| Objective: Most acute leukemia, a common malignant disease of hematopoietic system, can initially achieve complete remission after conventional chemotherapies, but considerable patients relapse eventually, even after receiving modern high-dose chemotherapy and hematopoietic stem cells transplantation. Bone marrow minimal residual disease (MRD) is regarded as the origin of the relapse. The residual leukemic cells cannot survive without the support of bone marrow hematopoietic microenvironment, and the abnormal microenvironment in acute leukemia might participate in the development of MRD by multiple mechanisms, So modifying the acute leukemic hematopoietic microenvironment may be a new strategy in the treatment of acute leukemia. Stromal cell derived factor-1 (SDF-1), a significant component in hematopoietic microenvironment, is mostly synthesized and secreted by bone marrow stromal cells (BMSCs). Human SDF-1, whose gene locates at chomosome ten, and classified as a member of chemokine CXC subfamily according to its amino acid sequence, plays important roles in the migration and location of hematopoietic stem and cancer cells through binding to its special receptor CXCR4 on the surface of hematopoietic cells. It also contributes to the expansion of tumor cells. Inhibiting the actions of SDF-1 has been proved to have some effects on reducing the tumor cells survival in vitro, but those data are obtained by administrating its receptor blockage directing against CXCR4. The effects of inhibiting SDF-1 expression of BMSCs on acute leukemic cells remain unclear. We observed the effects of inhibiting SDF-1 expression by RNAi on acute leukemic Jurkat cells co-cultured with BMSCs through transferring SDF-1 specific siRNA expressing plasmid into cultured human acute leukemic BMSCs, and explored the approach of curing acute leukemia by modifying hematopoietic microenvironment for the sake of abolishing MRD.Methods: In present experiment, BMSCs from acute leukemia at CR whthin one year were separated and cultured refer to Dexter-type long-term marrow cultures (D-LTMC).The expression level of SDF-1 in the supernatant of cultured BMSCs was measured by enzyme-linked immunosorbent assay (ELISA). SDF-1 special siRNA expressing plasmid was transfected into BMSCs with Lipofectamine 2000, and the positive clones(named as SCI) were selected by using G418. SDF-1 mRNA and protein expression level of BMSCs after RNAi were measured by semi-quantitative RT-PCR and enzyme-linked immunosorbent assay(ELISA), The BMSCs transfected with control siRNA expressing plasmid (named as SC2) and untransfected cells (named as SC3) from the same sample were taken as control. The effects of inhibiting SDF-1 expression by RNAi on Jurkat cells co-cultured with BMSCs were evaluated on the basis of the variation of cell adhesion, cell proliferation, cell cycle, cell apoptosis, the expression level of PCNA,Bcl-2,Bax,Fas and FasL and the drug sensitivity of Jurkat cells. Cell adhesion assay and cell proliferation assay were measured by cell counting, cell cycle, cell apoptosis, PCNA,Bcl-2,Bax,Fas and FasL expression and drug sensitivity were analysed by flow cytometry, TdT-mediated dUTP-X nick end-labeling (TUNEL), immunocytochemistry (ICC) and MTT assay, respectively. The Jurkat cells co-cultured with acute leukemic BMSCs transfected with the SDF-1 special siRNA expressing plasmid were assigned to experiment group (Group A), and the Jurkat cells co-cultured with untransfected acute leukemic BMSCs (Group B) were taken as control.Result: Main correlated results were as follows:1. Over-expression of SDF-1 in acute leukemia: The content of SDF-1 in cultured supernatant of BMSCs from acute leukemia at CR whthin one year was (2891 ± 305 )pg/ml, that of BMSCs from normal or no series hematopoietic system disease was (1583 + 157 )pg/ml, the former significantly higher than the latter(P<0.05).2. SDF-1 mRNA and protein expression level after RNAi: BMSCs from an acute leukemia patient at CR were transfected with plasmid expressing siRNA targeting SDF-1 or control (non-silencing) siRNA. Stable clones were obtained after G418 selection for further analysis. Compared with SC2 and SC3, the SDF-1 mRNA level of SCI was decreased significantly, but no obvious change was seen between SC2 and SC3, and the optical density ratio of RT-PCR products of SDF-1/ P -actin of SCI, SC2 and SC3 were 0.20, 1.46 and 1.48, respectively. The SDF-1 protein level of cultured supernatants of SCI, SC2 and SC3 were (384±41)pg/ml, (2428 ± 299)pg/ml,and (2474±271)pg/ml, respectively, theresults(SCl vs SC2 P<0.05, SCI vs SC3 PO.05, SC2 vs SC3 P>0.05) were consistent with those of mRNA level.3. The effects of inhibiting SDF-1 expression by RNAi on Jurkat cells co-cultured with BMSCs:3.1 Cell adhesion: After Jurkat cells were co-cultured with BMSCs for 24 h, the suspended and total number of Jurkat cells were determined by cell counting. The adhesion rates were calculated as follows: (total cells number - suspended cells number)/ (total cells number) X 100%.The adhesion rates in Group A and Group B were 28.8% ±2.6% and 57.4% ±3.8%, respectively. Down-regulated SDF-1 expression of acute leukemic BMSCs significantly reduced the adhesion of Jurkat cells to stromal cells layer (P<0.05).3.2 Cell proliferation: The numbers of viable Jurkat cells (trypan blue exclusion cells) at co-cultured 0, 24, 48, 72 and 96 h were counted, and population double time (PDT) was calculated according to Patterson formula:Td=Tlg2/lg(Nt/N0). In Group A, the PDT of Jurkat cells (42 hours) was prolonged compared with that of Group B (29 hours). Inhibiting SDF-1 expression of BMSCs made co-cultured Jurkat cells proliferate slow.3.3 Cell cycle: After Jurkat cells were co-cultured with BMSCs for 3 days, cell cycle of Jurkat cells was measured by flow cytometry. The results showed that the percentage of G0/Gi, S and G2/M phase of co-cultured Jurkat cells in Group A were 28.47%±2.39%, 25.57%±1.90% and 45.96%±3.24%, respectively, while those in Group B were 19.43%± 2.80%, 74.48% + 3.23% and 6.09% ± 1.96%, respectively. Silence of SDF-1 gene expression in BMSCs made co-cultured Jurkat cells to be arrested in Go/Gi and G2/M phase while decreased in S phase (PO.05).3.4 Cell apoptosis: After Jurkat cells were co-cultured with BMSCs for 3 days, TUNEL positive cells rates in Group A(15.2%±0.8%) were higher than those in Group B(5.4%±0.7%), there were significant statistical differences between them (P<0.05). The results suggested that inhibiting SDF-1 expression of BMSCs could induce co-cultured Jurkat cells apoptosis.3.5 PCNA,Bcl-2,Bax,Fas and FasL expression level. After Jurkat cells were co-cultured with BMSCs for 3 days, The PCNA positive cells rates of co-cultured Jurkat cells in Group A(79.7%±2.6%) were lower than that in Group B(91.4%±2.9%) , there was significant statistical differences between them (P<0.05). While almost all Jurkat cellsexpressed Bcl-2,Bax,Fas and FasL, but there were differences in positive degree. The differences were analyzed by Iamge Pro Plus software and expressed as total positive area (lira2) x mean optical density/count of cells. The expression degrees(xlO3/cell) of Bcl-2,Bax,Fas and FasL of co-cultured Jurkat cells in Group A were 82.5 ±6.0, 166.3 + 5.7, 59.7±5.4 and 115.4±5.2, respectively, while those in Group B were 148.3 + 5.6, 64.8± 5.6, 126.4 ±5.8 and 58.4 ±6.0, respectively. Expression degree of Bcl-2 and Fas was weaker in Group A than in Group B (F<0.05), however, expression degree of Bax and FasL was stronger in Group A than in Group B (P<0.05). The results suggested that inhibiting SDF-1 expression of BMSCs could make PCNA Bcl-2 and Fas expression level of co-cultured Jurkat cells decrease and Bax and FasL expression level increase.3.6 Drug sensitivity. Viable cells rates of co-cultured Jurkat cells after treated with different concentrations(0, 10, 100, 1000 and 10000 nM) of doxorubicin for 48h were measured. The Inhibition concentration 50% (IC50) values of doxorubicin were 585 nmol/L (Group A) and 6162 nmol/L(Group B), respectively. Down-regulating SDF-1 expression of BMSCs enhanced the sensitivity of co-cultured Jurkat cells to doxorubicin.Conclusion: SDF-1 is over-expressed in acute leukemia, RNAi might reduce effectively the expression of SDF-1, and inhibiting SDF-1 expression could reduce residue and expansion of leukemic cells. Therefore, inhibiting SDF-1 expression of BMSCs by RNAi may be helpful in therapy of marrow residual disease and become a new method of curing acute leukemia. |
PDF Full Text Request