经不同途径区域灌注氟尿嘧啶在肝癌、肝组织及血浆中的浓度差异

背景与目的:肝动脉、肝门静脉灌注区域化疗是肝癌的重要治疗手段,本研究探讨区域性灌注化疗时氟尿嘧啶(fluorouracil,5-FU)在大鼠肝癌和肝脏组织及血浆中的分布,为临床肝脏肿瘤化疗提供参考。方法:将24只荷瘤大鼠随机分为4组,分别经外周静脉(尾静脉)、肝动脉、肝门静脉或结扎肝动脉后经肝门静脉灌注5-FU,剂量为20 mg/kg。采用高效液相色谱法测定肝癌、肝脏组织及血浆中5-FU的含量,并计算药物在血浆、肝脏和肝癌组织间的穿透比率。结果:结扎肝动脉的肝门静脉组5-FU浓度在肝脏和肝癌组织中最高,分别为(22.1±9.5)μg/g和(16.4±7.2)μg/g;其次为肝动脉组;肝门静脉组5-FU浓度在肝癌组织中的浓度较低,为(8.9±3.7)μg/g;外周静脉组5-FU浓度在肝脏和肝癌组织中的药物浓度均为最低,肝癌组织中的浓度仅为(4.3±2.2)μg/g。在血浆中的5-FU浓度正好相反,外周静脉组浓度最高(26.8±12.5)μg/m L,肝动脉组(16.4±9.7)μg/m L、结扎肝动脉的肝门静脉组(15.9±10.1)μg/m L和肝门静脉组(14.9±8.5)μg/m L等3组浓度相近,均明显低于外周静脉组(P<0.05)。5-FU的肝癌/血浆穿透比率依次为结扎肝动脉的肝门静脉组(103.47%),肝动脉组(92.94%),肝门静脉组(59.58%)和外周静脉组(16.08%)。结论:与外周静脉注射全身化疗比较,区域性灌注化疗可显著提高肝癌和肝脏组织中的药物浓度,同时减少化疗药物在外周血中的分布,其中经结扎肝动脉的肝门静脉灌注和经肝动脉灌注是肝癌区域性化疗2种较好的途径。     

肝脏肿瘤血供

肝脏肿瘤血供方式与肿瘤的诊断和治疗密切相关。研究表明:(1)实验性动物肝癌早期血供来自肝动脉或/和门静脉,随肿瘤趋向成熟呈动脉供血为主;(2)95％以上的肝细胞癌为几乎完全由肝动脉供血的多血供肿瘤;(3)转移性肝癌血供依原发灶不同差异较大,从多血供到无血供,其相当部分血供来自门静脉;(4)肝脏良性肿瘤亦多为肝动脉供血为主的多血供肿瘤,与恶性肿瘤不同,其血管结构类似于正常;(5)肝动脉结扎和栓塞后,肝癌血供的主要变化是侧支循环的建立和门静脉供血的增加。     

入肝门静脉动脉化对肝部分切除大鼠肝细胞能量代谢的影响

目的 在大鼠肝部分切除基础上建立入肝门静脉动脉化加门腔分流术的动物模型,研究大鼠术后早期肝脏细胞能量代谢的变化.方法 90只Sprague-Dawley大鼠分为3组:肝切除组+门静脉动脉化组、单纯肝切除组及单纯门静脉动脉化组,每组30只.观察术前及术后5 min吲哚靛青绿15 min储留率(indocyanine green retention rate at 15 rain,ICGR15)、术后5 min及2 h动脉血酮体比率(arterial blood ketone body ratio,AKBR)及肝组织能荷(energy charge,EC)的变化情况.结果 肝部分切除或单纯门静脉动脉化手术均导致术后大鼠ICGR15明显升高(P<0.01),单纯门静脉动脉化大鼠肝脏吲哚靛青绿15 min储留率显著低于肝部分切除大鼠(P<0.01).肝切除附加门静脉动脉化后,大鼠术后ICGR15明显低于单纯肝部分切除组(P<0.01).肝部分切除或单纯行门静脉动脉化手术均导致术后大鼠AKBR及EC明显降低(P<0.01),单纯门静脉动脉化组大鼠术后2 h开始出现回升,而肝部分切除大鼠仍持续下降.肝部分切除附加门静脉动脉化组AKBR及EC术后均明显高于单纯肝部分切除组(P<0.01).结论 肝部分切除或门静脉阻断的手术操作均明显降低肝脏细胞能量代谢水平,入肝门静脉动脉化能有效促进肝部分切除术后大鼠残肝细胞能量代谢水平的恢复.     

原发性肝癌的栓塞化疗

原发性肝癌的首选疗法仍是手术切除,但大多数肝癌患者常伴有严重肝硬变或肿瘤已达中晚期而失去手术切除时机.自Goldstein(1976)首先用肝动脉栓塞治疗肝癌以来,经20余年的观察和改进,取得了良好的疗效,目前经肝血管栓塞化疗已成为多数不能切除的中晚期肝癌的首选疗法.1 栓塞化疗的途径、方法及机理1.1 经肝动脉栓塞化疗 研究表明,正常肝脏的血供20%～25%来自肝动脉,75%～80%来自门静脉;而肝癌的血供90%以上来自肝动脉,门静脉血供在10%以下,门静脉主要供给肿瘤边缘、子结节及小肝癌.中国人原发性肝癌80%发生在肝硬变的基础上,肝血流明显减少,肝硬变发生肝癌时,肝动脉扩张增粗,癌灶内大量小动脉新生,交织成团,使肝癌动脉血供骤增.肝动脉阻断后,正常肝组织血供减少约35%,肝细胞损伤轻微且较快恢复正常,而肝癌血供减少90%以上,导致癌细胞大部分甚至全部坏死,这是肝动脉栓塞治疗肝癌的理论基础.     

活体成像系统检测携荧光素酶增殖缺陷型腺病毒在小鼠体内的表达和分布

目的:研究携带荧光素酶的增殖缺陷型腺病毒(Ad-luciferase,Ad-Luci)经不同途径注射后小鼠体内荧光素酶的表达和分布,为肿瘤的腺病毒载体基因治疗提供参考。方法:建立人胰腺癌PANC-1细胞裸鼠移植瘤模型,Ad-Luci病毒单次或多次瘤体内注射,或者肌内、尾静脉和腹腔注射Ad-Luci病毒至正常小鼠,采用IVIS Lumina活体成像系统观察荧光素酶在小鼠体内的分布和表达,并观察Ad-Luci病毒的毒性作用。结果:单次瘤内注射Ad-Luci荧光素酶在瘤内持续表达15 d,多次瘤内注射后表达时间更长。肌内注射组荧光素酶随时间延长表达减弱,注射后48 h无荧光素酶表达。尾静脉注射组荧光素酶大部分聚集在肝脏,注射后18 d仍有表达。腹腔注射组4 h后可见荧光素酶的表达,但7 d后无表达。Ad-Luci各注射组小鼠均未出现明显的毒性反应。结论:腺病毒携带外源基因经瘤内注射可在瘤内表达较长时间,多次注射可延长表达时间;经尾静脉注射后主要聚集在肝脏,且表达时间较长。     

肝脏海绵状血管瘤的双重供血及外科处理

13年来,住院治疗的68倒成人肝脏海绵状血管瘤病人,男31例,女37例.年龄36~67岁,平均43岁.肿瘤直径3.5~29cm.手术包括单纯肿瘤切除,肝脏楔形切除,肝叶切除,肝动脉TH胶栓塞或99.8%酒精注射.TH胶异位栓塞致死1例.肝动脉和门静脉造影显示:肝脏海绵状血管瘤系肝动脉和门静脉双重供血.肝动脉栓塞,硬化疗法和结扎的适应证;有症状的巨大海绵状血管瘤,手术切除困难或有较大的手术危险性者.     

腹腔内注射IL-2重组腺病毒、IL-3重组腺病毒增强化疗药物抗白血病作用的研究

对实验性白血病小鼠模型大剂量化疗后,直接腹腔注射IL-2重组腺病毒(Ad-IL-2)和/或IL-3重组腺病毒(Ad-IL-3),发现腹腔注射对照腺病毒载体小鼠虽经大剂量化疗,仍有大量白血病细胞浸润至骨髓、血管、肝脏及脾脏。而腹腔注射Ad-IL-2或Ad-IL-3组小鼠肿瘤生长缓慢,注射Ad-IL-2组小鼠脾NK、CTL活性显著提高,注射Ad-IL-3组小鼠腹腔巨噬细胞数量及杀伤活性明显提高,联合应用Ad-IL-2,Ad-IL-3组小鼠抗白血病作用最为明显,骨髓中虽然仍见白血病细胞,但可见较多正常造血细胞,且肝、脾中未见白血病细胞浸润。表明腹腔内注射Ad-IL-2和Ad-IL-3可显著增强大剂量化疗对白血病的治疗效果。     

经肝动脉门静脉双途径灌注化疗治疗直肠癌肝转移一例

男性患者,55岁,直肠腺癌根治术后50天,复查CT扫描:肝右叶多个低密度影占位病变,结合临床考虑系转移性肝癌。入院后剖腹探查见肝右叶多个.肝左叶两个直径0.3～2.0cm,的散在分布、大小不等的质硬结节。即经胃网膜右动脉、静脉分别置入国产皮下植入式药泵各一具进入肝固有动脉和门静脉。术后病理报告:肝转移性腺癌。用CF/5-FU疗法:前四天经肝动脉,后三天经门静脉给药:CF(醛氢叶酸钙)200mg/日,2小时滴完;5-FU500mg/日,开始滴注CF 1小时后注射,28天为一周期。两周期后复查CT:肝脏大小及形态未见异常,肝实     

热碘油栓塞阻断兔肝VX2肿瘤血供的研究

[目的]应用多普勒超声观察热碘油栓塞阻断兔肝VX2肿瘤血供的效果及其对肝脏血流动力学指标的影响.[方法]30只载瘤兔,随机分为3组.分别采用37℃生理盐水(对照)、37℃碘油及60℃热碘油经兔肝动脉灌注治疗,1周后多普勒超声观察肿瘤血供、肝动脉最大血流速度(Vmax)、阻力指数(RI)、门静脉平均血流速度(Ⅴ)、门静脉血管内径的变化.[结果]治疗前,所有病灶能量及彩色多普勒均可检测出瘤内及瘤周较丰富血流信号,治疗后显示60℃热碘油组瘤内及瘤周血流信号均明显减弱,部分消失.治疗后60℃热碘油组肝动脉血流速度与37℃碘油组比明显降低(P=0.004),与对照组比阻力指数增大(P=0.021),门静脉血流速度及内径与其他两组比尤明显变化(P均＞0.05);60℃热碘油组存活期41.0±3.0d与对照组31.5±3.0d相比有显著差异(P=0.006).[结论]热碘油栓塞可更有效地阻断兔肝VX2肿瘤供血,延长荷瘤兔生存期.     

原癌基因c-met在肝脏离断门静脉分支结扎大鼠术后剩余肝中的表达

目的观察大鼠肝脏离断+门静脉分支结扎术后剩余肝组织中c-met基因和蛋白的表达及其在术后剩余肝快速再生中的作用。方法将雄性SD大鼠96只应用计算机产生随机化数字完全随机分为肝脏离断+门静脉分支结扎术组、单纯门静脉分支结扎术组和假手术组。检测术后24 h、72 h、7 d和10 d时各组大鼠剩余肝组织中c-met基因和蛋白的表达。结果肝脏离断+门静脉分支结扎术组术后剩余肝组织中c-met mRNA和蛋白的表达均增加,并于7(10.09±1.41)d达到峰值,明显高于单纯门静脉分支结扎术组的(3.49±0.94)(P0.05)。结论肝脏离断+门静脉分支结扎术后大鼠剩余肝组织中原癌基因c-met表达明显升高。     

不同途径免疫的AFP基因修饰DC瘤苗体内抗肿瘤效应的研究

目的:评价bcl-2反义寡核苷酸对肾细胞癌细胞Bcl-2蛋白表达抑制及诱导凋亡作用.方法:合成与已知人类基因无同源性的bcl-2反义寡核苷酸(AS1与翻译起始端互补,AS2与编码区互补),以阳离子脂质体Lipofectin为转染载体,MTS比色实验测定细胞活率,反转录-聚合酶链反应(RT-PCR)测定bcl-2 mRNA的表达,Western杂交法测定Bcl-2蛋白表达,碘化丙啶(PI)染色流式细胞术测定凋亡细胞.结果:所测定的5个肾癌细胞株均有稳定的bcl-2 mRNA表达;转染的反义寡核苷酸对ACHN细胞Bcl-2蛋白的表达有明显抑制作用,而正义寡核苷酸无明显影响,AS2的抑制作用大于AS1;反义寡核苷酸可诱导ACHN细胞的凋亡,AS1和AS2的诱导率分别为32.1%和43.2%.结论:bcl-2反义寡核苷酸可抑制肾癌细胞Bcl-2蛋白的表达,并进而诱导细胞的调亡.     

Bcl-2反义寡核苷酸诱导胃癌细胞凋亡的研究

目的　探讨反义寡核苷酸（ＡＳＯＤＮ） 对ｂｃｌ２ 基因的表达调控及诱导胃癌细胞凋亡的作用。方法　应用ＭＴＴ方法比较两条人工合成的ＡＳＯＤＮ 直接作用及其以脂质体（ＤＯＴＡＰ） 为载体对胃癌细胞的抑制效果。应用流式细胞术和ＲＴＰＣＲ 方法检测ＡＳＯＤＮ 对ｂｃｌ２ 蛋白和ｍＲＮＡ 表达量的调控。应用相差显微镜、电子显微镜、流式细胞术等方法,观察ｂｃｌ２ ＡＳＯＤＮ 诱导ＢＧＣ８２３ 胃癌细胞凋亡的效果。结果　ＭＴＴ实验显示两条ＡＳＯＤＮ 抑制胃癌细胞增殖的作用呈浓度和时间依赖性,且靶位点于ｍＲＮＡ蛋白编码区（ＡＳＯＤＮ２） 和以ＤＯＴＡＰ 为载体的ＡＳＯＤＮ 对胃癌细胞的增殖抑制效果为佳。ＡＳＯＤＮ作用于胃癌细胞,降低ｂｃｌ２ 蛋白和ｍＲＮＡ 表达。ｂｃｌ２ ＡＳＯＤＮ处理胃癌细胞,观察到肿瘤细胞缩小、凋亡小体出现、染色质浓缩、凋亡峰等凋亡特征性改变。结论　ｂｃｌ２ ＡＳＯＤＮ可降低ｂｃｌ２ ｍＲＮＡ和蛋白表达,诱导胃癌细胞凋亡     

Bcl-2反义寡核苷酸增强阿糖胞苷诱导原代白血病细胞凋亡的研究

背景与目的:有研究证实,针对bcl-2mRNA翻译起始区和蛋白编码区的2个有效反义作用靶点的反义寡核苷酸能增强HL-60和K562细胞对阿糖胞苷(Ara-C)、柔红霉素、足叶乙甙的敏感性。本研究观察这两个新靶点的反义寡核苷酸对Ara-C诱导原代培养的急性白血病(AL)、慢性淋巴细胞白血病(CLL)细胞凋亡的影响。方法:用细胞记数法观察细胞的生存情况;免疫荧光标记法通过流式细胞仪检测细胞Bcl-2蛋白水平;用姬姆萨染色法观察并用流式细胞仪检测细胞凋亡。结果:靶向bcl-2mRNA翻译起始区与靶向蛋白编码区的两个反义寡核苷酸分别与Ara-C联合作用AL、CLL细胞48h,细胞的活性受到明显的抑制,分别同无关寡核苷酸(NS-ODN)联合Ara-C组、单用Ara-C组进行比较,差异有显著性(P<0.05)。这两个不同靶点的反义寡核苷酸分别与Ara-C联用均能明显下调AL、CLL细胞Bcl-2蛋白的表达,并且联合作用AL、CLL细胞48h的凋亡细胞百分率分别与NS-ODN联合Ara-C组、单用Ara-C组进行比较,差异有显著性(P<0.05)。与靶向bcl-2mRNA翻译起始区的反义寡核苷酸比较,靶向蛋白编码区的反义寡核苷酸提高AL细胞对Ara-C的敏感性作用要强些(P<0.05)。结论:针对bcl-2mRNA翻译起始区和蛋白编码区两个靶点的反义寡核苷酸能增强Ara-C诱导AL、CLL细胞的凋亡。     

XIAP反义寡核苷酸抑制胶质瘤细胞增殖和诱导凋亡的体外实验研究

目的观察X连锁凋亡抑制蛋白（XIAP）反义寡核苷酸对人脑胶质瘤细胞系BT325的增殖及凋亡的影响。方法将XIAP全硫代反义寡核苷酸，通过脂质体途径转染BT325脑胶质瘤细胞。用CCK-8实验检测细胞杀伤率，RT-PCR和Western blot技术检测XIAP的mRNA和蛋白表达，JC—1检测细胞线粒体膜电位，流式细胞仪检测细胞凋亡率，比色法检测Caspase-3活性。结果XIAP反义寡核苷酸能够抑制BT325脑胶质瘤细胞的增殖；XIAP反义寡核苷酸使XIAP的mRNA和蛋白表达均明显下调，mRNA较对照组减少了67．8％，蛋白较对照组减少了63．3％；反义寡核苷酸使线粒体膜电位下降，诱导BT325细胞凋亡，反义寡核苷酸组的凋亡率为54．7％，错义链组为14．O％，两组比较，差异具有显著性（P〈0．05）；反义寡核苷酸使BT325细胞Caspase-3活性明显增高，与对照组比较差异有显著性（P〈0．05）。结论XIAP可能参与了脑胶质瘤细胞BT325的增殖和凋亡，下调XIAP可抑制BT325细胞增殖及促进其凋亡。     

Significance of COX-2 expression in human renal cell carcinoma cell lines

Accumulating evidences indicate that cyclooxygenase (COX)-2 plays an important role in tumorigenesis in many human cancers. Yet the relationship between COX-2 and human renal cell carcinoma (RCC) remains unclear. The aim of our study was to evaluate COX-2 expression in human RCC cell lines and its role in tumorigenesis of human RCC. Among the human RCC cell lines (SMKT-R4, OS-RC-2, ACHN) and normal renal cell line RPTEC, COX-2 overexpression was found in OS-RC-2 cells both at mRNA and protein levels. COX-2 sense- and antisense-orientated vectors were constructed and transferred into RCC cells. Significant suppression of cellular proliferation was demonstrated in OS-RC-2 antisense transfectants, whereas promotion was found in SMKT-R4 sense transfectants by colony-forming assay despite the observation that COX-2 specific inhibitor NS398 exhibited similar IC50 among RCC cell lines by MTT assay. In comparison with parent cells and sense transfectants, significant suppression of COX-2 expression and PGE2 production and increase in butyrate-induced apoptosis were observed in OS-RC-2 antisense transfectants by Western blot, ELISA assay and FACS analysis, respectively. Furthermore, tumor growth and angiogenesis of OS-RC-2 antisense transfectants in nude mice was significantly suppressed and the survival time of these mice was significantly prolonged. Our study demonstrates that COX-2 is overexpressed in OS-RC-2 RCC cell line and plays an important role in tumorigenesis of the cells in vivo, which implies that COX-2 may be a therapeutic target for COX-2-expressing RCC, and that suppression of COX-2 expression by antisense-based strategy may have potential utility in treatment of COX-2-expressing RCC.     

Inhibition of proliferation of human small cell lung cancer cells expressing an autocrine system for gastrin releasing peptide by antisense oligodeoxynucleotides to gastrin releasing peptide receptor

The objectives of this study were to investigate the effect of antisense (AS) oligodeoxynucleotides (ODNs) directed against gastrin releasing peptide (GRP) receptor mRNA on proliferation of human small cell lung cancer (SCLC) NCI-H345 cells which express the autocrine system for GRP. The methods used were to expose human SCLC cell lines to antisense ODNs or sense ODNs and to measure their proliferation by spectrophotometric assay or viable cell counts. Our results demonstrated that the single or combined AS ODNs against GRP receptor inhibited proliferation of human SCLC NCI-H345 cells significantly by 37% (P<0.01), but did not inhibit proliferation of either human bronchial epithelial BEAS 2B cells or human SCLC NCI-N417 cells, neither of which express the GRP autocrine system. The sense controls did not significantly inhibit proliferation compared with no treatment controls. Specificity was also demonstrated by the observation that cells exposed to AS ODNs had a decrease in GRP receptor expression as measured by specific binding of 34% (P<0.01), and when all three AS ODNs were used, binding was decreased by 60% (P<0.03). Furthermore, AS ODNs decreased by 75% the maximum percentage of cells responding to GRP in an intracellular calcium release assay. Our conclusions are that antisense ODNs directed against a GRP receptor which is involved in an autocrine loop in human SCLC cells inhibited proliferation of these cells by their impact on reducing GRP receptor expression. Further development of means of increasing AS ODN specificity and effectiveness in human SCLC cell is warranted.     

沉默EZH2基因表达对肾癌细胞生物学行为的影响

目的 研究RNA干扰靶向沉默EZH2基因表达对ACHN肾癌细胞株增殖、细胞周期和凋亡的影响。方法 应用Western blot法检测EZH2蛋白在人正常近端肾小管上皮细胞株HK-2和肾癌细胞株786-0和ACHN中的表达。脂质体法介导化学合成EZH2 siRNA转染ACHN细胞株,Western blot法检测转染后ACHN细胞EZH2蛋白的表达情况,CCK-8法检测转染后ACHN细胞增殖率,流式细胞术检测转染后ACHN细胞周期分布和凋亡情况。结果 肾癌细胞株786-0和ACHN中EZH2蛋白的表达水平明显高于人正常近端肾小管上皮细胞HK-2。RNA干扰EZH2基因可成功地敲减ACHN细胞EZH2蛋白的表达。EZH2-siRNA干扰后,实验组ACHN细胞生长明显受抑制,在转染后48和72 h细胞增殖受抑制效应著（P＜0.05）。细胞周期分析显示：siEZH2转染组G1期ACHN细胞比例呈增加趋势,而S期和G2期细胞比例呈减少趋势,其中在转染后48h G1期ACHN细胞比例显著增加,而S期和G2期细胞比例明显下降（P＜0.05）;凋亡分析显示：siEZH2转染组ACHN细胞凋亡率随转染后时间的延长而呈增加趋势,在转染后48和72 h细胞凋亡率增加最显著（P＜0.05）。结论 沉默EZH2基因表达可靶向肾癌ACHN细胞周期中G1/S限制点而阻滞细胞周期进展,同时可有效抑制肾癌细胞增殖和促进细胞凋亡。     

G3139 induces cell death by caspase-dependent and -independent apoptosis on human melanoma cell lines

G3139 is an 18-mer phosphorothioate oligodeoxynucleotide (ODN) which has been targeted on the initiation codon region of the bcl-2 gene. Currently, clinical trials on G3139 for diverse tumors are underway in phase II and phase III. However, basic investigations of bcl-2 antisense ODN (G3139) and reverse ODN (G3622) have not been fully examined. In this report, we investigate cell death caused by G3139 and G3622 and the impact of antisense ODN in melanoma cell lines. We confirmed that G3139 reduced the level of bcl-2 protein and both G3139 and G3622 inhibited cell proliferation and induced apoptosis. G3139 was noted to produce a more intense effect than G3622. Although the general caspase inhibitor, Z-VAD-fmk, prevented apoptosis incompletely, the inhibition ratio of both ODNs was approximately equivalent. Our results suggested that inhibition of cell proliferation by ODNs is produced by apoptosis, but that the apoptotic pathway is not fully induced by the caspase-dependent pathway. Upon examination of the intracellular apoptotic protein dynamics, AIF localized within the mitochondria was translocated to the cytosol within 24 h, and subsequently to the nuclei after 48 h of treatment with G3139. Our results imply the following: the transfection of ODNs can induce apoptosis, the anti-tumor effect of G3139 is better than G3622, and the difference in the anti-tumor effect is specifically based upon the reduction of expression of the target DNA in malignant tumors. We consider that antisense ODNs may be an important tool for anti-tumor chemotherapy and the targeting of specific DNA is important in enhancing the anti-proliferative effect against tumors.