Analysis of Related Early Warning Indexes of Cytokine Release Syndrome in Multiple Myeloma Patients after CAR-T Treatment / 中国实验血液学杂志

OBJECTIVE@#To investigate the relationship between the levels of ferritin, C-reactive protein (CRP), lactate dehydrogenase (LDH) and interleukin-6 (IL-6) in peripheral serum and cytokine release syndrome (CRS) in patients with relapse and/or refractory multiple myeloma (R/R MM) after receiving chimeric antigen receptor T cells (CAR-T) immunotherapy.@*METHODS@#Twenty-eight patients with R/R MM were treated with 1×10@*RESULTS@#Among the 28 patients, 27 cases (96.4%) developed CRS, 24 cases (85.7%) in 1-2 grade CRS and 3 cases (10.7%) in 3-5 grade. The severity grade of CRS of 27 patients was positively correlated with the peak values of ferritin, CRP, LDH, and IL-6 in peripheral blood (r@*CONCLUSION@#After receiving CAR-T cellular immunotherapy, the incidence of CRS in patients with R/R MM is higher, but most of them are in grade 1 or 2. The severity of CRS is positively correlated with the levels of ferritin, CRP, LDH and IL-6 in peripheral blood.

Effect of OCT4A Gene on the Biological Characteristics of K562 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate biological effects of OCT4A gene on K562 cells and explore the molecular mechanism of K562 cell apoptosis.</p><p><b>METHODS</b>Two recombinant lentiviral vectors were constructed, which could stablely up- regulate and down- regulate OCT4A protein. Recombinant lentivirus was generated by co-transfection of three-plasmids and transfec-ted into K562 cells. The experiments were divided into 5 groups: normal, pLVX-OCT4A-ZsGreen1, pLVX vector control, PLB-OCT4A shRNA and non-specific shRNA groups. Western blot was applied to detect the expression of OCT4A protein, the cell counting kit-8 was applied to evaluate the effect of OCT4A on proliferation of K562 cells. The apoptosis and differentiation of K562 cells were detected by flow cytometry with AnnexinV/7-AAD double staining. The mRNA expressions of caspase-3,BIM,BCL-xL,BAX in K562 cells were determined by real time PCR.</p><p><b>RESULTS</b>The OCT4A fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR), the 2 lentiviral vectors were successfully constructed. In comparson with those in the control group, the expression of OCT4A protein of pLVX-OCT4A-ZsGreen1 group was significantly increased, but decreased in PLB-OCT4A shRNA group. CCK-8 assay showed that the higher the content of OCT4A protein, the faster the cell proliferation. The apoptosis rate was (3.48±0.52)% of pLVX-OCT4A-ZsGreen1 group, which was lower than that of control group, while the apoptosis rate PLB-OCT4A shRNA group was (7.25±0.57)%, which was higher than that of control group (P<0.05), however, the K562 cells differentiation was not influenced(P>0.05). Compared with control group, the gene expression of Caspase-3,BIM and BAX was down-regulated(P>0.05), but a significant up-regulation of BCL-xL gene expression was observed(P<0.05).</p><p><b>CONCLUSION</b>Two lentiviral vectors have been successfully constructed, which can stably up- and down- regulate the expression of OCT4A in K562 cells respectively. OCT4A can promote the K562 cell proliferation and inhibit the apoptosis, the mechanism may be related with up-regulation of BCL-xl expression.</p>

A Modified Protocol for Rapid Establishing Mouse Model with Ph Positive Acute Lymphoblastic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate a modified protocol of establishing mouse Ph ALL model so as to provide a more convenient and more powerful tool for Ph ALL studies.</p><p><b>METHODS</b>Immature B cells from BALB/c mice were transfected with the Mig190 retrovirus and infused into irradiated syngeneic mice. The immunophenotype was identified by flow cytometry, the BCR-ABL was identified by RT-PCR and Western blot. Leukemia cells isolated from sick mice were re-infused into syngeneic mice without irradiation.</p><p><b>RESULTS</b>The acute lymphoblastic leukemia was established in mice after Mig190 retroviral transfection, the lymphoblasts were observed by blood smears, the immunophenotype of these leukemic cells was CD19, and the BCR-ABL was detected by RT-PCR and Western blot, the immunophenotype was conformed as Ph ALL, the isolated leukemia cells infused into mice rapidly developed B-ALL.</p><p><b>CONCLUSION</b>A new protocol is established to generate mice with Ph ALL. This mouse model can provide a more simple and effective tool in comparison with the conversional protocols.</p>

Effect of Steadily Down-regulating VE-Cadherin Expression on Susceptibitity of K562 Cells to Chemotherapy / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of steadily down-regulating the expression of VE-cadherin on the chemotheraputic sensitivity of K562 cells, and explore its possible mechanism.</p><p><b>METHODS</b>Specifically targeting interference sequences carrying human VE-cadherin were designed, the recombinant lentiviral vector containing the IRES-GFP and NEO segment was constructed; recombinant lentivirus was generated by three-plasmids packing system, and transfected into K562 cells, then the cells steadily down-regulated were sorted. CCK-8 assay was performed to evaluate the VE-cadherin of chemotherapeutic (Imatinib) sensitivity of K562 cells. The apoptosis was analyzed by flow cytometry with Annexin V/7-AAD double labeling. The expressions of CD133 and ALDH1 mRNA were determined by real time PCR. The protein expressions of VE-cadherin, BCR-ABL and β-catenin were analyzed by Western blot.</p><p><b>RESULTS</b>The recombinant lentiviral vector pLB-shVEC-NEO-IRES-GFP was successfully constructed, packed into the lentivirus, then the K562 cells steadily down-regulating VE-cadherin expression was obtained. When VE-cadherin was down-rengulated in K562 cells, the proliferation rate was reduced while the the apoptosis rate was increased; the mRNA levels of CD133 and ALDH1 also were reduced; BCR-ABL fusion protein was not obviously changed; the total β-catenin protein, as well as the nuclear β-catenin protein were decreased in the K562/shVEC cells. Conclution: K562 cells are more susceptible to chemotherapy when VE-cadherin is down-regulated, that may be realized via reducing the stability and the nuclear transfer of β-catenin protein.</p>

Construction of A Lentiviral Vector Carrying CUEDC1 Gene and Its Effect on the Proliferation and Colony-formating Ability of MOLT-4 Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To construct a lentiviral vector carrying human CUEDC1 gene, to establish leukemic cell line MOLT-4 stably expressing recombinant plasmid, to analyze the expression of CUEDC1 in MOLT-4 cells and to investigate its effect on the proliferation of MOLT-4 cells.</p><p><b>METHODS</b>The CUEDC1 gene was amplified by RT-PCR, and then was subcloned into the lentiviral vector pCDH to generate a lentiviral vector pCDH-CUEDC1. Recombinant lentivirus was generated by co-transfection of 3 plasmids, and transfected into MOLT-4 cells. The Real-time PCR and Western blot were respectively applied to detect the expression of CUEDC1 mRNA and protein, the CCK-8 and colony formation assay were used to evaluate the effect of CUEDC1 on proliferation of MOLT-4 cells.</p><p><b>RESULTS</b>The recombinant lentiviral vector pCDH-CUEDC1 had been constructed successfully. After infection of MOLT-4 cells with the lentivirus, the recombinant plasmid could stably up-regulate the expression of CUEDC1 and protein. The CCK-8 detection and colony formation assay showed that exogenous CUEDC1 could significantly promote cell growth and the colony formation of MOLT-4 cells.</p><p><b>CONCLUSION</b>The recombinant lentiviral vector carrying human CUEDC1 has been successfully constructed, exogenous CUEDC1 can significantly promote cell growth and the colony formation of MOLT-4 cells.</p>

Establishment and Identification of Mouse Model of Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish a mouse model of Phacute lymphoblastic leukemia (ALL) for providing a valuable tool to facilitate the researches on PhALL.</p><p><b>METHODS</b>CML-like mice were generated by transfection to bone marrow cells of BABL/c with Mig190 retrovirus. The PhALL mouse model was established by infusion of sorted CML like mouse-derived BCR-ABLB cells into the mice of same linege. Immonophenotypes, BCR-ABL transcription and expression of these leukemic cells were detected by flow cytometry, RT-PCR and Western blot respectively.</p><p><b>RESULTS</b>CML-like presentation appeared in the mice after Mig190 virus transfection. After infusion with sorted BCR-ABLB cells, all the receipted mice eventually presented the clinical signs of acute leukemia. Flow cytometry analysis showed that these leukemic cells were positive for CD19; both RT-PCR and Western blot indicated that this mouse model is consistent with PhALL phenotypecally and molecalarlly.</p><p><b>CONCLUSION</b>A novel method to establish PhALL mouse model has been developed successfully, and this model can provide useful tool for the study of PhALL.</p>

Effect of BRD4 Inhibitor GSK525762A on Proliferation and Apoptosis of SUP-B15 Cells In Vitro and Its Possible Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of BRD4 inhibitor GSK525762A on the proliferation and apoptosis of Philadelphia chromosome-positive acute lymphoblastic leukemia SUP-B15 cells and its mechanism.</p><p><b>METHODS</b>SUP-B15 cells were treated with different concentration of GSK525762A, the proliferation-inhibition curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis were detected by flow cytometry with Annexin V and 7-AAD staining. The transcripts of anti-apoptotic genes C-MYC, CDK6 and BCL-2 were detected by real-time PCR.</p><p><b>RESULTS</b>GSK525762A could inhibit significantly SUP-B15 cell proliteration in dose-and time-dependent manner; GSK525762A treatment could induce apoptosis of SUP-B15 cells. The levels of C-MYC,CDK6 and BCL-2 mRNA transcripts in SUP-B15 cells were reduced in GSK525762A-treated group.</p><p><b>CONCLUSION</b>The GSK525762A can remarkably inhibit proliferation and induce apoptosis of SUP-B15 cells. The down-regulation of apoptosis-related genes C-MYC,CDK6 and BCL-2 may be involved in the process of apoptosis.</p>

Effects of PTPN11 on the Biological Characteristics of AML Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect the expression of PTPN11 gene in acute myeloid leukemia (AML) cell line and to explore the effects of PTPN11 over expressing on proliferation and apotosis of AML cell lines.</p><p><b>METHODS</b>The expression of PTPN11 in AML cell lines（HEL,U937, K562, KG-1, HL -60） was detected by RT-PCR, Q-PCR and Western blot. The PTPN11 gene was amplified by RT-PCR. PTPN11 DNA fragement and the lentiviral vector PCDH-CD513B were digested by BamHI and EcoRI, and then ligated by T4 DNA ligase. Recombinant lentivirus was generated by co-transfection of three-plasmids into 293FT cells using lipofectamine 2000. Then Q-PCR and Western blot were used to detect the expression of PTPN11 in the lentivirus infected HEL and U937 cells. The CCK-8 and Annexin V/7-AAD assays were performed to evaluate effects of PTPN11 on proliferation, apoptosis of HEL and U937 cells.</p><p><b>RESULTS</b>All 5 AML cell lines expressed the PTPN11 gene, restriction analysis and gene sequencing confirmed that recombinant lentiviral vector was successfully constructed. After transfection of cells with the lentivirus, the recombinant plasmid could stably up-regulate the expression of PTPN11. Analysis of the proliferation and apoptosis of transfected AML cells indicated that as compared with the control group, the OD values of over-expression group were significantly higher and the apoptotic rates were significantly lower (P<0.05).</p><p><b>CONCLUSION</b>PTPN11 is expressed in all the 5 AML cell lines. The lentiviral expression vector carrying human PTPN11 and the engineered HEL and U937 cell lines stably up-regulating PTPN11 gene expression are successfully obtained. Over-expression of PTPN11 promotes the proliferation of AML cell lines and inhibit then apoptosis.</p>

Effect of ADAM10 Inhibitor GI254023X on Proliferation and Apoptosis of Acute T-Lymphoblastic Leukemia Jurkat Cells In Vitro and Its Possible Mechanisms / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of ADAM10 inhibitor GI254023X on the proliferation and apoptosis of acute T-lymphoblastic leukemia Jurkat cells and its mechanisms.</p><p><b>METHODS</b>Jurkat cells were treated with different concentrations of GI254023X, the proliferation-inhibition curve was assayed and plotted by CCK-8 method, the cell viability and apoptosis was detected by flow cytometry with Annexin V and 7-AAD staining, the cleavage of Notch1 protein was determined by Western blot, the transcripts of anti-apoptotic genes BCL-2, MCL-1, BCL-xl and Notch1 target gene Hes-1 were detected by real-time PCR.</p><p><b>RESULTS</b>The GI254023X obviously inhibited the proliferation of Jurkat cells in concentration-dependent manner. As compared with the control group, the apoptosis of cells increased along with increment of GI254023X concentration. Compared with control group, the expression of Cleaved Notch1 was down-regulated while the expression of Notch1 was up-regulated in a time-dependent manner after the treatment with GI254023X. The levels of MCL-1 and Hes-1 mRNA transcripts in Jurkat cells were reduced in GI254023X treated group, but did not show obvious effect on the level of BCL-2 and BCL-xl mRNA transcripts.</p><p><b>CONCLUSION</b>GI254023X can remarkably inhibit proliferation and induce apoptosis of Jurkat cells. The inhibition of Notch1 activation and the down-regulation of apoptosis-related gene MCL-1 may be involved in the process of apoptosis.</p>

Effect of BRD4 inhibitor GSK525762A on proliferation and apoptosis of KU812 leukemic cells and its mechanism / 中国实验血液学杂志

This study was purposed to investigate the effect of bromodomain-containing protein 4 (BRD4) inhibitor GSK525762A on the proliferation and apoptosis of chronic myeloid leukemia blast crisis KU812 cells and its mechanism. KU812 cells were treated with different concentrations of GSK525762A (100, 250, 500, 1 000, 2 500 and 5000 nmol/L) and the inhibitory effects of drug on KU812 cell proliferation after 48 and 72 hours were detected by using CCK-8 assay. KU812 cells were treated with 3 different concentrations of GSK525762A (1.0, 2.5 and 5 µmol/L) and the cell apoptosis after 72 hours were assayed by using flow cytometry. KU812 cells were treated with DMSO and 2.5 µmol/L GSK525762A, and the mRNA levels of C-MYC, BCL-2, CDK6, BCL-xL, BAK and BAX were determined by using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results showed that GSK525762A could significantly inhibit the proliferation of KU812 cells and the inhibitory effect on KU812 cell proliferation was dependent on the dose-course and time-course of GSK525762A treatment. GSK525762A treatment could induce apoptosis of KU812 cells in a dose-dependent manner. After GSK525762A treatment, the mRNA levels of proliferation-promoting genes ( C-MYC and CDK6) and pro-survival genes ( BCL-2 and BCL-xL) decreased, while the transcription level of pro-apoptosis genes BAK and BAX increased, as compared to that of the control group. It is concluded that GSK525762A can inhibit the proliferation of KU812 cells and induce cell apoptosis possibly through depressing the transcription of C-MYC, BCL-2, CDK6 and BCL-xL gene, and down-regulating BAK and BAX transcription.

Establishment of mouse mesenchymal stem cells overexpressing CXCR4 gene and evaluation of their functions / 中国实验血液学杂志

This study was purposed to establish the mesenchymal stem cells (MSCs) stably overexpressing mouse CXC chemokine receptor type 4 (CXCR4) gene and to explore their function. The recombinant lentiviral vector LV-CXCR4-IRES-EGFP with packaging plasmid pSPAX2 and envelope plasmid pMD.2G were co-transfected into 293FT packaging cell line using lipofectamine 2000 to produce the recombinant lentiviral vectors. The recombinant viruses were harvested and concentrated by using ultracentrifugation. Mouse bone marrow MSC were infected with the viral supernatants. Variable methods were used to optimize the transduction condition. EGFP expression was visualized using fluorescence microscope and efficiency of infection was determined by flow cytometry (FCM). Proliferation and apoptosis were detected by proliferation curve and FCM, respectively. Migration capacity was assessed by a chemotaxis assay using transwell. Expression of EGFP were detected by fluorescence microscopy in MSCs after infection. The results showed that through optimization of infection condition, the recombination lentiviral vectors had higher infection efficacy; after infection for 72 h, the higher expression of EGFP could be observed under fluorescence microscope; the expression of CXCR4 protein on MSC surface in CXCR4-MSC group significantly increased compared with those in the control group. Meanwhile, over-expression of CXCR4 had no effect on their capacity of proliferation and did not induce apoptosis. Moreover, CXCR4 enhanced the migration of cells in the transwell induced by SDF-1 gradient compared with the EGFP control group. It is concluded that the lentiviral vector can not only infect mouse MSCs efficiently, but also can make CXCR4 express stably in MSC.

Construction of lentiviral vector carrying human OCT4A gene and establishment of leukemic cell line K562 stably expressing OCT4A / 中国实验血液学杂志

This study was purposed to construct a lentiviral vector carrying human OCT4A gene and green fluorescent protein (GFP) , and infect the leukemic cell line K562, observe the expression of OCT4A in K562 cells. According to the sequence of OCT4A mRNA which was found in GenBank, the special primer sequences were synthesized. The OCT4A gene was amplified by RT-PCR, and then cloned into the pCR-Blunt vector. The OCT4A DNA fragment was subcloned into the lentiviral vector pLVX-IRES-ZsGreen1 which was restricted by EcoR1 to generate a lentiviral vector pLVX-OCT4A-ZsGreen1. The sequence of the recombinant plasmid was identified by DNA sequencing. Recombinant lentivirus was generated by co-transfection of three-plasmids into 293FT cells using lipofectamine 2000 and transfected into K562 cells. Real-time PCR and Western blot were applied to detect the expression of OCT4A mRNA and protein, CCK-8 and colony formation assay were performed to evaluate the effects of OCT4A on proliferation of K562 cells. The results showed that the recombinant lentiviral vector pLVX-OCT4A-ZsGreen1 was successfully constructed. The virus titers were (1.43 ± 0.25) × 10(8) U/ml. After infection of K562 cells with the lentivirus, the recombinant plasmid could stably up-regulate the expression of OCT4A gene and protein according the real-time PCR and Western blot detection results. CCK-8 and colony formation assay showed that exogenous OCT4A gene could significantly promote cell growth and the colony formation of K562 cells. It is concluded that the recombinant lentiviral vector pLVX-OCT4A- ZsGreen1 carrying human OCT4A gene is successfully constructed; K562 cells which stably up-regulates the expression of OCT4A mRNA are obtained, the results of this study provide fundamental basis for further study on mechanism of OCT4A in human leukemia development.

Effect of AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside on proliferation, differentiation and apoptosis in U937 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) on proliferation, differentiation and apoptosis of U937 cells and explore its possible mechanism.</p><p><b>METHODS</b>U937 cells were cultured with different concentrations of AICAR for 24 h and 48 h. Cell proliferation was evaluated. Cell growth curve was analyzed by CCK-8; cell apoptosis was analyzed by cell morphology, Annexin V/7-AAD double labeling. The differentiation of U937 cells was evaluated by expression of CD11b. The Bcl-xL, Bax, Bim, caspase-3 mRNA expressions of U937 cells were determined by real time PCR.</p><p><b>RESULTS</b>AICAR significantly inhibited the growth of U937 cells in a time-and dose-dependent manner, with a 24 h IC50 value of 1.1 mmol/L and 48 h of 0.9 mmol/L. 1.0 mmol/L AICAR didn't induce differentiation of U937 cells with the increase of CD11b expression for 24 h (P > 0.05). The U937 cells apoptosis was confirmed by cell morphology and Annexin V/7-AAD labeling. AICAR induced apoptosis of U937 cells and the apoptosis rate was (6.81 ± 1.16)% at 1 mmol/L AICAR higher than control group (2.74 ± 0.32)% without AICAR for 24 h treatment (P < 0.05). The real time PCR assay revealed that as compared with control group, the expression of Bim and caspase-3 mRNA were increased, while Bcl-xL and Bax were unchanged on the AICAR treatment.</p><p><b>CONCLUSION</b>AICAR can effectively inhibit proliferation and induce apoptosis of U937 cells. However, it has no significant effect on differentiation of U937 cells. The mechanism may be related with up-regulating Bim and Caspase-3.</p>

Construction of shRNA expression vector targeting AATF and establishment of stably transfected U937 cells / 中国实验血液学杂志

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 homology, 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 constructed 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.

Construction of hybridoma cells with IL1RAP as a new marker for leukemia stem cells and detection of its monoclonal antibody / 中国实验血液学杂志

This study was aimed to prepare and identify human monoclonal antibody against IL-1 receptor accessory protein (IL1RAP), which is a new identified surface marker for leukemia stem cell (LSC), BALB/c mice were immunized with recombinant hu-IL1RAP and the spleen cells from immunized mice were fused with SP2/0 myeloma cells by conventional hybridoma technique. Positive hybridoma cells were selected and cultured. ELISA and Western blot were used to detect the type, titer and sensitivity of antibody. Peripheral blood mononuclear cells were isolated and used to test the antibody specificity. The results showed that 8 hybridoma cell lines able to stably secrete IL1RAP monoclonal antibodies were obtained and named 3H6E10, 4B6A6, 8G11B5, 9E9F2, 10D8A7, 1C7H7, 1D7G11 and 2D3D3 respectively. These monoclonal antibodies belonging to IgG1/κ type could specifically bind to IL1RAP from peripheral blood mononuclear cells. It is concluded that the hybridoma cell lines with stable secretion of IL1RAP monoclonal antibodies is successfully constructed, thus providing novel ways to effectively clear LSC in the future.

Source and effect of IL-22 in graft versus host disease mouse after allo-genetic bone marrow transplantation / 中国实验血液学杂志

This study was purposed to explore the cellular source of IL-22 in graft versus host disease (GVHD) mouse following allo-genetic 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.

Effect of interfering the β-catenin by lentiviral vector-mediated RNAi on the biological behavior of mouse bone marrow mesenchymal stem cells / 中国实验血液学杂志

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 plasmids and lentiviral packaging plasmids 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 capability 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 (interference 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 interference 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 interference group decreased significantly, while the migration capability of MSC in control group was not changed obviously. 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.

Cloning of mouse adam10 gene promoter and construction and identification of dual luciferase reporter system / 中国实验血液学杂志

This study was aimed to clone mouse adam10 gene promoter and construct its dual luciferase report vector, and to investigate its transcriptional activity. Total DNA was extracted from mouse brain and used for amplifying the fragment containing adam10 gene promoter by PCR. The amplified product was inserted into pGL-4.10 vector to construct pGL4.10-adam10. The pGL4.10-adam10 and control plasmid pGL4.74 were co-transfected into HEK293 FT cells by lipofectamine 2000. The activity of adam10 gene promoter was assayed by luciferase system. The results showed that the recombinant plasmid pGL4.10-adam10 containing promoter of mouse adam10 was correctly constructed. The method was optimized by changing ratio of two plasmids. Moreover, the transcriptional activity of pGL4.10-adam10 stimulated by ionomycin increased. It is concluded that the dual luciferase reporter system is successfully established, which is useful in bioluminescence imaging technology in vitro. The effect of ionomycin can enhance the transcriptional activity of adam10 gene promoter.