有关中药黄芪药理研究的若干问题的探讨

1原文研究要点 作者为了研究中药黄芪(AMB)提取物对体外培养的肝癌细胞的抑制活性,将ANMB经粉碎、浸提、超速离心、减压浓缩、冷冻干燥得到AMB提取物.然后,以AMB提取物按不同时间处理人肝癌SMMC-7721细胞,再以MTT法检测其对人肝癌SMMC-7721细胞线粒体代谢活性的影响,以细胞记数法观察了其对细胞增殖的影响.     

土贝母水提物对体外培养人肝癌细胞增殖及代谢的影响

目的研究中药土贝母(BPF)水提物对体外培养的人肝癌细胞的抑癌活性.方法 BPF经粉碎、浸提、超速离心、减压浓缩、冷冻干燥得BPF水提物.人肝癌SMMC-7721细胞经BPF提取物处理不同时间后以MTT法测其对人肝癌细胞线粒体代谢活性的影响,以细胞记数法观察了其对细胞增殖的影响.结果①经200 mg/L BPF提取物处理48 h,人肝癌SMMC-7721细胞线粒体活性为对照组的31.3%,处理72 h为21.4%;②以100mg/L BPF提取物处理7d中,人肝癌细胞数分别为0.57,0.316,0.29,0.285,0.57,1.287,1.2,对照FCS组分别为1.82,3.67,6.2,9.4,10.1,11,9,NON组分别为1.7,3.35,5.6,8.4,8.9,9.3,7.6,实验组人肝癌细胞数不仅低于对照组,而且低于处理前细胞数(P＜0.01);③经BPF提取物处理24 h后,即使更换为正常培养基,人肝癌细胞数在96 h的恢复培养中基本无增加.结论中药BPF提取物能显著抑制体外培养的肝癌细胞增殖和降低线粒体代谢活性.     

麝黄消瘤方抑制肝癌细胞侵袭粘附作用的实验研究

目的：观察麝黄消瘤方（SHF）兔含药血清对SMMC-7721肝癌细胞侵袭粘附能力的影响及其机制的探讨。方法：以体外培养的肝癌细胞株为研究对象，将肝癌细胞株分别接种于中药组、对照组血清中培养，绘制细胞生长曲线，计算细胞生长抑制率。以流式细胞仪（FCM）测SHF对肝癌细胞表面细胞间粘附分子-1（ICAM-1）表达的影响。以免疫组化法观察SHF对肝癌细胞抑癌基因nm23-H1蛋白水平的表达。以细胞侵袭实验观察SHF对与纤维连接蛋白（FN）粘附的SMMC-7721细胞侵袭运动能力及其生长状况。以岍法测量SHF对肝癌细胞与FN粘附的影响。结果：中药组血清较对照组明显抑制肝癌细胞的体外生长，在第5天抑制率最高达66．25％；中药处理的肝癌细胞ICAM-1表达明显低于对照组，nm23-H1蛋白水平明显高于对照组。SHF可抑制肝癌细胞的侵袭运动能力及其与FN之间的粘附。结论：SHF可抑制SMMC-7721细胞的侵袭粘附能力，其作用与中药血清上调nm23-H1蛋白水平和降低ICAM-1表达等有关。     

美洲大蠊提取物诱导人肝癌细胞SMMC-7721凋亡及作用机制

目的 探讨美洲大蠊提取物对人肝癌细胞SMMC-7721增殖、凋亡的影响并探讨其相关机制.方法 取对数生长期状态良好的SMMC-7721细胞分为三组,观察组常规培养24h后加入美洲大蠊提取物,使终浓度分别为300、100、33.3 μg/mL,顺铂组加入顺铂20 μg/mL作用细胞48 h,对照组不做任何处理.采用MTT比色法检测细胞增殖抑制率;采用流式细胞仪分析细胞凋亡率;JC-1染色法检测细胞线粒体膜电位变化;分光光度法检测Caspase-3活性变化.结果 观察组和顺铂组细胞抑制率明显高于对照组,IC50为100 μg/mL,并呈现时间一剂量依赖关系(P＜0.05);观察组和顺铂组细胞凋亡率明显高于对照组,呈现剂量依赖性(P＜0.05).凋亡过程中线粒体膜电位下降,Caspase-3活性升高(P＜0.05).结论 美洲大蠊提取物具有抑制人肝癌细胞SMMC-7721增殖并诱导其凋亡作用,线粒体途径可能是其诱导肝癌细胞凋亡的主要机制之一.     

茜草蒽醌对SMMC-7721肝癌细胞的抑制作用及分子机制

目的探讨茜草提取物蒽醌单体对人SMMC-7721肝癌细胞的抑制作用及分子机制。方法将茜草蒽醌作用于体外培养的人肝癌SMMC-7721细胞，采用MTY法检测其对肝癌细胞生长的抑制作用，应用TRAP-PAGE银染半定量法和RT—PCR半定量法检测端粒酶活性及端粒酶亚单位即端粒酶相关蛋白1（TP1）基因、端粒酶RNA（hTR）基因、端粒酶催化亚单位（hTERT）基因变化。结果茜草蒽醌能抑制肝癌细胞的生长，呈现与浓度、时间的依赖趋势；降低端粒酶活性，使hTERT基因的表达量明显下降（P〈0．01），而hTR、TP1基因的表达无显著变化（P〉0．05）。结论茜草蒽醌能显著抑制肝癌细胞的生长并抑制端粒酶的活性，可能与其下调hTERT基因的表达有关。     

肝癥口服液含药血清对转化生长因子α诱导人肝癌细胞SMMC-7721增殖和凋亡的研究

目的观察肝疒徵口服液含药血清对转化生长因子(TGFα)诱导人肝癌细胞增殖和凋亡的影响.方法采用血清药理学方法,以病理鼠血清作对照,应用M TT比色法观察中药鼠血清对TGFα诱导人肝癌细胞SMMC-7721增殖的抑制作用.用流式细胞术检测中药血清对肝癌细胞凋亡和细胞周期的影响.结果中药血清对TGFα诱导的肝癌细胞SMMC-7721增殖有明显的抑制作用,抑制效应呈时间依赖性.中药血清能促进肝癌细胞凋亡,使细胞滞留于G0/1期,降低S期细胞百分比和增殖指数 .结论肝疒徵口服液含药血清能够抑制TGFα诱导的人肝癌细胞增殖,促进其凋亡.     

盐酸青藤碱诱导人肝癌细胞SMMC-7721凋亡及其死亡受体DR4、DR5表达的变化

目的观察人肝癌细胞SMMC-7721凋亡过程中死亡受体DR4、DR5的表达,探讨盐酸青藤碱诱导人肝癌细胞凋亡的作用机制。方法体外培养人肝癌细胞SMMC-7721,经不同浓度盐酸青藤碱作用后,采用MTT法检测细胞增殖情况;Hoechst染色荧光显微镜观察细胞凋亡;用Western blotting方法检测人肝癌细胞DR4、DR5的表达。结果盐酸青藤碱对人肝癌细胞的生长有明显的抑制作用,并呈剂量、时间依赖性,不同浓度组之间差异均有统计学意义(P0.05);Hoechst33258染色可见凋亡细胞皱缩,核碎裂,也可见典型凋亡小体;在蛋白水平,盐酸青藤碱处理后SMMC-7721细胞死亡受体DR4、DR5的表达水平较未处理组显著增加(P0.05)。结论盐酸青藤碱可抑制人肝癌细胞SMMC-7721的细胞增殖,促进其凋亡,此作用可能与细胞内死亡受体DR4和DR5表达上调有关。     

白藜芦醇在人γδT细胞杀伤肝癌细胞中的作用研究

目的探讨白藜芦醇对人外周血γδT细胞体外增殖及该细胞对肝癌细胞SMMC-7721细胞活性的影响。方法分离健康人外周血单个核细胞(PBMC),放入含有IL-2、帕米膦酸的RPMI 1640培养基进行培养并诱导γδT细胞,实验组给予白藜芦醇共同培养,对照组不加药物,在此基础上,两组均加入肝癌细胞SMMC-7721作为靶细胞,继续培养。在培养第10天后,用台盼蓝染色计数细胞,采用流式细胞术检测各组γδT细胞的纯度及其颗粒酶B和CD107a、穿孔素的表达;用CCK-8试剂盒检测γδT细胞对肝癌细胞SMMC-7721的体外杀伤效应。结果两组γδT细胞纯度均达到70%以上,实验组γδT细胞较对照组纯度显著更高(P0.01);且实验组γδT细胞扩增倍数,穿孔素、CD107a和颗粒酶B的表达及对肝癌细胞SMMC-7721的杀伤效应均显著高于对照组(P0.01)。结论白藜芦醇能促进γδT细胞增殖,并可能通过上调颗粒酶B和穿孔素的表达增强其对肝癌细胞SMMC-7721的杀伤效应。     

肝疒徵口服液含药血清对转化生长因子α诱导人肝癌细胞SMMC-7721增殖和凋亡的研究

目的:观察肝疒徵口服液含药血清对转化生长因子(TGFα)诱导人肝癌细胞增殖和凋亡的影响.方法:采用血清药理学方法,以病理鼠血清作对照,应用M TT比色法观察中药鼠血清对TGFα诱导人肝癌细胞SMMC-7721增殖的抑制作用.用流式细胞术检测中药血清对肝癌细胞凋亡和细胞周期的影响.结果:中药血清对TGFα诱导的肝癌细胞SMMC-7721增殖有明显的抑制作用,抑制效应呈时间依赖性.中药血清能促进肝癌细胞凋亡,使细胞滞留于G0/1期,降低S期细胞百分比和增殖指数 .结论:肝疒徵口服液含药血清能够抑制TGFα诱导的人肝癌细胞增殖,促进其凋亡.     

热休克蛋白70激发树突状细胞抗肝癌作用的实验研究

目的探讨以树突状细胞（DC）为载体，用肝癌细胞提取的热休克蛋白70-肿瘤肽复合物（HSP-PC）刺激DC，观察经活化后的DC对肝癌细胞SMMC-7721的杀伤作用。方法用脐带血在体外培养诱导DC，用从SMMC-7721提取的HSP70—PC与DC共同培养，检测其对肝癌细胞的杀伤活性。结果经HSP70-Pc刺激后的DC对SMMC-7721的杀伤能力明显高于单纯DC对肿瘤细胞的杀伤能力。结论用HSP70—PC刺激的DC经体外实验证实具有明显的抗肿瘤效应，为临床肿瘤免疫治疗的开展提供了有效的数据。     

Inhibitory activity of polysaccharide extracts from three kinds of edible fungi on proliferation of human hepatoma SMMC-7721 cell and mouse implanted S180 tumor

lNTRODUCTlONThepolysaccharidesfromediblefungi(e.g.LE[1],FV[z's])weremacromolecularsubstanceswithstrongantigenicityandwerealsoverifiedtohaveantitumoractivityagainstS-l80implantedtumorinmiceinvivoandthatfromABwereshowntohaveantiinfectionofvirusandanticancerationinvivo.Thereferencesabouttheeffects0fp0lysaccharideextractsfromediblefungioncancercellsinvitr0wereverylimited.InthisreporthumanhepatomaSMMC-772lcellswereusedasamodelt0detecttheanticanceractivityofpolysaccharideextractsfromthethree…     

CXCR4抑制剂AMD3465调节肺癌细胞株增殖、侵袭的实验研究

目的探讨CXCR4抑制剂AMD3465对肺癌细胞株增殖、侵袭的影响。方法培养肺癌细胞株A549并分为AMD3465组、对照组;分别用含有三种不同剂量(50、100、200 mg/L)AMD3465的DMEM处理以及用不含药物的DMEM处理。处理后12、24、48 h,检测细胞的增殖活力和侵袭数目;处理后48 h,检测细胞中增殖及侵袭基因的mRNA表达量。结果处理后12、24、48 h,三种不同剂量(50、100、200 mg/L)AMD3465组细胞的OD值均显著低于对照组、侵袭数目均显著少于对照组,AMD3456剂量较低时OD值、侵袭数目与对照组差距均大于AMD3456剂量较高时(P0.05);处理后48 h,50 mg/L AMD3465组、100 mg/L AMD3465组、200 mg/L AMD3465组细胞中Cyclin D1、PCNA、Ki-67、Cat B、MMP2、MMP9、MMP13的mRNA表达量均显著低于对照组;AMD3456剂量较低时细胞中Cyclin D1、PCNA、Ki-67、Cat B、MMP2、MMP9、MMP13的mRNA表达量与对照组差距均大于AMD3456剂量较高时(P0.05)。结论 CXCR4抑制剂AMD3465能够有效抑制肺癌细胞株的增殖、侵袭。     

Effects of hydroxyapatite nanoparticles on proliferation and apoptosis of human hepatoma BEL-7402 cells

AIM: To study the effect of hydroxyapatite (HAP) nanoparticleson human hepatoma cell line BEL-7402 in vitro.METHODS: The human hepatoma cell line BEL-7402 wascultured and treated with HAP nanoparticles at variousconcentrations. Growth suppression was detected with MTTcolorimetric assay, cell apoptotic alterations were evaluatedby cytochemical staining (Hoechst 33258), transmissionelectron microscopy (TEM), and flow cytometry (FCM).RESULTS: HAP nanoparticles inhibited the growth ofhepatoma cells in a dose-dependent manner, with IC50 valuesof 29.30 mg/L. Treated with 50-200 mg/L HAP nanoparticlesfor 48 h, BEL-7402 cells apoptosis with nuclear chromatincondensation and fragmentation as well as cell shrinkageand the formation of apoptotic bodies were observed undercytochemical staining and transmission electron microscopy.FCM analysis showed hypodiploid peaks on histogram, theapoptotic rates at the concentrations of 50, 75, 100, 150and 200 mg/L of HAP nanoparticles were 20.35±2.23%,25.35±1.92%, 29.34±4.61%, 44.92±3.78 % and53.64±3.49%, respectively, which were all significantlyhigher than that of control group 2.23±0.14%. There wasa significant correlation between HAP nanoparticleconcentration and apoptotic rate (r=0.994, P＜0.01).CONCLUSION: HAP nanoparticles not only inhibitproliferation but also induce apoptosis of human hepatoma cell line BEL-7402 in vitro.     

The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 cell line

AIM：The goal of this study was to characterize the AFP receptor,its possible signal transduction pathway and its proliferative functions in human hepatoma cell line Bel 7402.METHODS:Cell proliferation enhanced by AFP was detected by MTT assay,^3H-thymidine incorporation and S-stage percentage of cell cycle analysis .With radioactive labeled 125 I-AFP for receptor binding assay;cAMP accumulation,Protein kinase A activity were detected by radioactive immunosorbent assay and the change of intracellular free calcium([Ca^2+]i) was monitored by scanning fluorescence intensity under TCS-NT confocal microscope.The expression of oncogenes N-ras,p53,and P21 ras in the cultured cells in vitro were detected by Northern blotting and Western blotting respectively.RESULTS:It was demonstrated that AFP enhanced the proliferation of human hepatoma Bel 7402 cell in a dose dependent fashion as shown in MTT assay,^3H-thymidine incorporation and S-phase percentage up to 2-fold.Two subtypes of AFP receptor were identified in the cells with Kds of 1.3×10^-9 mol.L^-1 and 9.9×10^-8molL^-1 respectively.Pretreatment of cells with AFP resulted in a significant increase (625%)in cAMP accumulation ,The activity of protein kinase A activity were increased up to 37.5,122.6,73.7and 61.2%,at treatment time point 2,6,12and 24hours.THe level of intracellular calcium were elevated after the treatment of aplha-fetoprotein and achieved to 204% at 4 min The results also showed that AFP(20mg.L^-1),could upregulate the expression of N-ras oncogenes and p53 and p21^ras in Bel 7402 cells,Inthe later case,the alteration were 81.1%(12h)and 97.3%(12h)respectively compared with control.CONCLUSION:These results demonstrate that AFP is a potential growth factor to promote the proliferation of human hepatoma Bel 7402 cells,Its growth-regulatory effects are mdeiated by its specific plasma membrane receptors coupled with its transmembrane signaling transduction through the pathway of cAMP-PKA and intracellular calcium to regulate the expression of oncogenes.     

丙戊酸钠对人胰腺癌细胞PaTu8988增殖的影响及量效关系研究

目的 探讨丙戊酸钠(VPA)对人胰腺癌PaTu8988细胞增殖和细胞周期的影响.方法 应用0.2、1.0、5.0 mmoL/L的VPA干预人胰腺癌PaTu8988细胞24、48 h,采用WST-8法检测细胞存活率,流式细胞仪检测细胞周期.以培养基中单加二甲基亚砜为空白对照组,单加PBS为PBS组.结果 VPA干预24 h后,VPA 5.0 mmol/L组的细胞生长抑制率为18.9%,显著高于对照组、PBS组及VPA 0.2、1.0 mmol/L组(0、4.4%、6.8%、6.1%,P值均<0.05);干预48 h后,VPA 1.0、5.0 mmol/L组的细胞生长抑制率分别为12.9%、25.9%,显著高于对照组、PBS组、VPA 0.2 mmol/L组(0、6.2%、4.6%,P值均<0.01).VPA干预24 h后,VPA 1.0、5.0 mmol/L组G2期细胞比例分别为(26.57 ±1.88)%、(34.11±4.74)%,显著高于PBS组、对照组、VPA0.2 mmol/L组[(10.72±2.02)%、(13.53±2.28)%、(13.81±2.40)%,P值均<0.01];VPA干预48 h后的细胞周期变化与24 h一致.结论 VPA呈时间及剂量依赖性抑制人胰腺癌PaTu8988细胞的增殖,诱导细胞阻滞在G2期.     

Effects of Ginkgo biloba extract on cell proliferation and cytotoxicity in human hepatocellular carcinoma cells

AIM: To study the effect of Ginkgo biloba extract (EGb 761) containing 22-27% flavonoids (ginkgo-flavone glycosides) and 5-7% terpenoids (ginkgolides and bilobalides) on cell proliferation and cytotoxicity in human hepatocellular carcinoma (HCC) cells. METHODS: Human HCC cell lines (HepG2 and Hep3B) were incubated with various concentrations (0-1 000 mg/L) of EGb 761 solution. After 24 h incubation, cell proliferation and cytotoxicity were determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and lactate dehydrogenase (LDH) release, respectively. After 48 h incubation, the expression of proliferating cell nuclear antigen (PCNA) and p53 protein was measured by Western blotting. RESULTS: The results showed that EGb 761 (50-1 000 mg/L) significantly suppressed cell proliferation and increased LDH release (P<0.05) in HepG2 and Hep3B cells compared with the control group. The cell proliferation of HepG2 and Hep3B cells treated with EGb 761 (1 000 mg/L) was 45% and 39% of the control group (P<0.05), respectively. LDH release of HepG2 cells without and with EGb 761 (1 000 mg/L) treatment was 6.7% and 37.7%, respectively, and that of Hep3B cells without and with EGb 761 (1 000 mg/L) treatment was 7.2% and 40.3%, respectively. The expression of PCNA and p53 protein in HepG2 cells treated with EGb 761 (1 000 mg/L) was 85% and 174% of the control group, respectively. CONCLUSION: Ginkgo biloba extract significantly can suppress proliferation and increase cytotoxicity in HepG2 and Hep3B cells. Additionally, Ginkgo biloba extract can decrease PCNA and increase p53 expression in HepG2 cells.     

Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

INTRODUCTIONBone morphogenetic proteins (BMPs) were first identified in the 1960s[1]. BMPs are multi-functional growth factors that belong to the transforming growth factor beta (TGF-β) superfamily[2]. Mature BMPs are 30-38 kDa proteins that utilize BMP …     

全反式维甲酸联合奥沙利铂对人SMMC-7721肝癌细胞株的作用

目的:观察全反式维甲酸(ATRA)联合奥沙利铂(L-OHP)对人SMMC-7721肝癌细胞增殖及凋亡的影响.方法:选用ATRA(10-5mol/L)及不同浓度的L-OHP(10mg/L、20mg/L、40mg/L),并选用10-5mol/L的ATRA分别联合L-OHP(10mg/L、20mg/L、40mg/L)作用于SMMC-7721肝癌细胞24h、48h、72h;采用MTT比色法观察其对SMMC-7721肝癌细胞的生长抑制作用,倒置显微镜下观察细胞形态变化;采用流式细胞术分析药物作用48h时SMMC-7721细胞的周期分布和凋亡的情况.结果:ATRA及不同浓度L-OHP单药及联合均可显著抑制SMMC-7721肝癌细胞的生长,细胞形态改变,凋亡比例增加,并呈剂量-时间依赖性;两药联合较单药相比作用明显增强(P<0.01);ATRA联合20mg/LL-OHP与ATRA联合40mg/LL-OHP相比,作用细胞48h及72h时,对细胞生长抑制作用无统计学差异;两药联合较单药相比,细胞凋亡率明显增加,细胞阻滞S期增强(P<0.01).结论:ATRA可抑制肝癌SMMC-7721细胞的生长并诱导细胞凋亡,与L-OHP联用后作用增强并可减少奥沙利铂用量,具有协同作用.     

土贝母苷甲对人肺腺癌细胞增殖及凋亡的影响

目的探讨土贝母苷甲对人肺腺癌细胞(A549)增殖及凋亡的影响。方法应用MTS法检测土贝母苷甲不同浓度、不同时间对A549细胞增殖的影响;使用1μg/mL土贝母苷甲处理A549细胞48 h作为处理组,对照组加入培养基培养48h;使用流式细胞仪分别检测两组细胞的周期分布及细胞凋亡率;并使用Western blot检测两组细胞凋亡相关通路Caspase蛋白的表达。结果MTS实验结果显示各组细胞随着土贝母苷甲浓度的增加A549细胞的增殖速度降低,并在培养48h后,各组细胞的增殖能力出现显著差异(P<0.05);流式细胞仪检测土贝母苷甲处理A549细胞48 h后比对照组细胞凋亡率显著增高(P<0.05);处理组A549细胞更多的阻滞于G0/G1期(P<0.05);Western blot检测各组细胞Caspase通路蛋白表达显示处理组比对照组Caspase-9、Caspase-3和Parp蛋白表达增加(P<0.05)。结论土贝母苷甲可抑制人肺腺癌细胞增殖,并诱导人肺腺癌细胞凋亡,具有一定的抗肿瘤作用,为中医药土贝母在临床应用中奠定基础。