药物诱导细胞凋亡治疗肝癌

细胞凋亡(apoptosis)是细胞自然衰老、死亡的一种形式,是一切生物正常胚胎发生过程和人类发育过程细胞清除的正常途径。这一过程的紊乱,将导致人类发生多种疾病。许多研究资料表明,肿瘤的发生与细胞凋亡有密切关系,细胞凋亡异常在肿瘤发生和发展过程中具有非常重要的病理生理意义,细胞凋亡参与肿瘤的起始过程,并对癌症的发生起负相调控作用,Dive认为肿瘤的化疗、放疗的目的就是诱导肿瘤细胞凋亡。因此,在肝癌的治疗中不断地探讨新方法新途径,包括化疗药物、放射线、细胞因子、激素和基因编码等。近年来大量实验研究发现,肝癌细胞可以通过人为地触发细胞凋亡而被清除。其中药物诱导细胞凋亡对今后肝癌治疗研究提供了诱人的前景。不同种类的化学药物,生物药物和中药对不同种类肿瘤敏感细胞有促进凋亡作用。部分化疗药物、生物药在体外实验中能诱导肝癌细胞凋亡,如顺铂、环磷酰胺、阿霉素、细胞因子等。下面就药物诱导细胞凋亡治疗肝癌研究简要综述如下。     

扶正抗癌冲剂对体外胃癌细胞的抑制作用研究

目的研究中药扶正抗癌冲剂对体外胃癌细胞有无直接抑制或杀伤作用．方法采用体外培养的ＭＫＮ４５，ＭＫＮ２８及ＳＧＣ７９０１三种胃癌细胞株，以扶正抗癌冲剂配制药液（１６１５ｍｇ／ｍｌ含量）的不同稀释度，对３种胃癌细胞体外作用观察，并设丝裂霉素阳性对照组及单纯培养液加生理盐水阴性对照组．结果扶正抗癌冲剂对３种胃癌细胞株均有一定抑制作用，对低分化ＭＫＮ４５胃癌细胞抑制作用最明显，在１∶２稀释度４８ｈ抑制作用最强，抑制率达７４７％，持续到１２０ｈ抑制率仍在７１０％，与正常对照组比较有显著差异（Ｐ＜００５）．在电镜下观察被抑制的ＭＫＮ４５胃癌细胞发生线粒体肿胀、内质网水肿、胞浆脂肪空泡样变性、核仁缩小等变化．结论扶正抗癌冲剂对ＭＫＮ４５低分化胃癌细胞有较强的抑制和杀伤作用，并能使胃癌细胞结构改变．     

三氧化二砷对人肝癌细胞株的影响

三氧化二砷是中药砒石的主要成分,治疗急性早幼粒细胞白血病已获得满意疗效,其完全缓解率在初治患者达73%,复发患者也达52%,最长缓解期超过10 a.本药静脉给药为宜,不产生明显毒副作用[1],并且在治疗白血病时与其他化疗药之间无交叉耐药性[2].为进一步拓展药用价值,扩大其肿瘤谱及临床治疗应用谱,我们探讨三氧化二砷对人肝癌细胞株生长的抑制作用及其机制,为其用于肝癌治疗提供理论依据.     

MGr1抗体筛选文库获得胃癌耐药相关的新基因片段

目的制备胃癌多药耐药相关性单克隆抗体(mAb),并以该抗体筛选肿瘤耐药细胞表位文库,以获得肿瘤耐药相关的新基因片段.方法采用杂交瘤技术,以胃癌耐药细胞株SGC7901/ VCR为免疫原制备胃癌多药耐药相关性,mAbMGr1,并以MGr1作为探针对肿瘤耐药细胞表位文库反复进行筛选;对稳定阳性重组子所携带的cDNA片段进行正反向测序,并将测序结果输入GeneBank进行同源性分析;原位杂交与Northern blot检测阳性克隆在不同组织细胞中的表达.结果经过21次融合获得了26株培养上清能与SGC7901/ VCR结合的杂交瘤克隆,经免疫组化和单克隆抗体逆转耐药实验发现其中一株mAb在SGC7901/ VCR膜表面及胞内的表达显著高于其相应的药物敏感细胞SGC7901,并能部分逆转SGC7901/ VCR对ADR和5-FU的耐药性. Western blot表明该mAb对应的抗原Mr 800 000～110 000,与Pgp和MRP不同,该株mAb被命名为MGr1. 以MGr1筛选文库,获得了5个能与MGr1抗体结合的携带阳性重组子的菌落,经反复筛选后,证实有一个稳定的阳性克隆;正反向测序结果表明该阳性cDNA片段长度为310 bp,GeneBank检索显示该片段无任何同源性;原位杂交表明该基因在肝、食管、胃、大肠的正常组织均未见表达,Northern blot证实该基因在胃癌细胞SGC7901,胃癌耐药细胞SGC7901/ VCR,食管癌细胞EC109,肝癌细胞HCC7402中均有表达,但在SGC7901/ VCR中的表达量显著高于其他细胞.结论成功地制备了胃癌耐药相关性mAbMGr1. 以MGr1抗体筛选肿瘤耐药细胞表位文库获得了一个与胃癌耐药相关的新基因片段.     

促胃液素及其受体拮抗剂丙谷胺对人胃癌细胞系生长的影响

目的观察和分析五肽促胃液素ＰＧ及其受体拮抗剂丙谷胺ＰＧＭ对人胃癌细胞系ＭＧＣ生长的影响,为临床应用促胃液素受体拮抗剂协助治疗胃癌提供依据．方法选用５ｍｇ／Ｌ,１０ｍｇ／Ｌ,１５ｍｇ／Ｌ,２０ｍｇ／Ｌ４种浓度的ＰＧ和３０ｍｇ／Ｌ的ＰＧＭ分别作用于体外培养的浓度为２５×１０８／Ｌ的ＭＧＣ,分别培养２４,４８,７２ｈ,于酶标仪上选用波长５４０ｎｍ测定吸光值Ａ,并对数据进行比较分析．结果４种浓度的ＰＧ作用于ＭＧＣ,ＭＧＣ连续３ｄ的生长状态与对照组无明显差异,而ＭＧＣ在ＰＧＭ作用下,其连续３ｄ的平均Ａ值分别为００２９,００４６和００８４,而未被ＰＧＭ作用的ＭＧＣ的平均Ａ值分别是０１０１,０１１５和０１８２,ＭＧＣ在ＰＧＭ作用下生长明显低于对照组（Ｐ＜００５）．结论外源性的ＰＧ对ＭＧＣ无营养促进作用,而ＰＧＭ能抑制ＭＧＣ的生长     

培养胎肝细胞上清对暴发性肝衰竭小鼠存活率的影响

目的探讨胎肝细胞悬液治疗严重肝病的机理以及使用培养胎肝细胞分泌上清（ＦＨＣＳ）替代的可能性．方法采用Ｄ＿氨基半乳糖（Ｄ＿ｇａｌｎ）／内毒素（ＬＰＳ）诱发小鼠暴发性肝衰竭（ＦＨＦ），观察ＦＨＣＳ的保护作用．结果ＦＨＣＳ能显著提高ＦＨＦ小鼠的存活率（Ｐ＜００１），降低血清转氨酶（Ｐ＜００５），减轻肝细胞坏死程度，并促进肝细胞再生．结论ＦＨＣＳ对ＦＨＦ有较好的保护与治疗作用，可望为已被迫放弃的胎肝细胞悬液输注疗法开辟新的途径     

人大肠癌细胞株的转移潜能及其相关特性的比较

目的比较两种人大肠癌细胞株的转移潜能及其相关性．方法利用裸鼠实验性肝转移模型比较人大肠癌ＨＴ２９与ＬｏＶｏ细胞株的转移潜能；定量检测两种细胞株层粘连蛋白受体（ＬＮＲ）、上皮性钙调蛋白（Ｅｃａｄ）及Ⅳ型胶原酶的表达；用原位分子杂交及免疫组化检测两细胞株ＣＤ４４Ｖ６在蛋白质水平及ｍＲＮＡ水平的表达．结果ＬｏＶｏ细胞株为人大肠癌高转移株，裸鼠实验性肝转移率为１００％；ＨＴ２９细胞株转移相对较低，裸鼠肝内转移率为４２９％．定量检测发现，ＬｏＶｏ细胞几乎不表达Ｅｃａｄ，ＨＴ２９细胞膜上弱表达Ｅｃａｄ．ＬｏＶｏ表达ＬＮＲ与Ⅳ型胶原酶高于ＨＴ２９．ＬｏＶｏ细胞内ＣＤ４４Ｖ６的表达在蛋白质水平与ｍＲＮＡ水平均高于ＨＴ２９细胞．结论ＬｏＶｏ细胞株为人大肠癌高转移株，ＨＴ２９细胞株转移力相对较低；ＬＮＲ、Ｅｃａｄ、Ⅳ型胶原酶及ＣＤ４４Ｖ６可作为大肠癌细胞转移潜能的相关指标．     

微载体培养人肝细胞及肝非实质细胞

目的研究用微载体培养人肝细胞及肝非实质细胞的方法．方法采用体外简易两步灌流和差速离心法获取胎肝细胞和肝非实质细胞，在综合限定条件下进行微载体ｃｙｔｏｄｅｘ３粘附培养，并对培养肝细胞的形态及合成葡萄糖和白蛋白的功能进行动态测定．结果分离肝细胞及肝非实质细胞在接种时即呈明显的聚集倾向，将其与微载体混合振荡孵育２０ｍｉｎ后，二者极易相粘附．在被覆聚羟乙基异丁烯酸的培养瓶内，使用激素限定条件培养液培养约４８ｈ，典型的多细胞聚集球形体得以形成．该形态特征以及白蛋白、葡萄糖合成能力可保持１月结论使用微载体培养人肝细胞和肝非实质细胞具有多种优点，有广泛的应用价值．     

抗癌药物与钙拮抗剂合用对体外人胃癌细胞的影响

目的和方法：本文报道了抗癌药物与５－氟脲嘧啶（５－ＦＵ）、阿霉素（ＤＯＸ）及长春新碱（ＶＣＲ）与钙拮抗剂维拉帕米（ＶＰＭ）、尼卡地平（ＮＩＣ）、汉防已甲素（ＴＥＴ）、硝苯吡啶（ＮＩＦ）及硫氮草酮（ＤＩＬ）单独和联合应用对体外人胃癌细胞株ＭＫＮ４５、ＭＫＮ２８及ＭＧＣ８０３细胞增殖影响的研究结果。结果：不同浓度的５种钙拮抗剂单独处理各胃癌细胞株，发现随着浓度升高，胃癌细胞增殖受到的抑制作用也增强，其中ＶＰＭ、ＮＩＣ和ＴＥＴ较为显著。在联合用药实验中，无毒剂量ＶＰＭ、ＮＩＣ与低浓度ＤＯＸ、５－ＦＵ、ＶＣＲ合用时，可使这些化疗药物抑制胃癌细胞增殖的作用增强２．７～７．５倍。小剂量的ＴＥＴ可增强ＤＯＸ对３种胃癌细胞株的细胞毒作用，亦可增强ＶＣＲ、５－ＦＵ对某些细胞株的作用。而ＮＩＦ和ＤＩＬ对抗癌药物细胞毒性的增强作用不显著。结论：结果提示ＶＰＭ、ＮＩＣ及ＴＥＴ等钙拮抗剂能增强ＤＯＸ、５－ＦＵ和ＶＣＲ对体外人胃癌细胞的抗癌活性     

复方中药体外保护肝细胞的机制

目的探讨复方中药制剂体外保护肝细胞的机制.方法用MTT法、荧光染色、透射电镜、DNA琼脂糖凝胶电泳及流式细胞术等方法观察不同浓度中药复方制剂(包括黄芩、黄柏、板蓝根、山豆根等)抑制TNF-α+Act-D诱导正常大鼠肝细胞凋亡的作用,并检测各组清蛋白的分泌结果自组中药复方制剂能明显改善肝细胞的凋亡状况,出现了典型的形态学和生化学改变,但存在药物浓度上的差异.中药组(0.2,2 mg.L-1)的凋亡率(10.4%±1.2%,13.9±0.4%)低于凋亡模型组(vs17.4%±1.9%,n=3,P＜0.05),中药组(20mg.L-1)的凋亡率(18.30%±1.15%)与凋亡模型组相比没有显著性差异(P＞0.05).除此之外,自组中药复方制剂还促进肝细胞清蛋白的分泌,但存在作用时间和药物浓度的交互作用.结论复方中药制剂能够抑制正常肝细胞凋亡并促进其清蛋白分泌.     

Inhibitory activity of polysaccharide extracts from three kinds of edible fungi on proliferation of human hepatoma SMMC-7721 cell and mouse implanted S180 tumor

lNTRODUCTlONThepolysaccharidesfromediblefungi(e.g.LE[1],FV[z's])weremacromolecularsubstanceswithstrongantigenicityandwerealsoverifiedtohaveantitumoractivityagainstS-l80implantedtumorinmiceinvivoandthatfromABwereshowntohaveantiinfectionofvirusandanticancerationinvivo.Thereferencesabouttheeffects0fp0lysaccharideextractsfromediblefungioncancercellsinvitr0wereverylimited.InthisreporthumanhepatomaSMMC-772lcellswereusedasamodelt0detecttheanticanceractivityofpolysaccharideextractsfromthethree…     

Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

AIM To evaluate antihepatoma effect ofantisense phosphorothioate oligodeo-xyribonucleotides(S-ODNs)targeted to alpha-fetoprotein(AFP)genes in vitro and in nudemice.METHODS AFP gene expression was examinedby immunocytochemical method or enzyme-linked immunosorbent assay.Effect of S-ODNson SMMC-7721 human hepatoma cell growth invitro was determined using microculturetetrazolium assay.In vivo antitumor activitiesof S-ODNs were monitored by measuring tumorweight differences in treated and control micebearing SMMC-7721 xenografts.Induction of cellapoptosis was evaluated by fluorescence-activated cell sorter(FACS)analysis.RESULTS Antisense S-ODN treatment led toreduced AFP gene expression.Specificantisense S-ODNs,but not control S-ODNs,inhibited the growth of heaptoma cells in vitro.In vivo,only antisense S-ODNs exhibitedobvious antitumor activities.FACS analysisrevealed that the growth inhibition by antisenseS-ODNs was associated with their cell apoptosisinduction.CONCLUSION Antisense S-ODNs targeted toAFP genes inhibit the growth of human hepatomacells and solid hepatoma,which is related totheir cell apoptosis induction.     

Effect of gastrin and anti-gastrin antibodies on proliferation of hepatocyte cell lines

Gastrin (G-17) and its precursor glycine-extended gastrin (G-17-gly) have been shown to be trophic to some gastrointestinal tumors. This in vitro study assessed the effect of G-17, G-17-gly, anti-gastrin antibodies (anti-G-17), and the CCK-B receptor antagonist PD135,158 on three hepatoma cell lines (PLC/PRF/5, HepG2 and MCA-RH7777) and an embryonic liver cell line (WRL68). The pancreatic adenocarcinoma cell line AR42J was used as a positive control. G-17 and G-17-gly caused significant proliferation of AR42J and WRL68 cell lines. G-17-gly but not G-17 induced significant proliferation of the PLC/PRF/5 cell line. Anti-G-17 and PD135,158 significantly inhibited unstimulated AR42J and WRL68 cell lines. Anti-G-17 also inhibited the proliferative effects of G-17 and G-17-gly on AR42J, WRL68, and PLC/PRF/5 cell lines, whereas PD135,158 inhibited the proliferative effect of G-17 only. G-17 and G-17-gly as well as anti-G-17 and PD135,158 had no effect on HepG2 and MCA-RH77777 cell lines. It is concluded that G-17-stimulated proliferation is mediated via the CCK-B receptor and G-17-gly via a separate, as yet uncharacterized, receptor. There may therefore be a role for gastrin in embryonic hepatocellular proliferation and perhaps also in the proliferation of some hepatocellular tumors.     

Effect of liposome-entrapped methotrexate on ehrlich ascites tumor cells and uptake in primary liver cell tumor

Summary A therapeutically useful concentration (0,5 mg/ml) of Methotrexate was prepared in negatively charged artificial liposomes (lecithine: cholesterol: dicetylphosphate=5:5:1 molar ratios). Entrapment yield after separation on Sepharose 6B is nearly 100%. In contrast to free Methotrexate the liposome entrapped folic acid antagonist is eliminated only slowly by the kidneys and after intravenous application it is unevenly distributed in the body of mice, the highest concentrations being found in liver and spleen. Daily injections (7.5 mg/kg/day) of entrapped Methotrexate for 5 days into the tail vein of Ehrlich ascites tumor bearing mice reduced both tumor cell count and the production of ascites fluid about fourfold as compared to mice receiving the same dose of Methotrexate in the free form.Six hours after intravenous application of liposome entrapped Methotrexate the tissue concentration in normal liver is ca. 20-fold higher than when the same amount is applied in the free state. On the other hand, only a little uptake of liposome entrapped Methotrexate was detected in the tissue of nitrosamine-induced primary liver tumor when compared to normal liver tissue of rats.