Influence of Toxoplasma gondii on in vitro proliferation and apoptosis of hepatoma carcinoma H7402 cell

Objective:To discuss the influence of tachyzoite of Toxoplasma gondii（T.gondii） RH strain on proliferation and apoptosis of hepatoma carcinoma（HCC） H7402 cell.Methods:The HCC H7402 cell in logarithmic phase and tachyzoite of T.gondii RH strain in different concentrations（1×107/mL,2×107/mL.4×107/mL,8×107mL and 16×107/mL） were co-cultured.CCK-8was utilized to determine the inhibition rate of T.gondii tachyzoite on H7402 cell growth.Flow cytometry was used to detect the change of cell cycle.RT-PCR method was used to detect the expression of cyclinB1 and cdc2-two genes related to cell cycle.Western blot method was used to detect the expression of apoptosis-related proteins Caspase-3 and Bcl-2.Results:The tachyzoite of T.gondii RH strain can inhibit the proliferation of HCC H7402 cells.The inhibition rate of tumor cell growth increased with the increase of concentration of T.gondii tachyzoite.With the increase of concentration of T.gondii tachyzoite,the proportion of G0/G1 phase of H7402 cell increased,the proportion of S phase decreased,and PI value decreased accordingly.The expression of cyclinB1 and cdc2 genes decreased with the increase of the concentration of T.gondii tachyzoite.With the increase of the concentration of tachyzoite of T.gondii RH strain,the expression quantity of Caspase-3 in H7402 cell increased,but the expression quantity of Bcl-2protein decreased.Conclusions:T.gondii can inhibit the in vitro proliferation of HCC H7402 cell,and induce its apoptosis.This effect shows a trend of concentration-dependent increase.Moreover,it is related to the down-regulation of cyclinB1 and cdc2（cell cycle-related genes）,the increase of apoptosis-related protein Caspase-3.and the decreasc of Bcl-2 expression.

Ethanol extract of Ardisiae Japonicae Herba inhibits hepatoma carcinoma cell proliferation in vitro through regulating lipid metabolism

Objective:The aim of this study is to discover the possible working mechanisms of Ardisiae Japonicae Herba(AJH)on hepatoma carcinoma(HCC).Methods:In this study,ethanol extract of AJH was prepared and used to treat HCC cell in vitro.Furthermore,a genomic wide RNA sequencing(RNA-seq)was performed to screen deregulated genes in HCC cells after the treatment of AJH extract.The gene and protein expression related to lipid metabolism in HCC cells were also investigated to validate the results obtained from RNA-seq.Results:AJH extract could inhibit HCC cell proliferation in vitro.RNA-seq analysis has identified 1,601 differentially expressed genes(DEGs,fold change≥2.0 or fold change≤0.5,P<0.05)in HCC after AJH extract treatment,which included 225 up-regulated genes and 1,376 down-regulated genes.KEGG pathway analysis of DEGs demonstrated that lipid metabolism was a potential pathway related to AJH treatment.In agreement with the RNA-seq data,q PCR and Western-blot analysis indicated that expression of genes and proteins related to lipid metabolism(SREBP1,ACC,ACLY and FASN)were significantly downregulated in AJH treatment group as compared with the control group.Furthermore,AJH extract could also decrease lipid contents and cellular free fatty acid levels in HCC cells.Conclusion:Ethanol extract of AJH could inhibit HCC cell proliferation in vitro,the possible mechanism may be related to the inhibition of lipid metabolism.

Combined serum hepatoma-specific alpha-fetoprotein and circulating alpha-fetoprotein-mRNA in diagnosis of hepatocellular carcinoma

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, its prognosis is poor, and early detection is of utmost importance. Although alpha-fetoprotein (AFP) is a useful marker for detecting and monitoring HCC development, the false-negative or false-positive rates with AFP alone may be as high as 30%-40% for patients with small HCCs. To enhance the specificity and accuracy of AFP measurements for HCC, we analyzed AFP expression states in livers, detected the hepatoma-specific AFP (HS-AFP) fraction and AFP-mRNA from peripheral blood mononuclear cells, and explored their clinical implications for HCC diagnosis. METHODS: AFP expression and hepatocyte distributions in liver specimens were investigated by an immunohistoche- mical assay. Total RNAs were extracted from circulating blood, synthesized to cDNA through random primers and reverse transcriptase, and fragments of the AFP gene were amplified by a nested-PCR assay. The HS-AFP fraction was separated by lectin-affinity chromatography and its level was detected by radioimmunoassay. RESULTS: The incidence of AFP was 73.3% in HCC tissues and its expression in HCCs with moderate or low differentiation was significantly stronger than that of HCCs with high differentiation (P<0.05). The incidence of AFP gene fragments was 100% in HCCs, and 60% in paracancerous tissues (P<0.01). In the HCC and liver cirrhosis groups, the incidence of HS-AFP was 91.7% and 18% (P<0.01), and of AFP-mRNA was 56.7% and 16% (P<0.01), respectively, and neither was found in controls.HS-AFP or AFP-mRNA was not significantly related to size or number of HCC, but to its differentiation, metastasis, and relapse (P<0.05). CONCLUSIONS: Different AFP expression is present in different parts of HCC tissues. HS-AFP and AFP-mRNA fragments improve sensitivity and specificity, and both are useful markers to diagnose HCC or monitor metastasis and relapse.

Current and future treatments for hepatocellular carcinoma

Hepatocellular carcinoma(HCC) represents a unique challenge for physicians and patients.There is no definitively curative treatment.Rather,many treatment and management modalities exist with differing advantages and disadvantages.Both current guidelines and individual patient concerns must be taken into account in order to properly manage HCC.In addition,quality of life issues are particularly complex in patients with HCC and these concerns must also be factored into treatment strategies.Thus,considering all the options and their various pros and cons can quickly become complex for both clinicians and patients.In this review,we systematically discuss the current treatment modalities available for HCC,detailing relevant clinical data,risks and rewards and overall outcomes for each approach.Surgical options discussed include resection,transplantation and ablation.We also discuss the radiation modalities:conformal radiotherapy,yttrium 90 microspheres and proton and heavy ion radiotherapy.The biologic agent Sorafenib is discussed as a promising new approach,and recent clinical trials are reviewed.We then detail currently described molecular pathways implicated in the initiation and progression of HCC,and we explore the potential of each pathway as an avenue for drug exploitation.We hope this comprehensive and forward-looking review enables both clinicians and patients to understand various options and thereby make more informed decisions regarding this disease.

Role of liver resection in the management of multinodular hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the third leadingcause of cancer related deaths worldwide. Various treatment modalities have been applied to HCC depend-ing on the tumor load, functional capacity of the liver and the general condition of the patient. According to Barcelona Clinic Liver Cancer staging strategy and The American Association for the Study of Liver Disease guidelines, surgical resection is not advocated in the tretment of multinodular HCC. Despite this, many recent clinical studies show that, resection can achieve good results in patients with multinodular HCC and 5-year survival rate around 40% can be reached. If resection or transplantation is not performed, these patients are usually managed with palliative procedures such as transarterial chemoembolization, radioembolization and cytotoxic chemotherapy and 5-year survival of this group of patients will be extremely low. Although survival rates are lower and complications may be increased in this group of patients, liver resection can safely be performed in selected patients in experienced centers for the management of multinodular HCC.

Percutaneous Radiofrequency Ablation of Hepatocellular Carcinoma against the Diaphragm: Is Artificial Ascites Necessary?

Purpose: To evaluate the utility of artificial ascites induction for radiofrequency ablation (RFA) of peridiaphragmatic hepatocellular carcinoma (HCC) through retrospective cohort analysis comparing characteristics and complications of peridiaphragmatic HCC without the use of artificial ascites to non-peridiaphragmatic HCC. Materials and Methods: IRB approval was obtained. From September 2003 to December 2008, 150 consecutive patients with hepatic tumors received percutaneous RFA. 110 patients had presumed HCC, and of those 21 had lesions abutting the diaphragm. Of the remaining 89 patients with non-peridiaphragmatic HCC lesions, 21 were randomly selected for the comparison group. RFA volume, major and minor complication rates, pain, technical success, and recurrence rates were compared between the two groups. Results: There was no statistical difference in RFA volume (P = 0.962), overall major complication rate (P = 0.343) and minor complication rate (P = 0.118) between the two groups. However, the peridiaphragmatic group that underwent RFA with a clustered-needle demonstrated a statistically significant higher proportion of major complications compared to the non-peridiaphragmatic clustered-needle group (P = 0.033). There was no statistical difference in pain severity (P = 0.8802) or pain location (P = 0.15). There was no statistical difference in technical success rates (P = 1), local tumor progression rates (P = 1), or time to local tumor recurrence (P = 0.67). Conclusion: Artificial ascites induction for RFA of HCC lesions adjacent to the diaphragm may not be necessary, although clustered electrode technique should be avoided in this location as they present with a higher complication rate.

Inhibition of apoptosis by oncogenic hepatitis B virus X protein: Implications for the treatment of hepatocellular carcinoma

Hepatitis B virus X protein(HBx) plays an important role in the development of hepatocellular carcinoma(HCC). In addition, hepatoma upregulated protein(HURP) is a cellular oncogene that is upregulated in a majority of HCC cases. We highlight here recent findings demonstrating a link between HBx, HURP and anti-apoptosis effects observed in cisplatin-treated HCC cells. We observed that Hep3B cells overexpressing HBx display increased HURP mRNA and protein levels, and show resistance to cisplatin-induced apoptosis. Knockdown of HURP in HBx-expressing cells reverses this effect, and sensitizes cells to cisplatin. The anti-apoptotic effect of HBx requires activation of the p38/MAPK pathway as well as expression of SATB1, survivin and HURP. Furthermore, silencing of HURP using short-hairpin RNA promotes accumulation of p53 and reduces cell proliferation in SK-Hep-1 cells(p53^(+/–)), whereas these effects are not observed in p53-mutant Mahlavu cells. Similarly, HURP silencing does not affect the proliferation of H1299 lung carcinoma cells or Hep3 B HCC cells which lack p53. Silencing of HURP sensitizes SK-Hep-1 cells to cisplatin. While HURP overexpression promotes p53 ubiquitination and degradation by the proteasome, HURP silencing reverses these effects. Inoculation of SK-Hep-1 cancer cells in which HURP has been silenced produces smaller tumors than control in nude mice. Besides, gankyrin, a positive regulator of the E3 ubiquitin ligase MDM2, is upregulated following HURP expression, and silencing of gankyrin reduces HURP-mediated downregulation of p53. In addition, we observed a positive correlation between HURP and gankyrin protein levels in HCC patients(r^2 = 0.778; n = 9). These findings suggest a role for the viral protein HBx and the host protein HURP in preventing p53-mediated apoptosis during cancer progression and establishment of chemoresistance.

Pylephlebitis and liver abscess mimicking hepatocellular carcinoma

OBJECTIVE: To characterize the clinical and radiographic findings in patients with pylephlebitis and liver abscess with an emphasis on the findings that help to differentiate this disorder from portal vein occlusion associated with hepatocellular carcinoma. METHODS: We analyzed the clinical findings and radiographic images of four patients with pylephlebitis and liver abscess(es) who had been misdiagnosed as having hepatocellular carcinoma with portal vein thrombosis. Their medical records were reviewed in terms of clinical presentation, physical findings, laboratory data, treatment, and follow up. RESULTS: All patients undergoing color duplex ultrasonography had an echogenic thrombus within an expanded portal vein with negative color-flow findings within the thrombus. Contrast enhanced CT in all the patients demonstrated portal vein thrombosis associated with 'liver masses'. An intra-abdominal site of infection responsible for the subsequent ascending infection of the portal vein and liver was not identified in any patient on initial CT scan. At presentation, all patients were febrile and three of them had an elevated white blood cell count as well. All patients showed abnormalities of liver function. CONCLUSIONS: Liver abscess(es) associated with pylephlebitis may mimic hepatocellular carcinoma with portal vein thrombosis. Clinical features that help to distinguish the two entities include presence or absence of fever, elevated white blood cell count, elevated alpha-fetoprotein, cirrhosis, and risk factors for hepatocellular carcinoma.

Hepatocellular carcinoma with child Pugh-A Cirrhosis treated with stereotactic body radiotherapy

AIM To evaluate the control, survival, and hepatic function for Child Pugh(CP)-A patients after Stereotactic body radiotherapy(SBRT) in hepatocellular carcinoma(HCC).METHODS From 2009 to 2016, 40 patients with Barcelona Liver Clinic(BCLC) stages 0-B HCC and CP-A cirrhosis completed liver SBRT. The mean prescription dose was 45 Gy(40 to 50 Gy in 4-5 fractions). Local relapse, defined as recurrence within the planning target volume was assessed with intravenous multiphase contrast computed tomography or magnetic resonance imaging every 4-6 mo after completion of SBRT. Progression of cirrhosis was evaluated by CP and Model for End Stage Liver Disease scores every 3-4 mo. Toxicities were graded per the Common Terminology Criteria for Adverse Events(v4.03). Median follow-up was 24 mo.RESULTS Forty-nine HCC lesions among 40 patients were analyzed in this IRB approved retrospective study. Median tumor diameter was 3.5 cm(1.5-8.9 cm). Six patients with tumors ≥ 5 cm completed planned selected transarterial chemoembolization(TACE) in combination with SBRT. Eight patients underwent orthotropic live transplant(OLT) with SBRT as a bridging treatment(median time to transplant was 12 mo, range 5 to 23 mo). The Pathologic complete response(PCR) rate in this group was 62.5%. The 2-year in-field local control was 98%(1 failure). Intrahepatic control was 82% and 62% at 1 and 2 years, respectively. Overall survival(OS) was 92% and 60% at 1 and 2 years, with a median survival of 41 mo per Kaplan Meier analysis. At 1 and 2 years, 71% and 61% of patients retained CPA status. Of the patients with intrahepatic failures, 58% developed progressive cirrhosis, compared to 27% with controlled disease(P = 0.06). Survival specific to hepatic failure was 92%, 81%, and 69% at 12, 18, and 24 mo. There was no grade 3 or higher toxicity. On univariate analysis, gross tumor volume(GTV) < 23 cc was associated with freedom from CP progression(P = 0.05), hepatic failure-specific survival(P = 0.02), and trended with OS(P = 0.10).CONCLUSION SBRT is safe and effective in HCC with early cirrhosis and may extend waiting time for transplant in patients who may not otherwise be immediate candidates.

Rapid Development of Hepatocellular Carcinoma after Eradication of Hepatitis C Virus with Directly Acting Antiviral Treatment

Hepatitis C is a major risk factor for the development of hepatocellular carcinoma (HCC), arising typically on a background of liver cirrhosis. Treatment of hepatitis C has been revolutionized by the addition of oral direct-acting antivirals (DAAs) with sustained virological response (SVR) rates above 90%. There is a recent concern under debate about the increased risk of early HCC recurrence in patients with chronic hepatitis C who were treated with direct-acting antivirals. Nonetheless, these reports mostly focused on patients who were cirrhotic and were already treated for HCC. We report 4 cases of treatment naive, chronic hepatitis C patients who were treated with DAAs and rapidly developed infiltrative HCC despite achieving SVR. Moreover, in our scenario, one of the patients appeared clinically non-cirrhotic until he developed HCC.

Use of a New Algorithm with an Internally Cooled Electrode for Radiofrequency Ablation of Small Hepatocellular Carcinomas

Purpose: To investigate the efficacy of a new algorithm to increase the volume of tissue ablation via gradual ramp-up of power using an internally cooled electrode for ablating hepatomas 3 cm or less. Materials and Methods: 44 patients with 62 hepatomas were treated from March 4, 2004 to May 24, 2009. Ablation with a gradual ramp-up of power was performed using a single needle with an internally cooled electrode. Evaluation for tumor response was performed with 4-phase CT at 24 hours and 3 months. All immediate and follow-up complications were recorded. Results: Complete tumor ablation was achieved in 86%. The ablation volumes were 16 cm3 +/- 12 cm3 for tumors 3 +/- 12 cm3 for tumors 2 - 3 cm. Out of 68 total ablation sessions, there were 2 major complications (pleural effusions) requiring intervention (thoracentesis). Conclusion: Compared with existing techniques using a constant full-power setting, ablation of small hepatomas using an algorithm of gradual ramp-up of power provides comparable rate of complete tumor ablation, adequate ablation volumes, and a low rate of complications that require treatment.

吡喹酮通过5-HT2B受体对肝癌细胞恶性生物学行为的影响

目的探讨吡喹酮(praziquantel,PZQ)对肝癌细胞增殖、迁移和凋亡的影响及其作用机制。方法体外培养Hep3B人肝癌细胞株和Hepa1-6小鼠肝癌细胞株,分为正常对照组、PZQ处理组、5-羟色胺2B(5-HT2B)受体抑制剂组(RS127445组)、5-HT2B受体抑制剂+PZQ处理组(RS127445+PZQ处理组)。采用实时荧光定量PCR(qRTPCR)检测Hep3B人肝癌细胞株和Hepa1-6小鼠肝癌细胞株中5-HT2B受体mRNA相对表达水平,CCK-8法检测细胞增殖情况,划痕实验检测细胞迁移能力,流式细胞术检测细胞凋亡率,western blot法检测Bax、Bcl-2凋亡相关蛋白表达量。结果Hep3B人肝癌细胞株和Hepa1-6小鼠肝癌细胞株5-HT2B受体mRNA相对表达水平正常对照组为1.02±0.09和1.01±0.20,PZQ处理组为1.36±0.16和1.66±0.16,经PZQ处理后5-HT_(2B)受体mRNA相对表达水平均增加(t=3.22、5.07,P均<0.05)。PZQ处理组两种细胞株48 h细胞增殖率为(74.00±4.58)%和(77.00±5.29)%,低于正常对照组(t=9.88、7.47,P均<0.01);72 h细胞增殖率为(71.00±6.08)%和(67.33±7.57)%,低于正常对照组(t=7.87、6.00,P均<0.05)和RS127445+PZQ处理组(t=5.48、3.48,P均<0.05)。PZQ处理组两种细胞株48 h细胞迁移率为(52.91±3.15)%和(17.28±1.78)%,低于正常对照组(t=7.86、13.46,P均<0.01);72 h细胞迁移率为(58.79±3.25)%和(22.29±5.87)%,低于正常对照组(t=11.65、9.57,P均<0.05)和RS127445+PZQ处理组(t=3.13、6.97,P均<0.05)。PZQ处理组两种细胞株72 h细胞凋亡率为(16.13±0.66)%和(20.70±2.85)%,高于正常对照组和RS127445+PZQ处理组(t=27.82、5.65、9.54、4.10,P均<0.01);Bax相对蛋白表达水平分别为1.70±0.18和2.23±0.14,高于正常对照组(t=2.83、7.89,P均<0.05)和RS127445+PZQ处理组(t=9.40、5.25,P均<0.05);Bcl-2相对蛋白表达水平分别为0.52±0.17和0.53±0.02,低于正常对照组(t=3.57、8.39,P均<0.05)和RS127445+PZQ处理组(t=12.09、6.12,P均<0.05)。结论PZQ可通过5-HT2B受体对肝癌细胞的增殖、迁移和凋亡造成影响。

PNPLA3基因高表达对非酒精性脂肪性肝病模型细胞增殖和甘油三酯的影响

目的探讨patatin样磷脂酶域3(PNPLA3)高表达对非酒精性脂肪性肝病(NAFLD)细胞模型细胞增殖和甘油三酯(TG)的影响。方法将人肝癌细胞(SMMC-7721细胞)分为对照组、模型组、PNPLA3过表达组、PNPLA3过表达空载组。模型组:经游离脂肪酸诱导建立NAFLD细胞模型,PNPLA3过表达组、PNPLA3过表达空载组:在造模后经慢病毒原液感染、嘌呤霉素筛选获得。用定量反转录聚合酶链反应(qRT-PCR)、MTT比色法和TG测试技术检测细胞中PNPLA3表达情况、细胞存活率和胞内TG含量。结果qRT-PCR结果显示,模型组PNPLA3基因的表达量高于对照组(P<0.01),PNPLA3过表达组PNPLA3基因表达量高于模型组(P<0.01)。MTT结果显示,模型组细胞相对存活率高于对照组(P<0.05),PNPLA3过表达组细胞相对存活率高于模型组(P<0.01)。TG检测结果显示:模型组TG含量高于对照组(P<0.01),PNPLA3过表达组中胞内TG含量高于模型组(P<0.05)。结论成功构建PNPLA3过表达NAFLD稳转株,PNPLA3的过量表达有助于细胞脂质堆积并增强细胞相对存活率。

河蚌多糖水提取物对多种恶性肿瘤细胞增殖和迁移的影响

目的探讨河蚌多糖水提取物体外抗肿瘤活性。方法通过离心、浓缩和冷冻干燥的方法制备出可溶性的河蚌多糖水提物,分别采用CCK-8实验、细胞划痕实验和Transwell迁移实验检测河蚌多糖水提取物对肝癌细胞Hep3B、恶性黑色素瘤细胞A375和肺癌细胞PC9增殖、迁移能力的抑制作用。结果CCK-8实验表明,河蚌多糖水提取物具有时间-浓度依赖性抑制上述3种恶性肿瘤细胞的增殖;划痕实验和Transwell迁移实验结果表明,与对照组相比,河蚌多糖水提取物能够有效抑制上述3种恶性肿瘤细胞的迁移(P均<0.05)。结论河蚌多糖水提取物可以有效抑制多种恶性肿瘤细胞增殖和迁移,为其后续开发和应用提供一定的参考价值。

肝内胆管细胞癌与肝细胞癌病灶MRI增强扫描表现差异分析

目的分析总结肝内胆管细胞癌(ICC)与肝细胞癌(HCC)病灶增强磁共振成像(MRI)表现差异,为鉴别诊断提供依据。方法2019年12月~2023年7月我院诊治的原发性肝癌(PLC)患者120例,均接受MRI增强扫描,观察病灶强化特点,包括动脉期强化特征、动态强化模式、有无血流灌注异常、有无肝胆期靶征、病灶信号是否均匀、有无远端胆管扩张、有无包膜等特征。应用受试者工作特征曲线(ROC)下面积(AUC)分析MRI增强扫描特征鉴别诊断ICC与HCC的效能。结果在120例PLC患者中,经组织病理学检查诊断ICC者23例和HCC者97例;ICC病灶动脉期边缘强化、动态逐渐强化、肝胆期靶征和远端胆管扩张发生率分别为69.6%、56.5%、65.2%和39.1%,均显著高于HCC病灶的44.3%、17.5%、9.3%和3.1%,而病灶包膜发生率为4.3%,显著低于HCC病灶的45.4%(P<0.05);综合扫描特征,本组病例经MRI诊断ICC者31例,HCC者89例,其鉴别诊断ICC与HCC的敏感度为78.3%,特异度为86.6%,阳性预测值为58.1%,阴性预测值为94.4%。结论术前,可根据MRI增强扫描特征鉴别ICC与HCC提供帮助,为临床做出合理的处置方案提供依据。

白芍总苷对肝癌耐药细胞BEL-7402/ADM耐药性的逆转作用及机制探讨

目的观察白芍总苷(TPG)对肝癌耐药细胞BEL-7402/ADM耐药性的逆转作用,并探讨其作用机制。方法体外培养人肝癌ADM敏感细胞BEL-7402(亲本组)和ADM耐药细胞BEL-7402/ADM(耐药组),将BEL-7402/ADM细胞转染HDAC3基因siRNA干扰质粒(干扰组)及空载质粒(空载组)24 h,用白芍总苷孵育BEL-7402/ADM细胞48 h(用药组)。取亲本组、耐药组、用药组细胞,分别以不同浓度ADM作用48 h;MTT法测算增殖抑制率,计算各组ADM的半数抑制浓度(IC_(50))。取亲本组、耐药组、空载组、干扰组及用药组细胞,分别采用RT-q PCR法和Western blot法检测细胞多药耐药基因MDR1和去乙酰化酶(HDAC3)mRNA和蛋白。结果亲本组、耐药组和用药组细胞ADM的IC_(50)分别为0.139、8.807、4.890 nmo L/L;用药组细胞对ADM耐药的逆转倍数为1.80倍,逆转率为45.23%。MDR1 mRNA及蛋白相对表达量耐药组、空载组>用药组>干扰组>亲本组(P均<0.05或0.01),HDAC3 mRNA及蛋白相对表达量耐药组、空载组>干扰组>亲本组、用药组(P均<0.05或0.01)。结论白芍总苷通过抑制HDAC3的表达,引起MDR1表达下调,从而逆转BEL-7402/ADM细胞的耐药性。

黄芪总黄酮对肝癌HepG-2细胞凋亡的诱导作用

目的:探讨黄芪总黄酮(TFA)对人肝癌HepG-2细胞生长抑制及凋亡诱导作用及其机制。方法:以不同浓度TFA处理HepG-2细胞,MTT法测定细胞增殖抑制率;流式细胞仪(FCM)检测细胞凋亡率;Westernblot检测凋亡相关蛋白Survivin、Bc1-2、Bax的表达。结果:TFA能够抑制体外培养的人肝癌细胞HepG-2生长、诱导细胞凋亡,其作用具有量效关系和时效关系。Western blot检测显示,随着TFA浓度的增加,细胞凋亡抑制蛋白Survivin和Bcl-2的表达量明显降低,而细胞凋亡促进蛋白Bax的表达量显著增加。结论:TFA对HepG-2细胞具有抑制增殖和诱导凋亡的作用,其机制与下调细胞凋亡抑制蛋白Survivin与Bcl-2的表达和上调细胞凋亡促进蛋白Bax的表达有关。

BC047440-siRNA对肝癌HepG2细胞增殖抑制及其分子机制的初步探讨

目的利用小干扰RNA(small interfering RNA,siRNA)抑制肝癌相关基因BC047440的表达,观察其受抑制后对细胞增殖能力的影响并初步分析其抑制增殖的分子机制。方法设计针对BC047440基因的siRNA,构建pGenSil-1-BC047440-siRNA真核表达载体。分别用脂质体法将介导siRNA的真核绿色荧光表达载体pGenSil-1-BC047440-siRNA、空载体质粒pGenSil-1转染人肝癌HepG2细胞建立稳定细胞株;随后实验分为3组:转染siRNA表达载体组(PP2)、转染空质粒组(PP1)和未处理的亲本HepG2细胞组(PP0)。采用荧光定量PCR检测BC047440mRNA表达变化;CCK-8分析细胞增殖活性;平板克隆形成实验检测单个细胞的增殖能力;最后选取NF-κB下游增殖相关基因c-myc、bcl-2、cyclin D1,用荧光定量PCR检测其mRNA水平的变化。结果成功转染并获得PP1、PP2组抗性克隆,PP2组与PP0组比较,BC047440mRNA水平明显降低,抑制率约为75%;细胞增殖实验中PP2组细胞增殖能力明显降低,平板克隆形成实验结果显示PP2组克隆形成率为(8.42±0.82)%,明显低于PP1组[(39.31±5.39)%]和PP0组[(40.96±3.21)%](P<0.01)。检测NF-κB下游增殖相关的几个基因时发现PP2组c-myc mRNA表达明显降低,约为PP0组的12/25;其余基因无明显变化。结论干扰BC047440基因表达可以抑制肝癌细胞的增殖,初步揭示BC047440基因可以对c-myc起正向调控作用,从而促进肝癌细胞的增殖,提示抑制BC047440基因可能成为控制人肝癌细胞生长的有效靶点。

杜仲叶绿原酸提取物联合西红花苷对肝癌细胞胆固醇代谢的影响

目的观察杜仲叶绿原酸提取物(CAEF)联合西红花苷对人肝癌细胞Hep G2胆固醇代谢的影响,并探讨其可能的作用机制。方法将人肝癌细胞Hep G2随机分为空白对照组、油酸诱导组、辛伐他汀组、CAEF组、联合用药组。油酸诱导组、辛伐他汀组、CAEF组、联合用药组分别加入油酸诱导脂肪变性模型;同时,辛伐他汀组加入10μmol/L的辛伐他汀10μL,CAEF组加入25 mg/L的CAEF 10μL,联合用药组加入含1μmol/L西红花苷、30μmol/L绿原酸、10μmol/L京尼平苷、10μmol/L槲皮素的混合液10μL;空白对照组仅加入等体积培养基。取各组干预48 h细胞,行油红O染色,检测细胞内中性脂滴含量。取干预24、48 h细胞上清液,检测TC、TG含量。采用RT-PCR法检测胆固醇代谢相关基因(ABCA1、SREBP2、CYP7A1、LXRα、AMPK2α、HMGCR)表达,免疫细胞化学法和Western blotting法检测HMGCR蛋白表达。结果辛伐他汀组、CAEF组、联合用药组干预48 h,细胞内中性脂滴含量明显降低,其作用强度依次为联合用药组>CAEF组>辛伐他汀组(P均<0.05);与对照组比较,CAEF组、联合用药组干预24、48 h时细胞外液TC、TG含量明显增加,细胞外液TC、TG含量依次为联合用药组>CAEF组>辛伐他汀组(P均<0.05)。CAEF组、联合用药组ABCA1、CYP7A1、AMPK2αmRNA表达明显升高,SREBP2、HMGCR mRNA表达明显降低(P均<0.05);此外,联合用药组LXRαmRNA表达明显降低(P<0.05)。辛伐他汀组、CAEF组、联合用药组HMGCR蛋白表达明显降低,以联合用药组降低最明显(P均<0.05)。结论 CAEF联合西红花苷可协同抑制肝癌细胞脂质积聚,其作用机制可能与调控肝癌细胞胆固醇代谢相关基因和蛋白的表达有关。