Effect of hypoxia inducible factor1-α inhibitor on reversal of multidrug resistance of K562/A02 cell line

Objective To study the reversal effect of the hypoxia inducible factor( HIF)-1α inhibitor,YC-1 ,on muitidrug resistance of K562/A02 cells and its mechanism. Methods Pre- and post- incubation with adriamycin (ADM) alone or in combination with YC-1 for 48 h, the proliferation capacity of K562/A02 and K562 cells were evaluated by MTT assay. The apoptosis rate of K562/A02 cells after treated with 0,5,10 and 20 μmol/L YC-1 alone or in combination with 1 mg/L ADM and intracellular ADM concentration were analyzed by flow cytometry(FCM). The mRNA levels of HIF-1α and mdr1 genes were determined by semi-quantitative RT-PCR. The protein levels of HIF-1α and P-glycoprotein (P-gp) were detected by Western blot. Results The IC50 of ADM for K562 and K562/A02 cells were ( 1.56 ± 0.07 ) mg/L and (42.98 ±3.15) mg/L respectively. The resistance of K562/A02 cells to ADM was 27.55- fold higher of that of K562cells. After treatment with YC-1 (5μmol/L, 10μmol/L, 20 μmol/L) for 48h, the resistances of K562/A02cells to ADM were 24.63-, 16.38- and 10.71- fold increase respectively. After treatment of K562/A02 cell with YC-1(0 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmoL/L) alone or in combination with 1 mg/L ADM for 48 h, the apoptotic rates were ( 1.9 ± 0. 9) %, (4.9 ± 0. 9 ) %, ( 5.8 ± 1.1 ) %, and ( 9.3 ± 1.4 ) % and(2.3 ± 0.7 ) %, (8.2 ± 1.2) %, ( 19.0 ± 1.7 ) %, and ( 34.5 ± 2.4 ) % respectively. The intracellular flucorescence intensity of ADM were 232 ±33, 1300 ±219, 1961 ±240 and 3342 ±269 in the combined treatment group. With the increase in YC-1 concentration, the levels of mdr1 mRNA reduced, while that ofHIF-1α mRNA had no obvious change.Furthermore.the expressions of HIF-1α and P-gp were also decreased in K562/A02 cells.Conclusion YC-1,as a HIF-1 inhibitor,cau reverse multidrug resistance of K562/A02cells through down-regulating HIF-1α and p-gp.     

低氧诱导因子-1α抑制剂逆转K562/A02细胞多药耐药机制研究

目的 探讨低氧诱导因子抑制剂3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazoh(YC-1)对人白血病耐阿霉素细胞K562/A02的耐药逆转作用,并探讨逆转机制.方法 采用MTT法检测不同浓度YC-1和阿霉素(ADM)单独或联合使用48 h对K562/A02细胞和K562细胞增值抑制效应及耐药逆转效应;用流式细胞术检测0、5、10和20 μmol/L YC-1单独或联合1 mg/L阿霉素作用K562/A02细胞48 h后细胞凋亡率及联合应用后细胞内阿霉素浓度;半定量RT-PCR检测各组细胞低氧诱导因子-1α(HIF-1α)、mdr1基因mRNA表达变化;Western blot方法检测各组细胞HIF-1α、P-糖蛋白(P-gp)表达变化.结果 K562与K562/A02细胞对阿霉素的IC50值分别为(1.56±0.07)mg/L和(42.98±3.15)mg/L,耐药倍数为27.55倍.予5、10和20μmol/L YC-1作用后,K562/A02细胞对阿霉素的耐药倍数分别为24.63、16.38和10.71倍;0、5、10和20μmol/L YC-1单独或联合1 mg/L阿霉素处理K562/A02细胞48 h后,凋亡率分别为(1.9±0.9)%、(4.9±0.9)%、(5.8±1.1)%和(9.3±1.4)%与(2.3±0.7)%、(8.2±1.2)%、(19.0±1.7)%和(34.5 ±2.4)%.0、5、10和20 μmol/L YC1联合1 mg/L阿霉素处理K562/A02细胞48 h后细胞内阿霉素荧光强度分别为232±33、1300±219、1961±240和3342±269;随着YC-1浓度增加,HIF-1α mRNA表达没有明显差异,mdr1 mRNA逐渐下调,HIF-1±和P-gp表达均下调.结论 YC-1可以通过抑制HIF-1α蛋白表达,下调mdr1 mRNA水平和P-gp水平,增加细胞内阿霉素药物浓度,部分逆转K562/A02细胞耐药.     

鼠尾草酸逆转K562/A02细胞多药耐药的机制研究

Objective To investigate the effects of carnosic acid(CA)on reversal of the muhidrug resistance(MDR)of human leukemia cell line K562/A02 and its mechanism.Methods MTT assay was used to determine the sensitivity of K562/A02 cells to adriamycin(ADM)pre-and post-treated with CA.Flow cytometry(FCM)and laser scanning confocal microscopy(LSCM)were used to measure intracellular fluorescence intensity and concentration of ADM respectively.The expression level of mdr1 was detected by semi-quantitative RT-PCR.P-glycoprotein(P-gp)expression was detected by FCM and Western blot.Resuits CA decreased,IC50 of ADM in K562/A02 cells from 16.31 μg/mL to 1.35μg/mL,being a 12.08fold decrease.The intracellular ADM fluorescence intensity of K562/A02 was increased from 17.05 t0 60.53after treated with CA(P<0.01).In living K562/A02 ceils,after treated with CA,the diffuse distribution of intracellular ADM was recovered in both nuclear and cytoplasm,and the concentration of intracellular ADM increased from 4.93μg/mL to 15.43μg/mL.RT-PCR assay showed that CA inhibited the expressions of mdr1 mRNA in K562/A02 cells(P<0.01).Mean fluorescence intensity of P-gp detected by FCM in CA-treated K562/A02 was decreased to 22.80 as compared with that in untreated K562/A02 cells(44.40,P<0.05).Conclusion CA can reverse the MDR of K562/A02 cells in vitro.The mechanism may be associated with down-regulation of mdr1 and inhibition of P-gp function.     

鼠尾草酸逆转K562/A02细胞多药耐药的机制研究

Objective To investigate the effects of carnosic acid(CA)on reversal of the muhidrug resistance(MDR)of human leukemia cell line K562/A02 and its mechanism.Methods MTT assay was used to determine the sensitivity of K562/A02 cells to adriamycin(ADM)pre-and post-treated with CA.Flow cytometry(FCM)and laser scanning confocal microscopy(LSCM)were used to measure intracellular fluorescence intensity and concentration of ADM respectively.The expression level of mdr1 was detected by semi-quantitative RT-PCR.P-glycoprotein(P-gp)expression was detected by FCM and Western blot.Resuits CA decreased,IC50 of ADM in K562/A02 cells from 16.31 μg/mL to 1.35μg/mL,being a 12.08fold decrease.The intracellular ADM fluorescence intensity of K562/A02 was increased from 17.05 t0 60.53after treated with CA(P<0.01).In living K562/A02 ceils,after treated with CA,the diffuse distribution of intracellular ADM was recovered in both nuclear and cytoplasm,and the concentration of intracellular ADM increased from 4.93μg/mL to 15.43μg/mL.RT-PCR assay showed that CA inhibited the expressions of mdr1 mRNA in K562/A02 cells(P<0.01).Mean fluorescence intensity of P-gp detected by FCM in CA-treated K562/A02 was decreased to 22.80 as compared with that in untreated K562/A02 cells(44.40,P<0.05).Conclusion CA can reverse the MDR of K562/A02 cells in vitro.The mechanism may be associated with down-regulation of mdr1 and inhibition of P-gp function.     

尼洛替尼联合5-溴汉防己甲素逆转K562/A02细胞耐药的研究

Objective To investigate the reversible effect of nilotinib,BrTet(5-bromotetrandrine)and their combination on multidrug resistance cell line K562/A02 and its mechanism.Methods Cell proliferation inhibition wag assessed by MTT method and cell apoptosis by flow cytometry(FCM).The expression of mdr1 mRNA was determined by RT-PCR,and the expression of P-gp was assessed by Western blot.Results After 48 h 5 nmol/L nilotinib or 0.5μmol/L BrTet treatment,IC50 of daunorubicin(DNR)to K562/A02 was 4.52 mg/L or 5.41 mg/L respectively;While on combinative treatment,its IC50 decreased to 2.98mg/L.Nilotinib or BrTer alone was not able to increase the DNR induced apoptosis rate of K562/A02 cell (P>0.05),while on combination treatment the apoptosis rate increased remarkably.After 48 h 5 nmol/Lnilotinib or 0.5 μmol/L BrTet treatment alone,gray-scale value of mdr1 mRNA was 0.48±0.04 or 0.64±0.01,respectively;while on combinative treatment the value decreased to 0.35±0.04.The P-gp expression level in K562/A02 cells was 0.61±0.05.or 0.52±0.02 when treated with 5 nmol/L nilotinib or 0.5 μmol/L BrTet alone for 48 h.but on combination treatment,the level decreased to 0.44±0.03.Conclusion Nilotinib or BrTet alone can partially reverse drug resistance of K562/A02 cells.The mechanism may be associated with the decrease of mdr1 mRNA and P-gp expression and increase of the apoptosis rate.And there is a synergistic action with these two agants in combination.     

尼洛替尼联合5-溴汉防己甲素逆转K562/A02细胞耐药的研究

Objective To investigate the reversible effect of nilotinib,BrTet(5-bromotetrandrine)and their combination on multidrug resistance cell line K562/A02 and its mechanism.Methods Cell proliferation inhibition wag assessed by MTT method and cell apoptosis by flow cytometry(FCM).The expression of mdr1 mRNA was determined by RT-PCR,and the expression of P-gp was assessed by Western blot.Results After 48 h 5 nmol/L nilotinib or 0.5μmol/L BrTet treatment,IC50 of daunorubicin(DNR)to K562/A02 was 4.52 mg/L or 5.41 mg/L respectively;While on combinative treatment,its IC50 decreased to 2.98mg/L.Nilotinib or BrTer alone was not able to increase the DNR induced apoptosis rate of K562/A02 cell (P>0.05),while on combination treatment the apoptosis rate increased remarkably.After 48 h 5 nmol/Lnilotinib or 0.5 μmol/L BrTet treatment alone,gray-scale value of mdr1 mRNA was 0.48±0.04 or 0.64±0.01,respectively;while on combinative treatment the value decreased to 0.35±0.04.The P-gp expression level in K562/A02 cells was 0.61±0.05.or 0.52±0.02 when treated with 5 nmol/L nilotinib or 0.5 μmol/L BrTet alone for 48 h.but on combination treatment,the level decreased to 0.44±0.03.Conclusion Nilotinib or BrTet alone can partially reverse drug resistance of K562/A02 cells.The mechanism may be associated with the decrease of mdr1 mRNA and P-gp expression and increase of the apoptosis rate.And there is a synergistic action with these two agants in combination.     

HO-1基因表达对伊马替尼耐药的慢性髓系白血病细胞增殖的影响

目的 通过氯高血红素(Hemin)诱导伊马替尼耐药慢性髓系白血病(CML)细胞株K562/A02-IM 中血红素加氧酶-1(HO-1)基因表达,探讨HO-1基因对伊马替尼耐药CML细胞增殖的影响,为治疗CML多药耐药及新药的开发提供思路和实验依据.方法 采用RT-PCR法检测20例CML伊马替尼耐药患者骨髓细胞中HO-1基因的表达,半定量RT-PCR法和Western blot法分别检测不同剂量Hemin处理K562/A02-IM细胞不同时间后HO-1的表达,通过Annexin V/PI双染色法检测细胞凋亡情况,采用MTT法检测Hemin诱导及锌原卟啉抑制HO-1表达与细胞存活率的关系.结果 RT-PCR结果显示耐药患者骨髓细胞中HO-1基因阳性表达,半定量RT-PCR和Western blot法显示,不同浓度的Hemin(0、10、20及40 μmol/L)处理K562/A02-IM细胞16 h后,HO-1的表达量随Hemin浓度的升高而增加,存在剂量依赖关系,而20 μmol/L Hemin分别处理K562/A02-IM细胞0、8、16、24 h后,HO-1的表达肇在处理16 h组最高.Annexin V/PI法检测显示,0、10、20及40 μmol/L Hemin作用于K562/A02-IM细胞16 h后细胞凋亡率分别为(17.61±0.01)%、(12.13±0.11)%、(7.94±0.03)%和(4.62±0.15)%,其抗凋亡的作用呈剂量依赖性;20 μmol/L Hemin作用K562/A02-IM细胞8、16及24 h的细胞凋亡率分别为(14.72±0.05)%、(8.15±0.07)%和(16.37±0.13)%.MTT法检测显示与对照组相比,Hemin诱导K562/A02-IM细胞HO-1基因表达促进了细胞的增殖,且作用存在剂量依赖关系;而锌原卟啉抑制HO-1的表达,促进了细胞的凋亡(P<0.05).结论 CML伊马替尼耐药患者骨髓细胞HO-1基因阳性表达,HO-1 是一种可诱导表达型基因,具有抗细胞凋亡及促进细胞增殖的作用,抑制HO-1 表达可能成为治疗CML耐药的新方法.

Abstract：

Objective To investigate the effect of heme oxygenase-1 (HO-1 ) expression on cell growth and apoptosis in imatinib resistant chronic myeloid leukemia (CML) cells ( K562/A02-IM) , and explore the relationship between HO-1 gene and CML. Methods The expression of HO-1 in 20 drug-resistant CML patients was detected by RT-PCR. Different concentrations of hemin were used to induce HO-1 expression of K562/A02-IM, HO-1 expression at different time was detected by RT-PCR and Western blot analysis. Cell apoptosis was detected by Annexin V/PI staining, and MTT assay was used to detect viability of K562/ A02-IM cells after induction or inhibition of HO-1 gene by hemin and zinc protoporphyrin (ZPP). Results RT-PCR showed that HO-1 was expressed in the bone marrow mononuclear cells (BMMNCs). When treated with hemin at different concentrations (0, 10, 20, 40 μmol/L) for 16 h, the expression of HO-1 in K562/ A02-IM was increased in a dose-dependent manner, and peaked at 20 μmol/L of hemin for 16 h. The apoptosis rates were (17.61 ±0.01)%, (12. 13 ±0.11)%, (7.94 ±0.03)% and (4.62 ±0. 15)% at 0,10, 20 and 40 μmol/L of hemin respectively for 16 h and were (14. 7 ± 0.05) % , (8. 1 ± 0. 07) % and (16. 3 ± 0. 13)% at 20 μmol/L of hemin treatment for 8,16, and 24 h respectively. Hemin induced apoptosis of K562/A02-IM cells in a dose-dependent manner. The expression of HO-1 was induced in K562/A02-IM cells in a dose-dependent manner, and the survival of K562/A02-IM cells was significantly increased as compared to that of control group. When HO-1 was inhibited by ZPP, the cells survival was sharply decreased compared to that of the control group (P<0.05). Conclusion HO-1 was expressed in the BMMNCs. It is a kind of molecules whose expression can be induced and can promote the growth of drug-resistant cells. Inhibition of HO-1 expression probably be used for the treatment of drug-resistant CML.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

伴有阻塞性睡眠呼吸暂停综合征的急性脑梗死患者血浆溶血磷脂含量的测定

Objective To observe the changing characteristics of plasma lysophosphatidic acid (LPA) or acidia phospholipid (AP) levels in patients with obstructive sleep apnea syndrome-associated(OSAS)acute cerebral infarction and to explore the pathophysiological mechanisms of OSAS-related stroke so as to provide basis for clinical antithrombotic therapy. Methods Thirty-six patients of OSAS, 32 patients of OSAS-related acute stoke and 36 patients of acute stoke without OSAS diagnosed by clinical and accessory examinations were enrolled in the current study. Thirty-eight age-matched healthy subjects were recruited as controls. The changes of the plasma LPA and AP levels were measured. Results Within 24 hours after symptom onset, the plasma LPA and AP levels in the OSAS-related acute cerebral infarction group (LPA(3. 78 ±0. 56) μmol/L; AP(7. 63 ± 1. 38) μmol/L) were significantly higher than those in the OSAS group(LPA(3. 17 ±0. 65) μmol/L; AP(6. 60 ± 1. 20) μmol/L) ,the not OSAS-related acute cerebral infarction group (LPA (3. 40 ± 0. 59)μmol/L; AP (6. 41 ± 1. 37)μmol/L) and the control group (LPA(2.76±0.45)μmol/L;AP(4.52±0. 83) μmol/L (P < 0. 01)) . The levels of LPA and AP in the OSAS group and the not OSAS-related acute cerebral infarction group were significantly higher than those in the control group(P<0. 01). Seven days after symptom onset, the plasma LPA and AP levels in the OSAS-associated acute cerebral infarction group (LPA(3.08 ± 0. 58) μmol/L; AP(6. 15 ±1. 14)μmol/L) were still higher(P < 0. 01) . The plasma LPA levels were not significantly different among the OSAS-related acute cerebral infarction group, the not OSAS-related acute cerebral infarction group and the control group 21 days after symptom onset, whereas the plasma AP levels in the OSAS-related acute cerebral infarction group (5. 04 ± 0. 83) μmol/L were still significantly higher than those in the not OSAS-related acute cerebral infarction group (4. 57 ± 0. 94) μmol/L and the control group (P < 0.05). Conclusions The significantly elevated plasma LPA and AP levels in patients with OSAS suggested that platelets in vivo are in an activated state and in cerebral ischemia and hypoxia state, especially for the OSAS-related acute cerebral infarction patients. The activated state of platelet may persist for a long time, thus the time window for antithrombotic therapy may be longer.