丙戊酸钠对Kasumi-1细胞株细胞周期的影响

目的 观察丙戊酸钠(VPA)对急性髓性白血病(AML)Kasumi-1细胞周期的影响,并探讨其分子机制.方法 将对数生长期Kasumi-1细胞1×105/ml(观察组)分别经1、2、3 mmol/L VPA处理3 d;对照组不加药.RT-PCR法检测VPA不同浓度及不同作用时间(3 mmol/L作用0、1、2、3d)细胞周期调节因子cyclinE1、p21WAF1/CIP1、p27KIP1 mRNA及p21WAF1/CIP1蛋白变化.结果 观察组cyclinE1 mRNA表达明显降低(P<0.05),p21WAF1/CIP1 mRNA及其蛋白表达明显升高(P<0.05),且均呈剂量、时间依赖性;p27KIP1 mRNA表达水平无明显变化.结论 VPA可通过对Kasumi-1细胞周期蛋白及周期蛋白依赖性激酶抑制剂的影响调节细胞周期,使细胞阻滞于G0/G1期;本研究可为急性白血病的治疗提供理论依据.     

细胞周期依赖性蛋白激酶对体外培养人肝癌细胞侵袭力的影响

目的:探讨CDKs对体外培养的人肝癌细胞SMMC-7721侵袭力的影响及其分子机制.方法:将细胞分为A、B两组(A组用终浓度为0μmol/L Roscovitine.B组用终浓度为32μmol/L Roscovitine,均培养24 h),采用流式细胞术检测PACDKs特异性抑制剂Roscovitine干预后的人肝癌细胞SMMC-7721的细胞周期,并用Transwell小室、划痕实验、PCR技术分别检测处于不同细胞周期下的人肝癌细胞侵袭能力、水平运动能力及uPA、MMP-9mRNA表达.结果:经终浓度为32μmol/L的Roscovitine干预24 h后的人肝癌细胞SMMC-7721处于G0/G1期细胞比例迅速升高(72.19%±0.47%vs 59.22%±0.54%,P<0.05),细胞侵袭能力下降,穿膜细胞数明显减(71.40±5.59 vs 149.60±16.36,P<0.05);细胞水平运动能力明显下降(P<0.05);uPA mRNA的表达下降、但MMP-9mRNA的表达却无明显变化.结论:Roscovitine干预使人肝癌细胞SMMC-7721侵袭能力和水平运动能力下降,其机制可能与肝癌细胞周期时相分布发生改变和uPAmRNA的表达下降有关.     

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.     

曲古菌素A对食管癌EC1细胞增殖和细胞周期的影响及其分子机制

目的:探讨不同浓度曲古菌素A对食管癌细胞系EC1 细胞增殖、细胞周期的影响及其对细胞周期调控基因p21 WAF1/CIP1 表达的影响. 方法:用0.3,0.5,1.0 μmol/L 的TSA 处理EC1 细胞,MTT 检测TSA 作用24 、48 h 对EC1 细胞的抑制作用,流式细胞仪检测0.3,0.5,1.0 μmol/L 的TSA 作用24 h 后EC1 细胞周期的改变,Western blot 法检测p21 WAF1/CIP1 变化. 结果:TSA 在0.5 μmol/L 以上时对EC1 细胞有抑制作用;0.3 μmol/L TSA 处理细胞后细胞周期与对照组相比,无明显变化;0.5 μmol/L TSA 处理EC1 细胞后,G0/G1期细胞较对照组明显增加,S 期细胞较对照组明显减少(74.56% ±1.34% vs 62.12%±0.52%;14.52%±1.81% vs 27.50%±0.66%,均P <0.05);0.5,1.0 μmol/L TSA 处理细胞后p21 WAF1/CIP1 表达明显增加(均P<0.05). 结论:一定浓度的TSA 对人食管癌细胞EC 具有的增殖抑制作用,引起EC1 细胞发生G0/G1 期阻滞,其部分机制与p21 WAF1/CIP1 上调有关.     

PHB1对人肝癌细胞增殖的影响及其作用机制

目的探讨抗增殖蛋白1(prohibitin1,PHB1)对人肝癌细胞增殖的影响及其作用机制。方法构建PHB1真核表达重组质粒(pEGFP-PHB1)和PHB1靶向siRNA质粒(shRNA-PHB1)转染人肝癌细胞HepG2和SMMC-7721,诱导PHB1在人肝癌细胞中表达上调和下调,采用MTT、流式细胞学、荧光定量PCR和免疫印迹等技术检测人肝癌细胞增殖、细胞周期,以及细胞周期关键调控分子的表达情况。结果高表达PHB1可阻滞HepG2和SMMC-7721细胞于G0/G1期[(67.28±2.94)%比(56.71±2.56)%,t=6.64,P=0.00;(69.48±3.82)%比(60.43±2.59)%,t=4.80,P=0.00],使S期比例下降[(14.74±1.45)%比(24.13±1.92)%,t=9.54,P=0.00;(13.73±1.26)%比(25.50±2.30)%,t=10.99,P=0.00],抑制细胞增殖;周期调控分子p53和p21CIP1mRNA和蛋白质水平显著升高,而Cyclin A2、Cyclin E1和CDK2 mRNA和蛋白质水平显著降低(P0.01)。低表达PHB1可促使HepG2和SMMC-7721细胞趋于S期[(31.65±2.71)%比(24.68±1.28)%,t=5.69,P=0.00;(31.02±2.49)%比(25.88±2.40)%,t=3.64,P=0.005],促进细胞增殖;p53、p21CIP1、Cyclin A2、Cyclin E1、CDK2 mRNA和蛋白质水平与PHB1高表达时相反(P0.01)。结论高表达PHB1可以阻滞人肝癌细胞周期于G0/G1期,进而抑制细胞增殖,其作用机制可能与p53介导的G0/G1期相关细胞周期蛋白有关。     

survivin反义寡核苷酸对SMMC-7721细胞增殖和凋亡的影响

目的 观察survivin反义寡核苷酸(ASODN)对人肝癌细胞株SMMC-7721增殖和凋亡的影响.方法 人工合成survivin基因ASODN和正义ODN(SODN),并行硫代磷酸化修饰,通过脂质体途径转染SMMC-7721;分别用RT-PCR和Western blot检测survivin mRNA和蛋白表达;用MTT法检测ASODN对SMMC-7721增殖的影响;流式细胞仪检测细胞周期变化及细胞凋亡率;倒置显微镜观察细胞形态变化.结果 SMMC-7721可强表达survivin mRNA和蛋白;ASODN呈浓度依赖性抑制survivin mRNA和蛋白表达及SMMC-7721增殖,诱导细胞凋亡,使细胞阻滞于G2/M期.SODN对survivin mRNA和蛋白及SMMC-7721的增殖、细胞周期无明显抑制作用.结论 脂质体介导转染survivin ASODN可抑制细胞增殖、使细胞阻滞于G2/M期,从而促进细胞凋亡.     

p21基因转染人肝癌SMMC-7721 细胞对化疗药物敏感性的研究

目的探讨p21基因转染人肝癌SMMC-7721细胞（7721细胞）对化疗药物的敏感性，深入研究p21基因对肝癌细胞的治疗作用。方法应用磷酸钙介导p21基因转染人肝癌7721细胞；通过G418筛选稳定表达细胞株并扩大培养；采用MTT法检测肿瘤细胞增殖速度；流式细胞术检测细胞周期变化。结果经过3～4w G418筛选得到稳定表达的细胞株，p21基因稳定转染并联合化疗药物应用可明显抑制7721细胞的体外增殖，细胞周期明显改变，细胞从G1期到G2期发生阻滞。结论提示转染外源p21基因联合化疗药物可使7721细胞周期阻滞于G1期，并可抑制肿瘤细胞增殖。     

健脾理气药诱导人肝癌细胞SMMC7721凋亡的研究

目的观察健脾理气药的诱导凋亡效应,为其临床应用进一步提供依据.方法采用血清药理学方法研究中药的体外效应.应用四甲基偶氮唑蓝(MTT)比色法检测含中药兔血清对肝癌细胞的抑制效应,以Annexin V标记法、DNA含量测定、电子显微镜方法检测含中药血清诱导凋亡及细胞周期阻滞效应.免疫组化法观察含中药血清对P53,P21WAF1/CIP1蛋白的影响,RT-PCR法观察含中药血清对P21WAF1/CIP1 mRNA表达水平的影响.结果含中药血清有一定的抑制肝癌细胞作用,其作用3 d的抑制率为6.6%,作用6 d的抑制率为36.2%.含中药血清作用2 d诱导9.8%±4.0%的肝癌细胞凋亡,使细胞周期阻滞于S期,并上调P53蛋白、P21WAF1/CIP1 mRNA及蛋白的表达.结论健脾理气药具一定的诱导凋亡及抑制肝癌细胞效应,上调p53,p21WAF1/CIP1基因的表达为分子机制.     

丁酸钠对人胃癌MKN-28细胞增殖的抑制作用及其机制

目的 观察脱乙酰化酶抑制剂丁酸钠对人胃癌MKN-28细胞增殖的抑制作用,并探讨其作用机制.方法 MTT法观察丁酸钠对人胃癌MKN-28细胞的生长抑制作用;流式细胞术检测细胞凋亡及细胞周期;RT-PCR检测丁酸钠作用前后p21WAF1 mRNA的表达.结果 1.0、2.5、5.0 mmol/L丁酸钠作用24、48、72 h均可抑制 MKN-28细胞增殖,其效果具有时间、剂量依赖性(P＜0.05);丁酸钠1.0、2.5、5.0 mmol/L处理72 h后,MKN-28细胞G0/G1期细胞数量显著增加,S期细胞数量显著降低(P＜0.05);细胞凋亡率分别为13.7%±0.8%、20.8%±2.4%、33.6%±2.6%,与对照组2.8%±0.4%相比P均＜0.05;与对照组比较,丁酸钠处理后p21WAF1 转录水平上调.结论 丁酸钠可显著抑制人胃癌MKN-28细胞的生长,其可能的机制是通过上调p21WAF1基因的表达,诱导细胞凋亡,使细胞周期阻滞于G0/G1期.     

丹参酮ⅡA介导p38MAPK信号转导诱导人肝癌细胞凋亡

目的: 研究丹参酮ⅡA诱导人肝癌细胞凋亡和凋亡相关基因表达的p38MAPK信号转导通路, 揭示其抗肝癌的部分机制.方法: 4、8、16 mg/L丹参酮ⅡA分别作用人肝癌SMMC-7721细胞48 h后, 免疫荧光染色观察细胞凋亡情况; 琼脂糖凝胶电泳观察凋亡细胞特征性DNA条带; 流式细胞仪法(Flowcytometry, FCM)检测细胞凋亡和细胞周期; 荧光定量PCR检测Fas和Caspase-3基因mRNA的表达水平; 并比较阻断p38MAPK信号通路后丹参酮ⅡA对肝癌细胞凋亡和Fas和Caspase-3基因mRNA的表达.结果: 丹参酮ⅡA作用48 h后, 荧光显微镜下观察到经Hoechst染色的典型凋亡细胞. 琼脂糖凝胶电泳可见凋亡细胞DNA呈规律性的梯状条带. 4、8、16 mg/L浓度丹参酮ⅡA作用人肝癌细胞后的细胞凋亡率分别为12.83%±1.51%, 17.86%±2.70%和29.24%±7.58%, 与对照组6.30%±2.08%比较均有显著性差异( P<0.01); 阻断p38MAPK信号通路后, 凋亡率和G0/G1期细胞比例明显降低( P<0.01). 8 mg/L丹参酮ⅡA作用人肝癌细胞48 h后Fas mRNA和Caspase-3 mRNA的表达明显上升; 阻断p38MAPK信号通路后, 丹参酮ⅡA作用人肝癌细胞的Fas mRNA和Caspase-3 mRNA的表达明显下降.结论: 丹参酮Ⅱ A 能诱导人肝癌细胞株SMMC-7721凋亡, 阻滞肝癌细胞于G0/G1期. 通过p38MAPK信号转导通路上调Fas、Caspase-3 mRNA的表达可能是其诱导肝癌细胞凋亡的重要机制.     

Effects of tachyplesin on the regulation of cell cycle in human hepatocarcinoma SMMC-7721 cells

AIM: To investigate the effects of tachyplesin on the cell cycle regulation in human hepatcarcinoma cells. METHODS: Effects of tachyplesin on the cell cycle in human hepatocarcinoma SMMC-7721 cells were assayed with flow cytometry. The protein levels of p53, p16, cyclin D1 and CDK4 were assayed by immunocytochemistry. The mRNA levels of p21(WAF1/CIP1) and c-myc genes were examined with in situ hybridization assay. RESULTS: After tachyplesin treatment, the cell cycle arrested at G0/G1 phase, the protein levels of mutant p53, cyclin D1 and CDK4 and the mRNA level of c-myc gene were decreased, whereas the levels of p16 protein and p21(WAF1/CIP1) mRNA increased. CONCLUSION: Tachyplesin might arrest the cell at G0/G1 phase by upregulating the levels of p16 protein and p21(WAF1/CIP1) mRNA and downregulating the levels of mutant p53, cyclin D1 and CDK4 proteins and c-myc mRNA, and induce the differentiation of human hepatocacinoma cells.     

去甲斑蝥素诱导人肝癌细胞凋亡的研究

目的探讨去甲斑蝥素（NCTD）对人肝癌细胞SMMC-7721凋亡的影响。方法用MTT方法检测人肝癌细胞SMMC-7721的生长抑制率。应用流式细胞仪检测细胞周期和凋亡。结果 NCTD对人肝癌细胞SMMC-7721有生长抑制作用,随浓度升高、时间延长作用增强,呈剂量和时间效应关系。流式细胞仪示SMMC-7721细胞的S期细胞明显增多,G0/G1期细胞减少,凋亡率上升（P〈0.05或〈0.01）。结论 NCTD能显著抑制人肝癌SMMC-7721细胞的生长,其可能与抑制SMMC-7721细胞增殖,诱导细胞凋亡有关。     

RNA干扰p21对肝癌细胞SMMC-7721增殖的影响

目的:探讨RNA干扰技术沉默p21基因对肝癌细胞SMMC-7721增殖及恶性表型变化的影响.方法:通过慢病毒载体将p21小干扰RNA片段稳定转染入SMMC-7721细胞,通过RT-PCR,Western blot分别检MJp21 Mrna和蛋白表达变化,流式细胞仪检测7721-p2l RNAi组(感染p21 siRNA...     

Selective inhibition of cell growth by activin in SNU-16 cells

AIM: To investigate whether activin regulates the cell proliferation of human gastric cancer cell line SNU-16 through the mRNA changes in activin receptors, Smads and p21CIP1/WAF1. METHODS: The human gastric cancer cell lines were cultured, RNAs were purified, and RT-PCRs were carried out with specifically designed primer for each gene. Among them, the two cell lines SNU-5 and SNU-16 were cultured with activin A for 24, 48 and 72 h. The cell proliferation was measured by MTT assay. For SNU-16, changes in ActRⅠA, ActRⅠB, ActRⅡA, ActRⅡB, Smad2, Smad4, Smad7, and p21CIP1/WAF1 mRNAs were detected with RT-PCR after the cells were cultured with activin A for 24, 48 and 72 h. RESULTS: The proliferation of SNU-16 cells was down regulated by activin A whereas other cells showed no change. Basal level of inhibin/activin subunits, activin receptors, Smads, and p21CIP1/WAF1 except for activinβB mRNAs was observed to have differential expression patterns in the human gastric cancer cell lines, AGS, KATOⅢ, SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-638, SNU-668, and SNU-719. Interestingly, significantly higher expressions of ActRⅡA andⅡB mRNAs were observed in SNU-16 cells when compared to other cells. After activin treatment, ActRⅠA,ⅠB, andⅡA mRNA levels were decreased whereas ActRⅡB mRNA level increased in SNU-16 cells. Smad4 mRNA increased for up to 48 h whereas Smad7 mRNA increased sharply at 24 h and returned to the initial level at 48 h in SNU-16 cells. In addition, expression of the p21CIP1/WAF1, the mitotic inhibitor, peaked at 72 h after activin treatment in SNU-16 cells. CONCLUSION: Our results suggest that inhibition of cell growth by activin is regulated by the negative feedback effect of Smad7 on the activin signaling pathway, and is mediated through p21CIP1/WAF1 activation in SNU-16 cells.     

Effects of tachyplesin on proliferation and differentiation of human hepatocellular carcinoma SMMC—7721 cells

AIM: To investigate the antitumor activities of tachyplesin on human hepatocellular carcinoma (HCC) cells. METHODS: Tachyplesin, isolated from acid extracts of Chinese horseshoe crab (Tachypleus tridentatus) hemocytes, was used to treat the human HCC cell line SMMC-7721. Effects of tachyplesin on the proliferation of SMMC-7721 cells were measured with trypan blue dye exclusion test and HE staining. The morphology and ultrastructure of the cells were examined by light microscopy and transmission electron microscopy, respectively. The activities of gamma-glutamyltransferase (gamma-GT) and tyrosine aminotransferase (TAT) were assayed with biochemical methods. The levels of alpha fetoprotein (alpha-FP), proliferating cell nuclear antigen (PCNA), p21( WAF1/CIP1) and c-myc were examined by immunocytochemistry. RESULTS: After treatment with tachyplesin 3.0 mg/L, the proliferation of SMMC-7721 cells was inhibited significantly, with the cell growth inhibitory rate amounted to 55.57 % and the maximum cell mitotic index declined by 43.68 %. The morphology and ultrastructure underwent restorational alteration. The activity of gamma-GT declined while TAT activity increased obviously, and the levels of alpha-FP and PCNA decreased. Moreover, the expression of p21(WAF1/CIP1) protein was up-regulated and that of c-myc protein was down-regulated. CONCLUSION: Tachyplesin could effectively inhibit the proliferation of hepatocarcinoma cells, reverse the malignant morphological and ultrastructural characteristics, alter the levels of enzymes and antigens, regulate the expression of differentiation-associated oncogene and tumor suppressor gene, and induce hepatocarcinama cell differentiation.     

The effects of the histone deacetylase inhibitor valproic acid on cell cycle, growth suppression and apoptosis in multiple myeloma

The aim of this study was to evaluate the effects of valproic acid (VPA), as a histone deacetylase inhibitor, on myeloma cell lines and on sorted human bone marrow multiple myeloma cells. VPA induced accumulation of acetylated histones, potently inhibited proliferation in a dose-dependent manner and induced apoptosis in all myeloma cell lines tested as well as in sorted primary multiple myeloma cells. Cell cycle analysis indicated an arrest in G0/1 phase in response to VPA. Accumulation of p21 and reduced levels of cyclin D1 were detected. The production of vascular endothelial growth factor was significantly inhibited by VPA. These results provide the framework for clinical trials.     

p53-dependent G1 arrest involves pRB-related proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein.

The cell cycle regulatory tumor suppressor proteins p53 and pRB are targeted for inactivation by several tumor viruses, including the high-risk types of human papillomaviruses (HPVs) via interactions of the HPV E6 and E7 oncoproteins with p53 and pRB, respectively. p53 plays a central role in a signal transduction pathway that mediates G1 arrest after DNA damage, though the mechanism by which G1 arrest occurs has not been elucidated. The cyclin-associated protein p21waf1/cip1 has recently been shown to be induced by p53 and to inhibit cyclin complex-mediated phosphorylation of pRB in vitro. Thus, we investigated a possible role for pRB in the p53-mediated DNA damage response. After gamma-irradiation, cells expressing wild-type p53 arrested in G1, contained increased levels of WAF1/CIP1 mRNA, and demonstrated accumulation of hypophosphorylated pRB. In contrast, cell lines with abnormal p53 genes or with p53 functionally inactivated by the E6 oncoprotein of HPV16 (a high-risk HPV) failed to arrest in G1, did not elevate WAF1/CIP1 mRNA, and did not accumulate hypophosphorylated pRB. Despite apparently normal elevation of p53 protein and WAF1/CIP1 mRNA after irradiation, cells expressing HPV16 E7 also failed to arrest in G1 and did not accumulate hypophosphorylated pRB. Disruption of RB genes alone did not totally abrogate this G1 arrest. Our results suggest that p53 indirectly regulates phosphorylation of pRB and that pRB and/or other pRB-like molecules that bind to HPV16 E7 participate in the DNA damage-mediated G1 arrest signal. In the process of HPV infection, the HPV E6 and E7 oncoproteins may undermine this cell cycle checkpoint, contributing to the accumulation of genetic alterations during tumorigenesis.