Silencing Calreticulin Expression Inhibits Invasion Ability of SNK6 Cells in Vitro via Down-Regulating Expression of VEGF and MMP2/9 / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of steadily down-regulating the expression of calreticulin (CALR) on the invasion of natural killer/T-cell lymphoma SNK6 cells, and explore its possible mechanism.@*METHODS@#The sequences of specific short hairpin RNA (shRNA) targeting on human CALR were designed, and were inserted into pLKO.1-puro lentivirus vector, and the reconbinant lentivirus vector was obtained; the lentivirus particles were backed by three-plasmid system and transfected into SNK6 cells, the SNK6 cells stably down-regulating the CALR expression were sercened by puromytain, the CALR-silencing effect was verified by real-time PCR and Western blot. CCK-8 assay was used to evaluate the cell viability, The transwell invasion assays was used to analyse invasion of SNK6 cells. The mRNA expression of Calreticulin, MMP2, MMP9 and VEGF was determined by real time PCR, the protein expression of Calreticulin and GAPDH was analyzed by Western blot.@*RESULTS@#The recombinant lentiviral vector pLKO.1-puro-shCALR was successfully constructed, packed into the lentivirus, then the SNK6 cells stably down-regulating Calreticulin expression was obtained. When Calreticulin was down-rengulated in SNK6 cells, the proliferation rate was reduced and the invasion ability was decreased; the mRNA levels of VEGF and MMP-2/9 also were reduced.@*CONCLUSION@#The stable down-regnlation of CALR expression in SNK6 cells can attenuate the imvasiveness of SNK6 cells, which maybe related with transcriptional decrease of MMP2, MMP9 and VEGF.

Clinical results of multi-artery-graft and single-artery-graft off-pump coronary artery bypass grafting: a propensity score matching follow-up study / 上海交通大学学报（医学版）

Objective·To explore the difference between multi-artery-graft off-pump coronary artery bypass grafting (OPCABG) and single-artery-graft OPCABG on left main coronary artery or multivessel disease with propensity score matching. Methods·A total of 1578 patients with left main coronary artery or multivessel disease underwent isolated OPCABG were selected in Ruijin Hospital from January 2012 to September 2016. The propensity score methodology was used to obtain risk-adjusted outcome. Kaplan-Meier analysis was applied for estimation of freedom from major adverse cardiac and cerebrovascular events (MACCE) and readmission for heart disease. Independent predictor of MACCE were assessed by Cox regression analysis. Results·The average follow-up time was 28 months (7-55 months). There was no statistical difference in short-term clinical endpoints in hospital. In the follow-up results, multi-artery-graft OPCABG patients had statistical improvement in readmission for heart disease (2.7％ vs 12.7％, P=0.023), CCS class (1.2±0.4 vs 1.4±0.6, P=0.020) and patency rate of grafts in 1 year after operation (95.8％ vs 85.9％, P=0.025), compared with single-artery-graft OPCABG. There was no statistical difference in other endpoints. There was statistical improvement for multi-artery-graft OPCABG patients in freedom from readmission for heart disease (P=0.028). Female was an independent predictor of MACCE (95％ CI 0.117-0.906, P=0.032). Conclusion·Multi-artery-graft OPCABG appears to be safe and with good patency of grafts and clinical outcomes in treating left main coronary artery or multivessel disease. The follow up of female patients should be paid more attention.

Comparison of Laboratory Indexes and Therapeutic Efficacy between Two Kinds of Different RBC Transfusion in AIHA Patients / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of 2 kinds of red blood cells (RBC) on the laboratorial indexes and therapeutic efficacy of patients with autoimmune hemolytic anemia (AIHA).</p><p><b>METHODS</b>The clinical data of 120 patients with AIHA from June 2015 to June 2016 were analyzed retrospectively. These 120 patients were divided into A goup and B group. The patients in A group (60 cases) were infused with washed RBC, while the patients in B group (60 cases) were infused with WBC-deplated RBC. The changes of laboratotial indexes, clinical symptoms and incidence of adverse reactions in 2 groups before treatment and at 24 hourse after treatment as well as the therapeutic efficacy and improvement status of clinical symptoms were compared.</p><p><b>RESULTS</b>The RBC count and Hb level in 2 groups after treating for 1 day were significantly higher than those before treatment (P<0.01), while the TBIL level and reticulocyte (Ret) count were significantly lower than those before treatment (P<0.01). However, the RBC, Hb, TBIL levels and Ret count in 2 groups before and after treatment showed no statistical difference (P>0.05); the improvement of clinical symptoms, complex therapeutic efficacy and incidence of adverse reactions in 2 groups after treatment also showed no significantly difference (P>0.05).</p><p><b>CONCLUSION</b>Both washed and leukocyte-deplated RBC can alleviate the anemia in patients with AIHA in short-time, but the leucocyte-deplated RBC is more expensive, suggesting that the wased RBC is more practical for treatment of AIHA patients.</p>

Effects of mTOR Inhibitor Rapamycin on Burkitt's Lymphoma Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the effects of mTOR inhibitor rapamycin on proliferation, cell cycle and apoptosis of Burkitt's lymphoma cell line Raji and CA46 cells and its mechanism, so as to provide the experimental evidence for a therapeutic target of Burkitt's lymphoma.</p><p><b>METHODS</b>3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was performed to assess the inhibitory effect of rapamycin on proliferation of Burkitt's lymphoma cell line Raji and CA46 cells. The cell cycle distribution of Raji and CA46 cells was analyzed by flow cytometry with propidium iodide(PI) single staining. The cell apoptosis of Raji and CA46 cells was analyzed by flow cytometry with FITC Annexin V+PI double staining. The expressions of RPS6, p-RPS6, survivin and caspase-3 proteins were detected by Western blot after treating with rapamycin.</p><p><b>RESULTS</b>Rapamycin markedly inhibited the proliferation of both Raji and CA46 cells in a time- and concentration-dependent manners, showing good biological activity, the cell proliferation inhibition rate reached about 20% after treatment with 1 nmol/L rapamycin. After treatment with different concentrations of rapamycin for 24 and 48 hours, the proportion of both cells in G/Gphase in the treated groups was significantly increased in a time- and concentration-dependent manners in comparison with the solvent control group. With regard to the cells in S and G/M phase, the decreased population was accompanied by the increase of G/Gphase cells. After treatment with 100 nmol/L rapamycin for 48 hours, both Raji and CA46 cells demonstrated an apparent apoptosis,especially late apoptosis by flow cytometry with Annexin V+PI staining. After treatment with rapamycin, the expression of p-RPS6 and survivin of Raji and CA46 cells was obviously down-regulated, the expression of caspase-3 was obviously up-regulated in a time- and dose-dependent manners. However, rapamycin did not obviously affect the expression of RPS6.</p><p><b>CONCLUSION</b>The rapamycin can effectively inhibit cell proliferation, arrest Raji and CA46 cells in G/Gphase, and this effect associates with inhibiting the activation of mTOR/RPS6 signal pathway through down-regulating the expression of phosphorylated RPS6, i.e. mTOR downstream signal pathway. It also can induce apoptosis by down-regulating the expression of anti-apoptotic protein survivin and activating the intrinsic pro-apoptotic protein caspase-3.</p>

Lethal effect of cytotoxic lymphocytes against U266 cells induced by DCs modified with GM-CSF gene and pulsed with tumour antigen / 中国实验血液学杂志

The purpose of this study was to investigate the lethal effect of cytotoxic lymphocytes against U266 cells induced by DCs pulsed with multiple myeloma (MM) U266 lysate and transfected with GM-CSF recombinant adenovirus. The cytotoxic lymphocytes against U266 cells were induced by culturing with DCs, which pulsed with MM U266 antigens and transfected with GM-CSF recombinant adenovirus. The effect of cytotoxic lymphocytes against U266 cells were measured by LDH release detection. Experiments were divided into 3 groups: N-DC group as control in which DCs were normal; U-DC group in which DCs were pulsed by U266 soluble antigen, and G-U-DC group in which DCs were stimulated by U266 soluble antigen and GM-CSF transfected with Ad-CMV. The results showed that there was significant difference on killing rate against U266 cells between 3 groups (F = 10.939, p < 0.05). The killing rate of G-U-DC group was the highest (p < 0.001), and killing rate of U-DC group was higher than that of N-DC group (p < 0.001). It is concluded that the cytotoxic lymphocytes against U266 cells can be induced by DCs pulsed with U266 lysate, and the lethal effect of CTLs can be enhanced when DCs transfected by recombinant adenovirus with exogenous gene GM-CSF.

In vitro inducing effect of dendritic cells cotransfected with survivin and granulocyte-macrophage colony-stimulating factor on cytotoxic T cell to kill leukemic cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Survivin is a rather specific gene in tumor tissue. We transfected dendritic cells (DCs) with recombinant adenovirus (Ad) containing survivin gene and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and tested the inducing effect of the transfected DCs on cytotoxic T lymphocytes (CTL) to kill leukemic cells.</p><p><b>METHODS</b>After derived from the peripheral, DCs was assayed by mixed leukocyte reaction (MLR) tests. Lactate dehydrogenase (LDH) release test was used to evaluate cytotoxicity of CTL.</p><p><b>RESULTS</b>Expression of survivin in transfected DCs was confirmed by Western blotting analysis. GM-CSF expression was confirmed by enzyme-linked immunosorbent assay (ELISA). In MLR assay, DCs coinfected with Ad-survivin and Ad-GM-CSF induced higher allogeneic lymphocyte reaction than control DCs at ratios of 1:5, 1:10, 1:50 and 1:100. DCs coinfected with Ad-survivin and Ad-GM-CSF had much higher activity of CTL to HL-60 cells than DCs infected with Ad-survivin only, Ad-GM-CSF only, or control DCs. Levels of interleukin-12 (IL-12) and interferon gamma (IFN-gamma) in lymphocyte supernatants containing DCs coinfected with Ad-survivin and Ad-GM-CSF were significantly higher than those in the control group.</p><p><b>CONCLUSION</b>DCs coinfected with Ad-survivin and Ad-GM-CSF induce much higher anti-leukemic response in vitro than those infected with either factor. Therefore, adenovirus vectors containing survivin and GM-CSF genes may be promising vaccine candidates for leukemia therapy.</p>

Induction of anti-lymphoma cytotoxic T cell effect by dendritic cells transfected with recombinant adenovirus vectors carrying survivin gene / 中国实验血液学杂志

This study was purposed to investigate the immunological effects of modified dendritic cells (DCs) in inducing cytotoxic T cells (CTLs) effect against lymphoma cells. The DCs were derived from human peripheral blood and transfected with recombined adenovirus vector carrying survivin gene, Western blot was used to detect the expression of survivin, the lactate dehydrogenase (LDH) release test was used to determine the cytotoxicity of CTLs, the mixed lymphocyte reaction (MLR) was used to measure the ability to proleferate allo-lymphocyte by DCs, ELISA was used to assay IL-12 level in supernatant. The results showed that the expression of survivin in transfected dentritic cells was confirmed by Western blot analysis. In MLR assay, DCs transfected with Ad-survivin could induce higher allogeneic lymphocytes reaction at the ratios of 1:5, 1:10, 1:50 and 1:100. DCs transfected with Ad-survivin had much higher activity of CTL to CA46 cells than control DCs. The levels of IL-12 of supernatants containing DCs transfected with Ad-survivin were significantly higher than that in the control group. It is concluded that DCs transfected with Ad-survivin can induce CTL response in vitro against lymphoma cells.

Construction of recombinant adenovirus vector containing human survivin gene and its expression in dendritic cells / 中国实验血液学杂志

The study was aimed to construct the recombinant adenovirus vectors containing human survivin gene, and to investigate their expression in transfected dendritic cells. Full length cDNA encoding survivin was obtained by PCR amplification from plasmid pcDNA3.0-survivin. The PCR product was restricted, and then inserted into pShuttle-CMV. The plasmids of pShuttle-CMV-survivin were linearized with PmeI, and the fragment containing survivin was ligated with pShuttle-CMV and transfected into E. coli BJ5183. After homologous recombination in bacteria, the extracted plasmid from the positive bacteria were linearized with PacI, transfected into HEK293 cells with liposome Lipofectamine 2000. Then, the harvested adenovirus supernatants were transfected into dendritic cells. The results showed that the recombinant adenovirus-survivin was constructed successfully and its titer was about 2.65 x 10(9) pfu/ml. The expression of survivin in transfected dendritic cells was confirmed by Western blot analysis. It is concluded that the recombinant adenovirus vector containing human survivin was constructed successfully, which may provide preliminary laboratory evidence for anti-leukemia immunotherapy.