【作者】 郝胜华；

【导师】 黄祖发；

【作者基本信息】 中南大学 ， 器官移植， 2008， 博士

【摘要】 第一章健康人淋巴细胞P-gp的表达水平目的:对健康人群淋巴细胞P-gp表达水平进行观察,明确在健康人群的分布特征,为进一步对移植病人淋巴细胞P-gp表达水平变化的研究提供基础。方法:收集健康人63例淋巴细胞标本,年龄8-71岁,分别采用RT-PCR和流式细胞仪技术,对淋巴细胞P-gp的表达从基因和蛋白两个层面进行分析,并对不同性别、年龄和淋巴细胞亚型进行分组比较研究。结果:健康人群淋巴细胞有一定的P-gp表达,平均为12.5%,不同的性别分组和细胞亚型之间,P-gp的水平无明显的差别。14岁以下的儿童表达水平较低,约6.5%,与其他各年龄组相比有显著差异,而在大于14岁的各年龄组之间,淋巴细胞P-gp的水平基本相近。各分组MDR1 mRNA的半定量分析所得的结果与蛋白水平的表达基本相符,与内对照的β-actin mRNA相比,健康人MDR1 mRNA的表达呈现较低的水平。结论:正常情况下,淋巴细胞具有一定的P-gp表达,平均为12.5%。其表达水平在不同性别、细胞亚型和大多数年龄组之间没有明显的差别,处于一种稳定的、较低的表达状态。第二章移植病人淋巴细胞P-gp表达水平的改变目的:观察移植病人在使用免疫抑制剂后,淋巴细胞P-gp表达水平的改变,并探讨其可能的形成原因。方法:移植病人78例,分别在术后1月、3月、6月、12月采集淋巴细胞标本,采用RT-PCR和流式细胞仪技术,对淋巴细胞P-gp的表达从基因和蛋白两个层面进行分析,观察淋巴细胞表达水平的改变,并对不同性别、年龄、细胞亚型等进行分组研究,因病例限制,此部分研究不包括儿童年龄组(＜14岁)。结果:移植病人在接受免疫抑制剂治疗后,淋巴细胞MDR1的表达在基因和蛋白水平均明显的增高。MDR1 mRNA的表达水平为0.47,比用药前升高2.76倍(P＜0.05);P-gp的表达平均为37.5%,较用药前增高2.6倍(P＜0.05)。用药后第一个月,P-gp即有增高18.5%,3个月可达高峰,为40.8%,其后基本维持在此水平;不同性别之间无差别;各年龄组之间无差异。各亚型淋巴细胞P-gp水平均升高,其中CD+4T细胞明显高于其他各亚型。根据增高的程度,可分为三种情况:低度:表达水平＜20%,28例;中度:表达水平20-40%,35例;高度:表达水平＞40%,15例。中、高度的P-gp表达水平与正常对照和低度表达之间具有显著的差异性(P＜0.05)。结论:移植病人在使用免疫抑制剂后,大多数病人(64%)淋巴细胞P-gp的表达可出现明显的增高。说明免疫抑制剂的使用,可诱导P-gp表达水平的增高,而淋巴细胞P-gp表达水平的增高,势必将对环孢素的细胞内分布和免疫抑制作用产生重要的影响。第三章移植病人淋巴细胞P-gp高表达的诱导因素分析目的:通过对不同治疗方案淋巴细胞P-gp表达水平改变的分析研究,探讨诱导P-gp高表达的具体原因,方法:移植病人78例,按其免疫抑制治疗方案,分为下列各组:环孢素方案组:CsA+MMF+Pred;他克莫司方案组:TAC+MMF+Pred;强的松方案组:CsA/TAC+MMF+Pred;非强的松方案组:CsA/TAC+MMF。观察各不同治疗方案对淋巴细胞P-gp表达水平的影响,分析每种药物对P-gp的诱导作用。结果:手术后随诊一年半时间,在环孢素与他克莫司组,移植病人淋巴细胞P-gp的水平均表现出相应的增高,在两组之间并未观察到淋巴细胞P-gp表达的显著差别,说明这两种药物对P-gp的诱导效果相同;而在强的松和非强的松组之间,淋巴细胞P-gp的表达也未见明显的改变,说明强的松不是主要的诱导原因。结论:尽管同属于P-gp作用的底物性药物,但诱导移植病人淋巴细胞P-gp高表达的主要原因为环孢素和他克莫司,强的松并无明显的诱导作用。第四章淋巴细胞P-gp的表达水平与淋巴细胞内药物浓度的关系目的:探讨淋巴细胞P-gp水平与细胞内药物浓度的关系,阐明P-gp的高表达是降低细胞内药物浓度的主要原因。方法:根据淋巴细胞P-gp表达的水平,移植病人分为高、中、低三组,分别采集淋巴细胞标本,进行罗丹明123蓄积试验,判断不同水平P-gp对淋巴细胞内药物浓度的影响;并通过特异性的功能抑制,进一步证实P-gp的影响作用。结果:与健康人相比较,移植病人低表达组的细胞内药物浓度虽有降低,但无明显的差别;中度表达组表现出显著的差别;而高表达组则具有极显著的差别。在移植病人各组之间,中高组与低度组也存在明显的差异性。应用特异的功能抑制剂维拉帕米,可明显升高淋巴细胞内的药物浓度,说明细胞内药物浓度的降低,主要是因为P-gp高表达的所引起的。结论:P-gp的高表达,能够显著降低淋巴细胞内的药物浓度,而在移植病人淋巴细胞P-gp水平的增高,可能对环孢素的作用产生重大的影响。第五章P-gp表达水平对环孢素免疫抑制作用的影响目的:探讨不同表达水平的对环孢素免疫抑制作用的影响方法:收集移植病人淋巴细胞样本,根据P-gp表达水平的不同,分为高、中、低三组。采用有丝分裂原PHA刺激的淋巴细胞转化实验,以淋巴细胞分裂指数和IL-2为指标,观察环孢素对不同表达P-gp水平淋巴细胞活化的抑制作用。结果:在相同的浓度,环孢素对淋巴细胞的活化及细胞因子的抑制作用,随着细胞P-gp表达水平的增高而逐渐减弱。在10-20μg/L之间,可基本全部抑制PHA刺激的淋巴细胞的增殖反应和IL-2的产生,P-gp低度表达的淋巴细胞,尽管其增殖指数和IL-2的产生可分别升高为19%和20ng/L,但与正常对照相比较,尚无明显的差别。在中度和高度表达的患者,环孢素对淋巴细胞的活化抑制作用明显减弱,淋巴细胞的分裂指数可分别达34%和47%,IL-2产生可分别达32ng/L和45ng/L。通过应用P-gp特异的功能抑制剂维拉帕米后,各组间淋巴细胞活化率的差别不再有显著的差别。结论:淋巴细胞P-gp表达水平的增高,可通过降低细胞内的环孢素浓度,减弱环孢素对淋巴细胞的活化抑制作用,因此,P-gp的高表达有可能导致淋巴细胞对环孢素耐药性的产生。第六章P-gp表达水平与移植肾功能的关系的探讨目的:探讨淋巴细胞P-gp表达水平与移植物功能的关系,明确淋巴细胞P-gp的表达的改变,对移植物功能的影响。方法:移植病人78例,根据其淋巴细胞表达水平,分为高、中、低三组。严格定期随访一年,观察血尿素氮、肌酐值等生化指标;移植肾血流动力学指标;24小时尿量、下肢水肿等临床指标。每出现一次异常登记为不良事件一次。结果:78例病人随访一年,共计发生不良事件291人次,其中出现血尿素氮升高、血肌酐升高191人次;出现尿量明显减少、下肢水肿63人次;移植肾脏血流动力学指标的异常47人次。分析发现,淋巴细胞P-gp的表达水平与移植肾功能有显著的相关性,淋巴细胞P-gp低表达的病人,其移植肾脏功能明显好于淋巴细胞P-GP高表达的病人。结论:移植病人淋巴细胞表达水平的增高,将对环孢素的免疫抑制作用产生明显的影响,进而引起免疫抑制不足,导致移植物功能的受损。更多还原

【Abstract】 Part one The expression level of lymphocytes P-gp in healthy populationObjective:To investigate the P-gp expression level of lymphoctes in healty populatioon,in order to establish the diagnosis standards in overexpression cases.Methods:Collecting lymphoctye samples from 63 healthy cases,to analyze the expression level of P-gp using RT-PCR and FACScan technique,comparising the expression level among groups classified as sex,age,lymphocyte subtypes.Results:The average epression level of lymphocyte P-gpin healthy person is 12.5%.the diffrences among the sex groups and lymphocytye subtype groups are not distinct respcetively;no diffrence among age groups except the significant lower expression in＜14yr group;the results of RT-PCR is similar to that of FACScan.Conclusions:Physically,P-gp is positively experssed in lymphocytes with a 12.5%average level.No difference are found among groups classified as sex,lymphocyte subtype,age,except the＜4yr group expressing lower P-gp. Part two Alteration of P-gp expression in transplant recipientsObjective:To investigate the alteration of lymphocyte P-gp expression in transplant recipients.Methods:Collecting the lymphocyte samples from 78 recipients in 1st、3rd、6th、12th month respectively,analyze the expression level of P-gp,and comparise the difference among groups classified as sex、age、lymphocyte subtype.Results:Averagelly,the expression level of lymphocyte P-gp in transplant recipients increase initially at 1st month,to the peak level around 3rd month,after then,keep stable at this level.No difference is between sex groups and age groups;the P-gp expression level in CD+4T cell is significantlly higher than in B cell and CD+8T cell.Conclusions:Medication of immunosuppressant may induce the overexpression of lymphocyte P-gp in transplant recipients. Part three Analysis of the induction factors of P-gp overexpressionObjevtive:To make sure the drugs responsible for the induction of P-gp overexpression in transplant recipients.Methods:Analyzing the expression level of lymphocytes P-gp in transplant recipients medicated by different regimens respectively,to eluciate the role of particular immunosuppressant in the induction of P-gp expression.Results:The expression levels of P-gp are higher and stable in recipients medicated by calcineurin inhibitors with or without prednison, meaning that glycucorticoids play a minor role in the induction of P-gp even though it is a kind of P-gp substrates.Conclusions:Calcineurin inhibitors are major factors implicated in the induction of overexpression of lymphocyte P-gp in transplant recipients. Part four Infuence of P-gp overexpression in lymphocytes on the intracellular drug accumulationObjective:To evaluate the efflux of drug from lymphocytes by the overexpressing P-gp.Methods:Compare the accumulation of Rhodamine 123 in lymphocytes with different expression level of P-gp.Results:The fluorence intensity of Rhodamine123 in lymphocytes is reversly related to the expression level of P-gp.The accumulation of Rhodamine123 are significantly less in midlle and high groups than control and low groups.Conclusions:The overexpression level of P-gp increase the drug-efflux activity from lymphocytes and decrease the intracellular concentration of therapeutic agents. Part five Inhibition of P-gp overexpression in lymphocytes on CsA pharmacological effectsObjective:To evaluate the immunosuppression efficacy of CsA under the context of alteration of lymphocytes P-gp expression in transplant recipients.Methods:using lymphocyte culture experimental system,to analyze the influence of P-gp expression level on CsA efficacy to suppress the lymphocyte proliferation and cytokine secretion by PHA application.Results:The inhibition effect of CsA on PHA-stimulusing prolieration and cytokine secretion of lymphocytes is markdly decreased in lymphocytes with middle and high P-gp level.Conclusions:The overexpression of P-gp in lymphocytes decreases immunosuppression efficacy of CsA. Part six Relation between P-gp overexpression and graft functionObjective:Analyzing the graft functions in recipients with different P-gp expression level,to investigate the relation between P-gp overexpression and graft function.Methods:78 recipients being followed up for one year,the graft function is evaluated by laboratory test including serum Crea,UN;graft hemodynamic criteria;edema,oligouriea.Each of the single alteration and occurrence is recorded as one ill-episode.Results:The sum of ill-episode during follow-up is 291.the incidence of ill-episode are significant higher in middle and high P-GP level groups.Conclusion:The level of lymphocyte P-gp is reversely related to the graft function,and can be used as a mark of diagnosis and prognosis for graft dysfunction.更多还原