多发性骨髓瘤骨髓微环境中脑源性神经营养因子促进血管新生的作用及调控机制的研究
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摘要
第一部分慢病毒shRNA干扰骨髓瘤细胞表达BDNF抑制体内肿瘤生长和局部血管新生
目的通过慢病毒shRNA载体干扰骨髓瘤细胞BDNF的表达,建立人骨髓瘤裸鼠模型,在体内实验中研究BDNF对骨髓瘤肿瘤生长和血管新生的作用
方法构建慢病毒载体pGCSIL-eGFP-shBDNF并转染至骨髓瘤RPMI 8226细胞,流式细胞分选术筛选eGFP阳性稳定转染BDNFshRNA的骨髓瘤细胞,Western blot验证shRNA对BDNF蛋白表达的干扰作用；分离培养正常人骨髓间充质细胞,流式细胞术鉴定其表面分子；诱导BMSCs向成骨细胞、成脂肪细胞和成软骨细胞分化,通过茜素红染色、油红O染色及阿辛蓝染色鉴定其多能定向分化的能力。将稳定转染BDNFshRNA/Control shRNA的RPMI 8226细胞单独或与骨髓间充质细胞混合后接种裸鼠皮下,建立骨髓瘤移植瘤老鼠模型。活体荧光显微镜下连续监测骨髓瘤细胞的体内生长,观察肿瘤大小,总体存活时间,通过免疫组化染色CD34计算肿瘤微血管密度(MVD),Western blot检测肿瘤组织BDNF、VEGF的蛋白表达。
结果转染慢病毒载体的RPMI 8226细胞经流式分选后,表达eGFP的BDNF shRNA组和control shRNA组转染细胞纯度分别达91.87%和90.50%,Western Blot结果证实转染BDNF shRNA的RPMI 8226细胞BDNF蛋白表达显著抑制；9名健康人来源的BMSCs均表达CD44、CD105和CD29,但不表达CD14,CD45,并且BMSCs在不同条件下经诱导能向成骨细胞、成脂肪细胞以及成软骨细胞定向分化；人骨髓瘤裸鼠模型中,BMSCs能促进骨髓瘤细胞在体内生长,加速肿瘤形成；对于RPMI 8226细胞移植瘤模型,shBDNF RPMI 8226组较Control RPMI 8226组移植瘤平均体积(578.10±138.27 mm3 vs 1100.01±132.38 mm3,P<0.001)降低,并伴随局部微血管密度减少(6.40±3.81/HRP vs 12.73±5.89/HRP,P<0.001)以及总体生存时间延长；同样,对于骨髓瘤-间充质细胞混合移植瘤,ShBDNF RPMI 8226/BMSCs组较Control RPMI8226/BMSCs组肿瘤平均体积(1076.51±161.00mm3 vs 1674.49±174.71 mm3,P<0.001)亦降低。微血管密度(22.13±8.56/HRP vs 30.26±12.53/HRP,P<0.001)亦减少并且总体生存时间延长；Western Blot证实干扰骨髓瘤BDNF的表达能降低肿瘤组织VEGF的分泌。
结论骨髓瘤分泌的BDNF在体内骨髓瘤的生长及局部血管新生过程中起重要作用,可能成为多发性骨髓瘤治疗的新靶点。
第二部分骨髓微环境中骨髓瘤细胞分泌的BDNF促进骨髓间充质细胞分泌VEGF
目的体外实验探讨BDNF/TrkB途径在骨髓微环境中对多发性骨髓瘤血管新生的作用。
方法分离培养正常人原代骨髓间充质细胞,在体外应用BDNF作用于正常人骨髓间充质细胞,或将稳定转染BDNF/Control shRNA的RPMI 8226细胞与正常人骨髓间充质细胞在transwell小室中培养,ELISA法检测BDNF对BMSCs分泌VEGF的影响,Western blot法分别检测BMSC细胞TrkB、STAT3、p-STAT3的蛋白表达量,凝胶电泳迁移实验(EMSA)检测核因子AP-1的DNA结合活性。
结果BDNF能促进BMSCs表达TrkB；沉默BDNF表达的RPMI 8226细胞与BMSCs共培养时,BMSCs表达的TrkB蛋白水平较对照组降低；BDNF以时间和剂量依赖性的方式促进BMSCs分泌VEGF这种作用能被TrkB抑制剂K252α所阻断；BMSCs与RPMI 8226细胞共培养能促进VEGF的分泌,共培养上清的VEGF水平是BMSCs及RPMI 8226细胞单独培养上清中VEGF水平之和的3.1倍,K252α亦能部分抑制这种作用,而当BMSCs与shBDNF RPMI 8226细胞共培养时,VEGF水平降低36.89%。BDNF能促进BMSCs内AP-1的DNA结合活性和STAT3的磷酸化水平,而阻断STAT3途径能明显下调BDNF对BMSCs分泌VEGF的促进作用。
结论骨髓瘤-间充质细胞共培养系统中,骨髓瘤细胞分泌的BDNF是通过活化受体TrkB促进骨髓基质细胞内STAT3的磷酸化,从而上调促血管新生因子VEGF的分泌。
第三部分脑源性神经营养因子-促进多发性骨髓瘤细胞分泌基质金属蛋白-9
目的探讨脑源性神经营养因子(BDNF)通过核转录因子NF-κB诱导多发性骨髓瘤(MM)细胞分泌基质金属蛋白酶(MMP)-9的分子机制。
方法培养人MM细胞株RPMI 8226,使用明胶酶谱法检测RPMI 8226细胞分泌的活性MMP-2、MMP-9水平的改变,用化学发光凝胶电泳迁移率分析(EMSA)法检测RPMI 8226细胞中NF-κB活化水平的改变。
结果以25、50、100和200μg/L浓度的BDNF刺激RPMI 8226细胞24小时后,其活性MMP-9的分泌水平分别为2.03±0.48、2.99±0.46、4.63±0.62和5.62±1.29gμg/L,均明显高于对照组(P<0.01),而MMP-2的表达在各浓度组之间的差异无统计学意义(P>0.01)。当BDNF作用浓度为100μg/L时,预先分别用1mmol/L的NF-κB抑制剂四氢化吡咯二硫代氨基甲酸酯(PDTC)和500nmol/L的BDNF高亲和力受体TrkB的酪氨酸抑制剂K252a处理15min和1h后,K252α和PDTC(P<0.001)均能显著抑制RPMI 8226细胞MMP-9的分泌(光密度值分别为867.52±101.81、727.98±92.05,对照组为1159.01±233.15,与对照组比,P值均小于0.01)。随着BDNF浓度的升高,NF-κB的活化作用明显增强,于BDNF浓度为100gμg/L时加K252a预处理1h,6h,12h,24h的3个时间点均观察到K252α能明显抑制BDNF对NF-κB的活化作用(P<0.05),且随着BDNF作用时间的延长而增强。
结论BDNF能促进MM细胞血管新生的能力可能是通过NF-κB信号转导途径激活MMP-9而实现的。
PRATⅠ
Silencing of BDNF expression by shRNA inhibits in vivo multiple myeloma growth and angiogenesis in the bone marrow milieu
Objective To evaluate the effect of MM derived BDNF on tumor growth and angiogenesis in human multiple myeloma xenograft animal model by using lentiviral based shRNA targeting BDNF expression.
Methods RPMI 8226 cells were transfected with lentiviral based shBDNF-pGCSIL-eGFP or control vector. Pure populations of transfected RPMI 8226 cell were isolated via a flow cytometry, and specific BDNF knockdown was verified by western blot. BM stromal cells (BMSCs) were isolated from health donors and their phenotypes were identified with flow cytometer;Confirmation of differentiation of the BMSCs to adipocytes, osteocytes and chondrocytes were performed by staining with alizarin red, oil red O and alcine blue respectively. BDNF shRNA RPMI 8226 cells were inoculated subcutaneously with or without BMSCs in the hind neck of irradiated nude mice; Tumor volume was measured by in-vivo optical imaging and capliper; Microvessel density (MVD) was assessed by immunohistochemical analysis for CD34 expression; BDNF and VEGF expression in tumor were analyzed by western blot.
Results The purity of transfectants was determined by flow cytometric analysis of enhanced GFP reporter protein, the selected BDNF shRNA and control shRNA RPMI-8226 cells could reach up to 91.87% and 90.50% when compared with parental RPMI-8226 cells. The silencing capacity of BDNF shRNA was confirmed by western blot;BMSCs, isolated from nine healthy donors, expressed CD44、CD 105 and CD29, but not CD14,CD45 and characterized for adipocytes, osteocytes and chondrocytes differentiation capacity; In human myeloma xenograft model, compared with control group, shBDNF RPMI 8226 group has decreased tumor volume(578.10±138.27 mm3 vs 1100.01±132.38 mm3, P <0.001)and microvessel density(6.40±3.81/HRP vs 12.73±5.89/HRP,P<0.001),and prolonged overall survival; Moreover, the shBDNF RPMI 8226/BMSCs group has dcreased tumor volume(1076.51±161.00mm3 vs 1674.49±174.71 mm3,P<0.001)and microvessel density(22.13±8.56/HRP vs 30.26±12.53/HRP,P<0.001),and prolonged overall survival as well.It also demonstrated that silencing of MM-derived BDNF expression significantly inhibited VEGF expression by tumor immunohistochemistry. Conclusion:Our studies demonstrate that BDNF, as a potential stimulator of angiogenesis, contributes to MM tumorgenesis and may be a new target for MM therapy.
PRAT II
MM derived BDNF promotes stroma-derived VEGF secretion in the bone marrow milieu
Objective To evaluate the involvement of BDNF/TrkB pathway in MM-BMSCs interaction that mediates bone marrow angiogenesis in MM.
Methods BMSCs were isolated from 9 healthy donors,and RPMI 8226 cells were transfected with lentiviral based BDNF/Control shRNA vector. An indirecte co-culture system between RPMI 8226 MM cells and BMSCs was established by using a 0.4μm transwell inserts.ELISA was used to evaluate the effect of BDNF on VEGF secretion from BMSCs. TrkB、STAT3 and p-STAT3 in BMSCs were detected by western blot and DNA-binding of AP-1 was detected by Electrophoretic mobility shift assay (EMSA).
Results TrkB was consistently expressed by represent BMSC cultures and induced by BDNF stimulation, whereas TrkB expression was inhibited when BMSCs were co-cultured with BDNF knockdown MM cells compared to control group.Stimulation of BMSCs with exogenous BDNF induced a time-and dose-dependent increase in VEGF secretion, which was completely abolished by K252a, an inhibitor of TrkB.When RPMI-8226 cells were cultured with BMSCs, VEGF secretion was up-regulated 3.1-fold over the sum of basal concentrations in monoculture controls. This up-regulation of VEGF secretion is partially abrogated by K252a and inhibited when BMSCs co-culturing with shBDNF RPMI 8226 cells. Western blot and EMS A determined BDNF induced AP-1 tranloction and STAT3 phosphorylation respectively, and blocking STAT3 activity with AG490 lead to down-regulation of VEGF.
Conclusion Our studies demonstrate that BDNF mediates MM-BMSCs interactions via selective activation of specific receptors TrkB and downstream signal transducer STAT3 in regulating VEGF secretion.
PARTⅢ
Brain-derived neurotrophic factor promotes the secretion of MMP-9 in human myeloma cell through modulation of nucleus factor-κB
Objective:To explore the mechanism of brain-derived neurotrophic factor (BDNF) promoting human multiple myeloma (MM) cells secreting matrix metalloproteinase-9 (MMP-9). Methods:Gelatin zymograph of culture supernatants was performed to visualize the content of MMPs in myeloma RPMI 8226 cells stimulated by BDNF.NF-κB activity was determined by chemilumiescent electrophoretic mobility shift assay (EMSA). Results: Treatment with 25,50,100 and 200μg/L BDNF for 24 h significantly enhanced the level of MMP-9 secreted by RPMI 8226 cells in a dose-dependent manner (2.03±0.48, 2.99±0.46,4.63±0.62 and 5.62±1.29μg/L, respectively vs 1.00μg/L of the control. P<0.01), while that of MMP-2 was not changed significantly (P>0.05).The BDNF-induced activity on of MMP-9 was inhibited by pretreatment with pyrrolidine dithiocarbamate(PDTC), a NF-κB inhibitor, or K252a, a specific tyrosine inhibitor of TrkB which is the receptor for BDNF.Pretreated with 1 mmol/L PDTC or 500 nmol/L K252a significantly down-regulated MMP-9 secreted by the 100μg/L of BDNF stimulated RPMI 8226 cells (the optical density values were 867.52±101.81 and 727.98±92.05,respectively, vs 1159.01±233.15 of the control).The activity of NF-κB was enhanced by BDNF in a dose-dependent manner, and pretreatment with K252a could significantly inhibit this activation at 1,6,1 2 and 24 h (P<0.05) in a time-dependent manner.
Conclusion:BDNF plays an important role in the angiogenesis of MM to promote the up-regulation of MMP-9,which may be induced by enhanced NF-κB activity in MM cells.
目的通过慢病毒shRNA载体干扰骨髓瘤细胞BDNF的表达,建立人骨髓瘤裸鼠模型,在体内实验中研究BDNF对骨髓瘤肿瘤生长和血管新生的作用
方法构建慢病毒载体pGCSIL-eGFP-shBDNF并转染至骨髓瘤RPMI 8226细胞,流式细胞分选术筛选eGFP阳性稳定转染BDNFshRNA的骨髓瘤细胞,Western blot验证shRNA对BDNF蛋白表达的干扰作用；分离培养正常人骨髓间充质细胞,流式细胞术鉴定其表面分子；诱导BMSCs向成骨细胞、成脂肪细胞和成软骨细胞分化,通过茜素红染色、油红O染色及阿辛蓝染色鉴定其多能定向分化的能力。将稳定转染BDNFshRNA/Control shRNA的RPMI 8226细胞单独或与骨髓间充质细胞混合后接种裸鼠皮下,建立骨髓瘤移植瘤老鼠模型。活体荧光显微镜下连续监测骨髓瘤细胞的体内生长,观察肿瘤大小,总体存活时间,通过免疫组化染色CD34计算肿瘤微血管密度(MVD),Western blot检测肿瘤组织BDNF、VEGF的蛋白表达。
结果转染慢病毒载体的RPMI 8226细胞经流式分选后,表达eGFP的BDNF shRNA组和control shRNA组转染细胞纯度分别达91.87%和90.50%,Western Blot结果证实转染BDNF shRNA的RPMI 8226细胞BDNF蛋白表达显著抑制；9名健康人来源的BMSCs均表达CD44、CD105和CD29,但不表达CD14,CD45,并且BMSCs在不同条件下经诱导能向成骨细胞、成脂肪细胞以及成软骨细胞定向分化；人骨髓瘤裸鼠模型中,BMSCs能促进骨髓瘤细胞在体内生长,加速肿瘤形成；对于RPMI 8226细胞移植瘤模型,shBDNF RPMI 8226组较Control RPMI 8226组移植瘤平均体积(578.10±138.27 mm3 vs 1100.01±132.38 mm3,P<0.001)降低,并伴随局部微血管密度减少(6.40±3.81/HRP vs 12.73±5.89/HRP,P<0.001)以及总体生存时间延长；同样,对于骨髓瘤-间充质细胞混合移植瘤,ShBDNF RPMI 8226/BMSCs组较Control RPMI8226/BMSCs组肿瘤平均体积(1076.51±161.00mm3 vs 1674.49±174.71 mm3,P<0.001)亦降低。微血管密度(22.13±8.56/HRP vs 30.26±12.53/HRP,P<0.001)亦减少并且总体生存时间延长；Western Blot证实干扰骨髓瘤BDNF的表达能降低肿瘤组织VEGF的分泌。
结论骨髓瘤分泌的BDNF在体内骨髓瘤的生长及局部血管新生过程中起重要作用,可能成为多发性骨髓瘤治疗的新靶点。
第二部分骨髓微环境中骨髓瘤细胞分泌的BDNF促进骨髓间充质细胞分泌VEGF
目的体外实验探讨BDNF/TrkB途径在骨髓微环境中对多发性骨髓瘤血管新生的作用。
方法分离培养正常人原代骨髓间充质细胞,在体外应用BDNF作用于正常人骨髓间充质细胞,或将稳定转染BDNF/Control shRNA的RPMI 8226细胞与正常人骨髓间充质细胞在transwell小室中培养,ELISA法检测BDNF对BMSCs分泌VEGF的影响,Western blot法分别检测BMSC细胞TrkB、STAT3、p-STAT3的蛋白表达量,凝胶电泳迁移实验(EMSA)检测核因子AP-1的DNA结合活性。
结果BDNF能促进BMSCs表达TrkB；沉默BDNF表达的RPMI 8226细胞与BMSCs共培养时,BMSCs表达的TrkB蛋白水平较对照组降低；BDNF以时间和剂量依赖性的方式促进BMSCs分泌VEGF这种作用能被TrkB抑制剂K252α所阻断；BMSCs与RPMI 8226细胞共培养能促进VEGF的分泌,共培养上清的VEGF水平是BMSCs及RPMI 8226细胞单独培养上清中VEGF水平之和的3.1倍,K252α亦能部分抑制这种作用,而当BMSCs与shBDNF RPMI 8226细胞共培养时,VEGF水平降低36.89%。BDNF能促进BMSCs内AP-1的DNA结合活性和STAT3的磷酸化水平,而阻断STAT3途径能明显下调BDNF对BMSCs分泌VEGF的促进作用。
结论骨髓瘤-间充质细胞共培养系统中,骨髓瘤细胞分泌的BDNF是通过活化受体TrkB促进骨髓基质细胞内STAT3的磷酸化,从而上调促血管新生因子VEGF的分泌。
第三部分脑源性神经营养因子-促进多发性骨髓瘤细胞分泌基质金属蛋白-9
目的探讨脑源性神经营养因子(BDNF)通过核转录因子NF-κB诱导多发性骨髓瘤(MM)细胞分泌基质金属蛋白酶(MMP)-9的分子机制。
方法培养人MM细胞株RPMI 8226,使用明胶酶谱法检测RPMI 8226细胞分泌的活性MMP-2、MMP-9水平的改变,用化学发光凝胶电泳迁移率分析(EMSA)法检测RPMI 8226细胞中NF-κB活化水平的改变。
结果以25、50、100和200μg/L浓度的BDNF刺激RPMI 8226细胞24小时后,其活性MMP-9的分泌水平分别为2.03±0.48、2.99±0.46、4.63±0.62和5.62±1.29gμg/L,均明显高于对照组(P<0.01),而MMP-2的表达在各浓度组之间的差异无统计学意义(P>0.01)。当BDNF作用浓度为100μg/L时,预先分别用1mmol/L的NF-κB抑制剂四氢化吡咯二硫代氨基甲酸酯(PDTC)和500nmol/L的BDNF高亲和力受体TrkB的酪氨酸抑制剂K252a处理15min和1h后,K252α和PDTC(P<0.001)均能显著抑制RPMI 8226细胞MMP-9的分泌(光密度值分别为867.52±101.81、727.98±92.05,对照组为1159.01±233.15,与对照组比,P值均小于0.01)。随着BDNF浓度的升高,NF-κB的活化作用明显增强,于BDNF浓度为100gμg/L时加K252a预处理1h,6h,12h,24h的3个时间点均观察到K252α能明显抑制BDNF对NF-κB的活化作用(P<0.05),且随着BDNF作用时间的延长而增强。
结论BDNF能促进MM细胞血管新生的能力可能是通过NF-κB信号转导途径激活MMP-9而实现的。
PRATⅠ
Silencing of BDNF expression by shRNA inhibits in vivo multiple myeloma growth and angiogenesis in the bone marrow milieu
Objective To evaluate the effect of MM derived BDNF on tumor growth and angiogenesis in human multiple myeloma xenograft animal model by using lentiviral based shRNA targeting BDNF expression.
Methods RPMI 8226 cells were transfected with lentiviral based shBDNF-pGCSIL-eGFP or control vector. Pure populations of transfected RPMI 8226 cell were isolated via a flow cytometry, and specific BDNF knockdown was verified by western blot. BM stromal cells (BMSCs) were isolated from health donors and their phenotypes were identified with flow cytometer;Confirmation of differentiation of the BMSCs to adipocytes, osteocytes and chondrocytes were performed by staining with alizarin red, oil red O and alcine blue respectively. BDNF shRNA RPMI 8226 cells were inoculated subcutaneously with or without BMSCs in the hind neck of irradiated nude mice; Tumor volume was measured by in-vivo optical imaging and capliper; Microvessel density (MVD) was assessed by immunohistochemical analysis for CD34 expression; BDNF and VEGF expression in tumor were analyzed by western blot.
Results The purity of transfectants was determined by flow cytometric analysis of enhanced GFP reporter protein, the selected BDNF shRNA and control shRNA RPMI-8226 cells could reach up to 91.87% and 90.50% when compared with parental RPMI-8226 cells. The silencing capacity of BDNF shRNA was confirmed by western blot;BMSCs, isolated from nine healthy donors, expressed CD44、CD 105 and CD29, but not CD14,CD45 and characterized for adipocytes, osteocytes and chondrocytes differentiation capacity; In human myeloma xenograft model, compared with control group, shBDNF RPMI 8226 group has decreased tumor volume(578.10±138.27 mm3 vs 1100.01±132.38 mm3, P <0.001)and microvessel density(6.40±3.81/HRP vs 12.73±5.89/HRP,P<0.001),and prolonged overall survival; Moreover, the shBDNF RPMI 8226/BMSCs group has dcreased tumor volume(1076.51±161.00mm3 vs 1674.49±174.71 mm3,P<0.001)and microvessel density(22.13±8.56/HRP vs 30.26±12.53/HRP,P<0.001),and prolonged overall survival as well.It also demonstrated that silencing of MM-derived BDNF expression significantly inhibited VEGF expression by tumor immunohistochemistry. Conclusion:Our studies demonstrate that BDNF, as a potential stimulator of angiogenesis, contributes to MM tumorgenesis and may be a new target for MM therapy.
PRAT II
MM derived BDNF promotes stroma-derived VEGF secretion in the bone marrow milieu
Objective To evaluate the involvement of BDNF/TrkB pathway in MM-BMSCs interaction that mediates bone marrow angiogenesis in MM.
Methods BMSCs were isolated from 9 healthy donors,and RPMI 8226 cells were transfected with lentiviral based BDNF/Control shRNA vector. An indirecte co-culture system between RPMI 8226 MM cells and BMSCs was established by using a 0.4μm transwell inserts.ELISA was used to evaluate the effect of BDNF on VEGF secretion from BMSCs. TrkB、STAT3 and p-STAT3 in BMSCs were detected by western blot and DNA-binding of AP-1 was detected by Electrophoretic mobility shift assay (EMSA).
Results TrkB was consistently expressed by represent BMSC cultures and induced by BDNF stimulation, whereas TrkB expression was inhibited when BMSCs were co-cultured with BDNF knockdown MM cells compared to control group.Stimulation of BMSCs with exogenous BDNF induced a time-and dose-dependent increase in VEGF secretion, which was completely abolished by K252a, an inhibitor of TrkB.When RPMI-8226 cells were cultured with BMSCs, VEGF secretion was up-regulated 3.1-fold over the sum of basal concentrations in monoculture controls. This up-regulation of VEGF secretion is partially abrogated by K252a and inhibited when BMSCs co-culturing with shBDNF RPMI 8226 cells. Western blot and EMS A determined BDNF induced AP-1 tranloction and STAT3 phosphorylation respectively, and blocking STAT3 activity with AG490 lead to down-regulation of VEGF.
Conclusion Our studies demonstrate that BDNF mediates MM-BMSCs interactions via selective activation of specific receptors TrkB and downstream signal transducer STAT3 in regulating VEGF secretion.
PARTⅢ
Brain-derived neurotrophic factor promotes the secretion of MMP-9 in human myeloma cell through modulation of nucleus factor-κB
Objective:To explore the mechanism of brain-derived neurotrophic factor (BDNF) promoting human multiple myeloma (MM) cells secreting matrix metalloproteinase-9 (MMP-9). Methods:Gelatin zymograph of culture supernatants was performed to visualize the content of MMPs in myeloma RPMI 8226 cells stimulated by BDNF.NF-κB activity was determined by chemilumiescent electrophoretic mobility shift assay (EMSA). Results: Treatment with 25,50,100 and 200μg/L BDNF for 24 h significantly enhanced the level of MMP-9 secreted by RPMI 8226 cells in a dose-dependent manner (2.03±0.48, 2.99±0.46,4.63±0.62 and 5.62±1.29μg/L, respectively vs 1.00μg/L of the control. P<0.01), while that of MMP-2 was not changed significantly (P>0.05).The BDNF-induced activity on of MMP-9 was inhibited by pretreatment with pyrrolidine dithiocarbamate(PDTC), a NF-κB inhibitor, or K252a, a specific tyrosine inhibitor of TrkB which is the receptor for BDNF.Pretreated with 1 mmol/L PDTC or 500 nmol/L K252a significantly down-regulated MMP-9 secreted by the 100μg/L of BDNF stimulated RPMI 8226 cells (the optical density values were 867.52±101.81 and 727.98±92.05,respectively, vs 1159.01±233.15 of the control).The activity of NF-κB was enhanced by BDNF in a dose-dependent manner, and pretreatment with K252a could significantly inhibit this activation at 1,6,1 2 and 24 h (P<0.05) in a time-dependent manner.
Conclusion:BDNF plays an important role in the angiogenesis of MM to promote the up-regulation of MMP-9,which may be induced by enhanced NF-κB activity in MM cells.
引文
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