Zhen-Yu Li

Xuzhou Medical College, Suchow, Jiangsu, China

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Publications (49)28.05 Total impact

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    ABSTRACT: A large number of studies have suggested that cytotoxic T lymphocyte antigen-4 (CTLA-4) also plays an important role in acute graft-versus-host disease (GVHD). However, the mechanism of CTLA-4 in regulating acute GVHD is still unknown. In the present study, we indicated that compared to healthy controls, CTLA-4 plasma and relative mRNA levels in patients with acute GVHD were decreased and then markedly elevated post 28 days' treatment. CTLA-4 levels in patients with I - II grade acute GVHD were higher than those with III - IV grade acute GVHD either before or after treatment. Upregulation of CTLA-4 significantly increased the luciferase activity and phosphorylation degree of STAT3. Meanwhile, T cell activation was remarkably inhibited and the levels of IFN-γ, IL-17, IL-22 were declined. Thus CTLA-4 might be involved in the pathogenesis of acute GVHD and it might down-regulate T helper 1 cells by increasing STAT3 expression in acute GVHD.
    Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 11/2015; DOI:10.1016/j.bbmt.2015.11.003 · 3.40 Impact Factor
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    ABSTRACT: Objective: To investigate the effect of alantolactone on perliferation and apoptosis of multiple myeloma (MM) RPMI-8226 cells, and to explore its possible mechism in vitro and in vivo. Methods: The RPMI-8226 cells were treated with alantolactone (1, 2.5, 5, 7.5 and 10 µmol/L) for 48 h, cell viability was detected by CCK-8 assay and the value of IC50 was calculated; The RPMI-8226 cells were treated with alantolactone (2.5, 5 and 7.5 µmol/L) for 48 h, the apoptotic rate was detected by flow cytmetry with Annexin V/PI staining; the expression level of cleaved caspase-3 and phosphorylation of ERK were measured by Western blot; the nude mice was used to further confirm the proapoptotic effect of alantolactone on MM cells in vivo. Results: The alantolactone inhibited RPMI-8226 cell viability remarkably with a dose-dependent manner; the IC50 value of RPMI-8226 cells at 48 h was 4.32 ± 0.15 µmol/L; the apoptotic rate increased observably with a dose-dependent manner; the levels of cleaved-caspase-3 increased and the phosphorylation of ERK decreased significantly; as compared to control, the volum of tumor was much smaller, the expression levels of Ki67 and p-ERK decreased. Conclusion: The alantolactone can efficiently inhibit the proliferation and induce the apoptosis of multiple myeloma RPMI-8226 cells in vitro and in vivo through inhibiting the activation of ERK signal pathway.
    Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 11/2015; 23(5):1336-1340.
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    ABSTRACT: Previously, we demonstrated the importance of T-cell immune response cDNA 7 (TIRC7) in acute immune thrombocytopenia (ITP). As the downstream molecule of TIRC7, cytotoxic T lymphocyte antigen-4 (CTLA-4) has been verified its negative regulation of acute ITP. This study aimed to investigate the exact role of CTLA-4 and its relationship with TIRC7 in acute ITP. 37 patients with acute ITP were enrolled and received dexamethasone (40mg/day) for 4 consecutive days. Patients who had platelet counts more than 50×10(9)/L or less were defined as responders or non-responders after treatment. The plasma, protein and mRNA levels of CTLA-4 and TIRC7 were monitored by ELISA, western blot and q-PCR, respectively. After high-dose dexamethasone therapy, CTLA-4 levels were significantly elevated not only in acute ITP patients (P<0.001; P<0.0001) but also in acute ITP responders (P<0.0001; P<0.0001). The levels of CTLA-4 were negatively correlated with the levels of TIRC7 before and after treatment; IFN-γ (Th1), IL-17 (Th17) and IL-22 (Th22) levels were all elevated, which were decreased after treatment not only in patients with acute ITP (P<0.01) but also in acute ITP responders (P<0.01). CTLA-4 level might reflect treatment efficacy and it might be associated with the pathogenesis of acute ITP. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Thrombosis Research 07/2015; 136(4). DOI:10.1016/j.thromres.2015.07.017 · 2.45 Impact Factor
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    ABSTRACT: Antithymocyte globulin (ATG) combined with cyclosporine A (CsA) has been widely used as a standard regimen in the treatment of aplastic anemia (AA), especially in severe aplastic anemia (SAA). Abnormally activated T cells might be the immune pathogenesis of AA. T cell immune response cDNA 7 (TIRC7) has been demonstrated its essential role in T cell activation; however, little is known about the role of TIRC7 in AA. In this study, we documented that TIRC7 levels in CsA group were higher than that in ATG + CsA (AC) group only in the follow-up phase (P < 0.05; P < 0.05); nevertheless, TIRC7 levels in SAA group were elevated than non severe aplastic anemia group not only in the treatment phase (P < 0.05; P < 0.05) but also in the follow-up phase (P < 0.05; P < 0.01). The trend of changes of T helper (Th) 1, Th17 and Th22 levels before and after treatment was similar to the changes of TIRC7 levels in either AC group or CsA group. Thus, TIRC7 might be involved in the pathogenesis of AA and AC might down-regulate Th1 cells by modulating the expression of TIRC7 in AA.
    Medical Oncology 07/2015; 32(7):647. DOI:10.1007/s12032-015-0647-2 · 2.63 Impact Factor
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    ABSTRACT: Acute graft-versus-host disease (aGVHD) has become the important complication post-allogeneic hematopoietic stem cell transplantation. Abnormally activated T cells might play an important role in the pathogenesis of aGVHD. But its exact mechanism remains poorly understood. T cell immune response cDNA 7 (TIRC7) has been identified to be essential in T cell activation; however, the role of TIRC7 in aGVHD remains unclear. The purpose of this study was to measure the expression of TIRC7 and T helper (Th) cells in patients with aGVHD before and after treatment. We showed that TIRC7 levels in aGVHD patients were higher than those of healthy controls and markedly declined after treatment. The levels of IFN-γ (Th1), IL-17 (Th17), and IL-22 (Th22) were in accordance with the grade of aGVHD. In addition, TIRC7 levels were also associated with the severity of aGVHD. In conclusion, TIRC7 might be involved in the pathogenesis of aGVHD and TIRC7 level might be an indicator to evaluate the response of patients with aGVHD to treatment.
    Annals of Hematology 01/2015; 94(6). DOI:10.1007/s00277-015-2300-8 · 2.63 Impact Factor
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    ABSTRACT: Chronic myeloid leukemia (CML) is a clonal disease from hematopoietic stem cells. Surviving leukemia stem cells (LSCs) and progenitor cells are a potential source for CML relapse and progression. Recent data reported that IL-1 receptor accessory protein (IL1RAP) gene was differentially expressed in CML versus normal stem and progenitor cells. However, whether the level of IL1RAP is associated with clinical phases of CML, and correlations between IL1RAP expression and detections of diagnosis is still unclear. Here we demonstrated that IL1RAP was up-regulated in CD34+ and CD34+CD38- cells which highly enriched with stem cells. Furthermore, IL1RAP expression in CD34+CD38- cells was tightly consistent with the generation of BCR-ABL fusion gene and Philadelphia chromosome. Importantly, we found that the level of IL1RAP increased with disease progression from chronic phase (CP) into accelerated phase (AP) and blast phase (BP), which was investigated not only in new diagnosed CML patients but also in patients treated with tyrosine kinase inhibitors (TKI) and hydroxyurea. Negative correlation was detected between IL1RAP expression and neutrophil (NE), whereas no relation was found in white blood cell (WBC), lymphocyte (LY), red blood cell (RBC), platelet (PLT), age or gender of CML patients. In conclusion, we identified IL1RAP as a surface marker of LSCs may be a potential indicator for CML clinical phases.
    International Journal of Clinical and Experimental Medicine 12/2014; 7(12):4787-98. · 1.28 Impact Factor
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    ABSTRACT: Beta-catenin is a key regulator of leukemia stem cell maintenance and drug resistance. Herein, we investigated the protective effects of the stromal cell-mediated VE-cadherin-β-catenin signal on Ph+ leukemia cells during imatinib treatment. We found stromal cells could desensitize imatinib and up-regulate VE-cadherin expression on Ph+ leukemia cells (K562 and SUP-B15 cells), which further stabilized and activated β-catenin. Knockdown of VE-cadherin with shRNA diminished the β-catenin protein and partly resensitized Ph+ leukemia cells to imatinib despite the presence of stromal cells, suggesting VE-cadherin is a potential target in the treatment of Ph+ leukemia.
    Leukemia Research 10/2014; 38(12). DOI:10.1016/j.leukres.2014.09.012 · 2.35 Impact Factor
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    ABSTRACT: Arginine kinase (AK) is a key enzyme in cellular energy metabolism of invertebrates. There are two conserved amino acid residues D14 and R138 in dimeric AK which form inter-subunit hydrogen bond. In S. japonicus AK, mutations in these residues caused pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Mutations (R138G, R138A and D14G) abolished D14 and R138 interaction disrupted the structure or conformation of S. japonicus AK. These R138G, R138A and D14G mutations changed their native assembles of dimeric AK and caused them in a partially unfolded state. The partially unfolded state of these mutant AKs made them prone to aggregate under environmental stress. The D14E/R138K and R138K mutant AKs showed similar characteristics to those of WT AK for forming the interaction which could replacement roles of D14 and R138 interaction. These results suggested that D14 and R138 interaction is involved in AK's activity, substrate synergism and structural stability.
    International journal of biological macromolecules 02/2014; 66. DOI:10.1016/j.ijbiomac.2014.02.039 · 2.86 Impact Factor
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    ABSTRACT: To explore gene expression of NANOG in T-cell acute lymphoblastic leukemia (T-ALL) cell lines and the effects of NANOG gene down-regulation on apoptosis of leukemia cells. Real-time PCR (RT-PCR) and Western blot were used to detect the expression level of NANOG gene and protein in MOLT-4, CCRF-HSB2 and Jurkat cells. To test the efficiency of RNA interference, MOLT-4 cells were firstly infected by lentiviral vectors, which were successfully constructed with NANOG specific shRNA. NANOG expression levels were subsequently re-evaluated by RT-PCR and Western blot. The percentages of early apoptotic cells (Annexin V⁺/7-AAD⁻) and late apoptotic cells (Annexin V⁺/7-AAD⁺) were analyzed by flow cytometry. The expression of apoptosis-related genes was also detected. Both NANOG gene and protein expression was positive in MOLT-4 and CCRF-HSB2 cells. The lentiviral vectors pLB-shNANOG-1, pLB-shNANOG-2, and pLB-shcontrol were successfully constructed, as evidenced by the viral titers (1.83-3.12)×10⁸ IU/ml. The experimental data on infection of MOLT-4 cells with such lentiviral vectors revealed that both shRNA interfering sequences (shNANOG-1 and shNANOG-2) could stably down-regulate NANOG gene and protein expressions. The percentages of early apoptotic cells in groups of shNANOG-1[(8.06±1.61)%]and shNANOG-2[(5.67±1.59)%]were significantly increased as compared to that of MOLT-4 group[(1.13±0.40)%]or sh-control [(1.15±0.49)%](P<0.05). However, no statistical difference among them was observed for late apoptotic cells (P>0.05). The gene expression of TP53, PMAIP1, and CASP9 of either shNANOG-1 or shNANOG-2 group was augmented as compared to that of MOLT-4 group or sh-control (P<0.05). Reversely, a significant down-regulation of Bcl-2 gene expression was observed (P<0.05). NANOG can be expressed in various human T-ALL cell lines. Down-regulation of NANOG can trigger leukemia cellular apoptosis through mitochondria-dependent apoptosis pathway.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 12/2013; 34(12):1001-5. DOI:10.3760/cma.j.issn.0253-2727.2013.12.001
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    ABSTRACT: This study was aimed to investigate the effect of Wnt/β-catenin signaling pathway on the biologic behavior of mouse bone marrow mesenchymal stem cells(mBM-MSC) by constructing a RNAi lentiviral vector specific to β-catenin. Three pairs of shRNA coding sequences directed against different sites of β-catenin mRNA were designed and were linked into lentiviral vector plasmid PLB for constructing the PLB-β-catenin/shRNA1, PLB-β-catenin/shRNA2 and PLB-β-catenin/ shRNA3. Those plasimds and lentiviral packaging plasimds were co-transfected into the packaging cells 293FT, then the virus particles were collected and the viral titer was assayed after concentration, and these viral particles were infected to MSC. The flow cytometry was used to sort GFP (+)cells, Western blot and RT-PCR were used to verify the inhibitory effect of those cells on expression of β-catenin gene in cells, CCK-8 method was used to detect the cell proliferation level, Annexin-V/7-AAD was used to determine the cell apoptosis after interference, the cell scratch and transwell tests were used to detect the migration capalbility of MSC. The results showed that the efficient inhibition of β-catenin mRNA and protein expression, and the suppression of MSC proliferation were observed in group of PLB-β-catenin/shRNA2(inteference group), while there was no significant changes of MSC proliferation between negative group(PLB group) and the normal group (control group). The flow cytometric detection indicated that after induced by serum starvation for 72 h, the apoptosis of MSC increased in inteference group, but there was no difference between PLB and control groups(P > 0.05). The cell scratch and transwell tests demonstrated that the migration capability of MSC in inteference group dicreased significantly, while the migration capability of MSC in control group was not changed obvi-ously. It is concluded that the constructed specific RNAi lentivirus can effectively inhibit the expression of β-catenin gene, decrease expression level of β-catenin mRNA and protein. The Wnt/β-catenin signaling pathway plays an important role in biological behavior of BM-MSC.
    Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 11/2013; 21(6):1546-51.
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    ABSTRACT: This study was purposed to explore the cellular source of IL-22 in graft versus host disease (GVHD) mouse following allo-geneic bone marrow transplantation. BALB/c and C57BL/6 mice were used as recipients and donors, respectively. GVHD model was established by irradiated BABL/c mice inoculated with mixed suspension of C57BL/6 bone marrow cells and splenic lymphocytes. The mice were divided into normal group(normal), total body irradiated group(TBI), bone marrow cell-transplanted group(BMT), and the combination of bone marrow cell and splenic lymphocytes-induced GVHD group(BS). The level of IL-22 in plasma was detected by ELISA. The cellular source of IL-22 and IL-22(+) subsets were detected by flow cytometry. The results showed that compared with normal mice, the level of IL-22 in plasma from BS mice was the highest(P < 0.01). All the lymphocytes of spleen, lymph nodes and peripheral blood from BS mice could produce IL-22, in which the percentage of IL-22(+)CD4(+) T cell was higher than that of IL-22(+)CD8(+) T cells. Not only Th22 cells but also Th1 and Th17 cells were the cellular source of IL-22 in GVHD mice. It is concluded that the high level of IL-22 has been found in mice with GVHD, which mainly originates from IFN-γ(-)IL-17(-)IL-22(+) Th22 cells.
    Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 11/2013; 21(6):1526-9.
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    ABSTRACT: To investigate the effects of immature dendritic cells(imDC)expressing chemokine receptor-7(CCR7) on acute graft-versus-host disease(aGVHD) in allogeneic bone marrow transped (allo-BMT) mouse model. We constructed the lentiviral vectors carrying mouse CCR7 gene and infect imDC effectively in vitro. GVHD model was established with C57BL/6(H-2b)donor mice and BALB/c (H-2d) recipient mice. After irradiation, recipients were injected with donor bone marrow and spleen cells along with CCR7-modified dendritic cells. Mice were randomized into irradiation, transplant control, pXZ9-imDC(empty vector control) and CCR7-imDC groups. Survival, GVHD score, histopathological analysis and plasma levels of inflammatory cytokines were observed. The mean survival in irradiation, transplantation, pXZ9-imDC and CCR7-imDC groups were(8.20±1.48)d,(12.20±2.78)d, (20.70±6.01)d and (27.5±7.55)d respectively. The survival in CCR7- imDC group was significantly improved compared with other groups(P<0.05). GVHD scores in transplantation, pXZ9-imDC and CCR7-imDC groups were (6.90±1.66),(5.60±0.97) and (4.10±1.79) respectively. CCR7-imDC group had significantly lower GVHD score and minor tissue damages shown by histopathological analysis than the other groups. Plasma IFN-γ level increased and reached the peak at +10 day in transplant group, while it gradually decreased in pXZ9-imDC and CCR7-imDC groups, and then reached the nadir at +20 day post-allo-BMT, with the lowest level in CCR7-imDC group(P<0.01). Plasma IL-4 decreased in transplant group, while it gradually increased in pXZ9-imDC and CCR7-imDC groups and reached the highest level at + 10 day in CCR7- imDC group(P<0.01). The 95%-100% of H-2b positive cells in recipient mice on + 30 day post-allo-BMT demonstrated the complete donor- type implantation. Genetically modified immature DC by CCR7 gene could alleviate damages by GVHD and prolong survival of recipient mice after allo-BMT.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 09/2013; 34(9):782-787. DOI:10.3760/cma.j.issn.0253-2727.2013.09.010

  • Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 09/2013; 34(9):804-806. DOI:10.3760/cma.j.issn.0253-2727.2013.09.015
  • Yan-Jie Li · Zhen-Yu Li · Kai-Lin Xu ·
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    ABSTRACT: The studies in recent years have found that miRNA play an important role in various biological functions, such as cell differentiation, proliferation, apoptosis, migration, and survival. Many studies considered the abnormally expressed miRNA as significant regulator in the pathogenesis, progression and metastasis of malignant tumors. miRNA can funcion as oncogenes or tumor suppressors by regulating post-transcriptional factors effecting multi pathways and may even represent target for therapies. This review summarizes the current advancement about the roles of miRNA in the biology of multiple myeloma (MM), including focuses on miRNA signatures relevant to MM, functions of critical miRNA in the mechanisms and development of MM behind their deregulation, the role in the drug-resistance and perspective of their expression in MM.
    Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 09/2013; 21(5):1318-25.
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    ABSTRACT: To investigate the sensitivity of imatinib mesylate (IM) on Sup-B15 Ph⁺ acute lymphoblastic leukemia (ALL) cells knockdown of VE-cadherin (CD144), and to further explore its mechanism. CD144 in Sup-B15 leukemia cells was stably knockdowned via lentivirus-mediated RNA interference (named as Sup-B15/shVEC). The inhibitory effects of IM on Sup-B15/shVEC and Sup-B15 leukemia cells were measured by CCK-8 test, and the apoptosis of those cells was determined by AnnexinV/7-AAD dyeing using flow cytomery, the percentage of CD34⁺CD38⁻ leukemia cells also by flow cytomtery. ALDH1 mRNA levels were detected by real-time RT-PCR, and protein levels of CD144, CD133, Bcr-abl and β-catenin by Western blot. IM treatment presented inhibitory effects on Sup-B15/shVEC and Sup-B15 leukemia cells at multiple concentrations of IM. The IC50 of IM on Sup-B15/shVEC and Sup-B15 leukemia cells were 25.1μmol/L and 18.7μmol/L, respectively (P<0.05). After 48h of 20 μmol/L IM treatment, the percentages of apoptosis cell in Sup-B15/shVEC cells and Sup-B15 cell were (13.52±2.06)% and (3.03±0.72) %, respectively (P<0.05). The percentage of CD34⁺CD38⁻ cells in Sup-B15 cells was significantly higher than in Sup-B15/shVEC cells [(2.39±0.28)% vs (0.96±0.07)%, P<0.05). As compared to Sup-B15 cells, the transcription of ALDH1 in Sup-B15/shVEC was remarkably downregulated, and the CD133 protein level was also downregulated in Sup-B15/shVEC cells. Both cytoplamic and nucleic β-catenin protein levels (but not for Bcr-abl levels) decreased in Sup-B15/shVEC cells as compare to Sup-B15 cells. Knockdown of CD144 sensitized Sup-B15 Ph+ ALL cells to IM. The possible mechanisms underlying this phenomenon might be via inhibiting β-catenin nucleic translocation and facilitating β-catenin degradation.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 06/2013; 34(6):522-526. DOI:10.3760/cma.j.issn.0253-2727.2013.06.014
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    ABSTRACT: To investigate the change of CD4⁺CD25(high)CD127(low) regulatory T cells (Tregs) percentage in patients with primary immune thrombocytopenia (ITP) treated by different methods. One hundred and thirty-eight newly diagnosed adult ITP patients (57 male, median age 40 years, range 18-70 years) were enrolled in this study, who were randomisedly separated into three regiment groups, namely prednisolone (PSL, 1.5 mg/kg for 2-4 weeks and subsequently stepwise reduction) group enrolled 49 patients, dexamethasone [(one course of high-dose dexamethasone (HDD) 40 mg/day, d1-4] 45 patients, and rituximab plus HDD (rituximab 100 mg on days 7, 14, 21, 28 and HDD) group 44 patients. Peripheral blood was taken in ITP patients of each group before treatment, 14 d and 28 d after treatment. The percentages of CD4⁺CD25(high)CD127(low) Tregs in 30 healthy controls and 138 patients were analyzed by flow cytometry. Overall response (OR) rates of PSL, HDD and R+HDD groups at day 28 were 69.4%, 66.7% and 79.5% respectively with no difference. After the following 12 months, sustained response (SR) was more pronounced in R+HDD group compared to the other two groups (R+HDD vs PSL: 66.7% vs 37.8%, P<0.05; R+HDD vs HDD: 66.7% vs 22.7%, P<0.05). The percentage of CD4⁺CD25(high)CD127(low) Tregs in peripheral blood of ITP patients [(1.67±0.70)%] was significantly lower than in healthy control group; After treatment, the percentages of Tregs in peripheral blood of patients both at day 14 and 28 in R+HDD group remarkably decreased compared with before treatment [(4.28±1.09)% vs (1.68±0.68)%, P<0.05; (4.44±0.63)% vs (1.68±0.68)%]. The percentages of Tregs at day 14 in both other two groups decreased notably compared with before treatment. But the Tregs levels measured at day 28 in PSL and HDD groups were similar with before treatment. The percentage of CD4⁺CD25(high)CD127(low) Tregs in peripheral blood of ITP patients was lower than healthy individual. The higher SR of patients treated by R+HDD was related to its ability to up-regulate the percentage of CD4⁺CD25(high)CD127(low) Tregs.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 06/2013; 34(6):478-481. DOI:10.3760/cma.j.issn.0253-2727.2013.06.002
  • Wei Chen · Miao Li · Zhi-Ling Yan · Hai Chen · Bin Pan · Ling-Yu Zeng · Zhen-Yu Li · Kai-Lin Xu ·
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    ABSTRACT: Objective: To construct mouse CXC chemokine receptor type 4 (Cxcr4) gene overexpressing lentiviral vector and to evaluate its biological effect on mouse mesenchymal stem cells (MSCs). Methods: Cxcr4 gene was amplified and subcloned into pCR-Blunt vector. Cxcr4 gene and enhanced green fluorescent protein (EGFP) gene expressed bicistronic recombinant lentiviral vector LV-CXCR4-IRES-EGFP and control vector LV-IRES-EGFP were constructed, respectively. Both plasmids were co-transfected into 293FT packaging cell line with packaging plasmid pSPAX2 and enveloping plasmid pMD.2G using Lipofectamine 2000 to produce lentiviral virus, respectively. The recombinant viruses were harvested and the virus titer was determined by limiting dilution. Mouse MSCs were infected with viral supernatant. EGFP expression was visualized using fluorescence microscope and efficiency of infection was determined by flow cytometry (FCM). Cell counting kit-8 (CCK-8) was applied in mixed lymphocyte reaction (MLR) to evaluate the suppressive effect of MSCs on mice splenocyte proliferation in vitro. Wound healing ability of MSCs was measured by scratch experiment and migration capacity by a chemotaxis assay using a transwell assay. Results: The Cxcr4 fragment was amplified by reverse transcription polymerase chain reaction (RT-PCR) and verified by DNA sequencing. The restriction enzyme digestion experiment demonstrated that the recombinant lentiviral vector LV-CXCR4-IRES-EGFP and the control vector LV-IRES-EGFP were successfully constructed. Expression of CXCR4 was detected by fluorescence microscopy, which indicated that the lentiviral particles expressing CXCR4 were packaged. Furthermore, expression of EGFP were detected by fluorescence microscopy in MSCs after infection and the expression of CXCR4 protein on MSCs surface in CXCR4-MSC group was significantly increased comparing to those in the control group(P < 0.05).CXCR4-MSCs group and the control group were (90.3 ± 3.37)% and (1.53 ± 0.34)%, respectively. Meanwhile, overexpression of CXCR4 had no effect on their capacity of immune regulation when co-cultured with splenocyte(P > 0.05). Moreover, overexpression of CXCR4 can not only accelerated the wound healing after scratch, but also enhanced the migration ability of cells in the transwell induced by high concentration of SDF-1 in a dose-dependent manner compared with the EGFP control group. Conclusion: The CXCR4 expressing lentiviral vector LV-CXCR4-IRES-EGFP was successfully constructed. The lentiviral vector can not only efficiently infect mouse MSCs, but also stably express CXCR4 in MSCs. The MSCs modified with CXCR4 have biological characteristic in vitro.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 05/2013; 34(5):440-444. DOI:10.3760/cma.j.issn.0253-2727.2013.05.014
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    ABSTRACT: Objective: To compare the efficacy and safety of low-dose rituximab combined with different dosage of glucocorticoids for immune thrombocytopenia (ITP). Methods: Seventy-four patients (35 male, median age 34 years, range 18-70 years) including 60 newly-diagnosed, 6 persistent, 5 chronic and 3 refractory patients were enrolled in this study, and separated into control (36 cases) and experimental (38 cases) groups according to the dosage of glucocorticoids. Patients in both groups received dexamethasone 40 mg/day on days 1-4, followed by rituximab 100 mg on days 7, 14, 21, 28. The patients in experimental group also received decrements of prednisone 60, 30, 20, 10 mg/day on days 5-7, 8-14, 15-21, 22-28. The initial, long-term efficacy and safety were evaluated. Results: Platelet counts of all patients at day 4 remarkably increased, with the median platelet count from 11(1-26) × 10⁹/L to 84(23-132) × 10⁹/L in control group, and 10(2-20) × 10⁹/L to 80(22-115) × 10⁹/L in experimental group; the platelet counts of patients at day 14 in experimental group [163(19-262) × 10⁹/L] was higher than that of control group [98(18-238) × 10⁹/L] (P<0.05). The overall response (OR) rates at day 28 in experimental group (84.21%) was significantly higher than that of control group (66.67%, P = 0.03). There was no significant difference of sustained response (SR) rates in two groups (63.89%vs 65.79%, 58.33%vs 60.53%, P > 0.05) at six and twelve months follow-up points. Both groups showed similar incidence of adverse events, and no patients discontinued the treatment due to side effects. Conclusion: Low-dose rituximab and glucocorticoids was an effective method for ITP patients, and maintenance treatment with decrements of prednisone contributed to improving earlier CR rate.
    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 05/2013; 34(5):409-412. DOI:10.3760/cma.j.issn.0253-2727.2013.05.007
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    ABSTRACT: This study was aimed to construct the targeting AATF shRNA eukaryotic expression vector and establish the stably transfected U937 cell lines. The sequence of AATF mRNA was obtained from GenBank. After excluding homeology, three plasmid expression vectors coding shRNA targeting 228 ∼ 249, 303 ∼ 324 and 443 ∼ 464 of AATF gene sequence were synthesized. Two terminals of shRNA carried BamHI and HindIII restriction sites. The selected nucleotides were cloned into the plasmid pSilencer 3.1-H1 neo respectively, and the resultant recombinant plasmids were named as pSA-1, pSA-2, pSA-3. The sequences of the recombinant plasmids were identified by DNA sequencing. The recombinant plasmids were transfected into the cell line U937 by electroporation with Neon(TM) Transfection System. The transfected cells were persistently screened under G418 (500 mg/L), and isolated with a limited dilution for 8 weeks. The inhibition of AATF mRNA and protein expression was respectively detected by RT-PCR and Western blot. The results indicated that RNAi eukaryotic expression vectors targeting AATF had correct reading frame and nucleotide sequence. Real-time PCR revealed that AATF shRNA effectively silenced mRNA expression of AATF. Western blot analysis found that AATF shRNA obviously suppressed protein expression of AATF (P < 0.05). It is concluded that the shRNA eukaryotic expression vector has been successfully constucted which can inhibit the expression of AATF, and the establishment of stably transfected U937 cell lines provide a original route for exploring the mechanism of AATF in human Leukemia further.
    Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 05/2013; 21(3):567-570.
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    ABSTRACT: This study was purposed to investigate the B7-H3 expression in multiple myeloma cell lines and CD138 cells of patients with multiple myeloma, and explore its clinical significance. Three myeloma cell lines (RPMI8226, U266 and H929) were used. Forty-five patients with multiple myeloma were enrolled in the study. The expression of B7-H3 was detected by flow cytometry and RT-PCR. The relationship between B7-H3 and clinical prognostic factor was analyzed. The results showed that (1)In myeloma cell lines, high expression of B7-H3 was seen in RPMI8226 (92.30 ± 1.1)% and U266 (79.03 ± 1.2)% but not in H929 cell line (4.26 ± 0.2)%. (2) Exogenous IL-6 had no effect on upregulation of B7-H3 in myeloma cell lines. (3) In multiple myeloma patients, the proportions of B7-H3 positive cells in newly diagnosed, remission and relapsed patients were (48.58 ± 33.593)%, (22.16 ± 18.853)%, and (57.65 ± 28.296)%, respectively. The difference between the newly diagnosed and remission patients, and remission and relapsed patients was significant (P = 0.023, P = 0.004). (4)High B7-H3 expression was correlated with high numbers of bone destruction and high levels of serum calcium (P = 0.027, P = 0.046, respectively). It is concluded that the relation of B7-H3 molecule expression with prognosis of multiple myeloma may be negative, but with degree of bone destuction is positive, thus the high expression of B7-H3 may correlated with disease progression and bone destruction of patients with multiple myeloma.
    Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 05/2013; 21(3):637-642.