本文選題：腎移植 + 淋巴細胞��； 參考：《復旦大學》2014年博士論文

【摘要】：第一部分腎移植受者移植前外周血白細胞分型與腎移植預后關系目的：腎移植受者免疫狀態(tài)的監(jiān)測對評估其罹患排斥反應、腫瘤、感染或免疫抑制劑藥物中毒風險的高低十分重要。在腎移植臨床工作和科學研究的實踐中,對腎移植受者免疫狀態(tài)尤其是術后相關指標的系統(tǒng)評估方法正逐漸完善。群體反應性抗體的檢測已經(jīng)應用于移植前受者的風險評估,而探尋其他移植前免疫檢測指標也逐漸得到了學界的共識。腎移植受者在移植前淋巴細胞功能紊亂,表現(xiàn)為T細胞數(shù)顯著減少,CD4+/CD8±比例降低,Th1/Th2匕例升高,同時伴有初始T細胞和中央記憶T細胞細胞減少,CD4+CD28-T細胞比例增加,。此種狀態(tài)導致T細胞免疫的減弱,從而增加術后移植腎功能延遲恢復或急性排斥反應的發(fā)生率,影響腎移植預后。本部分旨在研究腎移植受者移植前白細胞及淋巴細胞分型與術后移植物功能恢復情況的關系,并探討其意義。方法：收集2012年12月至2013年12月于復旦大學附屬中山醫(yī)院泌尿外科腎移植中心行同種異體腎移植術尿毒癥患者的相關資料,檢測相關臨床生化指標,采用流式細胞術檢測淋巴細胞分型。根據(jù)腎移植受者術后6個月肌酐水平三分位數(shù)將患者分為術后6個月肌酐較低組(Group A:Cr≤95 μmol/L)、肌酐中等組(Group B:95Cr≤139μmol/L)、肌酐較高組(Group C:Cr139μmol/L),并采用單因素方差分析方法、單因素線性分析及多因素Logistic回歸分析比較移植前白細胞、淋巴細胞、CD3+ T細胞、CD3+CD4+ T細胞、CD3+CD8+ T細胞、B細胞、NK細胞數(shù)及CD3+/CD4+比值與術后6個月肌酐的關系。結果：術后6個月肌酐較低組移植前白細胞總數(shù)顯著低于肌酐較高組(5.36±0.67vs.7.3±1.32,p=0.010),移植前淋巴細胞數(shù)顯著低于肌酐較高組(0.93±0.46vs.1.86±0.47,p=0.005),移植前CD3+T細胞數(shù)顯著低于肌酐中等組及肌酐較高組(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細胞數(shù)顯著低于肌酐中等組及肌酐較高組(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細胞數(shù)顯著低于肌酐較高組(22.33±17.53 vs.59.98±25.64,p=0.010)。移植前CD19+B細胞數(shù)、NK細胞數(shù)及CD3+/CD4+比值在各組間無顯著性差異。單因素線性分析顯示移植前白細胞總數(shù)、淋巴細胞數(shù)、CD3+T細胞數(shù)、CD3+CD4+ T細胞數(shù)、CD3+CD8+ T細胞數(shù)與術后6個月肌酐值顯著正相關(p均0.05),移植前CD19+B細胞數(shù)、NK細胞數(shù)及CD3+/CD4+比值與術后6個月肌酐值無顯著相關性。進一步術后6個月肌酐較低組、肌酐中等組和肌酐較高組中對年齡、性別、高血壓、糖尿病、供腎類型、術前肌酐進行多因素Logistic回歸分析,顯示移植前白細胞總數(shù)在不同分組間不存在顯著性差異,而移植前淋巴細胞數(shù)、CD3+T細胞數(shù)、CD3+CD4+ T細胞數(shù)、CD3+CD8+ T細胞數(shù)在不同分組間存在顯著性差異(p均0.001)。結論：腎移植受者移植前外周血白細胞、淋巴細胞及其亞型水平與術后腎功能水平密切相關。移植前淋巴細胞數(shù)、CD3+T細胞數(shù)、CD3+CD4+ T細胞數(shù)、CD3+CD8+ T細胞數(shù)較高的腎移植受者,術后6個月血清肌酐趨于維持在較高水平。因此,腎移植受者移植前免疫功能狀態(tài)可能影響移植腎預后,該結論尚需進一步的研究證實。第二部分ESRD患者外周血白細胞分類變化及淋巴細胞分型的相關因素分析目的：腎移植受者在移植前ESRD期間因氧化應激、尿毒癥毒素潴留、代謝紊亂等,存在免疫激活與免疫抑制并存的狀態(tài),對各種免疫細胞功能有廣泛的影響,這種免疫功能紊亂可能影響移植腎短期及長期預后。本研究旨在比較ESRD患者與健康志愿者不同白細胞分類水平,分析ESRD患者淋巴細胞亞群水平的相關因素,探討影響腎移植受者移植前免疫細胞變化的因素。方法：分別收集2012年12月至2013年12月于復旦大學附屬中山醫(yī)院泌尿外科腎移植中心行活體親屬腎移植術之健康供腎者及2012年10月至2013年6月于復旦大學附屬中山醫(yī)院腎臟內(nèi)科住院之尿毒癥患者的相關資料,檢測相關臨床生化指標,同時采用流式細胞術檢測淋巴細胞分型。采用單因素方差分析、獨立樣本t檢驗和Kruskal-Wallis檢驗比較ESRD患者與健康志愿者白細胞分類水平,采用雙變量相關分析淋巴細胞亞群與其他臨床及實驗室指標的相關性,采用多因素線性回歸分析T細胞分型與其他臨床及實驗室指標的相關性。結果：在ESRD組和健康對照組間進行比較發(fā)現(xiàn)ESRD組外周血白細胞總數(shù)顯著高于健康對照組[(6.43±2.19)×109vs.(5.67±1.39)×109,p=0.025],中性粒細胞數(shù)顯著高于健康對照組[(4.34±1.99)×109vs.(3.28±1.26)×109,p=0.012],淋巴細胞數(shù)顯著低于健康對照組[(1.45±0.55)×109 vs.(1.88±0.42)×109,p=0.007],中性粒細胞/淋巴細胞比值顯著高于健康對照組(3.60±3.08 vs.1.83±0.85,p=0.007)。在ESRD組與健康對照組中對年齡、性別因素回歸后,白細胞總數(shù)在組間不存在顯著性差異,而中性粒細胞數(shù)(p=0.015)、淋巴細胞數(shù)(p=0.001)、NLR(p0.001)在組間仍有顯著性差異。將ESRD組患者根據(jù)不同透析方式分為未透析組、血透組和腹透組,發(fā)現(xiàn)健康對照組外周血淋巴細胞數(shù)分別顯著高于未透析組(1.88±0.42 vs.1.49±0.65,p=0.002)、血透組(1.88±0.42 vs.1.45±0.48,p=0.023)和腹透組(1.88±0.42 vs.1.39±0.48,p=0.001)；而不同透析組之間無顯著性差異。單因素相關性分析顯示ESRD患者外周血中性粒細胞數(shù)與糖尿病、CVD、血磷、NT-proBNP、hsCRP降鈣素原等呈顯著正相關,與白蛋白、轉鐵蛋白、25OH-vitD等呈顯著負相關(p均0.05)；淋巴細胞數(shù)與eGFR、血紅蛋白、前白蛋白、膽固醇、甘油三酯、LDL-C、二氧化碳、血鈣、轉鐵蛋白等呈顯著正相關,與p2-微球蛋白、尿素、肌酐、NT-proBNP、hsCRPiPTH、降鈣素原、促紅素等呈顯著負相關(p均0.05)；B細胞數(shù)與收縮壓、eGFR、血紅蛋白、白蛋白、前白蛋白、膽固醇、甘油三酯、LDL-C、轉鐵蛋白等呈顯著正相關,與年齡、β2-微球蛋白、尿素、肌酐、NT-proBNP、hsCRP、降鈣素原、促紅素等呈顯著負相關(p均0.05)。多因素線性回歸分析顯示CD3+T細胞數(shù)與收縮壓、BMI、肌酐、膽固醇、鈣呈獨立正相關,與吸煙史、舒張壓、β2-微球蛋白、白蛋白、尿素呈獨立負相關(p均0.05)；CD3+CD4+ T細胞數(shù)與收縮壓、LDL-C呈獨立正相關,與高血壓史、舒張壓、β2-微球蛋白、尿素、hsCRP呈獨立負相關(p均0.05)；CD3+CD8+ T細胞數(shù)與肌酐、膽固醇呈獨立正相關,與吸煙史、p2-微球蛋白呈獨立負相關(p均0.05)。結論：腎移植受者移植前免疫狀態(tài)和健康對照組不同,這可能與ESRD患者合并脂代謝紊亂、尿毒癥毒素潴留等因素有關。第三部分硫酸吲哚酚和硫酸對甲酚對淋巴細胞增殖影響研究目的：硫酸吲哚酚和硫酸對甲酚是新近發(fā)現(xiàn)的兩種與蛋白質(zhì)結合的尿毒癥毒素,在慢性腎臟病及腎移植受者中,它們與腎功能密切相關。研究表明這兩種毒素可以激活固有免疫細胞,但有關其對適應性免疫細胞影響的研究未見報道。本部分擬探討硫酸吲哚酚和對甲酚對淋巴細胞增殖或凋亡是否存在影響,并初步探討此種影響是否呈時間或濃度依賴性。方法：收集健康志愿者外周血標本,提取外周血單個核細胞后分離并培養(yǎng)淋巴細胞,在5% CO2、37℃C的環(huán)境下用合適的細胞培養(yǎng)液及適宜濃度的PHA培養(yǎng)淋巴細胞,并在培養(yǎng)體系中分別加入不同濃度的硫酸吲哚酚(10μg/ml、 25nμg/ml、50μ/ml)或不同濃度的對甲酚(0.10mmol/L、0.25mmol/L、 0.50mmol/L),與淋巴細胞共同孵育,同時設立對照組,共同孵育12h、24h、48h結束后收集各組細胞,進行細胞計數(shù)并用CCK-8 kit檢測各孔淋巴細胞吸光度,評價其增殖或凋亡水平。結果：在PHA刺激下,將IndS與外周血淋巴細胞共培養(yǎng)24h,結果顯示IndS50μg/ml組淋巴細胞增殖度顯著高于空白對照組及IndS組(p均0.05)；PC與外周血淋巴細胞共培養(yǎng)24h,結果顯示PC 0.50mmol/L組淋巴細胞增殖度顯著高于空白對照組及PC 0.10mmol/L組(p均0.01)。IndS與外周血淋巴細胞共培養(yǎng)12h后,IndS 10μg/ml組淋巴細胞增殖度顯著高于空白對照組(p0.05),然而與各個高濃度組無顯著性差異,高濃度IndS組與空白對照組亦無顯著性差異。IndS與外周血淋巴細胞共培養(yǎng)48h后,不同濃度IndS組淋巴細胞增殖度顯著高于空白對照組(p均0.01),各組間無顯著性差異。PC與外周血淋巴細胞共培養(yǎng)12h或48h后,不同濃度PC組淋巴細胞增殖度顯著高于空白對照組(p均0.01),各組間無顯著性差異。結論：在體外條件,適宜濃度PHA刺激下,硫酸吲哚酚和對甲酚會促進人外周血淋巴細胞的增殖,并且共培養(yǎng)24h時硫酸吲哚酚和對甲酚對淋巴細胞的促增殖作用呈濃度依賴性的,共培養(yǎng)過長或過段時間時促增殖作用不表現(xiàn)為濃度依賴性。
[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.

【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R699.2

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