Antiangiogenic activity of curcumin in hepatocellular carcinoma cells implanted nude mice

Antiangiogenic activity of curcumin on the tumor neogenesis was investigated by evaluating the density of neocapillaries induced by Hepatocellular carcinoma cells (HepG2) in mice, using intravital fluorescence videomicroscopy. Male BALB/c nude mice (20-25 g) were used, and a dorsal skin-fold chamber was implanted. HepG2 (30 microl of 2 x 10(6) cells) were inoculated on the upper surface of the skin within the chamber. The mice were divided into two groups as follows. Dimethyl sulfoxide solution (0.1%) was fed (HepG2 group, n=5) or curcumin solution (3000 mg/kg bw) was fed oral daily (HepG2-Cur group, n=5), one day after the inoculation of HepG. On days 7 and 14 post-tumor-inoculation, the tumor microvasculature was visualized by injecting 0.1 ml of 0.5% rhodamine B isothiocyanate-labeled dextran intravenously, and observed under an intravital fluorescence videomicroscope. Based on the recorded videoimage, the tumor neocapillary density and microvasculature were evaluated using a digital image analysis and correlated with the tumor area. The image analysis demonstrated that in the HepG2-group the neocapillary densities were significantly increased on day 7, and day 14, compared to the aged-matched Sham-group (P<0.05). In the HepG2-Cur group, the increase of tumor neocapillary density was attenuated significantly. It was suggested that high dose of curcumin might be an effective anti-angiogenic drug in the treatment against tumor.     

Both glypican-3/Wnt/β-catenin signaling pathway and autophagy contributed to the inhibitory effect of curcumin on hepatocellular carcinoma

Aim

The aim of this study is to investigate the role of glypican-3(GPC3)/wnt/β-catenin signaling pathway and autophagy in the regulation of hepatocellular carcinoma (HCC) growth mediated by curcumin.

The effects of tetrahydrocurcumin and green tea polyphenol on the survival of male C57BL/6 mice

The effect of feeding of two different antioxidants, tetrahydrocurcumin (TC) and green tea polyphenols (PPs) on the survival of male C57BL/6 mice was examined. Mice that started to receive diets containing TC (0.2%) at the age of 13 months had significantly longer average life spans (days, mean ± SD) than control mice (797.6 ± 151.2 vs.882 ± 154.6, both n = 50, controls vs. TC treated, plus 11.7%, P < 0.01). The 10% longest survival was also significantly greater in TC-treated mice (plus 6.5%, P < 0.01). In contrast, in mice that started to receive TC in their 19th month of life, no significant difference from the control mice was found for either the average life span or the 10% longest survival. In mice that received water containing PPs (80 mg/l), the average life span was also significantly longer than in the control mice (801 ± 121.5 vs. 852.7 ± 88.2, plus 6.4%, P < 0.05), although the 10% longest survival was not significantly different from that in the control mice (P > 0.05). The body weights of the TC (but not PP) fed mice, were slightly (2–4%) but significantly (P < 0.05) lower than the values for the corresponding ages in the control mice in the first six months of treatment. Thereafter, the difference in average body weight between the control and the TC-fed animals was totally lost. Although an additional contribution of an unintended slight decrease in food intake due to TC feeding (suspected due to the difference in body weight) is not excluded, we suggest that the feeding of nutritional antioxidants such as TC and PPs may have the potential to beneficially modify the life spans of animals.     

Metastatic human hepatocellular carcinoma models in nude mice and cell line with metastatic potential

Metastatic human HCC model is needed for the studies onmechanism and intervention of metastatic recurrence,Byusing orthotopic implantation of histologically intacttissues of 30 surgical specimens,a patient-likemetastatic model of human HCC in nude mice (LCI-D20)and a low metastatic model of human HCC in nude mice(LCI-D35) have been established.All mice withtransplanted LCI-D20 tumors exhibited extremely highmetastatic ability including spontaneous metastasis toliver,lungs,lymph nodes and peritoneal seeding.Remarkable difference was also found in expression ofsome of the invasiveness related genes and growthfactors between the LCI-D20 and LCI-D35 tumors.PAI-1increased gradually following tumor progression in LCI-D20 model,and correlated with tumor size and AFP level.Phasic expression of tissue intercellular adhesionmolecule-1 in this model was also observed.Using cornealmicropocket model,it was demonstrated that the vascularresponse induced by LCI-D20 tumor was stronger than thatinduced by LCI-D35 tumor.Similar report on metastatichuman HCC model in nude mice and human HCC cell linewith metastatic potential was rarely found in theliterature.This LCI-D20 model has been widely used forthe studies on intervention of metastasis,including anti-angiogenesis,antisense approach,metalloproteinaseinhibitor,differentiation inducer,etc.It is concluded thatthe establishment of metastatic human HCC model in nudemice and human HCC cell line with metastatic potentialwill provide important models for the in vivo and in vitrostudy of HCC invasiveness,angiogenesis as well asintervention of HCC recurrence.     

Expression of angiostatin cDNA in human hepatocellular carcinoma cell line SMMC-7721 and its effect on implanted carcinoma in nude mice

AIM: To transfect murine angiostatin cDNA into human hepatocellular carcinoma cell line SMMC-7721 and to investigate its effects on implanted carcinoma in nude mice. METHODS: A eukaryotic expression vector of pcDNA3.1-mAST containing murine angiostatin was constructed. Then pcDNA3.1-mAST plasmid was transfected into cell line SMMC-7721 by Lipofectamine. The resistant clone was screened by G418 filtration and identified by RT-PCR and Western blotting. Nude mice were divided into three groups of 10 each. Mice in blank control group were only injected with SMMC-7721 cells. Mice in vector control group were injected with SMMC-7721 cells transfected with pcDNA3.1 (+) vector, whereas mice in angiostatin group were injected with SMMC-7721 cells transfected with pcDNA3.1-mAST plasmid. Volume, mass and microvessel density (MVD) of the tumors in different groups were measured and compared. RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). pcDNA3.1-mAST was successfully transfected into SMMC-7721 cell line and showed stable expression in this cell line. No significant difference was observed in the growth speed of SMMC-7721 cells between groups transfected with and without angiostatin cDNA. Tumor volume, mass and MVD in the angiostatin group were significantly lower than those in the blank control group and vector control group (P<0.01). The inhibitory rate of tumor reached 78.6%. Mass and MVD of the tumors only accounted for 34.6% and 48.9% respectively of those in the blank control group. CONCLUSION: Angiostatin cDNA could be stably expressed in human hepatocellular carcinoma cell line SMMC-7721 without obvious inhibitory effects on the growth of SMMC-7721 cells. When implanted into nude mice, SMMC-7721 cells transfected with angiostatin cDNA show a decreased tumorigenic capability. It suggests that angiostatin can inhibit tumor growth through its inhibition on angiogenesis in tumors.     

姜黄素对非酒精性脂肪肝细胞模型保护作用以及机制研究

目的 探讨姜黄素对非酒精性脂肪肝病(NAFLD)细胞模型的保护作用以及保护机制。方法 体外用0.6 mmol/L 油酸诱导HepG2 细胞脂质沉积,建立NAFLD细胞模型。 将HepG2细胞分为对照组(Con)、模型组(OA)、姜黄素对照组和姜黄素干预组。采用Bodipy493/503荧光染色观察各组细胞内脂滴分布,使用透射电镜观察细胞线粒体超微结构变化,使用试剂盒检测培养上清液肿瘤坏死因子α和白介素-6(IL-6),采用DCFH-DA法检测HepG2细胞活性氧(ROS)生成量,采用Hoechst 33258染色检测HepG2细胞凋亡情况,采用Western blot法检测凋亡和炎症相关蛋白Bcl-2、Bax、NF-κB、Caspase-3/9和线粒体内细胞色素C(mCytc)。结果 与 Con组比,OA组 HepG2细胞脂质沉积明显增加,而姜黄素干预组细胞脂质沉积明显减少;OA组细胞线粒体明显损伤,而姜黄素干预组细胞线粒体损伤明显减轻;与Con组比,OA组 HepG2细胞上清液TNF-α和IL-6显著升高,而姜黄素干预组细胞上清TNF-α和IL-6明显降低;OA组HepG2细胞绿色荧光强度为(52.24±5.11)%,显著强于Con组【(6.71±2.31)%, P<0.05],而姜黄素干预的HepG2细胞绿色荧光强度降低; OA组 HepG2细胞凋亡率为(12.12±0.72)%,显著增高Con组【(2.04±0.57)%,P<0.05 ],而黄素干预组细胞凋亡率显著降低【(5.71±0.61)%,P<0.05】;与Con组比,OA组细胞Bax、NF-κB和cleaved-Caspase-3/9蛋白表达增强,而Bcl-2和mCytc表达减弱(P均<0.05),而姜黄素干预组细胞Bax、NF-κB和Caspase-3/9蛋白表达减弱,而mCytc和Bcl-2表达增强(P均<0.05)。结论 姜黄素可缓解NAFLD细胞脂肪变性,其作用机制可能与减轻炎症反应、抑制氧化应激损伤和细胞凋亡有关。     

Effects of curcumin on tumor angiogenesis and biomarkers, COX-2 and VEGF, in hepatocellular carcinoma cell-implanted nude mice

Anti-angiogenic activity of curcumin and effects of curcumin on angiogenic biomarkers, cycloxygenase (COX)-2 and vascular endothelial growth factor (VEGF) levels were investigated. One day after hepatocellular carcinoma cell (HepG2) cells (30 microl of 2 x 10(6) cells) were inoculated onto the upper layer of the skin-fold chamber (HepG2-group, n = 15), curcumin solutions of 300 and 3000 mg/kg BW were daily oral fed to HepG2-Cur-300 and HepG2-Cur-3000 groups (n = 30), respectively. Intravital fluorescence videomicroscopy was performed to monitor neocapillaries in the tumor on days 3, 7 and 14 post-tumor-inoculation, using RITC-dextran (0.1 ml of 0.5% injected intravenously). The tumor neocapillary density (NCD) was evaluated in correlation with the tumor area, using a digital image analysis. The results demonstrated that the NCD of HepG2-groups were significantly increased on day 7 and 14, compared to the aged-matched Sham-groups (p<0.001). The increased NCD on day 7 and 14 were attenuated significantly by daily treatment of curcumin solution (3000 mg/kg BW).The curcumin treatment reduced the tumor-induced over-expression of COX-2 and serum VEGF in HepG2 groups significantly (p<0.001), indicating that curcumin could inhibit tumor angiogenesis. This mechanism might be mediated through reduction of angiogenic biomarkers, COX-2 and VEGF.     

肺癌抑癌基因1对HepG2细胞生长的抑制效应

目的探讨肺癌抑癌基因1（TSLC1）对人肝癌细胞株HepG2生长的影响。方法RT-PCR法制备TSLC1全长cDNA并克隆至真核表达载体pCI-neo，稳定转染至肝癌细胞系HepG2中。以转染空质粒pCI-neo的HepG2细胞为对照组，野生型HepG2细胞为空白组，显微镜下观察细胞形态，MTT法检测细胞增殖，FACSort流式细胞仪检测细胞周期，AnnexinV／PI双染法检测细胞凋亡情况。结果实验建立了高表达TSLCI蛋白的稳定细胞株。实验组细胞呈多角形，聚集成团，细胞之间的黏附非常紧密，对照组和空白组细胞呈梭形，细胞与细胞之间较疏散。与对照组和空白组相比，实验组细胞株细胞生长速度减慢，增殖受到明显抑制，G0／G1期细胞为63．66%±3．83％，高于对照组（47．45％±0．91％）和空白组（54．47％±0．96％）；S期细胞数为22．90％±6．04％，低于对照组（36．58％±0．61％）和空白组（33．61％±2．99％），P〈0．01，实验组细胞周期发生了G0/G1期阻滞。实验组细胞早期凋亡率和晚期凋亡率分别为17．09%±0．20％和16．11％±0．40％，与对照组和空白组细胞相比均明显升高（P〈0．01）。结论TSLC1基因明显抑制HepG2细胞生长，并诱导细胞发生凋亡。     

色素上皮衍生因子抑制人肝癌裸鼠移植瘤生长

目的 观察人色素上皮衍生因子(PEDF)对血管生成的抑制作用,探讨PEDF在肝癌基因治疗中的应用前景. 方法构建色素上皮衍生因子慢病毒表达质粒pLenti-PEDF,经293T细胞包装,收集病毒上清液,感染肝癌细胞株HepG2.收集各组细胞的条件培养基,通过Westernblot分析各组细胞PEDF的表达情况,并通过人脐静脉血管内皮细胞增殖及迁移试验研究其体外生物学活性.人肝癌细胞HepG2移植到裸鼠皮下,研究Lenti-PEDF病毒对肝癌移植瘤生长的抑制作用,逆转录聚合酶链反应检测肿瘤组织中PEDF mRNA表达.多组间均数比较采用单因素方差分析,组间两两比较采用LSD法.结果 限制性内切酶酶切分析和测序结果均表明成功构建了PEDF慢病毒表达载体,以293T细胞包装的重组慢病毒能够高效感染肝癌细胞,肝痛细胞感染重组慢病毒后可高效表达PEDF并分泌到培养上清液中.体外研究结果表明,重组PEDF可明显抑制人脐静脉血管内皮细胞的增殖及迁移,抑制率分别达29%和48%,与空白对照组相比,差异有统计学意义(P值均<0.01).Lenti-PEDF病毒能明显抑制人肝癌裸鼠皮下移植瘤的生长(P<0.01).瘤内注射Lenti-PEDF病毒21 d后,逆转录聚合酶链反应在肿瘤组织检测到PEDF mRNA的过表达.结论 本研究初步提示PEDF能有效遏制肿瘤的血管生成和肿瘤生长,为肝癌的基因治疗提供了一条新的思路.     

姜黄素干预小鼠肠道菌群并抑制结直肠癌变的实验研究

目的拟通过监测姜黄素干预小鼠肠癌发生发展过程中肠道菌群的变化,探究姜黄素对小鼠肠道菌群的影响,以及可能的抗癌作用机制及作用靶点。 方法将25只6周龄C57BL/6小鼠随机分为基础饮食组（BD）5只,AOM/DSS造模组（MO）10只,姜黄素干预造模组（CU）10只,分别于实验前和处死前收集小鼠粪便进行DNA提取及高通量菌群测序。 结果T1点时（干预前）各组小鼠肠道菌群的多样性指数差异无统计学意义,T2点时（干预后）MO组的多样性指数更高,高于其他2组,与BD组差异有统计学意义（t=2.73,P=0.02）,而与CU组比较差异无统计学意义。T2点时各组的多样性指数均较T1点时明显升高,其中CU组与BD在T2点时多样性指数更接近,均小于造模组。造模前后菌群在属水平发生了较大变化,CU组较MO组肠道菌群变化要小,而且拟杆菌类和疣微菌门较MO组变化较大。MO小鼠体重增量平均要少于CU组,CU组小鼠结直肠肿瘤的成瘤数量、瘤体体积,明显少于MO组。 结论在小鼠结直肠癌发生过程中,姜黄素能够维持小鼠肠道菌群的稳定性,降低了小鼠肠道肿瘤发生率,可能为抑制结直肠癌发生发展的机制之一。     

抑癌基因Smad4对HepG2生长的影响及其机制探讨

目的研究抑癌基因Smad4对人肝癌细胞株HepG2．生长的影响，并探讨其作用机制。方法采用脂质体瞬时转染法转染PFTX-5Smad4质粒至人肝癌细胞株HepG2（实验组），以转染空质粒PFTX-5的HepG2细胞为对照组，野生型HepG2细胞为空白组，利用免疫印迹（Westernblot）和逆转录聚合酶链反应（RT—PCR）检测Smad4及血管内皮生长因子（VEGF）表达的变化；用MTT法检测细胞增殖及用FITC—AnnexinV、碘化吡啶（propidiumiodide，PI）双染后流式细胞仪（FCM）检测细胞周期和凋亡情况，Westernblot检测细胞周期素D1（CyclinD1）表达差异。结果PFTX-5Smad4瞬时转染到肝癌细胞后，实验组与对照组和空白组相比，Smad4mRNA和蛋白表达明显上升（P〈0．05），而VEGFmRNA和蛋白的表达显著降低（P〈0．05）。实验组细胞CyclinD1表达明显下降（P〈0．05），细胞周期时相分布出现合成前期（G。／G．期）阻滞。实验组细胞凋亡率明显升高（P〈0．01），增殖受到明显抑制。结论Smad4高表达可能通过降低肝癌细胞中VEGF的表达，抑制eyclinD1转录合成，并诱导强烈的G1期阻滞和细胞凋亡，对细胞的增殖有明显的抑制作用，为进-步研究肝癌生长增殖机制提供了实验基础。     

Quantitative assessment of cerebral neocapillary network and its remodeling in mice using intravital fluorescence videomicroscopy

To assess the responses of different growth factors on cerebral neocapillary density (NCD), cerebral angiogenesis was induced in mice using growth factors, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) at a concentration of 6 ng/ml each. Intravital fluorescence videomicroscopy was used to quantitatively evaluate microhemodynamic parameters such as diameter and red cell velocity. The gel–nylon mesh-sandwich system was implanted over the exposed cortex. After incubation for different periods of time (days 7, 14 or 28), fluorescein isothiocyanate (FITC)-labeled red cells were injected through a carotid artery and the neocapillaries on the upper surface of the nylon mesh were observed under a fluorescence videomicroscope. Based on the recorded videoimages, we evaluated the density, diameter and red cell velocity of the neocapillaries. The NCD in the bFGF group on day 7 was significantly higher than that in the PDGF group on day 7 (P < 0.01). The NCD (index) reached 100% on day 14, while it reduced significantly in both the groups on day 28. The neocapillary diameter was greater than that of the pre-existing capillaries on day 7. On day 14, a clear difference appeared in the capillary density between large and small vessels. The red cell velocity increased with the number of days after incubation. The response of cerebral neocapillaries to acetylcholine was measured after 28 days of incubation with growth factor bFGF and with PDGF. The red cell velocity increased significantly from its basal value in the PDGF group. These results suggest that the neocapillaries in the PDGF group matured earlier than those in the bFGF group.     

中药姜黄素对肿瘤细胞周期及自由基的影响

姜黄素是中药姜黄根茎中的主要成份,具有抗炎、抗氧化和抗肿瘤等药理作用。早期研究认为,姜黄素能抑制体外淋巴瘤细胞的生长和体内由TPA、PMA、AOM等诱导的动物肿瘤的形成。本文探讨了姜黄素对人结肠癌细胞LoVo体外生长、细胞周期及其细胞内自由基的影响。MTT结果表明姜黄素对LoVo细胞具有细胞毒作用。20μM、40μM浓度的姜黄素对细胞的抑制率分别是为41.9％、78.1％。5-10μM浓度抑制作用小,姜黄素对LoVo细胞的IC_(50)为21.8μM。姜黄素处理24h时,主要使细胞阻滞于S,G_2 M期,使此期细胞比例增加。而G_(0/1)期的细胞比例减少。处理48h后,G_2 M期的细胞比例反而降低,部分可能由于G_2 M期细胞并发凋亡所致。姜黄素对肿瘤细胞内自由基形成的抑制作用结果表明,浓度大于10μM的不同姜黄素处理组和对照组比较MDA含量明显下降(p<0.01),各处理组间则无显著性差异(p>0.05),而5μM姜黄素无抑制作用。结论:姜黄素可抑制结肠癌LoVo细胞的生长。其机制可能与干扰细胞周期及抑制细胞内自由基的生成有关。     

人肝癌裸鼠移植模型的研究进展

肝癌的基础与临床研究迫切需要能真正模拟肝癌在人体内自然生长、侵袭及转移全部过程的动物模型.目前,人肝癌裸鼠移植模型是人体外最接近人类肝癌的整体实验模型,并且造模时间短,成功率高,按移植部位可以分为皮下移植、原位移植、腹腔移植以及转移模型.影响裸鼠移植模型建立的因素主要有人肝癌细胞或外科标本的特性、移植部位及移植癌细胞数量、裸鼠的品系和周龄以及生长环境和其他因素的影响.     

Inhibitory effect of the angiogenesis inhibitor TNP-470 on tumor growth and metastasis in nude mice bearing human hepatocellular carcinoma

The antitumor and anti-metastatic effects of a potent angiogenesis inhibitor,O-(chloroacetyl-carbamoyl)fumagillol (TNP-470), was investigated in a highly metastatic model of human hepatocellular carcinoma—LCI-D20. Small pieces of LCI-D20 tumor tissue were implanted subcutaneously into the right axillary region of 24 nude mice; the mice were then randomized into two groups. To one group, TNP-470 30 mg/kg was given as a subcutaneous injection every other day from day 1 to day 15 and the mice were sacrificed on day 26. An antitumor effect of TNP-470 was clearly demonstrated by tumor weight (0.97±0.34 g compared to 2.04±0.34 g,P<0.001) and -Fetoprotein value (93±59 g/L compared to 769±282 g/L,P<0.001). There was also an anti-metastatic effect of TNP-470. Lung metastases developed in only 1 of 12 mice in the treated group, while they developed in 6 of mice of the control group. No severe side-effect of TNP-470 was found in this study. In vitro study revealed that the purified hepatoma cells were insensitive to TNP-470 (the 50% inhibitory concentration was 43 g/ml). These results suggest that the angiogenesis inhibitor TNP-470 has both strong antitumor and anti-metastatic effects on a human hepatocellular carcinoma model in nude mice.Abbreviations TNP-470 O-(chloroacetyl-carbamoyl) Fumagillol - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide This work was partly supported by the CHina Medical Board of New York, grant 93-583, and a Leading Speciality grant of Shanghai Health Bureau     

MMP-2和ICAM-1在裸鼠体内塞来昔布抑制肝癌组织中的表达

目的:研究机体内部选择性环氧合酶-2(COX-2)抑制剂对肝细胞癌的抑制作用.方法:将三种肝癌细胞株HepG2、BEL-7402和SMMC-7721分别接种于6周龄裸鼠肝脏被膜下;将接种了不同肝癌细胞株的裸鼠分别分为3组,阴性对照组给予生理盐水灌胃,实验组给予塞来昔布灌胃,阳性对照组进行生理盐水灌胃的同时使用阿霉素腹腔注射;3 wk后对裸鼠肝脏肿瘤取材、免疫组织化学法观察肿瘤组织中基质金属蛋白酶-2(MMP-2)及其抑制剂(TIMP-2)以及细胞间黏附因子-1(ICAM-1)的表达.结果:在肝脏被膜下接种了HepG2、BEL-7402和SMMC-7721肝癌细胞株的裸鼠中,应用塞来昔布的裸鼠肿瘤组织中MMP-2的表达下降(P<0.05),MMP-2的表达增加(P<0.05),TIMP-2/MMP-2比值增加.在肝脏被膜下接种了BEL-7402和SMMC-7721肝癌细胞株的裸鼠中,应用塞来昔布的裸鼠肿瘤组织中ICAM-1表达下降.结论:塞来昔布在机体内部可能具有抑制肝癌细胞转移和改善预后的作用.     

Effects and mechanisms of silibinin on human hepatocellular carcinoma xenografts in nude mice

AIM： To investigate the in vivo effects and mechanisms of silibinin on the growth of hepatocellular carcinoma （HCC） xenografts in nude mice. METHODS： Nude mice bearing HuH7 xenografts were used to assess the anti-HCC effects and mechanisms of silibinin. RESULTS： Silibinin resulted in a potent dosedependent reduction of HuH7 xenografts in association with a significant decrease in Ki-67 and α-fetoprotein production, nuclear NF-κB content, polo-like kinase 1, Rb phosphorylation, and E2F1/DP1 complex, but increased p27/CDK4 complex and checkpoint kinase 1 expression, suggesting that the in vivo effects of silibinin are mediated by inhibiting G1-S transition of the cell cycle. Silibinin-induced apoptosis of HuH7 xenografts was associated with inhibited survivin phosphorylation. Silibinin-reduced growth of HuH7 xenografts was associated with decreased p-ERK, increased PTEN expression and the activity of silibinin was correlated with decreased p-Akt production, indicating involvement of PTEN/PI3K/Akt and ERK pathways in its in vivo anti-HCC effects. Silibinin-reduced growth of HuH7 xenografts was also associated with a significant increase in AC-H3 and AC-H4 expression and the production of superoxide dismutase （SOD）-1.CONCLUSION： Silibinin reduces HCC xenograft growth through the inhibition of cell proliferation, cell cycle progression and PTEN/P-Akt and ERK signaling, inducing cell apoptosis, and increasing histone acetylation and SOD-1 expression.     

达肝素钠对肝癌生长转移抑制的实验研究

目的研究低分子肝素达肝素钠对肝癌生长转移的抑制作用。方法采用人肝癌裸鼠转移模型(LCI-D20)。40只模型鼠随机分成4组即对照组、化疗组(顺铂 氟尿嘧啶),达肝素钠组、联合组(顺铂、氟尿嘧啶与达肝素钠)。观察肿瘤大小和转移、抑瘤率、测血清甲胎蛋白(AFP)、肿瘤微血管密度(MVD)、CD31。结果对照组、化疗组、达肝素钠组、联合组的肿瘤体积分别为(25245±13367)mm3、(1610 ±1217)mm3、(5883±3131)mm3和(5556±2570)mm3;抑瘤率分别为0%、93.6%、76.7%和78.0%;MVD 分别为20.7±6.8、18.2±2.6、4.8±1.8和6.5±2.4;CD31分别为31.8±5.7、25.5±5.1、21.6±4.8和19.6±2.4;AFP分别为(121.8±31.4)ng/ml、(21.5±13.3)ng/ml、(75.6±29.7)ng/ml 和(55.8±38.0)mg/ml;肝转移率分别为80%、70%、20%和10%;肺转移率分别为70%、60%、20%和10%; 腹壁转移率分别为90%、60%、30%和30%;腹水形成率分别为20%、10%、0%和0%。化疗组、达肝素钠组、联合组分别与对照组比,对肝癌生长的抑制作用差异有统计学意义,F=9.191,P<0.01。达肝素钠对肝癌血管形成和转移有良好的抑制作用,与对照组及单纯化疗组比差异有统计学意义,F=4.937,P<0.01。结论低分子肝素达肝素钠通过抗肿瘤血管形成,对肝癌的生长与转移有抑制作用。     

Anticancer activity of resveratrol on implanted human primary gastric carcinoma cells in nude mice

AIM: To investigate the apoptosis of implanted primary gastric cancer cells in nude mice induced by resveratrol and the relation between this apoptosis and expression of bcl-2 and bax. METHODS: A transplanted tumor model was established by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice. Resveratrol (500 mg/kg, 1 000 mg/kg and 1 500 mg/kg) was directly injected beside tumor body 6 times at an interval of 2 d. Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated. We observed the morphologic alterations by electron microscope, measured the apoptotic rate by TUNEL staining method, detected the expression of apoptosis-regulated genes bcl-2 and baxby immunohistoch-emical staining and PT-PCR. RESULTS: Resveratrol could significantly inhibit carcinoma growth when it was injected near the carcinoma. An inhibitory effect was observed in all therapeutic groups and the inhibition rate of resveratrol at the dose of 500 mg/kg, 1 000 mg/kg and 1 500 mg/kg was 10.58%, 29.68% and 39.14%, respectively. Resveratrol induced implanted tumor cells to undergo apoptosis with apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation. The inhibition rate of 0.2 mL of normal saline solution, 1 500 mg/kg DMSO, 500 mg/kg resveratrol, 1 000 mg/kg resveratrol, and 1 500 mg/kg resveratrol was 13.68±0.37%, 13.8±0.43%, 48.7±1.07%, 56.44±1.39% and 67±0.96%, respectively. The positive rate of bd-2 protein of each group was 29.48±0.51%, 27.56±1.40%, 11.86±0.97%, 5.7±0.84% and 3.92±0.85%, respectively by immunohistochemical staining. The positive rate of bax protein of each group was 19.34±0.35%, 20.88±0.91%, 40.02±1.20%, 45.72±0.88% and 52.3±1.54%, respectively by immunohistochemical staining. The density of bcl-2 mRNA in 0.2 mL normal saline solution, 1 500 mg/kg DMSO, 500 mg/kg resveratrol, 1 000 mg/kg resveratrol, and 1 500 mg/kg resveratrol decreased progressively and the density of bax mRNA in 0.2 mL normal saline solution, 1 500 mg/kg DMSO, 500 mg/kg resveratrol, 1 000 mg/kg resveratrol, and 1 500 mg/kg increased progressively with elongation of time by RT-PCR. CONCLUSION: Resveratrol is able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated by down-regulating apoptosis-regulated gene bcl-2 and up-regulating the expression of apoptosis-regulated gene bax.