健脾理气方对HepG2人肝癌细胞株p53和bcl-2基因表达的影响

[目的]观察健脾理气方对HepG2人肝癌细胞株p53和bcl-2基因表达的影响。[方法]30只雄性SD大鼠随机分为健脾理气方（中药）组、0．85％氯化钠（对照）组和顺铂（化疗）组,每组10只。利用血清药理学方法制备药物血清,3组分别加入PRMI1640血清培养的HepG2人肝癌细胞株中,作用48h。应用免疫细胞化学方法和图像分析方法观察健脾理气方对HepG2人肝癌细胞株p53、bcl-2基因表达影响。[结果]①免疫细胞化学法显示抑癌基因p53主要定位在细胞核,癌基因bcl-2主要定位在细胞质内。药物血清作用后,p53表达明显增强;bcl-2表达明显降低。随着作用时间延长,2种基因表达改变更明显。②中药组与对照组p53和bcl-2光度（AOD）值差异有统计学意义（P〈0．01）。③化疗组作用结果与中药组相似。[结论]健脾理气方药物血清上调p53、下调bcl-2,达到抑制HepG2人肝癌细胞株的作用。为应用健脾理气方治疗肝癌提供实验依据。     

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.     

健脾理气法治疗26例Ⅱ期肝癌临床观察

根据上海医科大学附属肿瘤医院1980年统计,在1000例左右初诊肝癌病人中90％为中、晚期:当今肝癌尚缺乏有效的治疗手段,尽管手术疗效最好,但仅适用于5.3％肝癌病人,因此在我国运用中医中药治疗肝癌比较普遍。有人观察到中、晚期肝癌有90.3％的病人自始至终或在某一阶段表现为脾虚证,为此作者应用健脾理气中药治疗26例Ⅱ期肝癌病人,并与非健脾理气中药治疗进行比较。     

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.     

健脾消积汤对晚期原发性肝癌患者生存质量的影响

[目的]观察健脾消积汤治疗晚期原发性肝癌(HCC)对患者生存质量的影响。[方法]将60例HCC患者随机分成2组,各30例,2组均采用相同的西药保肝、对症支持疗法。治疗组同时加用健脾消积汤治疗。观察2组治疗前后生存质量量表EORTCQLQ-SF-36调查问卷评分和临床症状积分情况。[结果]2组生存质量量表EORTCQLQ-SF-36调查问卷评分比较,治疗组躯体功能、角色功能、情绪功能、物理症状及整体健康状况均优于对照组,差异有统计学意义(P0.05),而认知功能,社会功能差异无统计学意义(P0.05)。2组治疗前后临床症状缓解情况比较,治疗组治疗后疼痛、乏力、食欲不振、腹胀较治疗前有显著改善(P0.05);对照组治疗后较治疗前症状有所改善,但差异无统计学意义(P0.05),治疗组优于对照组(P0.05)。治疗组6、12个月生存率分别为74.1%、37.4%;对照组为69.2%、23.1%;2组间比较差异无统计学意义(P0.05)。[结论]健脾消积汤具有稳定瘤体、改善临床症状,缓解疼痛而达到"带瘤生存"的目的,提高生活质量,延长生存期。     

复方苦参碱注射液联合健脾理气方治疗晚期原发性肝癌33例

[目的]观察复方苦参碱（商品名：岩舒）注射液联合健脾理气方治疗晚期原发性肝癌患者的疗效。[方法]将66例晚期原发性肝癌患者随机分成2组，各33例。对照组采用一般护肝、支持、对症治疗；治疗组在上述治疗基础上加用复方苦参碱注射液静脉滴注及口服健脾理气方加减治疗。[结果]治疗组病情稳定率、疼痛缓解率、肝功能改善率、生活质量评分均高于对照组（P〈0．05），两组肿瘤缓解率、生存期差异无统计学意义（P〉0．05）。[结论]复方苦参碱注射液联合健脾理气方加减治疗中晚期原发性肝癌可进一步改善患者疼痛症状、肝功能，稳定病情，提高患者生活质量。     

健脾理气方预防肝硬化及癌变的实验研究

目的:观察健脾理气方对肝硬化及癌变的预防作用。方法:以DEN诱发大鼠肝硬化伴癌前期病变为模型,生理盐水为对照。观察项目包括病理形态与生化学指标。结果:用中药后肝纤维化程度减轻;肝细胞增生灶与假小叶减少,肝组织GGT阳性灶和AFP阳性细胞、肝组织Hyp和肝细胞DNA含量均减少;肝组织GGT比活性降低。结论:健脾理气方能同时延缓肝硬化的形成与阻制癌前期的病变,其防癌机制可能在于药物抑制了肝细胞DNA的复制。     

健脾消积汤对晚期原发性肝癌生活质量及免疫功能的影响

[目的]观察健脾消积汤对晚期原发性肝癌生活质量及免疫功能的影响。[方法]60例患者随机分成2组,各30例,均采用相同的西药保肝对症支持疗法;治疗组加用健脾消积汤治疗。[结果]治疗组与对照组临床证候总改善率为90.0%、63.3%;总有效率为83.3%、60.0%;生存期>12个月的生存率占63.3%、36.7%,2组比较差异均有统计学意义(P<0.05)。2组治疗前后血T淋巴细胞亚群CD4 、CD4 /CD8 比值均升高(P<0.05),CD8 降低(P<0.05,P<0.01);且2组比较差异有统计学意义(P<0.01)。[结论]健脾消积汤可提高晚期原发性肝癌患者机体免疫功能,改善临床症状,提高生活质量,延长生存期。     

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.     

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.     

消痰散结方对裸鼠人胃癌血管生成的影响

目的:探讨中药消痰散结方对实验性裸鼠人胃癌血管生成的影响.方法:建立裸鼠人胃癌原位种植模型,将动物随机分为空白对照组、中药组和联合组(n=10),中药组予消痰散结方0.2 mL灌胃,联合组同时予5-FU溶液0.2 mL腹腔注射.第11周结束实验,动物行超声探查,利用TOMTEC imaging system对超声图像进行三维重建,计算肿瘤组织的三维超声血管容积指数(Ⅵ),处死动物,取瘤组织,Envision法免疫组化染色CD34,计数微血管密度(MVD).结果:与空白对照组(2.14±0.43 g)相比,中药组(1.12±0.38 g,P<0.05)和联合组(0.77±0.26 g)瘤质量明显减低(P<0.05);联合组比中药组瘤质量明显减低(P<0.05).中药组和联合组的血流较空白对照组明显减少.中药组和联合组Ⅵ和MVD与空白对照组相比.明显减低(Ⅵ:18.87%±3.74%,14.97%±3.61% vs 31.00%±4.06%,P<0.05;MVD:27.3±8.8,25.8±8.6 vs 49.3±15.3,P<0.05).结论:消痰散结方可以明显抑制裸鼠人胃癌原位种植瘤的血管生成.     

高转移MHCC97H细胞裸鼠原位移植瘤姑息性肝癌切除模型的建立

目的 模拟临床肝癌切除病例构建姑息性肝切除裸鼠模型,用于研究手术对残癌的影响并探索干预策略. 方法 采用肝左叶单瘤源接种技术,构建高转移潜能MHCC97H细胞裸鼠原位移植瘤模型,成功建模后14 d行姑息性肝切除.姑息术后14d,分析肿瘤相关基因差异表达；观察35 d,检测肝内、肺脏及腹腔转移；剩余裸鼠用于观察生存期.计量资料比较用t检验,生存分析用Kaplan-Meier法(Log Rank检验),P＜0.05为差异有统计学意义. 结果 原位移植瘤模型成功率100％,成功构建裸鼠姑息性肝切除模型,无手术相关死亡裸鼠.姑息组裸鼠肝内转移结节多于假手术组[(11.7±4.7)个对比(6.3±2.8)个,t=-2.412,P＜0.05],腹腔转移结节多于假手术组[(9.8±3.4)个对比(5.2±2.6)个,t=-2.641,P＜0.05],肺转移结节也多于假手术组[(14.3±4.7)个对比(8.7±4.7)个,t=-2.348,P＜0.05].应用生物信息学技术进一步分析发现,肿瘤转移抑制蛋白1、肿瘤坏死因子β1、Smad2、白细胞介素1β及基质金属蛋白酶7基因在姑息组残癌基因功能网络中处于核心位置.姑息组裸鼠生存期为(60.8±2.7)d,假手术组裸鼠生存期为(51.3±1.4)d,差异有统计学意义(x2=12.850,P＜0.01).结论 单瘤源姑息性肝切除模型能有效模拟临床肝切除病例,为术后残癌生物学特性的研究及干预提供一个新的平台.     

Effects of thalidomide on angiogenesis and tumor growth and metastasis of human hepatocellular carcinoma in nude mice

AIM: To investigate the effects of thalidomide on angiogenesis, tumor growth and metastasis of hepatocellular carcinoma in nude mice. METHODS: Twenty-four nude mice were randomly divided into therapy group and control group, 12 mice in each group. Thalidomide dissolved in 0.5% sodium carboxyl methyl cellulose (CMC) suspension was administered intraperitoneally once a day at the dose of 200 mg/kg in therapy group, and an equivalent volume of 0.5% CMC in control group. Mice were sacrificed on the 30th d, tumor size and weight and metastases in liver and lungs were measured. CD34 and VEGF mRNA in tumor tissue were detected by immunohistochemistry and semi-quantitative RT-PCR respectively and microvessel density (MVD) was counted. Serum concentrations of TNF-α and ALT and AFP were also tested. RESULTS: MVD and VEGF mRNA in therapy group were less than those in control group (31.08±16.23 vessels/HP vs 80.00±26.27 vessels/HP, 0.0538±0.0165 vs 0.7373±0.1297, respectively, P<0.05). No statistical difference was observed in tumor size and weight and metastases in liver and lungs. TNF-α was significantly lower in therapy group than in control group (28.64±4.64 ng/L vs 42.69±6.99 ng/L, P<0.05). No statistical difference in ALT and AFP was observed between groups. CONCLUSION: Thalidomide can significantly inhibit angiogenesis and metastasis of hepatocellular carcinoma. It also has inhibitory effects on circulating TNF-α.     

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。     

Gene transfer with cationic lipid into human hepatocellular carcinoma in nude mice.

BACKGROUND/AIMS: Using a cationic lipid, gene transfection into the tumor of a human hepatocellular carcinoma model in nude mice was attempted in order to explore the possibility of its use in gene therapy. METHODOLOGY: A DNA-lipid complex was made by combining the cationic lipid distearyldimethyl ammonium bromide (DDAB) with pCMV sPORT expressing the reporter gene LacZ. The expression of this complex was first investigated in vitro against the human hepatocellular carcinoma cell line Li7HM. It was then injected directly into a hepatocellular carcinoma model tumor created by implanting Li7HM into the liver of BALB/c nu/nu mice, and the expression of LacZ was histologically evaluated. RESULTS: LacZ gene was expressed in Li7HM in vitro with the optimized DNA-lipid complex. Cell toxicity was not a problem. Expression of LacZ was also seen in the mouse hepatocellular model tumor into which the complex had been injected, indicating successful gene transfection with this method. CONCLUSIONS: Direct injection of a DNA-lipid complex into a mouse hepatocellular carcinoma model tumor is a safe and simple method of gene transfection, proving this to be a viable method of transfer for use in gene therapy.     

Effect of phosphorus-32 glass microspheres on human hepatocellular carcinoma in nude mice

AIM: To study the effects of phosphorus-32 glass microspheres ((32)P-GMS) on human hepatocellular carcinoma in nude mice. METHODS: Human liver cancer cell line was implanted into the dorsal subcutaneous tissue of 40 BALB/c nude mice. Then the 40 tumor-bearing BALB/ c nude mice were allocated into treatment group (n=32) and control group (n=8). In the former group different doses of (32)P-GMS were injected into the tumor mass, while in the latter nonradioactive (31)P-GMS was injected into the tumor mass. The experimental animals were sacrificed on the 14th day. The ultrastructural changes of tumor in both treatment group and control group were studied with transmission electron microscopy (TEM) and stereology. RESULTS: In treatment group, a lot of tumor cells were killed and the death rate of tumor cells was much higher (35-70%). Ultrastructurally, severe nuclear damage was observed in the death cells. The characteristics of apoptosis such as margination of heterochromatin was also found in some tumor cells. Besides, well differentiated tumor cells, degenerative tumor cells and some lymphocytes were seen. The skin and muscle adjacent to the tumor were normal. In control group, the tumor consisted of poorly differentiated tumor cells, in which there were only a few of dead cells(5%). Stereological analysis of ultrastructural morphology showed that Vv of nuclei (53.31+/-3.46) and Vv of nucleoli(20.40+/-1.84) in the control group were larger than those(30.21+/-3.52 and 10.96+/-2.52) in the treatment group respectively (P<0.01), and Vv of RER (3.21+/-0.54) and Vv of mitochondria (4.53+/-0.89) in the control group were smaller than those (8.67+/-1.25 and 7.12+/-0.95) in the treatment group respectively (P<0.01, 0.05). Sv of the membrane of microvilli and canaliculi (27.12 um(2)/100 um(3)+/-11.84 um(2)/100 um(3)) in the control group was smaller than that (78.81 um(2)/100 um(3)+/- 19.69 um(2)/100 um(3)) in the treatment group (P<0.01). But Vv of lipid particles (3.71+/-1.97) and Vv of vacuoles (5.72+/-1.58) were much larger than those (0.30+/-0.16 and 0.35+/-0.15) in the treatment group respectively (P<0.05, P<0.01). CONCLUSION: The experimental results indicate that local administration of (32)P-GMS can produce obvious effect on liver cancer cells and the anticancer effect of (32)P-GMS is directly proportional to the dose administrated. Ultrastructural stereology can also show the effect of (32)P-GMS on the normalization of tumor cells, which is beneficial to the prognosis and treatment of patients. Moreover, local administration of (32)P-GMS is also safe.