【作者】 曹伟杰；

【导师】 黄河；

【作者基本信息】 浙江大学 ， 内科学， 2010， 博士

【摘要】 霉酚酸(MPA)是霉酚酸酯(MMF)在体内代谢后的活性成分,其作用靶点是次黄嘌呤核苷酸脱氢酶(IMPDH),通过特异性抑制IMPDH,可以耗竭细胞内鸟嘌呤核苷酸和脱氧鸟嘌呤核苷酸,进而阻断DNA和RNA的合成。淋巴细胞高度依赖嘌呤从头合成途径,所以对MPA作用非常敏感。MMF做为一种强效安全的免疫抑制剂,目前广泛应用于实体脏器移植和异基因造血干细胞移植。在异基因造血干细胞移植中,MMF广泛用于预防排斥反应和急性移植物抗宿主病,也用于治疗移植物抗宿主病。而在异基因造血干细胞移植中,骨髓中的两种干细胞-造血干细胞和间充质干细胞起着极其重要的作用。造血干细胞是异基因造血干细胞移植中重建造血和免疫的种子细胞,而间充质干细胞不仅是造血微环境的重要组成细胞,而且是近年来异基因造血干细胞移植领域的研究热点之一。研究表明间充质干细胞共移植可以促进造血植入、降低移植物抗宿主病的发生,而且间充质干细胞还用于治疗激素耐药重度急性GVHD,Ⅱ期临床研究显示了良好的应用前景。MMF对这两种骨髓干细胞有什么作用?MMF应用的一个明显的不良反应是血液系统毒性,是不是与MMF对造血干细胞的作用有关?目前的相关研究还不能解释该问题。本研究旨在研究MPA对间充质干细胞和造血干细胞的作用,并探索其作用的机制,为临床更好应用MMF提供实验依据。MPA的作用靶点是IMPDH酶,IMPDH酶有两种亚型：IMPDH I和IMPDH II, IMPDH I组成性表达于所有细胞,而IMPDH II表达于部分细胞,与细胞的增殖和恶性转化有关。IMPDH I在胞内嘌呤合成中起主要作用,也是MPA作用的主要靶点。IMPDH I单核苷酸多态性(SNP)近来引起研究的关注,Wang等报道在肾脏移植中患者IMPDH I基因单核苷酸多态性与肾脏排斥反应相关。IMPDH I单核苷酸多态性会不会影响MPA在异基因造血干细胞移植中的作用?我们选择了IMPDH I基因在肾脏移植中与急性移植物失功相关的单核苷酸突变位点和在中国人群中突变发生率在20%以上的共4个单核苷酸突变位点,收集本骨髓移植中心自2001年至2009年移植供受者的基因组DNA,检测供受者IMPDH I基因单核苷酸多态性的分布频率,并分析其与异基因造血干细胞移植急性移植物抗宿主病的发生风险的关系。第一章霉酚酸对人骨髓来源的间充质干细胞作用研究本部分研究首先采用CCK-8方法分析霉酚酸对人骨髓来源的MSCs增殖的影响。结果显示：浓度1μmol/L的MPA对间充质干细胞具有生长抑制作用,且其抑制作用呈现时间浓度依赖效应。为研究MPA抑制MSCs增殖的机制,我们在培养体系内添加鸟嘌呤核苷(Guo, 100μmol/L),结果显示鸟嘌呤核苷逆转了MPA的抑制增殖作用,加Guo组与相应浓度的MPA组相比差别有统计学意义(P<0.05)。同时我们用Annexin V流式细胞分析技术检测MPA各浓度组的凋亡情况,在作用的48小时和7天时间,10μmol/L和100μmol/L MPA浓度组的凋亡情况与相应对照组相仿(P>0.05),表明MPA不诱导MSCs细胞凋亡。接下来我们用CCK-8方法检测T-淋巴细胞、成纤维细胞和MSCs对MPA作用的敏感性,结果显示在作用的第6天上述各组细胞的IC50分别为0.20μmol/L、1.54μmol/L和2.47μmol/L。为了揭示MSCs、成纤维细胞对MPA作用敏感的原因,我们用RT-PCR方法分析这两种细胞IMPDH I和IMPDH II的表达情况,结果显示这两种细胞均表达IMPDH I和IMPDHⅡ.接下来我们研究MPA对MSCs成骨分化、成脂分化和成软骨分化的影响。Von kossa染色和钙定量显示MPA抑制MSCs的成骨分化real-time PCR方法定量分析Osteopontin和BMP-2的表达,二者表达也呈剂量依赖性降低。添加鸟嘌呤核苷(100μmol/L)则逆转了MPA对MSCs成骨分化的抑制。为进一步研究MPA抑制MSCs成骨分化的具体机制,我们用real-time PCR方法定量检测Runx2、Osterix和ATF4,结果显示Runx2、Osterix在诱导7天和14天表达均降低,western-blot证实二者蛋白表达降低,添加Guo后表达得到恢复。MPA对MSCs成脂分化没有影响,油红O染色显示MPA各浓度组成脂出现,脂滴定量显示MPA各浓度组与对照组差别无统计意义(P>0.05), real-time PCR检测adipophilin和leptin显示,MPA作用下二者表达与对照组没有差别(P>0.05)。蕃红O染色和real-time PCR方法定量检测aggrecan和collagenⅡ显示MPA对MSCs成软骨分化没有影响。第二章霉酚酸对人造血干祖细胞作用研究集落形成细胞(colony-forming cell, CFC)是评估造血细胞功能的实验室金标准,是用来检测造血干祖细胞增殖分化的功能指标。本部分研究首先探讨MPA对造血干祖细胞集落形成的影响。在MPA浓度0.1-5μmol/L浓度范围内,MPA对集落细胞总数、红细胞集落形成单位数目(CFU-E)、爆式红细胞集落形成单位数目(BFU-E)、粒细胞-巨噬细胞形成单位数目(CFU-GM)均无影响,在10μmol/L时上述细胞集落总数和各系集落数目开始降低(P<0.05),50和100μmol/L时几乎消失；粒细胞-红细胞-巨噬细胞-巨核细胞集落形成单位数目(CFU-GEMM)则从5μmol/L时开始减少(P<0.05),50和5μmol/L时消失。在MPA各浓度组添加鸟嘌呤核苷(200μmol/L),集落细胞总数、CFU-E、BFU-E和CFU-GM数目均得到恢复(P<0.05)；对CFU-GEMM来说,10μmol／L组添加Guo组得到恢复,而50和100μmol/L组添加Guo未得到恢复,这可能与CFU-GEMM在诱导体系中数目较少有关。这说明Guo可以逆转MPA对造血干细胞集落形成的抑制。为进一步检测MPA对造血干祖细胞的作用,采用7-AAD/Annexin V、CD34染色,流式细胞术检测CD34+细胞细胞的凋亡情况。培养48小时后,10μmol/L组和100μmol/L组CD34+细胞中凋亡细胞的百分数分别为5.26±0.95和3.58±0.93,与对照祖4.78±1.46%相仿(P>0.05)；培养96小时后,10μmol/L组和1O0μmol/L组CD34+细胞中凋亡细胞分别为15.44±1.81和16.01±2.73,与对照祖13.77±3相仿(P>0.05)。说明MPA不能引起CD34+细胞发生凋亡。第三章Ⅰ型次黄嘌呤核苷酸脱氢酶的基因多态性在异基因造血干细胞移植急性移植物抗宿主病中的作用研究目前MMF在异基因造血干细胞移植中广泛应用于预防和治疗aGVHD,而研究已证实个体间IMPDH的活性存在存在较大差异,因此我们设想IMPDH I基因的单核苷酸多态性(single nucleotide polymorphism, SNP)可能影响IMPDH的活性,导致个体间MMF作用的差异,可能与GVHD的发生风险相关。我们选择本移植中心自2001年1月至2009年3月连续接受异基因造血干细胞移植的患者及其供者,共240对。其中包括接受无关供者移植(URD)的患者及其供者138对,接受HLA全相合同胞供者移植(Sib)的患者及其供者102对。留取供者和患者移植前外周血样,提取基因组DNA,我们选择检测文献报道IMPDH I基因在肾脏移植中与急性移植物失功相关的SNP位点和在中国人群中突变发生率在20%以上的共4个SNP位点：内含子7(IVS7)+125 G>A(rs2278293)；内含子8(IVS8)-106 G>A(rs2278294)；外显子15(Exon15) 1572 G>A (rs2228075)和5’侧翼序列C>T(rs714510),分析其与异基因造血干细胞移植急性移植物抗宿主病的关系。我们的结果证实患者在IMPDH I基因IVS8-106位点为G／G基因型的aGVHD的发生率明显高于基因型为A／A和G／A的患者,多因素分析亦证实患者在IMPDH I基因IVS8-106位点为G／G基因型是aGVHD发生的危险因素((RR=2.018,95%CI：1.354-3.009,P=0.001)。本研究未发现其他3个位点的基因多态性与aGVHD的发生风险相关,可能与本中心的GVHD的预防方案中,MMF使用的剂量远远低于器官移植中的剂量有关。更多还原

【Abstract】 Mycophenolic acid (MPA) is the active metabolite of Mycophenolate mofetil (MMF), which depletes guanosine and deoxyguanosine nucleotides by inhibiting inosine monophosphate dehydrogenase (IMPDH), thus blocks DNA and RNA synthesis. Lymphocytes are highly dependent on de novo purine synthesis, therefore are very sensitive to MPA. MMF is currently widely used in solid organ transplantation as well as allogeneic hematopoietic stem cell transplantation (allo-HSCT), as a potent, safe immunosuppressive agent. In allo-HSCT, MMF is applied in the prophylaxis of rejection and acute graft-versus-host disease (acute GVHD), as well as the treatment of graft-versus-host disease after transplantation. Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are two kinds of stem cell in bone marrow, which are vital to allo-HSCT. HSCs are responsible for hematopoietic and immune reconstitution after allo-HSCT, where as MSCs are not only the important components of hematopoietic microenvironment, but also one of the research hotspots in allo-HSCT recent years. Research shows that co-transplantation of MSCs can promote engraftment, decrease the incidence of graft-versus-host disease. Further more, MSCs have a promising prospect in treatment of steroid-refractory severe acute GVHD in phase II clinical trails. What effect dose MMF have on these two kinds of bone marrow derived stem cells? One of the main side-effects of MMF is hematologic toxicity, is that relevant to the effect of MMF on these stem cells?There is no research to explain this at present.In this study, we will examine the effect of MMF on HSCs and MSCs, and explore the mechanism. There are two isoforms of IMPDH, IMPDH I is constructively expressed in all cell types, whereas IMPDH II is only expressed in particular cell types and is related to cell proliferation and malignant transformation. IMPDH I plays a major role in cellular purine synthesis, and is also the main target of MPA. The single nucleotide polymorphism (SNP) of IMPDH I has attracted attention recently. Wang et al. reported that rs2278293 and rs2278294 are relevant to the rejection of renal transplantation. Searching through NCBI, we found that there are 5 SNPs of IMPDH I whose mutation rate are more than 20%. To determine whether IMPDH I SNP influence the effect of MPA on allo-HSCT, we collected the bone marrow and clinical data of donors and recipients in our bone marrow transplantation center.PART ONE Effect of MPA on human bone marrow derived MSCsFirst, we examined the effect of MPA on the proliferation of human bone marrow derived MSCs by CCK-8 assay. We found that In the range of 1μM to 100μM, MPA caused a significant subdued proliferation rate of MSCs in a concentration-and time-dependent manner. To study the mechanism of the inhibitory effect of MPA on the proliferation of MSCs, we added guanosine (100μmol/L) to the culture, and found that the proliferation rate was restored (P<0.05). Meanwhile, we examined the apoptosis of MPA treated MSCs by Annexin V apoptosis assay.48h and 7d of incubation with MPA at the concentration of 10μM and 100μM showed no significant difference in apoptosis compare to control group (P>0.05), indicating that MPA did not induce apoptosis of MSCs. Then we compared the sensitivity of T-lymphocytes, fibroflasts, and MSCs to MPA by CCK-8 assay. After 6 days of incubation with MPA, the IC50 ofthese cells are 0.20μmol/L,1.54μmol/L, and 2.47μmol/L, respectively. To study why MSCs and fibroblasts are sensitive to MPA, we analyzed the mRNA expression of IMPDH I and IMPDH II by RT-PCR. The results showed that they both expressed IMPDH I and IMPDH II, and the expression is independent of cell cycle.Next, we studied the impact of MPA on the osteogenic, adipogenic as well as cartilage differentiation. Von Kossa stainnging and Ca2+ quantification showed that MPA inhibited the osteogenic differentiation of MSCs, and real-time PCR detected a dose dependent decrease in expression of Osteopontin and BMP-2. The addition of guanosine(100μmol/L) reversed the inhibition of MPA on the osteogenic differentiation of MSCs.To further reveal the exact mechanism of the inhibitory effect of MPA on the osteogenic differentiation of MSCs, we detected the expression of Runx2, Osterix and ATF4 by real-time PCR. Results showed that the expression of Runx2 and Osterix reduced on 7d and 14d after the induction, which could be restored by adding guanosine. The results were confirmed by western-blot. Oil Red O staining and Quantification of lipid contents showed that MPA had no effect on lipid production during adipogenic differentiation(P>0.05), and the expression of adipophilin and leiptin detected by real-time PCR showed no change between MPA group and control group(P>0.05), indicating that MPA did not affect the adipogenic differentiation of MSCs. Safranine O staining and real-time detection of aggrecan and aggrecan expression showed that MPA did not affect the cartilage differentiation of MSCs.PART TWO Effect of MPA on hematopoietic stem (progenitor) cellsColony-forming cell (CFC) is an indicator used to determine the proliferation and differentiation of hematopoietic stem (progenitor) cells, thus performs as the golden criterion for the evaluation of the function of hematopoietic cells in laboratory. In this part, we first investigated the effect of MPA on the colony-forming of hematopoietic stem (progenitor) cells. At the concentration of 0.1-5μmol/L, MPA did not affect the total number of colony cells, and the number of colony forming unit-erythroid (CFU-E), burst forming unit-erythrocyte (BFU-E), and colony forming unit-granulocyte and macrophage (CFU-GM), which started to drop at the concentration of 1Oμmol/L (P<0.05), and almost disappeared at the concentration of 50 and 100μmol/L. The number of colony forming unit-granulocyte, erythroid, macrophage and megakaryocyte(CFU-GEMM) began to decrease at the concentration of 5μmol/L (P<0.05), disappeared at the concentration of 50 and 100μmol/L. Adding guanosine (200μmol/L) to MPA treated cells restored the number of total colony-forming cells as well as CFU-E, BFU-E, CFU-GM (P<0.05).Regard to the number of CFU-GEMM, it could be restored by adding guanosine to 10μmol/L MPA group, but not to 50 and 100μmol/L group, which may due to the small number of CFU-GEMM in inducing system. These results indicated that guanosine was able to reverse the inhibitory effect of MPA on colony forming of hematopoeitic stem cells. To further examine the effect of MPA on hematopoietic stem (progenitor) cells, we used 7-ADD/Annexin V, CD34 staining flow cytometry method to detect the apoptosis of CD34+ cells treated by MPA. After incubation with 10μmol/L and 100μmol/L MPA for 48h, the percentage of apoptosis cells in CD34+ cells were 5.26±0.95% and 3.58±0.93%, respectively, similar to control group 4.78±1.46%(P>0.05). After incubation with 10μmol/L and 100μmol/L MPA for 96h, the percentage of apoptosis cells in CD34+ cells were 15.44±1.81% and 16.01±2.73%, respectively, similar to control group 13.77±3%(P>0.05), indicating that MPA did not induce apoptosis in CD34+ cells.Part three The association of the polymorphisms of IMPDH I gene and acute graft-versus-host disease after related and unrelated hematopoietic stem cell transplantationMMF has been widely used in the prophylaxis and treatment of aGVHD in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, there is no regular monitoring drug concentration of MMF. Considerable variablity in MPA pharmacokinetics has been observed in transplant patients. The considerable variability in baseline IMPDH I activity and MPA response may logically be under the control of genetic variation within the IMPDH I gene or in gene expression. Analysis of genetic variants could provide the explantation for the variability of IMPDH I activity and MMF response in transplant patients. The entire study population consisted of 240 pairs of transplant recipients and their donors who were transplanted from 2001 to 2009 in our Bone Marrow Transplantation Unit, which consisted of 138 pairs of recipients and their unrelated donors and 102 pairs of recipients and their HLA-identical sibling donors. DNA was extracted from peripheral blood using DNA Isolation Kit following the recommendation of the manufacturer. Four Single-nucleotide polymorphisms (SNPs) of IVS7+125 G>A (rs2278293), IVS8-106G>A (rs2278294), Exon15 1572 G>A(rs2228075)和5’ flanking region C＞T (rs714510) in IMPDH I gene were analyzed by Multiplex SnaPshot; Our results showed that in both the unrelated transplantation cohort and the sibling transplantation cohort, the IMPDHⅠIVS8-106 G/G genotypes in recipients were significantly associated with a higher incidence of aGVHD. In the combined cohort, multivariate analysis confirmed that recipients with the IVS8-106 G/G genotype also had a higher risk of aGVHD(RR=2.018,95%CI:1.354-3.009, P=0.001). There was no significant association between IVS7+125, Exon15 1572 and 5’flanking region and the risk of aGVHD, which may because the relatively small dosage of MMF in HSCT in comparision to the dosage in solid organ transplantation,更多还原