Effects And Mechanisms Of Mycophenolic Acid On Bone Barrow Stem Cells In Vitro And The Study Of The Polymorphisms Of Target Gene Of Mycophenolic Acid

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,...