肝癌特异性GGT的发生及其定量分析的临床价值

目的探讨肝癌特异性ＧＧＴ的动态变化及其定量分析的临床价值。方法以化学致癌剂２－ＦＡＡ（００５％）制备鼠肝癌模型，观察肝癌ＧＧＴ的产生及其肝组织内分布，并对不同肝病患者血清肝癌特异性ＧＧＴ区带（ＨＳＢ）进行定量分析研究。结果在实验性鼠的诱癌过程中，鼠肝脏癌变组织中ＧＧＴ大量表达，出现早并具有胎肝ＧＧＴ的特点；人血清中ＨＳＢ的定量检测发现：肝癌组明显高于其他肝病组，肝外肿瘤及正常对照组（Ｐ＜０００１）；ＨＳＢ在小肝癌中的阳性率为７５％，在ＡＦＰ阴性肝癌中为７８３％。结论研究资料提示ＨＳＢ的定量检测其临床价值优于ＨＳＢ的定性分析，且有助于小肝癌和ＡＦＰ阴性肝癌的早期诊断。     

奥曲肽与奥美拉唑联合应用对肝癌H22生长的抑制作用

目的观察奥曲肽与奥美拉唑联合应用对肝癌H22生长的抑制作用。方法建立小鼠肝癌H22肉瘤移植模型后,随机分为阴性对照组、顺铂组、奥曲肽组、奥美拉唑组和奥曲肽＋奥美拉唑组（联合用药组）,给药后计算肿瘤生长抑制率和肿瘤微血管密度的变化。结果奥曲肽组与奥美拉唑组的抑瘤率分别为33.5%、29.6%,联合用药组的抑瘤率为37.1%,差异无统计学意义（P〉0.05）。与阴性对照组比较,奥曲肽组与奥美拉唑组能够降低小鼠肝癌H22的微血管密度,差异有统计学意义（P〈0.05）,联合用药降低更加明显,其中奥曲肽组与奥美拉唑组的微血管密度分别为（29.34±8.78）、（40.95±9.23）,联合用药组为（23.89±5.76）。结论奥曲肽联合奥美拉唑能够显著抑制肝癌H22的生长,联合用药可能具有协同效果。     

B超引导下肝癌射频消融术后观察与护理分析

目的探讨B超引导下射频消融术治疗肝癌患者的术后护理方法。方法对该院16例肝癌患者采用射频消融术进行治疗,观察术后恢复情况并总结护理方法。结果 16例患者均为第1次行经皮肝穿刺射频消融。1个月后B超复查,病灶均呈低密度改变,治疗效果良好。结论 B超引导下射频消融术治疗肝癌患者的术后加强病情观察及术后护理是保证治疗成功的关键。     

含精氨酸-甘氨酸-天冬氨酸肽白细胞介素-24突变体蛋白对肝癌细胞抑制作用

目的 观察含精氨酸-甘氨酸-天冬氨酸(RGD)肽模体的白细胞介素(IL)-24突变体蛋白(RGD-IL-24)对肝癌细胞的抑制作用.方法 肝癌HepG2细胞分别加入各10μl的RGD-IL-24(8 mg/L)、IL-24(8 mg/L)和磷酸盐缓冲液(PBS),噻唑蓝(MTT)比色法检测HepG2细胞生长抑制作用;DAPI染色检测HepG2细胞凋亡;免疫印迹法检测HepG2细胞bax、bcl-2及Caspase-3蛋白表达;黏附实验检测与HepG2细胞的靶向黏附.结果 RGD-IL-24、IL-24治疗4 d后HepG2细胞存活率分别为(0.219±0.015)、(0.397±0.009),与对照组(0.823±0.013)比较差异有统计学意义(P<0.01).RGD-IL-24、IL-24治疗组细胞凋亡率分别为(0.631±0.027)、(0.472±0.031),与对照组(0.082±0.013)比较差异有统计学意义(P<0.01).RGD-IL-24抑制HepG2细胞生长、诱导凋亡效果均比IL-24显著增强(P<0.05).与IL-24治疗组比较,RGD-IL-24治疗组HepG2细胞促凋亡蛋白bax增加、抗凋亡蛋白bcl-2减少、活化Caspase-3蛋白量增加.黏附实验证实RGD-IL-24与HepG2细胞的靶向结合作用增强.结论 含RGD肽模体的IL-24蛋白能通过与肝癌HepG2细胞的靶向结合增强其凋亡诱导作用.     

泛素偶联酶UBE2C／UbcH10荧光蛋白表达载体的构建及肝癌细胞中的表达

目的构建人泛素偶联酶UBE2C／UbcH10荧光蛋白真核表达载体,并转染肝癌细胞,筛选建立稳定转染细胞株。方法从构建好的pMD19-T／UbcH10载体中PCR扩增UbcH10基因,PCR产物双酶切后克隆入pEGIZP—N1真核表达载体中,构建真核表达载体pEGFP—N1／UbcH10,进行双酶切和测序鉴定。将构建好的真核表达载体pEGFP—N1／UbcH10和空载体pEGFP—N1利用脂质体Lip2000^TM分别转染进入肝癌细胞SMMC7721中,建立肝癌细胞SMMC7721的G418细胞死亡曲线,选择G418的筛选浓度,经G418结合荧光显微镜观察筛选稳定转染细胞。将筛选获得的肝癌细胞进行有限稀释法单克隆化,收集单个细胞克隆扩大培养备用。结果经双酶切和测序鉴定,带有荧光蛋白标记的真核表达载体pEGFP—N1／UbcH10构建成功,经脂质体介导真核表达载体pEGFP—N1／UbcH10和空载体pEGFP-N1成功转入肝癌细胞SMMC7721中,经800μg／mL G418筛选并进行单克隆化获得稳定转染的细胞株SMMC7721-pEGFP—N1和SMMC7721-pEGFP-N1／UbcH10,阳性转化率达到94％以上。结论人泛素偶联酶UBE2C／UbcH10稳定高表达肝癌细胞株的建立,为该蛋白在肝癌发生发展过程中的具体作用机制的研究奠定了基础。     

High tumor-infiltrating macrophage density predicts poor prognosis in patients with primary hepatocellular carcinoma after resection

Macrophages constitute a major component of the leukocyte infiltrate of tumors and perform distinct roles in different tumor microenvironments. This study attempted to investigate the prognostic values of tumor-infiltrating macrophages in patients with hepatocellular carcinoma after resection, paying particular attention to their tissue microlocalization. The CD68(+) macrophages were assessed by immunohistochemistry in tissues from 137 patients with hepatocellular carcinoma. Prognostic value of intratumoral, marginal, and peritumoral macrophage densities was evaluated by Kaplan-Meier analysis and Cox regression. Both intratumoral and marginal macrophage densities were associated inversely with overall survival (P = .034 and .004, respectively) and disease-free survival (P = .006 and .008, respectively). In contrast, peritumoral macrophage density was associated with neither overall survival nor disease-free survival. Intratumoral macrophage density emerged as an independent prognosticator of overall survival (hazard ratio = 1.721, P = .049) and disease-free survival (hazard ratio = 2.165, P = .007). Marginal macrophage density, but not intratumoral macrophage density, was associated with vascular invasion, tumor multiplicity, and fibrous capsule formation. Our results demonstrate that high macrophage infiltration predicts poor prognosis in patients with hepatocellular carcinoma. These results, together with our previous report showing the distinct activation patterns of macrophages in different areas of tumor tissue, implies that macrophages in those areas may use different strategies to promote the tumor progression.     

芬太尼对人肝癌细胞bel-7404生长与凋亡的影响

目的探讨芬太尼对人肝癌细胞bel-7404生长及凋亡的影响。方法体外培养人肝癌bel-7404细胞,分F1、F2、F13、F4,4个实验组和1个对照组,实验组分别在RPMI-1640培养液中加入5ng／ml（F1）、50ng／ml（F2）、500ng／ml（F3）、5000ng／ml（F4）芬太尼,对照组不加芬太尼,所有样本孵育24h后,用倒置显微镜观察细胞形态学改变,应用MTT法和克隆形成实验检测细胞的增殖活性,应用流式细胞仪检测细胞凋亡率与细胞周期。结果各实验组人肝癌bel-7404细胞MTT和克隆形成率明显低于对照组（P〈0．01）,细胞凋亡百分比显著高于对照组（P〈0．05）。芬太尼浓度≥50ng／ml时,随着浓度的增加,凋亡率逐渐增高,细胞周期中G0／G1期比例逐渐增加,S期细胞比例逐渐减少,与对照组比较差异具有统计学意义（P〈0．05）。结论芬太尼可剂量依赖性抑制人肝癌细胞bel-7404的生长,干扰肝癌细胞增殖周期,诱导肝癌细胞凋亡。     

PNP—TK融合自杀基因及PNP单自杀基因对肝癌细胞杀伤效率

目的分析PNP—TK融合自杀基因系统及PNP单自杀基因系统对肝癌细胞的杀伤作用及二者对肝癌细胞潜在的杀伤机制。方法构建融合基因表达我体pcDNA3．0／PNP—TK。经酶切、PCR及测序鉴定重组体。G418筛选获得稳定转染了pcDNA3．0／PNP-TK的HepG2细胞克隆。RT—PCR和Western Blotting检测融合基因在HepG2细胞中的表达。台盼兰排斥法检测细胞的生长曲线,MTT法检测细胞对相应前药的敏感性及分别在一种和两种前药作用下所导致的旁观者效应。结果融合基因片段PNP—TK正确插入了pcDNA3．0载体中,pcDNA3．0,PNP-TK在肝癌细胞株HepG2中实现了表达。抗性细胞克隆对相应的前药十分敏感。在两种前药的联合作用下。pcDNA3．0／PNP-TK所导致的旁观者效应明显强于只给予MeP-dR一种前药以及pcDNA3．0／PNP单基因系统所致的旁观者效应。结论具有双自杀基因功能的表达载体pcDNA3．0／PNP-TK对肝癌细胞的杀伤效果优于PNP单自杀基因系统。     

Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells

The effect of calmidazolium on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 1.5 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Calmidazolium induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was insensitive to L-type Ca²⁺ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), calmidazolium-induced [Ca²⁺]_i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change calmidazolium-induced [Ca²⁺]_i rises. At concentrations between 1 and 15 μM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via protein kinase C-regulated Ca²⁺ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.     

Mechanisms of carvedilol-induced [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> rises and death in human hepatoma cells

The effect of the cardiovascular drug carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether carvedilol altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Carvedilol at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 20 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Carvedilol induced Mn²⁺ quench of fura-2 fluorescence, implicating Ca²⁺ influx. The Ca²⁺ influx was sensitive to La³⁺, econazole, nifedipine, and SKF96365. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), carvedilol-induced [Ca²⁺]_i rises were abolished; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change carvedilol-induced [Ca²⁺]_i rises. At concentrations between 1 and 50 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 1 μM (but not 30 μM) carvedilol was fully reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Apoptosis was induced by 30 (but not 1) μM carvedilol. Collectively, in HA59T hepatoma cells, carvedilol induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase-C-independent manner and Ca²⁺ influx via store-operated Ca²⁺ channels. Carvedilol-caused cytotoxicity was mediated by Ca²⁺ and apoptosis in a concentration-dependent manner.