云南石仙桃氯仿萃取物活性部位对人肝癌细胞HepG2的周期抑制作用

目的通过体外实验研究云南石仙桃氯仿萃取物活性部位对人肝癌细胞株HepG2的细胞增殖抑制作用,并进一步研究其作用机理。方法采用噻唑兰(MTT)比色法测定对HepG2细胞生长增殖的影响;采用流式细胞仪技术和蛋白质电泳技术检测对HepG2细胞的细胞周期及相应的细胞周期蛋白表达的影响。结果云南石仙桃氯仿层活性部位通过下调周期蛋白Cyclin B1及其蛋白激酶p34cdc2的表达将HepG2细胞阻滞在G2/M期。结论云南石仙桃氯仿层活性部位对人肝癌细胞HepG2的细胞周期具有阻断作用。     

石斛及其伪品云南石仙桃的鉴别

本文通过以石斛及其伪品云南石仙桃的来源、药材性状、显微特征、理化鉴别的研究，实验证明应用上述方法，即操作简便又可作出准确的判断。此法可作为鉴别石斛及伪品的较可靠的依据。     

云南石仙桃不同提取部位镇痛作用研究

目的研究云南石仙桃不同提取部位的镇痛作用。方法采用小鼠热板法、小鼠电刺激法观察云南石仙桃不同提取物对小鼠的镇痛作用。结果云南石仙桃3个提取物均可明显抑制热刺激所致的疼痛反应,小鼠电刺激法结果表明云南石仙桃水提液能明显延长小鼠痛阈值。结论云南石仙桃具有镇痛作用。     

云南石仙桃的化学成分

目的分离并鉴定兰科植物云南石仙桃(Pholidota yunnanensisRolfe)全草的体积分数为60%乙醇提取物中的化学成分。方法采用硅胶柱色谱法、凝胶柱色谱法、反相硅胶柱色谱法以及高效液相色谱法进行分离和纯化,并根据理化常数和光谱分析进行结构鉴定。结果分离得到了6个化合物,分别为4(3羟基2甲氧基苯基)2丁酮(4(3 hydroxy 2 methoxyphenyl)2 butanone,Ⅰ)、4(3羟基苯基)2丁酮(4(3 hydroxyphenyl)2 butanone,Ⅱ)、(R)(+)lasiodiploidin(Ⅲ)、(-)松脂素((-)pinoresinol,Ⅳ)、(-)丁香脂素((-)syringaresinol,Ⅴ)和β谷甾醇(βsitosterol)(Ⅵ)。结论化合物Ⅰ和Ⅱ为首次从植物中分离得到,Ⅲ~Ⅴ为首次从该属植物中分离得到。     

乌龙茶及其有效成分对肝癌细胞HepG2的生长抑制作用

目的 通过探讨茶及其有效成分对肝癌细胞HepG2增殖的影响,为乌龙茶成分进一步开发提供有力的实验室证据.方法 体外培养人肝癌细胞株HepG2,加入浓度为25、50、100和200 mg/L的茶叶浸出液、茶多酚、茶色素、茶多糖、茶氨酸作用24h后,用水溶性四唑盐(WST-1)检测肝癌细胞的存活率.结果 乌龙茶及其有效成分对肝癌细胞HepG2进行干预后,各组肝癌细胞的增殖均被抑制,各组的吸光度值均低于阴性对照组,茶汤100 mg/L组和200 mg/L组、茶多酚各组和茶色素各组、茶多糖和茶氨酸50mg/L、100 mg/L和200 mg/L组,与阴性对照组比较,差异均有统计学意义(均P＜0.05).同时茶多酚25 mg/L组、茶色素25 mg/L组与茶汤25 mg/L组比较,差异均有统计学意义[(0.1658±0.0025)、(0.1913±0.0013)比(0.2005±0.0013),均P＜0.05];茶多酚50mg/L组、茶色素50mg/L组、茶多糖50 mg/L组、茶氨酸50 mg/L组与茶汤50 mg/L组比较,差异均有统计学意义[(0.1545±0.0041)、(0.1655±0.0039)、(0.1595±0.0019)、(0.1728 ±0.0040)比(0.1970 ±0.0078),均P＜0.05];茶多酚100 mg/L组(0.1370±0.0016)与茶汤100 mg/L组(0.1600 ±0.0037)比较,差异有统计学意义(P＜0.05).结论 乌龙茶及其有效成分对人肝癌细胞HepG2具有明显的抑制作用,表现出抗肝癌的作用.     

大蒜辣素对HepG2肝癌细胞增殖的抑制作用

目的:研究大蒜辣素(Allicin)对肝癌HepG2细胞增殖的影响。方法:采用高效液相色谱法提纯Allicin,运用细胞计数试剂盒(Cell counting kit-8,CCK-8)研究Allicin对HepG2细胞增殖的影响,运用流式细胞仪研究其对细胞凋亡、DNA代谢的影响,运用Western blot研究其对细胞凋亡基因表达的影响。结果:Allicin经HPLC法提纯后其纯度达到99%;Allicin能抑制HepG2细胞的增殖,导致细胞大量凋亡及死亡,凋亡率达到8.67%±3.2%,死亡率达到70.38%±1.8%;也导致细胞DNA代谢发生紊乱,使大部分细胞处于DNA合成的G0/G1期并使进入S期细胞含量减少,延缓细胞增殖。Western blot实验结果表明,Allicin能导致细胞凋亡相关蛋白大量表达。结论:Allicin能抑制肝癌细胞的增殖,导致细胞大量死亡并引发细胞凋亡,并引起细胞DNA代谢发生紊乱,细胞大部分被阻滞在DNA合成的G0/G1期,Western blot实验结果表明,细胞凋亡基因相关蛋白Bax大量表达,而Bcl-2表达降低。     

Notch1表达对肝癌细胞HepG2生物学行为的影响

目的研究Notch1基因对Hes1、Hes2表达水平以及肝癌细胞HepG2生物学行为的影响。方法将HepG2细胞分为Notch1受体胞内段质粒(A)组、Notch1干扰RNA(B)组、空载体(C)组和未处理HepG2细胞(D)组。采用MTT法检测各组细胞增殖能力,流式细胞术测定细胞周期,RT-PCR和Western blot法分别检测Notch1、Hes1、Hes2mRNA和蛋白的表达。结果与C组和D组相比,A组细胞增殖能力减弱,G1期细胞以及Notch1、Hes1、Hes2mRNA和蛋白表达均明显增加(P<0.05),而B组细胞增殖能力增强,G1期细胞以及Notch1、Hes1、Hes2mRNA和蛋白表达均明显减少(P<0.05)。结论上调Notch1的表达能促进Hes1与Hes2表达水平并抑制肝癌细胞HepG2增殖。     

贝伐单抗对肝癌细胞株HepG2增殖的抑制作用

目的通过体外实验探讨贝伐单抗(bevacizumab)对人肝癌细胞HepG2增殖的抑制作用及可能的分子机制。方法MTT法检测贝伐单抗对肝癌细胞增殖的抑制作用;流式细胞仪检测细胞凋亡;RT-PCR分析肝癌细胞株VEGF、Flt-1、KDR mRNA表达量的变化。结果不同质量浓度的贝伐单抗对肝癌细胞株HepG2的增殖有抑制作用;贝伐单抗可诱导肝癌细胞株HepG2的凋亡;贝伐单抗作用于肝癌细胞株HepG2后,其VEGF、KDR mRNA表达量均减少。结论贝伐单抗可能通过阻断VEGF的促增殖作用而抑制HepG2细胞增殖。     

奥曲肽对肝癌细胞SMMC-7721增殖的抑制作用

目的 :探讨奥曲肽抑制肝癌细胞生长的作用。方法 :人肝癌细胞SMMC 772 1培养于含 10 %胎牛血清、10 0U·ml-1青霉素、链霉素的RPMI16 40培养液中。以 4个稀释度 10 -5、10 -4、10 -3 、10 -2 g·L-1将奥曲肽加入培养液 ,于加药后第 4 8h行MTT比色实验检测生长抑制率。奥曲肽浓度为 10 -3 g·L-1时于0、6、12、2 4、36h行DNA染色分析细胞周期。结果 :MTT比色法测定显示奥曲肽抑制肝癌细胞的生长 ,在浓度为 10 -5、10 -4、10 -3 、10 -2 g·L-1作用 4 8h生长抑制率分别为 9.33%、 12 .70 %、 19.70 %、2 0 .93%。药物作用 12、2 4h ,G0 -G1期细胞比例增加 ,G2 -M期细胞比例减少。结论 :奥曲肽抑制体外培养的人肝癌细胞的增殖 ,机制可能是将肝癌细胞阻滞于G0 -G1期 ,阻止细胞进入G2 -M期。     

索拉菲尼对肝癌细胞的抑制作用及其对CDK5表达水平的影响

目的探讨索拉菲尼对体外培养的人肝癌HepG2细胞的增殖抑制作用及其对周期素依赖性蛋白激酶5表达的影响。方法体外培养人肝癌HepG2细胞株,采用MTT法检测不同浓度索拉菲尼对HepG2细胞增殖的影响;同时经细胞爬片后,采用免疫细胞化学法检测肝癌细胞内CDK5的表达情况。结果索拉菲尼可使人肝癌细胞增殖率明显下降,且呈明显的时间-剂量依赖效应;药物作用48h后,给药组CDK5的表达较对照组明显减少,其着色程度随药物浓度增高而逐渐变淡。结论索拉菲尼对人肝癌HepG2细胞增殖有抑制作用,并能下调肝癌细胞中CDK5的表达,从而起到抗肿瘤的治疗作用。     

槲寄生凝集素对肿瘤细胞细胞周期的影响

目的通过体外实验研究槲寄生凝集素对肿瘤细胞的细胞毒性作用。方法采用MTT比色分析法测定槲寄生凝集素注射液对肿瘤细胞生长曲线的影响 ;采用流式细胞仪技术检测槲寄生凝集素注射液对肿瘤细胞细胞周期的影响。结果和结论槲寄生凝集素注射液对肿瘤细胞有剂量依赖性杀伤作用     

自噬对肝癌HepG2细胞增殖及对细胞周期影响的研究

目的研究用诱导肝癌HepG2细胞自噬与抑制肝癌HepG2细胞自噬的方法检测自噬对肝癌HepG2细胞增殖的影响,阐明细胞自噬的发生与细胞周期的关系。方法常规培养肝癌HepG2细胞,应用MTT比色法和流式细胞仪检测肝癌HepG2细胞增殖抑制率及细胞周期各时相的变化。结果自噬诱导剂雷帕霉素组的肝癌HepG2细胞抑制率明显高于对照组（P〈0.05）,对照组的肝癌HepG2细胞抑制率明显高于自噬抑制剂3-MA组（P〈0.05）;流式细胞仪分析示自噬诱导剂雷帕霉素组细胞周期中G1期细胞增多,S期细胞减少,G2/M期细胞相对增多（P〈0.05）。自噬抑制剂3-MA组细胞周期中G1期细胞减少,S期细胞增多,G2/M期细胞相对减少（P〈0.05）。结论自噬对肝癌HepG2细胞增殖有抑制作用,自噬不利于肝癌HepG2细胞成活。肝癌HepG2细胞自噬的发生多停留在细胞周期的G1期。     

肿瘤细胞周期测定方法研究

肿瘤是一类与细胞周期密切相关的疾病。流式细胞术对肿瘤细胞周期的分析可以观察到肿瘤细胞倍体的变化,有助于肿瘤的早期发现和治疗。区分肿瘤细胞所处于的细胞周期,明确地了解每个时相所占细胞周期的比例,对于肿瘤药物的研发具有重要意义。该文着重对G₀期和G₁期,G₂期和M期,以及S期细胞的测定方法进行归纳、总结,并对这些方法的优缺点及适用条件进行阐述和总结,为肿瘤药物的研发及基础研究等方面的科研工作提供参考。     

Anthocyanins from roselle extract arrest cell cycle G2/M phase transition via ATM/Chk pathway in p53‐deficient leukemia HL‐60 cells

Cell cycle regulation is an important issue in cancer therapy. Delphinidin and cyanidin are two major anthocyanins of the roselle plant (Hibiscus sabdariffa ). In the present study, we investigated the effect of Hibiscus anthocyanins (HAs) on cell cycle arrest in human leukemia cell line HL‐60 and the analyzed the underlying molecular mechanisms. HAs extracted from roselle calyces (purity 90%) markedly induced G2/M arrest evaluated with flow cytometry analysis. Western blot analyses revealed that HAs (0.1–0.7 mg mL^?1) induced G2/M arrest via increasing Tyr15 phosphorylation of Cdc2, and inducing Cdk inhibitors p27 and p21. HAs also induced phosphorylation of upstream signals related to G2/M arrest such as phosphorylation of Cdc25C tyrosine phosphatase at Ser216, increasing the binding of pCdc25C with 14‐3‐3 protein. HAs‐induced phosphorylation of Cdc25C could be activated by ATM checkpoint kinases, Chk1, and Chk2. We first time confirmed that ATM‐Chk1/2‐Cdc25C pathway as a critical mechanism for G2/M arrest in HAs‐induced leukemia cell cycle arrest, indicating that this compound could be a promising anticancer candidate or chemopreventive agents for further investigation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1290–1304, 2017.     

Effect of active fraction of Eriocaulon sieboldianum on human leukemia K562 cells via proliferation inhibition,cell cycle arrest and apoptosis induction

Eriocaulon sieboldianum (Sieb. & Zucc. ex Steud.), a genus of Eriocaulon in the Eriocaulaceae family, is an edible and medicinal plant used in traditional Chinese medicine. It was processed into healthcare beverages for expelling wind-heat, protecting eyes, and reducing blood fat. Also, it has been used with other herbs as Traditional Chinese herbal compound to treat cancer as adjuvants in tumor therapy in China. However, the active fractions and precise cellular mechanisms of E. sieboldianum extract remain to be illustrated. The goal of this study was to investigate the effects of the active fraction of E. sieboldianum on the growth of K562 cells and understand the possible mechanisms of its action. Our findings suggested that the fraction E3 of E. sieboldianum could effectively inhibit the activity of Aurora kinase and induce apoptosis via blocking cell cycle, up-regulating the expression of proapoptotic proteins including p53 and Bax and reducing the expression of Bcl-2. The levels of Cytochrome C, cleaved caspase-9, cleaved caspase-3 and cleaved PARP were also found to be increased after treatment with fraction E3 of E. sieboldianum.This study could improve the development of E. sieboldianum and raise its application value in cancer adjuvant therapy. Considering it is both a dietary supplement and a traditional Chinese herbal medicine which exhibits anticancer activities, it can be developed into functional food.     

桑黄多糖P1对肝癌HepG2细胞周期和钙调蛋白信号通路的影响

目的探讨桑黄多糖P1抑制人源性肝癌Hep G2细胞增殖的作用机制。方法桑黄多糖P16.25~200 mg·L-1与Hep G2细胞作用48 h,MTT法检测细胞存活率。桑黄多糖P1 100和200 mg·L-1与Hep G2细胞作用48 h,流式细胞术检测细胞周期和凋亡;荧光分光光度法测定细胞内Ca2+浓度;实时荧光定量PCR检测细胞内钙调蛋白(Ca M)、钙调蛋白依赖性蛋白激酶Ⅱ(Ca MKⅡ)、表皮生长因子(EGF)及其受体(EGFR)、K-ras和c-fos基因表达;Western蛋白质印迹法检测细胞内Ca MKⅡ和Kras蛋白表达。结果桑黄多糖P1对Hep G2细胞存活的抑制率随药物浓度的增大而显著提高,100和200 mg·L-1时抑制率高达44.0%和61.3%。桑黄多糖P1 200 mg·L-1处理48 h S期细胞百分含量明显升高,由对照组的(23.3±3.4)%升高至(37.8±2.2)%(P<0.01),而G2/M期细胞百分含量明显降低,由对照组的(15.3±1.2)%降至(3.4±1.9)%(P<0.01);细胞凋亡率无明显变化。与对照组相比,桑黄多糖P1 100和200 mg·L-1处理48 h后Ca M,Ca MKⅡ,EGF,EGFR,K-ras和c-fos mRNA水平显著下降(P<0.01);Western蛋白质印迹检测结果显示,桑黄多糖P1作用48 h后Hep G2细胞内Ca MKⅡ和K-ras蛋白水平与对照组相比显著下降(P<0.01)。桑黄多糖P1 200 mg·L-1处理细胞后,细胞内Ca2+荧光强度显著升高,0~20 min内分别由951增至1430(P<0.01),而对照组一直维持在1150左右。结论桑黄多糖P1可能通过提高细胞内Ca2+浓度以及下调Ca M,Ca MKⅡ,EGF,EGFR,K-ras和c-fos基因的表达,诱导Hep G2细胞S期阻滞,从而抑制Hep G2细胞增殖。     

GL-V9, a newly synthetic flavonoid derivative, induces mitochondrial-mediated apoptosis and G2/M cell cycle arrest in human hepatocellular carcinoma HepG2 cells

We recently established that GL-V9, a newly synthetic flavonoid derivative, is an active cytotoxic component. In this study, we demonstrated that GL-V9 inhibited cells growth via inducing apoptosis and G2/M cell cycle arrest in human hepatocellular carcinoma HepG2 cells. Following the treatment of HepG2 cells with GL-V9, we observed poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3 and caspase-9, while caspase-8 remained unchanged. The expression ratio of Bcl-2/Bax was also decreased in GL-V9-treated cells. Meanwhile, the cell cycle-related proteins, such as cyclin B1, CDK1 and cdc25 were down-regulated in GL-V9-induced G2/M cell cycle arrest. Furthermore, we showed that GL-V9-induced apoptosis in HepG2 cells was achieved through mitochondrial pathway. It also regulated changes of mitochondrial membrane potential and increased the production of intracellular reactive oxygen species. Besides, the growth inhibitory effect of GL-V9 was examined in vivo using murine implanted tumor model. These studies indicate that GL-V9 shows promise as a therapeutic agent against human heptoma.     

3种方法提取洋葱中的类黄酮物质对肝癌细胞株HepG2的作用

目的研究3种方法提取洋葱中的类黄酮物质对肝癌细胞株HepG2的作用。方法采用水煎煮法、50%乙醇回流法及50%乙醇冷浸法从洋葱中提取类黄酮物质,将其分别加入处于对数生长期的HepG2肝癌细胞中培养,以PBS为对照组,20、30、40、50、70μg.ml-1的类黄酮物质为实验组,用MTT法分别测定给药前及给药72 h后各浓度组在490 nm处的吸光度。结果水煎煮法、50%乙醇回流法及50%乙醇冷浸法提取的类黄酮物质抑制HepG2肝癌细胞生长的起始浓度分别为40、50、40μg.ml-1,随着浓度的增加,抑制作用显著增强。结论洋葱中类黄酮物质对体外培养的HepG2肝癌细胞表现出明显的抗肿瘤活性,50%乙醇冷浸法所提类黄酮物质的抗肿瘤活性最强。     

Novel pyrazolopyrimidine urea derivatives: Synthesis,antiproliferative activity,VEGFR-2 inhibition,and effects on the cell cycle profile

A series of novel diaryl urea pyrazolopyrimidine derivatives was designed and synthesized. All the synthesized compounds were evaluated for cytotoxic activity by the National Cancer Institute. A significant antiproliferative activity at a 10-µM dose was shown by four compounds ( 5c, 5e, 5g , and 5h ), and they were accordingly evaluated at five concentrations. They showed a potent and broad-spectrum antiproliferative activity, with GI₅₀ values between 0.553 and 3.80 µM and TGI values in the range of 2.17–100 µM. These four compounds potently inhibited the vascular endothelial growth factor receptor-2 (VEGFR-2) with IC₅₀ values in the nanomolar range. Molecular docking attributed their potent VEGFR-2 inhibitory activity to their interactions with key amino acids in the VEGFR-2 active site. Their flow cytometric analysis showed that they exerted their cytotoxic activity by reduction of the cellular proliferation and by induction of cell cycle arrest at the G2/M phase. Additionally, they induced DNA degradation or fragmentation, confirming the role of apoptosis in the cancer cell death and cytotoxicity induced by these compounds.