Changes of IL-21 and Its Mediated JAK/STAT Signaling Pathway in Patients with Immune Thrombocytopenia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the correlation between JAK/STAT signaling pathways and pathogenesis of immune thrombocytopenia(ITP).</p><p><b>METHODS</b>Twenty-six newly-diagnosed ITP patients was included in this study. They all meet the clinical and hematological criteria for the diagnosis of ITP, and patients with coronary heart disease, severe refractory hypertension, diabetes or with severe liver or kidney function incompetence were ruled out. 24 healthy control without autoimmune diseases, viral infectious diseases and with normal liver and kidney functions were also included. The expressions of Jak3, p-Jak3 mRNA, Stat3, and p-Stat3 were tested and the changes in levels of IL-21 mRNA, IL-21 cell secretion after DEX intervention and AG490 blockade were measured.</p><p><b>RESULTS</b>Compared with the healthy control, patients with ITP had significantly high expressions of Jak3, p-Jak3 mRNA, Stat3 and p-Stat3 protein, which significantly reduced after AG490 blocking (P<0.01). The expression of IL-21 mRNA and the secretion of IL-21 obviously decreased after DEX intervention, but increased after AG490 blocking(P<0.01).</p><p><b>CONCLUSION</b>The pathogenesis of ITP associates with the activation of JAK/STAT signaling pathways, and IL-21-mediated JAK/STAT signaling pathways play regulatory role in ITP.</p>

Analysis of Curative Efficacy of Different Chemotherapy Regimen Combined with Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation on Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To evaluate the efficacy of autologous peripheral blood hematopoietic stem cell transplantation (auto-PBHSCT) on patients with multiple myeloma( MM) after Sequential different chemotherapy.@*METHODS@#Seven cases of patients with MM were included in the A group, and 14 cases of patients received 4-6 courses of chemotherapy with VAD and MP before transplantation were included in the B group and received 4-6 courses of chemotherapy with VTD and VD before transplantation. Auto-peripheral blood hematopoietic stem cell were mobilized by G-CSF. Condition regimen were melphalan(A group) or bortezomib combined melphalan(B group). IFN-α(A group) or Thalidomide(B group) was used as maintenance treatment after auto-PBHSCT.@*RESULTS@#Two cases of patients reached to complete remission (CR)(2/7,28.6%),1 case got very good partial remission (VGPR) (1/7,14.3%), 4 cases got partial remission(PR) (4/7,57.1%) in A group, and 9 cases got CR (9/14,64.3%), 3 cases got VGPR(3/14,21.4%), and 2 cases got PR(2/14,14.3%) in the B group before auto-PBHSCT. The CR and VGPR were significant difference between 2 groups (P<0.05). All the patients got hematopoietic recovery. In 2 groups, the median time of ANC recovery≥0.5×10/L was 13 (11-16) and 14（11-18）days, that of WBC recovery ≥4.0×10/L were 16(15-19) and 18(16-20)days, Plt recovery ≥ 50 ×10/L was 21 (18-25) and 21(17-25) days. Bone marrow showed CR in 21 to 28 days after transplantation. All of 7 cases of patients remised in 6 to 47 months after transplantation, and 4 cases died lastly and 3 cases failed to be followed up in A group. The median time of progression-free survival(PFS) was 36(6-47) months, and that of overall survival(OS) was 37(7-50) months. In B group, 2 cases of patients remissed in 5 and 17 months after transplantation, and did lastly, 1 case relieved in 12 months after transplantation and failed to be followed up. 1 case of patient relived in 46 months after transplantation, and then received the second auto-PBHSCT, and got CR for 105 months. Other 10 cases got CR, their median time of PFS was 45.5(4-105) months, the median time of overall survival(OS) was 45.5(4-105) months. The PFS and OS were very significant different between 2 groups (P<0.01).@*CONCLUSION@#Bortezomib-based chemotherapy, Auto-PBHSCT and maintenance treatment with thalidomide were favorable to the patients of MM for survival prolongation.

Retrospective Analysis of Genetics Abnormalities in Patients with Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To explore the characteristics of cytogenetics and molecular genetics in patients with multiple myeloma(MM).@*METHODS@#Fluorescence in situ hybridization(FISH) was used for molecular genetics analysis in 86 cases of newly diagnosed MM, at the same time the chromosome karyotype analysis was performed in 20 cases. Specimen were bone marrow cells.@*RESULTS@#FISH detection showed that 68 cases of MM (79.07%) had at least one type of the molecular genetic abnormalities. The positive rates of IgH rearrangement, 1q21 amplification, D13S319 deletion, RB1 deletion and.P53 deletion were 62.79%, 26.74%, 24.42% ,13.95% and 1.16%, respectively. The positive rate of IgH was significantly higher than that of any other probes(P<0.01). The positive rate of IgH was 79.41% in 68 cases. Out of which the positive rate of IgH single and combined with 1, 2, 3, 4 probes was 59.26%, 24.07%, 11.11%, 5.56% and 0 respectively. The positive rate of IgH only was very signficantly higher than that of combined with any other probes(P<0.01).The positive rate of 1q21 was 33.82% in 68 cases, Out of which the positive rates of 1q21 or combined with 1,2,3,4 probes was 21.74%, 43.48%, 21.74%,13.04% and 0 respectively, the 1q21 probe showed positive as combined with other probes(P<0.01), especially with IgH(P<0.05). The positive rates of D13S319 were 30.88% in 68 cases of patients, out of which the positive rates of D13S319 single or combined with 1, 2, 3, 4 probes was 14.29%, 28.57%, 42.86%, 14.29% and 0 respectively, the D13S319 combined with other probes appeared more significant positive(P<0.01), especially with 1 or 2 probes (P< 0.01). The positive rate of RB1 was 17.65% in 68 cases, the positive rate of RB1 singl or combined with 1, 2, 3, 4 probes were 0, 25%, 50%, 25% and 0, the RB1 appeared positive always combined with other probes, especially with D13S319 probe (P<0.01). The positive rate of P53 was 1.47%, as combined with RB1 and D13S319 probes. The chromosomal karyotyping showed that 3 cases carried abnormal chromosomal and 17 cases carried normal chromosome, Out of which 17 cases showed positive by FISH. There was a significant difference of sensitivity between FISH combined with chromosome karvotyping and single chromosome karvotype (P< 0.01).@*CONCLUSION@#The genetic abnormalies display obvious heterogenicity in MM. The sensitivity of FISH is higher than that of chromosomal karvotyping. If FISH and chromosome karvotyping are combined, the positive rate of abnormality can be raised.

Clinical Analysis of Adoptive Immunotherapy after Autologous Peripheral Blood Hematopoietic Stem Cell Transplantation in B Lymphocyte Malignant Lymphoma / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the efficacy of autologous peripheral blood hematopoietic stem cell transplantation(auto-PBHSCT) combined with adoptive immunotherapy for patients with B lymphocyte malignant lymphoma(ML).</p><p><b>METHODS</b>A total of 110 cases of ML treated with adoptive immunotherapy after auto-PBHSCT from January 2000 to December 2009 were enrolled in adoptive immunotherapy group (treated group), while 74 cases of ML treated without adoptive immunotherapy after auto-PBHSCT from January 1995 to December 1999 were used as control group. The efficacy of 2 groups were analyzed and compared, 110 case of ML in treated group included 78 cases of non-Hodgkin's lymphoma(NHL), 32 cases of Hodgkin's lymphoma(HL),74 cases of ML in control group included 52 NHL and 22 HL. All of the patients were treated sequentially with chemotherapy regimens for 6 courses. After that, all the patients received auto-PBHSCT. After hematopoietic reconstruction, the patients in treated group were given 6 courses of adoptive immunotherapy(rhIL-2 100 WU/day for 10 days monthly for each course), while the patients in control group were not given immunotherapy. All the patients were followed-up for more than 5 years.</p><p><b>RESULTS</b>There was one patient in each group, who died of liver failure and cerebral hemorrhage respectively within 3 and 2 months, and all the other patients achieved hematopoietic reconstruction. Following-up for 1, 3, 5 years, the disease-free survival (DFS) rate in treated group was 97.3%,93.6%,87.3% while 91.9%, 73.0%, 64.9% in control group. Following-up for 3 and 5 years, there was very significant difference in DFS between 2 groups(P<0.01). The 1,3 and 5 year DFS rate of patients in stage I/II and III/IV in the treated group were 100%,100%,91.7% and 96.5%,91.9%,86.0% respectively while DFS of control group was 100%, 93.3%, 86.7% and 89.8%, 67.8%, 59.3%, there was a significant difference in 3 and 5 years DFS of III/IV stage patients between 2 groups (P<0.01). The 1,3 and 5 year DFS rate of HL patients were 100%, 93.8%,84.4% in treated group and 100%,72.7%,59.1% in control group respectively. There was significant difference in 3 and 5 years DFS of HL between 2 groups (P<0.05). The 1,3 and 5 year DFS rate of stage I/II HL patients were 100%,100%,88.9% in treated group and 100%,100%,80.0% in control group. The 1,3 and 5 year DFS of HL patients in stage III/IV was 100%,91.3%,82.6% and 94.1%,64.7%,52.9% respectively. There was significant difference in 3 and 5 years DFS of III/IV stage of HL patients between 2 groups (P<0.05). The 1,3 and 5 year DFS rate of NHL patients is 96.2%, 93.6%,88.5% in treated group and 90.4%,73.1%,65.4% in control group respectively. There was a significant difference in 3 and 5 years DFS of NHL between 2 groups(P<0.01). The 1,3 and 5 year DFS rate of stage I/II NHL patients was 100%, 100%, 93.3.9% in treated group and 100%, 90%, 90.0% in control group, respectively. The 1,3 and 5 year DFS of NHL patients in stage III/IV is 95.2%, 92.1%,87.3% and 88.1%,69.0%, 59.5% respectively. There was significant difference in 3 and 5 years DFS of III/IV stage NHL patients between 2 groups (P<0.05).</p><p><b>CONCLUSION</b>Therapeutic efficacy is satisfactory for the patients of B lymphocyte ML treated with adoptive immunotherapy after auto-PBHSCT, especially benefited the patients of stage III/IV significantly.</p>

Relationship between Gene Polymorphism of HLA-A(*)/-B(*)-DRB1(*) and Aplastic Anemia in Chinese Han Population of Northwestern Plateau / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the correlation between the HLA genes and pathogenesis of aplastic anemia (AA), so as to find the susceptible AA genes.</p><p><b>METHODS</b>Polymerase chain reaction with specific sequence primers (PCR-SSP) method was used to detect the HLA typing of 50 AA patients and 183 normal healthy individuals as controls in Chinese Han population of northwestern plateau.</p><p><b>RESULTS</b>The frequency of HLA-A* 0201 (45.0%), B* 1501 (11.0%), B* 5501 (9.0%) and DRB1* 0901 (19.0%) gene frequences in AA patients were significantly higher than those in controls (Odds Ratio: OR=1.657, 2.138, 2.314 and 1.932, x2=4.882, 3.876, 3.863 and 4.473 (P<0.05). In contrast, A* 0301 gene frequency (4.0%) in AA was significantly lower than that in controls, OR=0.349, x2=4.154 (P<0.05). The male HLA-A* 0201 gene frequency was lower than that in female (38.2% vs 59.4%), and the difference was statistically significant (P<0.05). Concludsion: The HLA-A* 0201, B* 1501, B* 5501 and DRB1* 0901 genes may be considered as the risk markers while A* 0301 gene as a protective marker of AA, the HLA-A* 0201 also shows the sex differences.</p>

Effects of the interleukin-21 expression in patients with immune thrombocytopenia and its regulation by high-dose dexamethasone / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the correlation of immunologic thrombocytopenia(ITP) pathogenesis with the abnormal expression of IL-21, and to explore the association of high-dose dexamethasone (HD-DEX) treatment with the IL-21 expression.</p><p><b>METHODS</b>26 newly diagnosed ITP patients and 24 healthy controls were enrolled in this study. The mononuclear cells and serum were obtain from density gradient centrifugation in the newly diagnosed ITP patients before HD-DXM treatment, and the samples of healthy controls were also used for assays. The protein and mRNA expression of IL-21 on peripheral blood mononuclear cells(MNC) were determined by flow cytometry and real-time reverse-transcription polymerase chain reaction. Plasma levels of IL-21, IFN-γ and IL-4 were determined by enzyme-linked immunoabsorbent assay (ELISA).</p><p><b>RESULTS</b>IL-21 expression on mononuclear cells was significantly higher in ITP patients (13.07%) than that in normal controls (8.2%), the ratio of IL-21/GAPDH mRNA expression on MNC was significantly higher in ITP patients (9.524±0.97) than that in normal controls (3.701±0.60, P<0.01). After HD-DXM therapy, the ratio of IL-21/GAPDH mRNA decreased significantly (5.87±1.21) as compared with the level before treatment. Significantly high levels of serum IL-21, IFN-γ and lower IL-4 were found in ITP patients, as compared with healthy controls. Serum IL-21 and IFN-γ levels in ITP patients decreased significantly after HD-DXM administration (P<0.01), while post-treatment levels of IL-4 were increased significantly, compared with the levels before treatment (P<0.01).</p><p><b>CONCLUSION</b>Therapeutic effect of DXM on ITP associates with down-regalation of IL-21 expression. The increased expression of IL-21 involves in the pathogenesis of ITP.</p>

Effects of salidroside on proliferation of bone marrow mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to investigate the effect of salidroside on proliferation of bone marrow mesenchymal stem cells (MSC) and their secretion of stem cell factor (SCF). MSC were isolated and amplified in vitro via density gradient centrifugation and adherence screening method. MCS were identified by flow cytometry and osteogenic/adipogenic induction. The effects of salidroside on cell proliferation, cell cycle and the SCF secretion of MSC were detected by flow cytometry. The results showed that the salidroside could induce the proliferation of MSC, peaked at the concentration of 1.5 mg/ml and in a time-dependent manner (in 24 h, 48 h and 72 h). Salidroside at 1.5 mg/ml could more effectively increase the percentage of cells in S and G1/M phase. Co-cultured with salidroside at the concentration of 1.5 mg/ml for 48 h, the SCF and the expression levels of SCF mRNA in co-culture supernatant were both significantly increased (P < 0.01). It is concluded that salidroside in a range of certain concentration can obviously promote the proliferation of MSC and increase the expression and secretion of SCF.

Curative efficacy for nasal type extranodal NK/T-cell lymphoma by autologous peripheral blood stem cell transplantation after sequencing chemotherapy and radiotherapy / 中国实验血液学杂志

The purpose of this study was to explore the curative efficacy for nasal type extranodal NK/T-cell lymphoma by autologous peripheral blood stem cell transplantation (APBSCT) after sequencing chemotherapy and radiotherapy. A total 65 cases diagnosed as nasal type extranodal NK/T-cell lymphoma by pathology and immuno-histochemistry were treated with chemotherapy and radiotherapy in our hospital from January of 2000 to December of 2009. They were divided into observation group (34 cases) and transplantation group (31 cases). The 34 cases of observation group were ceased from treatment, the 31 cases in transplantation group received APBSCT after conditioning regimen with TBI combined VEMAC. Autologous peripheral blood stem cells were mobilized with chemotherapy combined rhG-CSF. The patients were followed up for 3-5 years. The results showed that some side-effects such as bone marrow suppression and injure of oral cavity mucosa were found in patients after sequencing chemotherapy and radiotherapy. All patients in transplantation group obtained hematopoietic reconstruction, and there were no any special side effect such as VOD. In transplantation group, the median time of ANC ≥ 0.5×10(9)/L was 14 (11-17) days, median time of WBC count ≥ 4.0×10(9)/L was 17 (16-20) days, median time of Plt count ≥ 50×10(8)/L were 25 (23-28) days. After chemotherapy and radiotherapy, effective rate of treatment was 91.2% in observation group, whereas was 90.3% in transplantation group, there were no obvious difference between two groups (P > 0.05). After following up about 1 year, effective rate of treatment was 76.5% in observation group, whereas was 96.8% in transplantation group, there were obvious difference between two groups (P < 0.05). After following up about 3 years and 5 years the disease-free survival (DFS) was 61.3%, 43.5% and 87.1%, 81.5% in observation group and transplantation group, there was significant difference between two groups (P < 0.05). It is concluded that treatment with APBSCT after sequencing chemotherapy and radiotherapy for nasal type extranodal NK/T-cell lymphoma may increase DFS efficiently.

Autophagy is involved in doxorubicin induced resistance of human myeloma cell line RPMI8226 / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the role of autophagy in doxorubicin (DOX)-induced resistance of human myeloma cell line RPMI8226.</p><p><b>METHODS</b>We established doxorubicin induced resistant subline of myeloma cell line RPMI8226/DOX by drug concentration step-elevation method. Resistant index of DOX was measured by MTT assay. Autophagy of myeloma cell lines RPMI8226/s and RPMI8226/DOX was detected by transmission electron microscopy, immunofluorescence (LC3-FITC) and western blot respectively. Apoptosis of RPMI8226/DOX cells induced by DOX combined with autophagic inhibitor hydroxychloroquine or 3-MA was identified by AnnexinV-FITC/PI double fluorescence dyeing.</p><p><b>RESULTS</b>Resistant index of RPMI8226/DOX was approximately 10.8 fold of that of RPMI8226/S. Electron microscopic studies revealed that most of RPMI8226/DOX cells displayed viable attributes and contained numerous autophagic vacuoles. Fluorescent images of RPMI8226/DOX cells showed a punctuate distribution in LC3 protein. Increased LC3-II protein in RPMI8226/DOX cells was determined by immunoblotting. There were no differences among 8 μmol/L HCQ (3.24±1.08)%, 10 mmol/L 3-MA (2.81±0.80)% or control \[(2.12±1.24)%\] (P>0.05) in terms of AnnexinV-FITC/PI double fluorescence dyeing; Compared with apoptosis of (9.75±2.15)%, (24.36±2.16)% and (40.51±3.14)% of RPMI8226/DOX cells under 2, 4 and 6 μmol/L DOX, apoptosis increased significantly after 24 h incubation under 2, 4 and 6 μmol/L DOX combined with 8 μmol/L HCQ as of \[(16.56±1.89)%, (36.44±2.91)% and (62.68±3.75)%, respectively\], or under 2, 4 and 6 μmol/L DOX combined with 10 mmol/L 3-MA as of \[(15.47±1.85)%, (39.28±3.06)% and (55.46±4.07)%, respectively\] (P<0.05).</p><p><b>CONCLUSION</b>Autophagy was involved in doxorubicin-induced resistance of myeloma cell line RPMI8226, DOX resistance in myeloma cells was reversed partly by autophagy inhibitor hydroxychloroquine or 3-MA, and autophagy may be one of mechanisms for drug resistance.</p>

Efficacy of adoptive immunotherapy after mixed hematopoietic stem cell transplantation on acute myeloid leukemia / 中国实验血液学杂志

The purpose of this study was to investigate the efficacy of treatment with haploidentical donor's lymphocyte infusion(hiDLI) combined with interleukin-2 (IL-2) after transplantation of autologous peripheral blood stem cells mixed with haploidentical allogeneic bone marrow (mix-HSCT) for acute myeloid leukemia (AML). 49 patients diagnosed as AML were enrolled in this study. After preconditioning with TBI plus VEMAC regimen, all patients received mix-HSCT. Autologous peripheral blood hematopoietic stem cells were mobilized with chemotherapy-combined G-CSF, and haploidentical allogeneic bone marrow cells were not mobilized with G-CSF. 33 patients in test group were treated with hiDLI plus IL-2 for 1-8 times after hematopoietic reconstruction, 16 patients in control group received mix-HSCT only. All the patients were followed-up for more than 3 years. The results showed that all the patients obtained hematopoietic reconstruction, and no graft-versus-host disease (GVHD) was found. In two groups, the median time of absolute neutrophil count (ANC) ≥ 0.5×10(9)/L was 14 (12 - 18) and 14 (11 - 16) days, and WBC count ≥ 4.0×10(9)/L was 17 (16 - 22) and 18(17 - 20) days, Plt count ≥ 50×10(8)/L were 25 (24 - 29) and 25 (23 - 26) days. 9 patients in test group formed mixed chimerism (46XX/46XY) and sustained about 3 - 12 months; disease-free survival (DFS) was 63.6%, 3 patients in control group formed mixed chimerism (46XX/46XY), persistent about 3-6 months; DFS was 50.0%. It is concluded that treatment with hiDLI plus IL-2 after mix-HSCT for AML patients may increase DFS efficiently.

Effects of interferon-γ on expression of adhesion molecules in human umbilical cord mesenchymal stromal cells / 中国实验血液学杂志

This study was purposed to investigate the effects of interferon (IFN)-γ on expression of adhesion molecules in mesenchymal stromal cells derived from human umbilical cord tissue (UC-MSC). The UC-MSC were isolated from human umbilical cord by tissue culture. The expressions of specific markers on UC-MSC were detected by flow cytometry in the physiological condition. The adipogenic and osteogenic induction of UC-MSC was detected by alizarin and Oil red O staining. UC-MSC were exposed to IFN-γ (100, 1 000, 10 000 U/ml) for 24 h, the expressions of CD54, CD58, CD44, CD49d, CD62p, CD62L, CD102 and CD106 on cell surface were detected using flow cytometry. The results showed that in physiological condition, UC-MSC extremely low expressed CD102, CD106, CD62P, CD62L, while the expression of CD54 was relatively high (41.58 ± 0.83)%. When stimulated by IFN-γ, the expression of CD102, CD106, CD62P, CD62L increased slightly, but still low (< 5%), while CD54 and CD58 upregulated concentration-dependently up to (59.66 ± 1.36)% and (43.96 ± 0.62)% respectively. The expression of CD49d upregulated to (51.33 ± 0.74)% when UC-MSC exposed to IFN-γ 100 U/ml. CD44 increased to (73.22 ± 1.93)% when UC-MSC exposed to IFN-γ 1 000 U/ml. It is concluded that IFN-γ can elevate significantly the expression of CD54, CD49d, CD44 and CD58, but has no significant effect on CD102, CD106, CD62P and CD62L expression on the surface of UC-MSC.

Expression of radioresistant genes survivin and HO-1 in mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to investigate the expression of radioresistant genes survivin and HO-1 in mesenchymal stem cells (MSC). Human bone marrow MSC were isolated and enriched by Fircoll density gradient centrifugation, then identified by flow cytometry. MSC were induced with dexamethasone, insulin, 3-isobutyl-1-methyl-xanthine (IBMX) and indomethacin to differentiate into adipocytes. Then the expression of survivin and HO-1 in MSC was detected by RT-PCR. The results indicated that the expressions of surface antigen CD34 and HLA-DR in MSC in vitro were negative while the expressions of CD44 and CD71 were positive. MSC could be differentiated into adipocytes by inductor. RT-PCR showed the expression of radioresistant genes survivin and HO-1 in MSC. It is concluded that MSC have lower sensitivity to radiation, which may associate with the expression of radioresistant genes survivin and HO-1 in MSC.

Biological effects of low dose X-irradiation on human bone marrow mesenchymal stem cells / 中国实验血液学杂志

Recent studies have shown that low dose X-irradiation shows specific effect different from high dose exposures. However, the biologic responses of bone marrow mesenchymal stem cells (BM-MSC) to low dose X-irradiation have rarely been described in the literature. This study was purposed to investigate the biologic responses of human bone marrow-derived MSC to low dose X-irradiation. The proliferation of cells was tested with MTT assay, so that the cell growth curve was drawn at 1 to 7 days. The expression of survivin mRNA was detected by RT-PCR assay; the BM-MSC DNA damage induced by X-irradiation were detected with mononuclear cell gel electrophoresis. The results indicated that the proliferative ability of BM-MSC exposed to low doses of X rays was obviously enhanced as compared with control group. The low dose X-irradiation caused the damage of DNA in X-ray dose dependent manner. X-irradiation enhanced expression of survivin in MSC. It is concluded that the low dose below 20 cGy of X-irradiation has a promoting effect on survivin expression in BM-MSC. Whether the high expression of survivin plays an important role to resist ionizing radiation needs to be further studied.

Bortezomib combined with autologous peripheral blood hematopoietic stem cell transplantation for therapy of patients with multiple myeloma / 中国实验血液学杂志

This study was aimed to evaluate the therapeutic efficacy of bortezomib combined with autologous peripheral blood hematopoietic stem cell transplantation (autoPBSCT) for patients with multiple myeloma (MM). 5 patients underwent autologous hematopoietic stem cell transplantation. Bortezomib treatment was supplied for patients before autoPBSCT and in the conditioning of transplantation, it was also used in maintaining treatment. Patients with transplantation adopted bortezomib plus melphalan conditioning regimen. The number of infused MNC and number of CD34(+) cells were 4.06×10(8) (4.09×10(8) - 4.37×10(8))/kg and 3.98×10(6) (2.49×10(6) - 8.2×10(6))/kg respectively. The results showed that hematopoiesis was reconstituted in 5 patients, with a neutrophil cell count more than 0.5×10(9)/L at day 14 (13 - 25 days) after transplantation and platelet count more than 50×10(9)/L at day 28 (21 - 41 days) after transplantation. Transplantation-associated death was not observed. 5 patients were disease-free survival. In conclusion, treatment of bortezomib combined with autologous peripheral hematopoietic stem cell transplantation is an effective method for patients with multiple myeloma. Use of bortezomib after transplantation might still be favourable to MM patients, for survival prolongation and life quality improvement.

X-ray induces autophagy in human mesenchymal stem cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the autophagy in human bone marrow mesenchymal stem cells (hBMMSC) exposed to irradiation.</p><p><b>METHODS</b>The apoptosis and necrosis rate were assessed by Annexin V and propidium (PI) staining in hBMMSC at 4h after irradiated with X-ray at 0, 2, 4, 8 and 10 Gy. The autophagy was observed by transmission electron microscopy. The mRNA expression of Beclin1 and microtubule-associated protein 1 light chain 3 (MAPLC3 or LC3) was analyzed by RT-PCR in hBMMSC at 4h after X-ray irradiation at 0, 8 and 10 Gy.</p><p><b>RESULTS</b>The apoptosis rate of hBMMSC was markedly decreased while the necrosis and death rate were slowly increased with the increase of irradiation dose when under 8 Gy. The apoptosis rate was significantly increased and reached a peak while the necrosis and whole death rate were obviously increased when irradiated with 10 Gy X-rays. In addition, the change of apoptosis rate was more significant than that of necrosis rate. By electron microscopy, a mass of autophagic vacuoles (autophagosome and autolysosome) were observed in irradiation and positive control groups, but were only occasionally seen in normal control group. The proportion of hBMMSC with autophagic vacuoles in 8 Gy irradiation group was higher than that in 10 Gy one. The mRNA expression of Beclin1 and LC3 in irradiation groups and positive control group was significantly higher than in normal control group, and so did in 8 Gy irradiation group than that in 10 Gy group.</p><p><b>CONCLUSION</b>Irradiation may induce the autophagy in hBMMSC, and autophagy could protect hBMMSC from irradiation injury in a certain dose range.</p>

Expression characteristics of SDF-1 receptor CXCR4 in mesenchymal stem cells derived from human umbilical cord tissue / 中国实验血液学杂志

The purpose of this study was to explore the expression characteristics of SDF-1 receptor, CXCR4, in mesenchymal stem cells (MSC) of different passages derived from human umbilical cord (hucMSC). The hucMSC were isolated from Wharton's jelly tissue of human umbilical cord by tissue culture. The expressions of specific marker in hucMSC were detected by flow cytometry. The adipogenic and osteogenic induction of hucMSC were detected by alizarin bordeaux and Oil red O staining. The expressions of CXCR4 protein in hucMSC of 2nd-5th passages were detected by flow cytometry, and cxcr4 mRNA levels in hucMSC of 2nd-5th passages were evaluated by real-time quantitative PCR. The results showed that the expression of CD44, CD13, CD71 were positive while CD38, CD117, HLA-DR were negative. After induced by osteogenic and adipogenic inductors, the lipid droplets and calcium nodals appeared in hucMSC, hucMSC stained with oil red O and alizarin red were shown to be positive. The cxcr4 was found in hucMSC of 2nd-5th passages, and their expressions were (89.82 ± 0.62)%, (86.87 ± 1.32)%, (80.50 ± 4.46)%, (70.10 ± 0.68)% respectively. The cxcr4 mRNA was found in hucMSC of 2nd-5th passages, and expression of cxcr4 of 3rd-5th passages were 0.5585 ± 00875, 0.6205 ± 0.1377, 0.4634 ± 0.0447 times of expression of 2nd passage respectively. It is concluded that the cxcr4 mRNA expresses in hucMSC of 2nd-5th passages, and declines when the number of passages increases. Compared with 2nd passage, cxcr4 mRNA levels in hucMSC of 3rd-5th passages decline, but the expression level of cxcr4 mRNA between hucMSC of 3rd-5th passages is stable.

Effect of cobalt protoporphyrin on hyperexpression of heme oxygenase-1 and secretion of IL-10 in rat bone marrow mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to investigate the effects of cobalt protoporphyrin (CoPP) on hyperexpression of heme oxygenase-1 (HO-1) and secretion of IL-10 in bone marrow-derived mesenchymal stem cells (BMMSC). The BMMSC were isolated from rats and cultured, then were randomly divided into 4 groups, and were induced in culture medium with CoPP of 0, 25, 50, and 75 µmol/L respectively. The expressions of CD34, CD45, CD44 and CD71 in BMMSC were detected by flow cytometry; the osteoblast and adipocyte induction of BMMSC was determined by alizarin red and oil red staining respectively; the proliferation status of BMMSC was assayed by MTT method; the HO-1 expression in test groups was detected by RT-PCR; the optimal concentration of CoPP for induction of HO-1 expression was screened. The ELISA kit was used to detect the HO-1 levels in each groups. The results showed that the expressions of CD34, CD45 in BMMSC were negative while the expressions of CD44 and CD71 in BMMSC were positive. After induced by osteoblast and adipocyte inductor, BMMSC were positive for alizarin red staining and oil red staining. Various concentrations of CoPP had no significant impact on cell proliferation. When the CoPP concentration of 50 µmol/L could induce strong expression of HO-1, expression level in test groups was significantly higher than that in control group, meanwhile the IL-10 secretion was significantly higher than that in control group. It is concluded that CoPP has no ability to promote cell proliferation, 50 µmol/L is the optimum concentration for up-regulation of CoPP-induced BMMSC HO-1 expression, the up-regulation of HO-1 expression can increased IL-10 secretion significantly.

Effects of peptidoglycan on proliferation and cell cycle of human bone marrow-derived mesenchymal stem cells / 中国实验血液学杂志

This study was aimed to explore the effects of peptidoglycan (PGN) on proliferation and cell cycle of human bone marrow mesenchymal stem cells (MSCs). MSCs were isolated from human bone marrow by density gradient centrifugation. The purity of MSCs with the spindle fibroblastic morphology was identified by microphotography and the phenotypes were detected by flow cytometry (FCM). MSCs incubated with different doses of PGN (1, 10, 20 μg/ml) were used as test groups, and those incubated without PGN were regarded as control group. The isolated and cultured MSCs were inoculated into 96-well plates according to a certain concentration. Cell cycle was measured by flow cytometry after incubated with PGN for 72 hours. The results showed that the cell proliferation index was significantly increased in dose and time dependent manners after MSCs was incubated with PGN. Its effects on the proliferation of MSCs were highest in 10 μg/ml group. Compared with the control group, PGN could significantly decrease proportion of MSCs in G₀/G₁ phase and increase them in S and G₂/M phases (p < 0.05). It is concluded that PGN can promote more MSCs to enter the DNA synthesis phase and proliferate many much MSCs in dose and time dependent manners.

Experimental study on influence of bone marrow mesenchymal stem cells on activation and function of mouse peritoneal macrophages / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the influence of bone marrow (BM) mesenchymal stem cells (MSCs) on macrophage activation after lipopolysaccharide (LPS) stimulation.</p><p><b>METHODS</b>Mouse BM MSCs were isolated and purified by adherence screening, and mouse peritoneal macrophages (MPM) were collected by sodium thioglycollate peritoneal injection, and the co-culture system was established by planting macrophages on the MSCs monolayer. The grouping of experiments: group A: MPM; group B: MPM + LPS; group C: MPM + LPS + MSC; group D: MPM + LPS + MSC supernatant. Cell culture supernatants were collected to detect the changes of TNF-alpha/TGF-beta and nitrogen monoxide (NO) after stimulating macrophages with LPS for 18 hours. At the same time Escherichia coli standard strain (ATCC25922) was added into the culture system and incubated for another 24 hours, macrophages were stained and phagocytosis were examined.</p><p><b>RESULTS</b>The concentrations of TNF-alpha and NO in culture supernatants were increased significantly to (147.4 +/- 37.1) pg/ml and (59.9 +/- 8.7) micromol/L respectively after macrophage activation, however, at the present of MSC, the concentration of TNF-alpha was dramatically decreased [(97.6 +/- 30.3) pg/ml, P = 0.032], and the concentration of NO was decreased to (50.9 +/- 29.5) micromol/L (P > 0.05). The concentrations of TNF-alpha and NO were further decreased after addition of MSC supernatants [(58.3 +/- 31.5) pg/ml and (-3.4 +/- 2.3) micromol/L respectively, P < 0.01]. There was no change in the phagocytic rate and phagoindex of macrophages after activation.</p><p><b>CONCLUSIONS</b>MSCs can inhibit the activation of mouse peritoneal macrophages after stimulating with LPS but has no influence on the phagocytosis.</p>

Effect of human bone marrow mesenchymal stem cells on T lymphocyte killing K562 cells / 中国实验血液学杂志

The objective of this study was to investigate the effect of human bone marrow mesenchymal stem cells (MSCs) on T lymphocyte killing K562 cells. MSCs were isolated from bone marrow and cultured, T cells were harvested by using nylon column method from peripheral blood. The T cells were co-cultured with MSCs, the phenotype expressions of T cell subsets were detected by flow cytometry. Killing effects of T cells (culture alone and co-culture with MSCs) on K562 cells were detected by LDH, expressions of IFN-gamma and IL-4 were detected by ELISA. The results showed that after T cells were co-cultured with MSCs for three days, the proportion of CD4+ and CD4+CD25+ T cells raised significantly (p<0.05) as compared with group of culture alone, but the proportion of CD8+ T cell were not significantly changed (p>0.05). In group of T cells co-cultured with MSCs, killing effects of T cells on K562 cells weakened, at the same time, expression of IFN-gamma decreased while expression of IL-4 increased. It is concluded that the MSCs weaken killing effects of T cells on K562 cells, which associates with increase of CD4+CD25+ T cell subsets and changes of IFN-gamma and IL-4 levels.