The Effect of Nano-apatite on the Expression of Telomerase Gene of Human Hepatocellular Carcinoma Cells

To investigate the effect of nano-apatite on the expression of the telomerase gene of human hepatocellular carcinoma cell lines and further explore the mechanism of the nano-apatite inhibiting cancer cells. Using the hybridization in situ method to detect the expression of the telomerase gene of human hepatocellular carcinoma cells treated with the nano-apatite for 4 h at 37 ℃ . The hybridization in situ showed that the cytoplasm of the positive cells was stained in nigger- brown. The positive cell rate of the control group was 88.49% , the cisplatin group was 25.6% , the nano-apatite group was 63.6% . The activity of telomerase gene was both obviously dedined comparing with the control group and the difference had significance （ p 〈 0. 05, p 〈 0.01 ）. The nanoapatite obviously inhabit the expression of the telomerase gene of human hepatocellular carcinoma cells.

Cyclin L2, a novel RNA polymerase Ⅱ-associated cyclin, is involved in pre-mRNA splicing and induces apoptosis of human hepatocellular carcinoma cells

We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

Holotransferrin Enhances Selective Anticancer Activity of Artemisinin against Human Hepatocellular Carcinoma Cells

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. （the blue-green herb） in the early 1970s, which has been confirmed for effectively treating malaria, Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill can- cer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransfer- fin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling （TUNEL） and col- ony formation assay, respectively. The results showed that artemisinin at various concentrations signifi- cantly inhibited growth, colony formation and cell viability of SMMC-7721 cells （P〈0.05）, likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells （P〈0.01）. Our data suggest that treatment with artemisinin leads to inhibition of viability and pro- liferation, and apoptosis of SMMC-7721 ceils. Furthermore, we observed that holotransferrin signifi- cantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.

EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

Objective： To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas （HCC） cells at the level of gene and protein. Methods： Three methods, representational difference analysis （RDA） of cDNA, microarrays and fluorescence in situ hybridization （FISH） were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results： Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion： Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.

Effects of SAHA on proliferation and apoptosis of hepatocellular carcinoma cells and hepatitis B virus replication

AIM: To investigate the effects of suberoylanilide hydroxamic acid(SAHA) on proliferation and apoptosis of a human hepatocellular carcinoma cell line(HepG2.2.15) and hepatitis B virus(HBV) replication.METHODS: HepG2.2.15 cells were treated with different concentrations of SAHA.Cell morphology was examined by confocal laser scanning microscopy,and cell proliferation was determined using a MTT colorimetric assay.Flow cytometry was used to detect apoptosis and determine cell cycle phase,while hepatitis B surface antigen and hepatitis B e antigen content were measured using chemiluminescence.Reverse transcription polymerase chain reaction was performed to measure HBV DNA in cell lysate.RESULTS: Cell proliferation rates were significantly reduced by the addition of SAHA.The inhibitory effect of SAHA on cell proliferation was both time-and dosedependent.After 24 h of treatment with SAHA,the early cell apoptotic rate increased from 3.25% to 21.02%(P = 0.041).The proportion of G0 /G1 phase cells increased from 50.3% to 65.3%(P = 0.039),while that of S phase cells decreased from 34.9% to 20.6%(P = 0.049).After 48 h of treatment,hepatitis B surface antigen and hepatitis B e antigen content increased from 12.33 ± 0.62 to 25.42 ± 2.67(P = 0.020) and 28.92 ± 1.24 to 50.48 ± 1.85(P = 0.026),respectively.Furthermore,HBV DNA content increased from 4.54 ± 0.46 to 8.34 ± 0.59(P = 0.029).CONCLUSION: SAHA inhibits HepG2.2.15 cell proliferation,promotes apoptosis,and stimulates HBV replication.In combination with anti-HBV drugs,SAHA may potentially be used cautiously for treatment of hepatocellular carcinoma.

The receptor for β2GPⅠon membrane of hepatocellular carcinoma cell line SMMC-7721 is annexin Ⅱ

AIM: To evaluate the receptor protein which can specifically bind to β2GPⅠon the membrane of hepatocellular carcinoma (HCC) cell line SMMC-7721, and to study the biological function of the receptor.METHODS: Through β2GPⅠ-affinity chromatography column, the peptid-polysome-mRNA complex, which can specially bind to β2GPⅠ, stayed with the column and was separated from the whole polysome of liver cells, and then eluted and collected. Using cDNA synthesis kit and cDNA PCR kit, the corresponding cDNA was obtained and sequenced. RT-PCR was used to amplify annexinⅡ, and flow cytometry was used to study the competitive binding of annexinⅡ with β2GPⅠto SMMC-7721.RESULTS: A total of 1.1 kb of the cDNA fragment of the specific binding protein of β2GPⅠon liver cell membrane was obtained. The sequence of cDNA shared high homology with human annexinⅡ (98%). AnnexinⅡ was expressed on the membrane of SMMC-7721, and could compete with β2GPⅠfor combining with SMMC-7721.CONCLUSION: The receptor for β2GPⅠon membrane of SMMC-7721 cells is annexinⅡ, which might bridge HBV to infect hepatocytes.

Inhibitory effect of metformin on the proliferation of human hepatoma HepG2 cells and its potential mechanism

Objective： This work aimed to study the inhibitory effect and the related mechanism of metformin （MET） on the proliferation of human hepatoma HepG2 cells. Methods： Human hepatoma HepG2 cells were treated with MET （0, 2, 10, and 50 mM）. The inhibitory effect of MET on the proliferation of HepG2 cells was determined by MTT method. The apoptosis of HepG2 cells was detected by flow cytornetry. The expression of cyclin D1 in HepG2 cells was examined by Western blot. ROS-DHE fluorescence probe was used to stain the reactive oxygen species （ROS） generated by HepG2 cells after treat- ment. Results： MET could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. MET promoted the apoptosis of HepG2 cells. In addition, MET suppressed the expression of cell cycle protein cyclin D1 and induced the produc- tion of ROS in HepG2 cells. Conclusion： MET can inhibit the proliferation of human hepatoma HepG2 cells and induce cell apoptosis. Meanwhile, MET has the ability to decrease the expression of cyclin D1 and induce ROS generation, which may be involved in the mechanism of inhibiting hepatoma cells proliferation.

人脐带间充质干细胞条件培养基及外泌体对肝癌细胞增殖、迁移、侵袭和凋亡的影响

背景:间充质干细胞可通过分泌含细胞因子、生长因子和外泌体的细胞外囊泡调节肿瘤微环境,对肿瘤细胞生物学行为进行精准调控。目的:研究人脐带间充质干细胞条件培养基及外泌体对肝癌细胞生物学特性的影响。方法:收集人脐带间充质干细胞培养上清,通过高速离心并过滤获得人脐带间充质干细胞条件培养基;收集人脐带间充质干细胞培养上清,通过超高速梯度离心法提取人脐带间充质干细胞外泌体。使用PKH26标记人脐带间充质干细胞外泌体,与肝癌细胞MHCC97-H共培养,荧光显微镜观察MHCC97-H细胞摄取外泌体情况。通过CCK-8增殖实验、Transwell迁移和侵袭实验以及流式凋亡实验评估人脐带间充质干细胞条件培养基、人脐带间充质干细胞外泌体对肝癌细胞生物学功能的影响。结果与结论:(1)人脐带间充质干细胞外泌体可被MHCC97-H细胞摄取且主要分布于细胞质中;(2)人脐带间充质干细胞条件培养基处理后,MHCC97-H细胞的增殖、迁移和侵袭能力显著增强(P<0.001,P<0.05,P<0.01),凋亡能力显著下降(P<0.001);而人脐带间充质干细胞外泌体处理后,MHCC97-H细胞的增殖能力下降(P<0.001),迁移、侵袭及凋亡能力显著增强(P<0.001);(3)结果表明,人脐带间充质干细胞条件培养基具有促进MHCC97-H细胞增殖、迁移、侵袭和抑制凋亡的能力;而人脐带间充质干细胞外泌体则具有促进MHCC97-H细胞迁移、侵袭及凋亡和抑制增殖的能力。

Optimization of the microwave-assisted extraction of phlorotannins from Saccharina japonica Aresch and evaluation of the inhibitory effects of phlorotannin-containing extracts on HepG2 cancer cells

The use of a microwave-assisted extraction （MAE） method for the extraction ofphlorotannins from Saccharinajaponica Aresch （S.japonica） has been evaluated with particular emphasis on the influential parameters, including the ethanol concentration, solid/liquid ratio, extraction time, extraction temperature, and microwave power. The MAE procedure was optimized using single-factor design and orthogonal array design （OAD）. The content of total phlorotannins in S. japonica was determined using a Folin-Ciocalteu （FC） assay. A maximum total phlorotannin content of 0.644 mg of phloroglucinol equivalent per gram of dry weight plant （mg PGE/g DW） was obtained using the optimized model, which included an ethanol concentration of 55%, solid/liquid ratio of 1：8, extraction time of 25 min, irradiation power of 400 W, and temperature of 60~C. Under similar conditions, the application of a conventional extraction method led to a lower phlorotarmin yield of 0.585 mg PGE/g WD. These results demonstrated that the MAE approach provided better results for the extraction ofphlorotarmins from S.japonica and was a promising technique for the extraction of phenolic compounds from S. japonica and other materials. In addition, screening tests for the inhibitory activity showed that the phlorotannin-containing extracts significantly inhibited the growth of human hepatocellular carcinoma cells （HepG2） by inducing their apoptosis. The morphological changes that occurred during cell apoptosis were characterized using Hoechst33258 staining.

重楼皂苷Ⅰ对肝癌细胞的增殖及凋亡的影响

探讨重楼皂苷Ⅰ(paris saponinⅠ,PSⅠ)在体外对人肝癌SMMC-7721细胞株增殖和凋亡的影响及相关机制.MTT法检测PSⅠ对肝癌SMM-C7721细胞株的增殖抑制作用,Hoechst 33528染色法观察细胞核的形态学变化,流式细胞术Propidium iodide(PI)染色检测细胞周期及凋亡的情况,免疫印迹的方法检测Fas、Bcl-2、Bax、细胞周期素D1(cell cycle regulatory factor D1,Cyclin D1)和细胞周期素E(cell cycle regulatory factor E,Cy-clin E)的表达情况.PSⅠ能时间和浓度依赖性的抑制肝癌SMMC-7721细胞的增殖,与对照组比较,差异有统计学意义(P<0.05).干预后的SMMC-7721细胞染色质浓缩,核碎裂,凋亡小体形成,呈典型的凋亡变化.细胞周期阻滞于G1期,并且有凋亡峰形成,呈浓度依赖性.PSⅠ能浓度依赖性地上调Fas和Bax的表达,下调Bcl-2、Cyclin D1和Cyclin E蛋白的表达水平.PSⅠ可能是通过阻滞肿瘤细胞的生长及诱导细胞凋亡等机制,从而抑制肝癌细胞的增殖.

甲基莲心碱体外抑制人肝癌细胞增殖和侵袭的作用机制研究

目的探讨甲基莲心碱(neferine,Nef)对人肝癌HepG2和Bel-7402细胞侵袭的影响和作用机制。方法人肝癌HepG2和Bel-7402细胞体外培养,经不同浓度的甲基莲心碱处理后,CCK-8法检测细胞的增殖,Transwell细胞体外侵袭实验观察Nef对细胞侵袭能力的影响;免疫印迹检测Rho相关蛋白的表达。结果 CCK-8结果显示,与对照组比较,甲基莲心碱干预组抑制人肝癌HepG2和Bel-7402细胞增殖,并呈剂量-效应关系(P<0.05);Transwell细胞体外侵袭实验显示,3μmol·L^(-1)甲基莲心碱明显抑制HepG2和Bel-7402细胞的侵袭;免疫印迹结果显示,3μmol·L^(-1)甲基莲心碱作用HepG2和Bel-7402细胞12 h后,RhoA、RhoC和ROCK表达明显降低。结论甲基莲心碱可体外抑制HepG2和Bel-7402细胞的增殖和侵袭,其抑制肝癌细胞的侵袭可能与抑制RhoA、RhoC和ROCK蛋白的表达有关。

黄芩素对人肝癌细胞株SMMC-7721体外迁移及侵袭的影响

目的:通过观察黄芩素对人肝癌细胞株SMMC-7721体外迁移侵袭能力及Ezrin、MMP-9、VEGF表达的影响,探讨黄芩素对人肝癌细胞株SMMC-7721体外迁移侵袭的作用及其可能机制。方法:体外培养SMMC-7721,经黄芩素干预后用划痕愈合实验和侵袭小室实验分别检测黄芩素对SMMC-7721细胞迁移运动能力和侵袭能力的影响;RT-PCR和免疫细胞化学法检测黄芩素作用于SMMC-7721细胞48h后对Ezrin、MMP-9、VEGF mRNA和蛋白表达的影响。结果:划痕愈合实验显示各浓度实验组划痕愈合宽度占原宽度的比例比对照组明显下降(P<0.05);体外侵袭实验显示实验组穿过人工基底膜胶的细胞数明显减少(P<0.05);实验组Ezrin、MMP-9、VEGF mRNA和蛋白表达均明显下调(P<0.05)。结论:黄芩素可能通过直接或间接下调Ezrin、MMP-9、VEGF mRNA和蛋白的表达而抑制人肝癌细胞株SMMC-7721体外迁移侵袭,传统中药黄芩可望为肝癌临床治疗提供新的安全、有效、价廉的治疗方法。

川芎嗪对人肝癌耐药细胞Bel-7402/DXR多柔比星蓄积的影响

目的考察中药川芎有效成分川芎嗪(tetramethylpyrazine,TMP)对人肝癌耐药细胞Bel-7402/DXR中多柔比星(DXR)蓄积的影响。方法将人肝癌细胞耐药株Bel-7402/DXR及其亲本细胞Bel-7402分为6组:亲本空白对照组(Parental)、耐药空白对照组(Resistance)、亲本多柔比星组(Parental+DXR)、耐药多柔比星组(Resistance+DXR)、TMP组(Resistance+DXR+TMP)、维拉帕米(VRP)阳性对照组(Resistance+DXR+VRP),荧光显微镜下观察细胞内DXR荧光强度,流式细胞术检测细胞内DXR的平均荧光强度。结果荧光显微镜结果显示,(Resistance+DXR)组、TMP组、VRP组,其细胞内DXR荧光强度分别占Parental+DXR组的(50.03±6.01)%、(119.34±5.4)%、(169.25±21.0)%;流式细胞术结果显示:Resistance+DXR组、TMP组、VRP组细胞内DXR平均荧光强度分别为Parental+DXR组的(82.08±6.98)%、(134.43±39.5)%、(262.74±47.18)%。结论TMP可使人肝癌耐药细胞Bel-7402/DXR中抗癌药多柔比星的蓄积增加,其机制可能与逆转P-gp对药物的外排功能有关。

白花丹醌对人肝癌细胞HepG2侵袭和凋亡的影响

目的探讨白花丹醌对人肝癌HepG2细胞侵袭和凋亡的影响。方法人肝癌HepG2细胞进行体外培养,经不同浓度的白花丹醌处理后,MTT法检测细胞的增殖抑制作用;Transwell细胞体外侵袭实验观察细胞的侵袭能力;流式细胞术Annexin V/PI双染法检测细胞凋亡情况;免疫组化法检测细胞内Bax、Bcl-2蛋白的表达水平。结果 MTT结果显示,与对照组比较,白花丹醌干预组抑制HepG2细胞增殖,并呈剂量-效应关系(P<0.05);Transwell细胞体外侵袭实验显示,随白花丹醌用药浓度的递增,细胞侵袭能力明显下降,尤其以4、8μmol·L^(-1)明显(P<0.01);流式细胞术结果显示,白花丹醌作用HepG2细胞48 h,4、8μmol·L^(-1)组细胞凋亡率均明显高于对照组;免疫组化显示,随着白花丹醌浓度增加,Bax蛋白表达增强,Bcl-2蛋白表达减弱,并呈剂量依赖性(P<0.01)。结论白花丹醌能够抑制HepG2细胞增殖,促进HepG2细胞凋亡,同时具有抑制HepG2细胞侵袭的能力。

载多西紫杉醇脂质微泡联合超声靶向微泡破裂对人肝癌细胞增殖和凋亡影响的初步研究

目的:初步研究载多西紫杉醇脂质微泡(docetaxel-loaded lipid microbubbles,LDLM)联合超声靶向微泡破裂(ultrasound targeted microbubbles destruction,UTMD)对人SMMC-7721肝癌细胞增殖和凋亡的影响。方法:体外培养人SMMC-7721细胞,确定适宜的多西紫杉醇浓度,细胞分为6组,比较各组的细胞增殖抑制率、超微结构改变、凋亡率和细胞周期分布。结果:载药微泡+超声组的细胞增殖抑制率明显高于其他组,与其他各组相比差异有统计学意义(P=0.000);电镜下观察载药微泡+超声组的凋亡细胞最多;流式细胞仪检测结果显示载药微泡+超声组的细胞凋亡率最高(P=0.000),LDLM+US组G0/G1期细胞比例明显减少,为(0.79±0.27)%(P=0.000),G2/M期细胞比例升高最明显,为(90.54±0.48)%(P=0.000)。结论:LDLM联合UTMD可抑制人SMMC-7721细胞的增殖、促进其凋亡,为后续进一步从蛋白、基因层面探索其作用的机制奠定了重要基础。

水蛭提取物对肝癌HepG2细胞DNA去甲基化作用研究

目的探讨水蛭提取物对肝癌HepG2细胞DNA甲基转移酶(DNMTs)表达的抑制作用。方法采用MTT法检测水蛭提取物对肝癌HepG2细胞的增殖抑制作用,根据回归方程计算出半数抑制浓度(IC50)。以1/2 IC50含药量的水蛭提取物处理肝癌HepG2细胞,并以5-脱氧杂氮胞苷(5-Aza-cdR)处理作阳性对照,处理72 h时采用逆转录-聚合酶链反应(RT-PCR)、免疫组化等方法检测肝癌HepG2细胞DNMT1、DNMT3a、DNMT13b mRNA及蛋白表达水平。结果肝癌HepG2细胞DNMT1呈高表达,DNMT3a、DNMT3b呈中低度表达。经水蛭提取物处理后肝癌HepG2细胞DNMT1、DNMT3a表达水平明显下降,DNMT3b基本不表达,与空白对照组比较差异有统计学意义(P<0.01),与5-Aza-cdR组比较,水蛭提取物组降低DNMT1作用弱于5-Aza-cdR,降低DNMT3a、DNMT3b作用明显优于5-Aza-cdR组(P<0.05)。结论水蛭提取物能抑制肝癌HepG2细胞DNTMs表达,参与DNA去甲基化作用可能是水蛭抗肿瘤的机制之一。

重组人血管内皮抑素体外对肝癌HCCLM6细胞转移侵袭的影响

目的研究重组人血管内皮抑素在体外对人肝癌HCCLM6细胞粘附、迁移及侵袭的抑制作用。方法分别以25、50、100、200、400μg/ml的重组人血管内皮抑素在体外处理HCCLM6细胞,采用MTT法检测药物对细胞增殖的影响,利用transwell小室及matrigel胶体外检测药物对细胞迁移、侵袭及粘附的影响。结果重组人血管内皮抑素作用HCCLM6细胞72h后对细胞增殖存在一定的抑制作用;重组人血管内皮抑素可明显抑制HCCLM6细胞与matrigel的黏附、对人工基底膜的侵袭力和迁移力,且抑制作用均呈剂量依赖性。结论重组人血管内皮抑素具有直接抑制人肝癌细胞体外侵袭转移和增殖作用,其机制有待进一步探讨。

二甲双胍对人肝癌细胞 HepG2增殖及脂肪酸合酶的影响

目的二甲双胍治疗的糖尿病患者癌症发生风险较其他药物治疗者显著降低。探讨二甲双胍在人肝癌细胞HepG2中的抗肿瘤活性与脂肪酸合酶的关系。方法选取不同浓度(1、5、10、15 mmol/L)二甲双胍处理HepG2细胞24、48、72 h,用CCK-8法检测其对细胞增殖的影响。同时设阳性对照(紫杉醇10μg/m L),阴性对照(二甲双胍0 mmol/L)。设0、5、10、15 mmol/L二甲双胍处理72 h,用Western blot检测腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)、磷酸化AMPK(P-AMPK)、脂肪酸合酶(fatty acid synthase,FASN)、磷酸化哺乳动物雷帕霉素靶蛋白(P-m TOR)、磷酸化蛋白激酶B(P-Akt)蛋白表达,RT-PCR检测FASN mRNA的表达水平。结果 1、5、10、15 mmol/L二甲双胍干预24、48、72 h,分别随二甲双胍浓度与处理时间的增加,HepG2细胞的增殖抑制作用逐渐增强(P<0.05),且呈时间和浓度依赖性。其中10 mmol/L二甲双胍处理后增殖抑制率在72 h接近50%、15 mmol/L二甲双胍处理后增殖抑制率均>50%。0、5、10、15 mmol/L二甲双胍处理HepG2细胞72 h后,P-AMPK的蛋白表达随药物浓度增高而上调,P-m TOR、FASN的蛋白表达随药物浓度增高而下调,三者在10、15 mmol/L二甲双胍作用后的表达较阴性对照的差异均有统计学意义(P<0.05);而P-Akt蛋白表达在二甲双胍各浓度间差异无统计学意义(P>0.05)。FASN mRNA的表达随药物浓度增加呈下降趋势,5、10、15 mmol/L二甲双胍作用后表达量均较阴性对照显著下降(P<0.05)。结论二甲双胍的的抗肿瘤作用与其激活AMPK、抑制m TOR和FASN有关。二甲双胍虽然抑制了m TOR的活化,但没有造成Akt的反馈性激活。

不同来源桑黄粗多糖诱导肝癌细胞HepG2 S期阻滞及凋亡的研究

通过MTT、流式细胞计数、qRT-PCR等方法,研究不同来源桑黄粗多糖对肝癌细胞HepG2的抑制作用,并探讨其作用机制。结果发现,来源于不同地区、不同寄主桑黄子实体粗多糖对肝癌细胞HepG2的增殖抑制作用差异显著,其中采自四川攀枝花紫油树(SH8)、福建未知树种(SH12)上的桑黄子实体粗多糖对HepG2有一定的促增殖作用;但大多数桑黄子实体粗多糖对HepG2有抑制作用,且随多糖浓度的增加抑制作用增强。采自新疆阿克苏桑树(SH1)桑黄子实体粗多糖(800μg/mL)/至3495%±450%,S期细胞比率从2727%±173%升高至6216%±312%,G2/M期细胞比率从1419%±088%降低至289%±197%;凋亡结果显示,SH1粗多糖(800μg/mL)作用48 h后,早期凋亡细胞比率从正常组026%±009%提高至881%±048%,晚期凋亡细胞比率从正常组026%±011%提高至5175%±282%,坏死细胞比率由正常组089%±007%提高至1701%±129%;qRT-PCR结果显示,SH1粗多糖处理后细胞内周期调控相关蛋白P21、P27、P57、Cyclin A、Cyclin B、Cyclin D、Cyclin E、CDK1、CDK3和CDK4在mRNA水平表达显著上调,CDK2表达显著下调;凋亡相关基因Bcl-XS、Bad、Bax、Bid和Bcl-2的表达水平也显著升高。结果表明,桑黄粗多糖通过调控激活Cyclin A-CDK3、Cyclin E-CDK3、CyclinD-CDK4复合物促进HepG2细胞由G1期进入S期,抑制Cyclins-CDK2复合物诱导HepG2细胞S期阻滞,同时上调促凋亡蛋白基因Bcl-XS、Bad、Bax、Bid的表达,诱导细胞凋亡。