCK-8 kit检测各孔淋巴细胞吸光度,评价其增殖或凋亡水平。结果：在PHA刺激下,将IndS与外周血淋巴细胞共培养24h,结果显示IndS50μg/ml组淋巴细胞增殖度显著高于空白对照组及IndS组(p均0.05)；PC与外周血淋巴细胞共培养24h,结果显示PC 0.50mmol/L组淋巴细胞增殖度显著高于空白对照组及PC 0.10mmol/L组(p均0.01)。IndS与外周血淋巴细胞共培养12h后,IndS 10μg/ml组淋巴细胞增殖度显著高于空白对照组(p0.05),然而与各个高浓度组无显著性差异,高浓度IndS组与空白对照组亦无显著性差异。IndS与外周血淋巴细胞共培养48h后,不同浓度IndS组淋巴细胞增殖度显著高于空白对照组(p均0.01),各组间无显著性差异。PC与外周血淋巴细胞共培养12h或48h后,不同浓度PC组淋巴细胞增殖度显著高于空白对照组(p均0.01),各组间无显著性差异。结论：在体外条件,适宜浓度PHA刺激下,硫酸吲哚酚和对甲酚会促进人外周血淋巴细胞的增殖,并且共培养24h时硫酸吲哚酚和对甲酚对淋巴细胞的促增殖作用呈浓度依赖性的,共培养过长或过段时间时促增殖作用不表现为浓度依赖性。
[Abstract]:Part 1 Relationship between peripheral blood leucocyte typing before transplantation in renal transplant recipients and prognosis of renal transplant recipients: the monitoring of immune status in renal transplant recipients is very important for assessing the risk of rejection, tumor, infection, or immunosuppressive drug poisoning. In the practice of renal transplantation and in the practice of scientific research, renal transplantation is subject to renal transplantation. The system evaluation method of the immune state, especially the related indexes after the operation is gradually improved. The detection of the group reactive antibody has been applied to the risk assessment of the recipients before transplantation, and the exploration of other pre transplant immune detection indicators has gradually gained the consensus of the academic community. The renal transplant recipients have the dysfunction of the lymphocytes before transplant, showing T cells. The decrease of the number, the decrease of CD4+/CD8 + ratio, the increase of Th1/Th2 dagger, the decrease of the initial T cells and central memory T cells, the increase in the proportion of CD4+CD28-T cells, which leads to the weakening of the T cell immunity, thus increasing the rate of delayed recovery of the renal function and the incidence of acute rejection after the operation, and affecting the prognosis of renal transplantation. The purpose of this study was to study the relationship between the pre transplant leukocyte and lymphocyte subtypes and the recovery of graft function after transplantation, and to explore its significance. Methods: to collect related data from December 2012 to December 2013 in the uremic patients with allograft renal transplantation in the Department of Urology, Zhongshan Hospital Affiliated to Fudan University, the renal transplantation center. The related clinical biochemical indexes were measured by flow cytometry. The patients were divided into lower creatinine group (Group A:Cr < 95 mol/L), the medium group of creatinine (Group B:95Cr < 139 mol/L), the higher creatinine group (Group C:Cr139 u mol/L), and the single cause, according to the three digits of creatinine level at 6 months after the operation of the renal transplantation recipients. The relationship between pre transplant leukocyte, lymphocyte, CD3+ T cell, CD3+CD4+ T cell, CD3+CD8+ T cell, B cell, NK cell number and CD3+/CD4+ ratio was compared with creatinine at 6 months after operation. Results: the total number of white blood cells in the lower creatinine group was significantly lower at 6 months after the operation. In the high creatinine group (5.36 + 0.67vs.7.3 + 1.32, p=0.010), the number of lymphocytes before transplantation was significantly lower than that in the higher creatinine group (0.93 + 0.46vs.1.86 + 0.47, p=0.005). The number of CD3+T cells before transplantation was significantly lower than that in the middle creatinine group and the higher creatinine group (vs. Group B:61.60 + 38.35 vs.122.76 + 61.98, p=0.027, vs. Group 61.60 + 38.35 132.99 + 43.94, P=0.012), the number of CD3+CD4+ T cells before transplantation was significantly lower than that in the medium creatinine group and the high creatinine group (vs. Group B:36.84 + 20.86 vs.70.86 + 29.39, p=0.013; vs. Goup C:36.84 + 20.86 vs.65.66 + 20.10). The number of cells before transplantation was significantly lower than that in the higher creatinine group (22.33 + 17.53 25.64, 25.64,). There was no significant difference between the number of cells and the number of NK cells and the ratio of CD3+/CD4+. Single factor linear analysis showed that the total number of leukocytes, the number of lymphocytes, the number of CD3+T cells, the number of CD3+T cells, the number of CD3+CD4+ T cells, the number of CD3+CD8+ T cells were significantly positively correlated with the creatinine value of the 6 months after the operation (P 0.05), and the number of CD19+B cells, the number of NK cells and the CD3+/CD4+ ratio before transplantation. There was no significant correlation between creatinine values at 6 months after the operation. A multiple factor Logistic regression analysis of age, sex, hypertension, diabetes, kidney type, and preoperative creatinine in the middle creatinine group and the higher creatinine group at 6 months after the operation, and in the higher creatinine group, showed that the total number of white blood cells before transplantation did not differ significantly between the different groups, but before transplantation. The number of lymphocytes, the number of CD3+T cells, the number of CD3+CD4+ T cells and the number of CD3+CD8+ T cells were significantly different between different groups (P 0.001). Conclusion: the level of lymphocyte and its subtype in renal transplant recipients is closely related to the level of postoperative renal function. The number of lymphocytes, the number of CD3+T cells, the number of CD3+CD4+ T cells before transplantation, and the number of CD3+CD4+ T cells. The serum creatinine level of CD3+CD8+ T cells tends to maintain at a high level at 6 months after the operation. Therefore, the immune function status before transplantation in renal transplant recipients may affect the prognosis of renal transplantation. This conclusion needs further research. The correlation of peripheral blood leucocyte classification and lymphocyte typing in the second part of ESRD patients is related. Factor analysis: renal transplant recipients have a state of coexistence of immune activation and immunosuppression in the period of pre transplant ESRD due to oxidative stress, uremia toxin retention and metabolic disorder, which have a wide influence on the function of various immune cells. This immune disorder may affect the short-term and long-term prognosis of the transplanted kidney. This study aims to compare ESRD The leucocyte classification level was different from the healthy volunteers, the factors related to the lymphocyte subsets in ESRD patients were analyzed, and the factors affecting the changes of immune cells before transplantation were discussed. Methods: from December 2012 to December 2013, the living relative kidney of the renal transplantation center in Department of Urology, Zhongshan Hospital Affiliated to Fudan University, was collected. Healthy donors and patients with uremia hospitalized from October 2012 to June 2013 in Zhongshan Hospital Affiliated to Fudan University in the Department of Nephrology, the related clinical biochemical parameters were detected, and flow cytometry was used to detect lymphocyte subtypes. Single factor analysis of variance, independent sample t test and Kruskal-Wallis test ratio were used. The leucocyte classification level of ESRD patients and healthy volunteers was compared with other clinical and laboratory indexes by the bivariate correlation analysis of lymphocyte subsets, and the correlation between T cell typing and other clinical and laboratory indicators was analyzed by multifactor linear regression. Results: ESRD was compared between the ESRD group and the healthy control group. The total number of white blood cells in peripheral blood was significantly higher than that of the healthy control group [(6.43 + 2.19) x 109vs. (5.67 + 1.39) x 109, p=0.025], and the number of neutrophils was significantly higher than that of the healthy control group [(4.34 + 1.99) * 109vs. (3.28 + 1.26) * 109, p=0.012], and the number of lymphocytes was significantly lower than that in the healthy group [(1.45 + 0.55) * 109 vs. (1.88 + 4.34), p=0.007], neutral particle size The ratio of cell to lymphocyte was significantly higher than that in the healthy control group (3.60 + 3.08 vs.1.83 + 0.85, p=0.007). There was no significant difference between the groups in the group ESRD and the healthy control group after the regression of sex factors, but the number of neutrophils (p=0.015), the number of lymphatic cells (p=0.001), and NLR (p0.001) still had significant differences between the groups. ES Group RD patients were divided into non dialysis group, hemodialysis group and abdominal dialysis group according to different dialysis methods. The number of peripheral blood lymphocytes in the healthy control group was significantly higher than that of the non dialysis group (1.88 + 0.42 vs.1.49 + 0.65, p=0.002), the hemodialysis group (1.88 + 0.42 vs.1.45 + 0.48, p=0.023) and the abdominal penetration group (1.88 + 0.42 vs.1.39 + 0.48, p=0.001), and different dialysis groups. The single factor correlation analysis showed that the number of neutrophils in peripheral blood of ESRD patients was positively correlated with diabetes, CVD, blood phosphorus, NT-proBNP, hsCRP calcitonin, and was negatively correlated with albumin, transferrin and 25OH-vitD (P 0.05); the number of lymphoblastic cells and eGFR, hemoglobin, prealbumin, cholesterol, glycerin, and glycerin were three. Ester, LDL-C, carbon dioxide, blood calcium and transferrin were positively correlated with p2- microglobulin, urea, creatinine, NT-proBNP, hsCRPiPTH, calcitonin, and erythropoietin (P 0.05), and B cells were positively correlated with systolic blood pressure, eGFR, hemoglobin, albumin, prealbumin, cholesterol, triglyceride, LDL-C, and transferrin. There was a significant negative correlation with age, beta 2- microglobulin, urea, creatinine, NT-proBNP, hsCRP, calcitonin, and erythropoietin (P 0.05). Multifactor linear regression analysis showed that the number of CD3+T cells was independent positive correlation with systolic blood pressure, BMI, creatinine, cholesterol and calcium, which had independent negative correlation with the history of smoking, diastolic pressure, beta 2- microglobulin, albumin and urea (P 0.05). The number of CD3+CD4+ T cells was independent positive correlation with systolic blood pressure and LDL-C, which had independent negative correlation with hypertension, diastolic pressure, beta 2- microglobulin, urea and hsCRP (P 0.05); the number of CD3+CD8+ T cells was independent positive correlation with creatinine and cholesterol, and was negatively correlated with the history of smoking and p2- microglobulin (P 0.05). Conclusion: renal transplant recipients were immunized before transplantation. The status and health control group are different, this may be associated with ESRD patients with lipid metabolism disorders, uremic toxin retention and other factors. Third part of the study of the effects of indolenol sulfate and sulfuric acid on the proliferation of lymphocyte proliferation: indolol sulphate and sulfuric acid to cresol are newly discovered two kinds of uremic toxin associated with protein, in slow They are closely related to renal function. The study shows that these two toxins can activate the innate immune cells, but there is no report on the influence of the immune cells on the adaptive immune cells. This part is to explore whether there is an effect of indolol sulfate and cresol on the proliferation or apoptosis of lymphocytes. Method: collect the peripheral blood samples from the healthy volunteers, extract the peripheral blood mononuclear cells and isolate and cultivate the lymphocytes. In the environment of 5% CO2,37 C C, the lymphocytes were cultured with appropriate cell culture solution and suitable concentration of PHA, and the different concentration of sulfuric acid was added to the culture system. Indoles (10 g/ml, 25N g/ml, 50 /ml) or different concentrations of cresol (0.10mmol/L, 0.25mmol/L, 0.50mmol/L) were incubated with lymphocytes. At the same time, a control group was set up to incubate 12h, 24h, 48h, and the cells were collected at the end of 48h. Cell counts were counted and the lymphocyte absorbency was detected by CCK-8 kit, and the proliferation or apoptosis level was evaluated. Results: the results showed that IndS and peripheral blood lymphocytes were co cultured with 24h under PHA stimulation. The results showed that the proliferation of lymphocyte in IndS50 mu g/ml group was significantly higher than that of blank control group and IndS group (P 0.05). PC and peripheral blood lymphocytes co cultured 24h. The results showed that the lymphocytic proliferation of PC 0.50mmol/L group was significantly higher than that of the blank control group and the PC restriction group. Group (P 0.01).IndS and peripheral blood lymphocytes co culture 12h, the proliferation degree of lymphocyte in IndS 10 g/ml group was significantly higher than that of blank control group (P0.05), but there was no significant difference with each high concentration group. There was no significant difference between the high concentration IndS group and the blank control group, and.IndS and peripheral blood lymphocytes were co cultured 48h, and the concentration of IndS groups in different concentrations was different. The proliferation of Ba cells was significantly higher than that in the blank control group (P 0.01). There was no significant difference between the groups of.PC and peripheral blood lymphocytes. The proliferation of lymphocytes in the PC group was significantly higher than that in the blank control group (P 0.01). There was no significant difference between the groups of PC groups. Phenolics and cresol can promote the proliferation of human peripheral blood lymphocytes, and the proliferation of IAA and cresol on lymphocyte is concentration dependent on co culture of 24h. The proliferation promoting effect of co culture for long or over time is not concentration dependent.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R699.2

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