The Effect of Doxycycline on the Expression of MMP-2 and MMP-9 in Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To investigate the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in multiple myeloma (MM) patients, and analyze the effect of doxycycline (DOX) on the expression of MMP-2 and MMP-9 in MM cells.@*METHODS@#The peripheral blood and bone marrow samples of MM patients were collected, and the patients were divided into three groups: newly diagnosed group, remission group and relapsed/refractory group, while the peripheral blood samples of 34 health people and the bone marrow samples of 17 IDA patients were selected as normal control and control group. The levels of MMP-2 and MMP-9 were detected by ELISA. The protein levels of MMP-2 and MMP-9 in H929 cells treated by different concentrations of DOX were analyzed by Western blot. After H929 cells was treated by Akt inhibitor MK-2206 2HCl in combination with DOX, Western blot was used to detect the levels of MMP-2 and MMP-9.@*RESULTS@#The levels of MMP-2 and MMP-9 in newly diagnosed MM patients were higher than those in control (P<0.05), while for the patients in the remission group were decreased, but still higher than those in control. The levels of MMP-2 and MMP-9 were increased again for the patients in relapsed/refractory group, and showed no significant difference as compared with those in newly diagnosed group. The levels of MMP-2 and MMP-9 could be inhibited by 10 mg/L and 15 mg/L DOX treated by H929 cell. The protein levels of MMP-2 and MMP-9 showed no altered in H929 cells treated by 5 nmol/L MK-2206 2HCl alone. DOX exerted more profound inhibitory effect to MMP-2 and MMP-9 expression in H929 cells when Akt inhibitor MK-2206 2HCl was combined with DOX.@*CONCLUSION@#The levels of MMP-2 and MMP-9 are increased in MM patients and related to the disease status of MM. DOX can inhibit the expression of MMP-2 and MMP-9 in MM cells, and antagonizing its activation of Akt signaling pathway can further enhance the inhibitory effect.

Effect of Doxycycline on Intrinsic Apoptosis of Myeloma Cell Line H929 and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the mechanism of the in vitro toxicity of doxycycline to myeloma cell line H929 and also the possible pathway involved its toxicity.@*METHODS@#Myeloma cell line H929 was treated with DOX, MEK inhibitor U0126 or RAS agonist ML-098, either alone or in combination. Then, the expression of p-MEK, caspase-3, caspase-9 and c-Jun in H929 were used to detected by Western blot; the cells proliferation and apoptosis were detected by CCK-8 assay and flow cytometry, respectively.@*RESULTS@#DOX significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK in H929 (P<0.05). MEK antagonist U0126 significantly increased the levels of cleaved caspase-3 and caspase-9, and down-regulated the level of p-MEK (P<0.05). After Dox combined with ML-098 treatment of H929 cells, the apoptosis rate of H929 cells was lower than that of DOX alone treatment group(P<0.05). Compared with DOX alone treatment group, the expressions of p-MEK and p-ERK1/2 in DOX+ML-098 combined treatment group were increased, and the levels of cleaved caspase-3,9 in H929 cells were decreased (P<0.05). The levels of c-Jun mRNA and protein increased in H929 when treated by DOX alone (P<0.05).@*CONCLUSION@#DOX can induce apoptosis of H929 via intrinsic apoptosis pathway, and MEK/ERK pathway and c-Jun possibly play a role in this process.

Increment of N-cadherin Expression in Bone Marrow Mesenchymal Stem Cells Accompanies by Imparment of Bone Marrow B Lymphocyte Production in MRL/lpr Lupus Mice / 中国实验血液学杂志

OBJECTIVE@#To investigate the hematological changes in MRL/lpr lupus mice and detect N-cadherin expression in their bone marrow mesenchymal stem cell (BMMSC).@*METHODS@#Peripheral blood cell count was analyzed. The ratio of each lineage in bone marrow was analyzed by flow cytometry. Bone marrow CFU-pre-B, BFU-E and CFU-GM were detected by colony formation assay. Expression of N-cadherin in BMMSC was analyzed by Western blot before and after treatment with the BMP/Smad pathway aganist BMP-2 and inhibitor Noggin.@*RESULTS@#Hemoglobin, red blood cell count and hematocrit decreased in lupus mice, compared with C57BL/6 mice. The ratio of B220+ lymphocyte and the number of CFU-pre-B in bone marrow of lupus mice decreased. Expression of N-cadherin in BMMSC of lupus mice was higher than that in the control group. Expression of N-cadherin decreased in BMP-2-treated BMMSC and increased after Noggin treatment.@*CONCLUSION@#Hematological changes in lupus mice include anemia and impairment of bone marrow B cell production. The expression of N-cadherin in BMMSC of lupus mice increases which maybe involved in abnormal hemogenesis in MRI/Ipr lupus mice.

IL-1β-Treated Bone Marrow Mesenchymal Stem Cells Enhances Osteogenetic Potential via NF-κB Pathway / 中国实验血液学杂志

<p><b></b>Obsjective:To investigate the effects of differentaction time of IL-1β on the osteogenic capacity of bone marrow mensenchymal cells(BMMSC) and the role of nuclear factor-κB (NF-kB) pathway.</p><p><b>METHODS</b>BMMSC isolated from normal donors was treated with IL-1β for 1 or 7 days, respectively. Alkaline phosphatase (ALP) and alizarin red(AR) stainings were used to detect the osteogenic differentiation potential of BMMSC. The mRNA expression of EphB4, IGF-1 and OPG in BMMSC was measured by real-time PCR. The immunohistochemistry was employed to measure the expression of bone morphgenetic protein-2(BMP-2) and p-Smad1/5/8 in BMMSC. Furthermore, the Western blot was used for the detection of iκBα and phospho-iκBα (p-ikBα) in IL-1β-treated BMMSC. And the results of IL-1β-treated BMMSC were compared with control group.</p><p><b>RESULTS</b>Compared with control group, the osteogenetic potential of IL-1β-treated BMMSC was enhanced, but the pro-osteogenic differentiation effect of IL-1β was remarkedly inhibited in the presence of NF-kB pathway inhibitor PDTC. The total ikBα level of IL-1β-treated BMMSC was lower (P<0.05), and phospho-iκBα (p-iκBα) level was higher (P<0.05). Besides, BMP-2 expression was higher (P<0.05) in the IL-1β-treated BMMSC, however, p-Smad1/5/8 protien level was not significantly different among IL-1β-treated for 1 d, 7 d and control groups (P<0.05). And the mRNA expression levels of IGF-1, EphB4 and OPG in BMMSC were up-regulated after IL-1β treatment (P<0.05). In addition, the osteoblastogenesis of BMMSC treated with IL-1β for 7 days was significantly different from those treated only for 1 day.</p><p><b>CONCLUSION</b>Prolonging IL-1β treatment can enhance the osteogenetic differentiation of BMMSC more significantly. And this osteogenetic alteration of BMMSC occurs via its NF-κB pathway, but not via BMP-2/Smad pathway.</p>

Clinical Efficacy of Decitabine Combined with Modified CAG Regimen for Relapse/Refractory Acute Myeloid Leukemia with AML1-ETO / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the clinical characteristics of patients with relapse-refractory acute myeloid leukemia(AML) with AML1-ETO, and therapeutic effcacy and side effects of decitabine combined with modified CAG regimen.</p><p><b>METHODS</b>Clinical data of 8 cases of AML with AML1-ETOfrom June 2015 to January 2016 were analyzed retrospectively, including age, sex, initial symptoms, peripheral blood and bone marrow characteristics and so on. at the same time, the therapeutic effcacy and side effects of decitabine combined with modified CAG regimen were evaluated. The 8 patient were with median age of 44.5(16-59) years.</p><p><b>RESULTS</b>Among these 8 patients, 1 patients were relapsed and other 7 patients were relapse/refractory patients, their median white blood cell count was 23.57(7.5-65.29)×10/L, median platelet count was 40(19-69)×10/L, median hemoglobin 1evel was 107(79-131) g/L, median lactate dehydrogenase level was 313.5(124.1-865.9) U/L at the initial diagnosis. The results showed that after treatment with decitabine combined with modified CAG, 7 patients achieved complete remission, 1 patient did not achieve remission, the overall remission rate was 87.5%(7/8). The main side effects of this regimen was myelosupp-ression, there were no new lung infection and other serious complications, 1 case without complete remission was treated with FLAG, and died of heart failure.</p><p><b>CONCLUSION</b>According to preliminary results of decitabine combined with modified CAG regimen for treatment of relapse/refractory AML patients with AMLl-ETOdisplays higer remission rate and lower side effects.</p>

Proteasome inhibitor bortezomib inducing apoptosis of K562 cells not affected by bone marrow mesenchymal stem cells in vitro / 中国实验血液学杂志

The study was aimed to investigate the effects of proteasome inhibitor bortezomib on the apoptosis of K562 cells in the presence of bone marrow mesenchymal stem cells, and explore its effect on expression of adhesion molecule VCAM-1 of both MSC and K562 cells. The K562 cells were co-cultured in direct contact with MSC, while the control cells were just cultured alone. Bortezomib was administered at a final concentration of 50 nmol/L. Cell apoptosis was assayed by flow cytometry with Annexin-V/PI double staining kit. The VCAM-1 gene expression was determined by reverse transcription polymerase chain reaction (RT-PCR). The results indicated that bortezomib could induce apoptosis of K562 cells in a time-dependent manner. K562 cells growing on the layer of MSC demonstrated the similar sensitivity to apoptosis induction of bortezomib. K562 cells which did not express VCAM-1 originally were induced to express VCAM-1 mRNA when co-cultured with MSC. This effect could be abrogated by bortezomib treatment. Furthermore, bortezomib significantly downregulated the VCAM-1 expression of MSC. It is concluded that the proteasome inhibitor bortezomib can induce apoptosis of K562 cells even though in presence of the MSC layer.

Effect of proteasome inhibitor on migration ability and hepatocyte growth factor expression of bone marrow mesenchymal stem cells in multiple myeloma patients / 中国实验血液学杂志

This study was aimed to investigate the effect of proteasome inhibitor bortezomib on the migration ability and hepatocyte growth factor (HGF) expression of bone marrow mesenchymal stem cells (MSC) in multiple myeloma patients. Transwell assay was employed to measure the migration ability of bone marrow MSC in vitro before and after treatment with bortezomib. The HGF mRNA expression level was determined by real-time quantitative PCR. The results indicated that after treated with bortezomib of concentrations of 2.5 nmol/L for 48 hours, the migration activity of MSC decreased significantly as compared with control cohorts (p < 0.05). The HGF mRNA level in MSC after bortezomib treatment was significantly lower than that of control group (p < 0.05). It is concluded that bortezomib can inhibit the migration and down-regulate HGF mRNA expression of bone marrow MSC in multiple myeloma patients.

Chemotaxis-related factors are expressed abnormally in bone marrow mesenchymal stem cells of multiple myeloma patients / 中国实验血液学杂志

This study was aimed to investigate the mRNA expression levels of hepatocyte growth factor (HGF), stromal cell-derived factor-1 (SDF-1), monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) in bone marrow mesenchymal stem cells (MSC) from multiple myeloma (MM) patients. The mRNA expression levels of HGF, SDF-1, MCP-1 and IL-8 in bone marrow MSC from 20 newly diagnosed MM patients were detected by real time quantitative RT-PCR and were compared with that in 9 controls. The results indicated that the mean mRNA expression level of HGF was up-regulated in MM patients, as compared with controls (p < 0.01). However, the mean mRNA expression level of SDF-1 mRNA was down-regulated in MM patients, as compared with controls (p < 0.05). There was no significant difference in the mRNA expression levels of MCP-1 and IL-8 between MM and control cohorts (p > 0.05). It is concluded that BM-MSC from MM patients express HGF, SDF-1, MCP-1, IL-8, but these chemotaxis-related factors expression of bone marrow microenvironment cellular component are dysregulated in MM patients, which may result from the interplay between MM cells and MSC.

Change of cytokine expressions on bone marrow mesenchymal stem cells in patients with bone marrow failure syndromes and its significance / 中国实验血液学杂志

This study was aimed to investigate the expressions of multiple cytokines on bone marrow mesenchymal stem cells (MSC) in patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS), and its significance. The semi-quantitative reverse transcriptase-PCR (RT-PCR) was used to detect the expressions of IL-1β, SCF, G-CSF at mRNA level in bone marrow MSC of patients with AA and MDS. The real time quantitative polymerase chain reaction (RQ-PCR) technique was used to detect the mRNA expression of TPO in bone marrow MSC of AA and MDS patients. The results indicated that the expression of SCF in AA group was much lower than that in the normal control group (p < 0.05), and the expression of TPO in AA group was higher than that in the normal control group (p < 0.05), while the expression of IL-1β of AA had no significant difference when compared with the normal control group (p > 0.05). Compared with normal control group, the expressions of SCF of MDS patients was lower (p < 0.05), but the expressions of IL-1β and TPO did not show significant difference (p > 0.05). The expressions of IL-1β, SCF and TPO were no significant difference between AA and MDS groups (p > 0.05). Neither the AA patients, MDS patients nor the normal control group had the expression of G-CSF. It is concluded that the expression of SCF and TPO in bone marrow MSC of AA patients are obviously abnormal, the expression of SCF is also abnormal in bone marrow MSC of MDS patients.

Human bone marrow stromal cells facilitate the cord blood CD34+ cells ex vivo expansion and short-term engraftment in NOD/SCID mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the potential of human bone marrow stromal cells (MSCs) as the feeding-layer to promote ex vivo expansion of cord blood CD34+ cells and engraftment of the expanded cells in NOD/SCID mice.</p><p><b>METHODS</b>Human MSCs were routinely isolated and cultured. MSCs at passage 3 were used as feeding-layer for the expansion of cord blood CD34+ cell in the presence of thrombopoietin (TPO), flt3/flk2 ligand (FL), stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF). The engraftment potential between unexpanded and expanded cord blood cells transplanted into NOD/SCID mice was compared.</p><p><b>RESULTS</b>The total nucleated cells (TNC), CD34 cells and colony forming units (CFUs) in the MSC feeding culture were increased by 111.6-, 19.3- and 58-fold after 1 week expansion and 532.8-, 41.3- and 563.5- fold increased after 2 weeks expansion respectively as compared with that in non MSC feeding culture. In transplant experiment, the percentage of human CD45+ cells (45.3% -59.1%) in bone marrow of recipient mice transplanted with the MSC feeding expanded cells was the highest in all the groups at six weeks after transplantation.</p><p><b>CONCLUSION</b>Human MSCs enhance CB CD34+ cells in vitro expansion and their capacity of short-term engraftment in NOD/SCID mice.</p>

Effect of co-culture of bone marrow mesenchymal stem cells and K562 cells on expression of interleukin 8 / 中国实验血液学杂志

To investigate the effects of interaction between human bone marrow mesenchymal stem cells (MSCs) and K562 cells on the expression of proangiogenic factor IL-8, the K562 cells were co-cultured in direct contact with MSCs or cultured in MSCs conditioned medium while the controlled K562 cells were cultured alone. The IL-8 gene expression of K562 cells and MSCs was determined by RT-PCR. The results indicated that the expression of IL-8 in K562 cells when co-cultured in direct contact with MSCs or cultured in MSCs conditioned medium was obviously higher than that of K562 cells cultured alone (p<0.01). MSCs co-cultured with K562 cells also had an increased level of IL-8 compared with MSCs cultured alone (p<0.01). It is concluded that the interaction between MSCs and K562 cells via direct contact and the cytokine network promotes expression of IL-8.

Effects of As2O3, dexamethasone and thalidomide on apoptosis and cytoplasmic Ca2+ of myeloma cell line U266 / 中国实验血液学杂志

To investigate the influence of As2O3, dexamethasone (Dex) and thalidomide (Thal) on apoptosis-induced myeloma cell line U266 cytoplasmic calcium concentrations ([Ca2+]i), U266 cells were incubated in the culture of RPMI 1640 with 15% FBS in 24-well plate and exposed to different concentrations of As2O3, Dex and Thal for 8 hours, respectively, then cell apoptosis was analyzed by fluorescence microscopy and flow cytometry (FCM) with Annexin V-FITC/PI double staining, and cytoplasmic free calcium were detected on FCM through Fluo-3/AM loading. The results indicated that (1) apoptotic cells were gradually increased with enhancement of As2O3, Dex and Thal concentrations; (2) apoptotic cell rates increased from 0.56% in control to 31.54%, 28.35% and 21.97% respectively after treatment with As2O3, Dex and Thal; (3) As2O3, Dex induced U266 cell apoptosis accompanied with raise of [Ca2+]i; (4) [Ca2+]i had no statistically significant changes in Thal-induced apoptotic U266 cells. It is concluded that the raise of [Ca2+]i is one of the mechanisms for As2O3 and Dex-induced U266 cells apoptosis, whereas Thal-induced U266 apoptosis has no significant relation to [Ca2+]i changes.

Expressions of IL-1?,IL-6,SCF,TPO on bone marrow stromal cells in patients with multiple myelo-ma / 肿瘤研究与临床

Objective To study multiple cytokine mRNA expressions of bone marrow stromal cells in patients with multiple myeloma(MM),as well as to explore the role of these cytokines in the occurrence and development of MM.Methods Semi-quantitiative RT-PCR was used to detect the mRNA expressions of IL- 1?,IL-6,SCF,TPO.Results The mRNA expression levels of IL-1? and IL-6 were higher than that of nor- mal controls and other hematological malignancies(P

Effects of proteasome inhibitor PS-341 on the multiple cytokine expressions of mesenchymal stem cells from bone marrow in patients with multiple myeloma / 中国实验血液学杂志

To explore the effects of proteasome inhibitor PS-341 on the cytokine expressions of mesenchymal stem cells (MSC) in patients with multiple myeloma (MM), MSCs of 11 patients were cultured in medium of RPMI 1640 containing 10% FBS. When cells grew to 5 x 10(5) - 1 x 10(6), cells were exposed to 50 nmol/L PS-341 for 4 hours, then harvested. The expressions of IL-6, IL-1beta and SCF were detected by RT-PCR. The results indicated that after treatment with PS-341 the expressions of IL-6, IL-1beta and SCF of MSCs decreased markedly, especially that of IL-1beta, compared with control (P < 0.05, P < 0.01, P < 0.05, respectively). There were obviously differences of IL-1beta expression between refractory/relapsed group and complete remission (CR) group and IL-1beta expression was inhibited more seriously in CR group, whereas there were no significant differences of IL-6 and SCF expression between two groups; IL-1beta expression of patients treated with PS-341 was not detected; there were not effects of IL-1beta expression on expressions of IL-6 and SCF. It is concluded that proteasome inhibitor PS-341 downregulated the expressions of IL-6, IL-1beta and SCF of MSCs in patients with MM.

Expression of HOXB4 in cord blood progenitor cells expanded in vitro / 中国实验血液学杂志

HOXB4, a member of homeobox gene family, is closely related to the self-renewing and proliferative ability of primitive hematopoietic stem/progenitor cells (PHSC/PHPC). This study was aimed to investigate the self-renewing level of cord blood progenitor cells (CBPC) expanded in vitro. The HOXB4 expression at mRNA level was assayed by using real time RT-PCR. The results indicated that as culture prolonged, the total cells, CD34(+) cells greatly increased, however the HOXB4 expression gradually declined, even down to undetectable level similar to that of mature lymphocytes. Meanwhile, it was shown that CD34(+) cells co-cultured with bone marrow mesenchymal stem cells (BM-MSC) could abate the decline of HOXB4 expression. It is concluded that the self-renewing potential of CD34(+) cells gradually decreased during expansion in vitro, co-culture with BM-MSC was helpful to CD34(+) cell expansion and slowed the loss trend of its self-renewal.