IM-94对肝癌细胞的杀伤作用的实验研究

目的 探讨IM-94对人肝癌细胞的杀伤作用。方法 采用MTT法分析IM-94对人肝癌细胞772l生长的影响,用流式细胞术检测细胞有丝分裂周期及细胞凋亡,用H．E．染色分析细胞的核分裂相。结果 IM-94对肝癌细胞有明显的抑制作用,从流式细胞术分析细胞阻滞在G2 M期,并诱发细胞凋亡,在24h后,出现凋亡小峰。结论 IM-94能阻滞细胞分裂,诱导肝癌细胞凋亡。     

Transport mechanism of ursodeoxycholic acid in human placental BeWo cells

Ursodeoxycholic acid (UDCA) is a first‐line drug to treat intrahepatic cholestasis of pregnancy (ICP). However, its effects on the fetus are not clearly known. To better guide its clinical use, we aimed to study the mechanism underlying the placental transport of UDCA. The uptake and efflux of UDCA across placental apical membranes were studied using BeWo cells; effects of different exposure durations, UDCA concentrations, temperatures, and inhibitors of transporters were studied. A transwell assay was performed, and UDCA concentration in both fetal and maternal sides was measured using LC–MS/MS. Higher unidirectional transport of UDCA was observed in the basolateral‐to‐apical direction than in the apical‐to‐basolateral direction. Ko143 and verapamil, which are typical inhibitors of efflux transporters, significantly increased UDCA transport from different directions. UDCA uptake from the apical membrane of BeWo cells was time‐dependent, but sodium‐independent. It was inhibited by inhibitors of energy metabolism and of organic anion transporters, indicating an active transport mechanism. UDCA uptake from the apical membranes of BeWo cells could be mediated by organic anion‐transporting polypeptides, whereas its efflux could be mediated by breast cancer resistance protein and multidrug resistant protein 3. The results of the present study may provide a basis for UDCA use in pregnancy.     

古抑菌素对人肝癌细胞作用的研究

目的:通过组蛋白去乙酰化酶(histone deacetylase,HDACS)抑制剂古抑菌素A(Trichostatin A,TSA)对人肝癌细胞HepG2(以下简称HepG2)和正常肝细胞LO2(以下简称LO2)增殖与凋亡作用的比较,探讨TSA对肝癌的作用机制.方法:应用光学显微镜、透射电镜、四氮唑蓝(MTT)法、免疫细胞化学法观察经不同浓度TSA处理后的HepG2和LO2的增殖、凋亡与凋亡相关蛋白的改变.结果:HepG2细胞经TSA处理后,电镜下超微结构发生了凋亡早期改变;小剂量TSA对HepG2细胞具有较强的抑制作用,对LO2细胞影响不明显,当TSA浓度达到1000 nmol/L以上时,对LO2表现出明显的细胞毒性作用;TSA可诱导HeptG2细胞凋亡,并增加凋亡相关蛋白Bax的表达.结论:TSA可明显抑制肝癌细胞HepG2的增殖,其机制可能与上调Bax的表达,诱导细胞凋亡有关.     

新型维甲酸衍生物ATPR大鼠体内组织分布的研究

目的建立大鼠组织中新型维甲酸衍生物4-氨基-2-三氟甲基苯基维甲酸酯(ATPR)浓度的测定方法,并研究其在大鼠体内的组织分布。方法 SD大鼠灌胃给药ATPR(20mg·kg-1)和静脉注射给药ATPR(7 mg·kg-1)后,分别于给药后2、4、7 h和5 min、1、5 h取各脏器组织,经预处理,HPLC法测定各组织中ATPR浓度。结果大鼠灌胃ATPR后,肠组织中药物浓度最高,其次在肝、脾、肺组织浓度较高,在心、肾、脂肪和脑组织中浓度较低。静注给ATPR后,肝组织中药物浓度最高,其次在脾、肺组织浓度较高,在心、肾、肠、脂肪和脑组织中浓度较低。结论两种给药途径给药ATPR后,肝组织的药物浓度均较高,提示ATPR在诱导肝癌细胞分化和抗癌细胞增殖方面可能会有较好的靶向作用。     

乙酰水杨酸增强多胺衍生物ANISpm抗肝癌作用及其机制研究

探讨乙酰水杨酸增强多胺衍生物ANISpm抗肝癌作用及其作用机制。采用MTT法检测细胞毒性;应用高内涵活细胞成像系统检测细胞内ANISpm含量的变化及对细胞凋亡和线粒体膜电位的影响;采用Western blotting方法检测细胞色素c,精脒/精胺乙酰转移酶,Caspase-3、-8、-9等蛋白表达的变化;应用高效液相色谱法检测细胞内多胺含量的变化。应用H22肿瘤移植模型评价对实体瘤生长的影响。实验结果显示,乙酰水杨酸体内外均具有增强ANISpm抗肝癌的作用,其机制为乙酰水杨酸通过上调肝癌细胞内精脒/精胺乙酰转移酶的活性,降低细胞内多胺含量从而增加了ANISpm的摄取,并通过ANISpm诱导肝癌细胞凋亡。该凋亡作用与ANISpm诱导细胞线粒体膜电位降低,促进细胞色素c释放以及活化Caspase-3、-8、-9等有关。     

应用丙戊酸钠调节组蛋白乙酰化修饰抑制肝癌细胞增殖的作用及机制

目的观察应用组蛋白脱乙酰酶抑制剂丙戊酸钠调节染色体组蛋白低乙酰化水平对肝癌细胞增殖的作用，并进一步检测Cyctin A、Cyclin D1、Cyclin E、P21^Waf/cip1蛋白及mRNA表达变化，探讨其分子作用机制。方法应用0．75～4．0mmol／L丙戊酸钠作用于肝癌细胞系HepG2细胞后，MTT法检测细胞生长抑制、细胞克隆形成试验观察克隆形成率、碘化丙啶标记流式细胞术检测细胞周期、间接免疫荧光法分析Cyclin A、Cyclin D1、Cyclin E、P21^Waf/cip1蛋白表达，RT—PCR检测分析Cyclin A、Cyclin D1、Cyclin E、P21^Waf/cip1 mRNA表达。结果0．75～4．0mmol／L丙戊酸钠作用24、48、72、96h，观察组出现了时间-剂量依赖趋势的生长抑制，同时细胞克隆形成率显著降低，对照组细胞增殖周期G1、S、M期所占比例未见明显改变，而观察组则随药物浓度、作用时间的不同而出现不同程度的细胞增殖周期G1期阻滞，G1期比例由55．4％～82．8％不等，差异有统计学意义（P〈0．01）。观察组Cyclin A、Cyclin D1蛋白及mRNA表达均被明显下调而P21^Waf/cip1蛋白、mRNA表达则被明显上调，与对照组比较差异均有统计学意义（均P〈0．01）；而Cyclin E蛋白和mRNA表达变化则未见明显差异（P〉0．05）。结论通过应用特异性组蛋白脱乙酰酶抑制剂调节组蛋白乙酰化修饰可明显抑制肝癌细胞生长、抑制细胞克隆形成、阻滞细胞增殖周期于G1期；丙戊酸钠作为组蛋白脱乙酰酶抑制剂可明显抑制肝癌细胞增殖，其作用机制是通过上调P21^Waf/cip1 mRNA蛋白表达，下调Cyclin D1、Cyclin A mRNA蛋白表达分别和／或协同实现。     

Enhancing effect of chemically conjugated deoxycholic acid on permeability of calcitonin in Caco‐2 cells

One approach for enhancing intestinal absorption of therapeutic peptides is chemical conjugation to bile acids. In this study, recombinant salmon calcitonin (sCT) was chemically modified by covalent attachment of deoxycholic acid (DOCA). Three different sCT‐DOCA conjugates, namely, sCT‐mono‐DOCA, sCT‐di‐DOCA, and sCT‐tri‐DOCA, were prepared and characterized for their structural and biological properties. The permeability of these conjugates in the gastrointestinal tract was evaluated using Caco‐2 cell monolayers to determine their potential as an oral dosage form. The conjugates were isolated by reversed‐phase fast protein liquid chromatography (FPLC) and confirmed by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Circular dichroism spectra in 50% trifluoroethanol aqueous condition showed the ordered secondary structure of sCT‐DOCA. The biological activities of sCT‐DOCA conjugates were evaluated by cyclic AMP assay using T‐47D cells, and the mean EC₅₀ values of sCT, sCT‐mono‐DOCA, and sCT‐di‐DOCA were 0.034, 0.076, and 0.46 nM, respectively. The transport experiments using the Caco‐2 cell monolayer showed that the permeability of sCT‐DOCA conjugates in buffer was not altered from the native sCT. However, the permeability of sCT‐DOCA conjugates was increased up to 2.5 times that of the native sCT when sCT‐DOCA was formulated in 1% dimethylsulfoxide (DMSO) solution. The conjugated DOCA of sCT‐DOCA significantly enhanced the absorption of sCT‐DOCA in the intestinal membrane when sCT‐DOCA conjugates were completely solubilized by DMSO. In conclusion, this study proposes that therapeutic peptides that have poor absorption profiles could potentially be developed into orally active drugs by conjugation with DOCA in the formulation with appropriate solubilizing agents. Drug Dev. Res. 64:129–135, 2005. © 2005 Wiley‐Liss, Inc.     

吡非尼酮对肝癌HepG2细胞增殖和凋亡的影响(“豪森杯”优秀论文三等奖)

目的：研究吡非尼酮（pirfenidone,PF）对人肝癌细胞系HepG2增殖和凋亡的影响。方法：CCK-8法测定不同浓度PF对HepG2细胞增殖活性的影响;Hoechst 33258荧光染色法观察PF处理后HepG2细胞形态的变化;流式细胞仪检测细胞凋亡率。结果：PF对HepG2细胞具有显著增殖抑制作用,并呈浓度和时间依赖性;Hoechst 33258染色可见PF处理后细胞出现典型的凋亡形态学变化;流式细胞仪检测结果显示,与空白组比较,PF处理后的HepG2细胞凋亡率显著增加（P﹤0.01）。结论：PF对人肝癌细胞系HepG2细胞增殖具有抑制作用,且与诱导HepG2细胞凋亡有关。     

Cytotoxic effects and pro-apoptotic mechanism of TBIDOM, a novel dehydroabietylamine derivative, on human hepatocellular carcinoma SMMC-7721 cells

We have investigated the antiproliferative effects of TBIDOM (N-(4-(2,2,2-trifluoroethyl) benzylidene) (7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl) meth-anamine) and have explored its possible mechanisms on human hepatocellular carcinoma SMMC-7721 cells. The proliferative status of cells treated with TBIDOM was measured by the colorimetric MTT assay. Cellular apoptosis was analysed using Hoechst 33342 staining and flow cytometry. Reduction of mitochondrial membrane potential (Delta psi(m)) was also detected by flow cytometry. Western blotting assay was used to evaluate the release of cytochrome c and expression of p53, Bcl-2 and Bax proteins. It was shown that TBIDOM displayed a significant inhibitory effect on growth of SMMC-7721 cells in a dose- and time-dependent manner. Hoechst 33342 staining and flow cytometry analysis showed an increase of apoptosis rate and decrease of mitochondrial membrane potential after SMMC-7721 cells were exposed to TBIDOM for 24 h. Pretreatment of SMMC-7721 cells with TBIDOM significantly induced a decrease of Bcl-2 protein expression and an increase of caspase-3 activity and Bax protein expression. The results indicated that TBIDOM could effectively inhibit proliferation by induction of apoptosis and could be a promising candidate in the development of a novel class of antitumour agent.     

lncRNA TP73-AS1对肝细胞癌细胞HepG-2凋亡的影响及其机制

目的探讨肝癌中长链非编码RNA TP73-AS1差异表达,及其对肝癌细胞凋亡的影响与作用机制。方法从TCGA数据库中下载并处理新一代测序(RNASEQ)数据,利用生物信息学方法获取显著的肝癌相关lncRNA与mRNA的共表达网络。将差异表达lncRNA映射入上述网络中,利用生物信息学方法预测肝癌相关lncRNA及其功能。通过MTT、AO/EB、Western blot方法对其功能学进行研究。结果共识别25 439对显著的lncRNA-gene共表达关系。在22个差异的lncRNAs被映射中,TP73-AS1度最高,该lncRNA的62个互作基因映射到病毒致癌作用(Viral carcinogenesis pathway)通路中,并能影响下游MAPK信号通路与p53信号通路。lncRNA TP73-AS1-siRNA影响HepG-2细胞增殖,上调促凋亡蛋白Bax表达,增加Caspase-3活性,下调抗凋亡蛋白Bcl-2表达,使p53蛋白表达升高。结论 lncRNA TP73-AS1通过p53途径调节肝细胞癌细胞系HepG-2凋亡,为长链非编码RNA成为临床肝细胞癌患者的有效治疗药物提供了实验依据。     

脱氧胆酸对胰岛素聚乳酸纳米粒在大鼠小肠吸收的影响

目的 :探讨脱氧胆酸 (DOC)对胰岛素聚乳酸纳米粒 (Ins PLA NP)在小肠吸收的影响。方法 :在正常大鼠小肠分别给予含与不含吸收促进剂DOC的Ins PLA NP后 ,观察降血糖效果。结果 :Ins PLA NP小肠直接给药表现出和缓持久的降血糖效果 ,而DOC在小肠给药中能明显加快Ins PLA NP的吸收并增强药效 ,给药后 0 5h血糖下降至用药前的 (35 49± 6 6 4) % ,显著的降血糖作用可维持 2h左右。结论 :DOC对于Ins PLA NP在小肠部位的吸收具有促进作用     

新鬼臼毒素衍生物ZM-16诱导K562/A02细胞凋亡及其机制

鬼臼毒素衍生物是目前抗癌药物研究的特点,目前临床上应用较为广泛的依托泊苷(VP-16)和替尼泊苷(VM-26)均为半合成鬼臼毒素衍生物[1].     

5-氨基水杨酸抗HT-29细胞增殖作用及部分机制

目的观察5-氨基水杨酸（5-aminosalicylicacid,5-ASA）对人结肠癌HT-29细胞增殖抑制作用及可能机制。方法采用MTT法测定5-ASA抗HT-29细胞增殖的量效和时效关系,采用TUNEL法和免疫细胞化学法探讨作用机制。结果模型对照组HT-29细胞增殖明显,不同剂量5-ASA对体外培养的结肠癌HT-29细胞增殖均具有明显抑制作用,呈剂量和时间依赖性;5-ASA在10-500μmol·L^-1剂量下,均可使HT-29细胞凋亡率增加,增殖细胞核抗原（PCNA）表达减弱;5-脂氧化酶（5-lipoxygenase 5-LOX）和环氧化酶-2（cycloxygenase-2cox-2）蛋白表达均逐渐降低,且5-LOX减弱程度大于COX-2。结论5-ASA具有抗HT-29细胞增殖作用,机制可能与阻断COX-2和5-LOX通路有关。     

Preparation of camptothecin-loaded targeting nanoparticles and their antitumor effects on hepatocellular carcinoma cell line H22

Abstract

Camptothecin (CPT) is an effective anticancer agent against various cancers but the clinical application is limited because of its poor water solubility, low bioavailability and severe toxic side effects. The aim of the present study was to evaluate the feasibility of using targeted NPs as a high-performance CPT delivery system that targets liver cancer cells through intravenous (i.v.) administration route. CPT was incorporated into biotin-F127-PLA or F127-PLA polymeric nanoparticles (NPs) by a dialysis method. The preparation of the targeting NPs was performed by conjugating biotin-F127-PLA NPs with anti-3A5 antibody. The antitumor effect of the CPT-loaded nanoparticles against H22 cells in vitro was determined using an MTT assay. Tissue distribution and tumor inhibition in vivo were also evaluated. Survivin mRNA expression was assessed by real-time polymerase chain reaction. Results showed that the targeted CPT NPs exhibited regular spherical shapes with a mean diameter of approximately 180?nm. In vitro release of the targeted CPT NPs exhibited an initial burst (40%) within 12?h, followed by a slow release. Cytotoxicity test against H22 cells indicated that the targeted CPT NPs exerted significant antitumor effects. Compared with free CPT and non-targeted CPT NPs, the targeted CPT NPs showed superior inhibition ratio against tumor in vivo, which may be associated with reduced survivin mRNA expression. The results suggested that the new targeted CPT NPs may be a promising injectable delivery system for cancer therapy.     

熊果酸在Caco-2细胞单层模型中的跨膜转运

熊果酸(Ursolic acid, UA),又名乌索酸,乌苏酸,属a-香树脂醇(a-amyri)型五环三萜类化合物.在自然界分布非常广泛,据目前所知,至少存在于26个科70多种天然植物中[1].大量的研究表明,UA具有广泛的药理作用和重要的生物活性,尤其在抗炎、护肝、抗肿瘤以及机体免疫调节等方面已经显现出令人关注的药理特性[2,3].体内药代动力学研究表明, UA的生物利用度低[4],口服吸收情况较差,而且其在体内吸收转运的方式和机制也并不清楚.Caco-2细胞系来源于人结肠类腺癌细胞,其结构和生化作用类似于人小肠上皮细胞,含有与小肠刷状缘上皮相关的转运系统以及一     

A kojic acid derivative promotes intrinsic apoptotic pathway of hepatocellular carcinoma cells without incurring drug resistance

Hepatocellular carcinoma is one of the most pervasive cancers with low prognosis, high frequency of recurrence, and metastasis. Studies conducted have focused on extricating novel strategies for successful treatment. Kojic acid and its derivatives are already proven to have depigmenting, anti‐inflammatory, and anti‐neoplastic properties. In the present study, kojic acid and its 10 distinct derivatives were tested on HEPG2 cell line for their possible anti‐cancer effect and seven of them were observed to be cytotoxic. Compound 6 was chosen to proceed as the IC₅₀ dosage for HEPG2 cells was lower in comparison with the other derivatives and kojic acid itself. Further experiments pointed out that intrinsic apoptotic pathway was triggered with the exposure of the cells to IC₅₀ concentration of the derivative as the treatment led to escalation of intracellular ROS, induction of TP53 gene, and activation of caspase 3/7. Pro‐apoptotic Jnk and Bax genes were not triggered suggesting that the apoptotic pathway advance through an alternative route. Complementary experiments are in need; howbeit, the current findings suggest that the derivative offers a novel promising approach against hepatocellular carcinoma as it is not detrimental to healthy cells within the concentrations applied, and it does not induce drug resistance.     

Induction of apoptosis in HepG2 human hepatocellular carcinoma cells by a novel derivative of ursodeoxycholic acid (UDCA)

The effects of ursodeoxycholic acid (UDCA) and its novel derivative, named as HS-1030, on the proliferation of HepG2, human hepatocellular carcinoma cells were investigated. Whereas UDCA had no significant effect in a concentration range we have tested, HS-1030 inhibited the proliferation of HepG2 cells in a concentration dependent manner. Surprisingly, HS-1030 had no effect on the proliferation of Human Chang liver cell which is a normal liver cell line. We also found that proliferation-inhibitory effect of HS-1030 was due to the induction of apoptosis of HepG2 cells, which was confirmed by observing the internucleosomal DNA fragmentation and morphological changes (i.e. cell shrinkage, nuclear condensation and the formation of apoptotic bodies). These results suggest that HS-1030 may be a good candidate as a drug for the treatment of liver cancer.     

5-氨基酮戊酸对人舌麟癌Tea8113细胞株体外光动力杀伤作用的基础研究

目的初步研究体外5-氨基酮戊酸介导的光动力疗法对人舌鳞癌Tea8113细胞的杀伤效应。方法体外培养Tca8113细胞,以5-氨基酮戊酸为光敏剂,半导体激光治疗仪给予光动力疗法,采用MTr法检测光敏剂不同孵育时间、不同光敏剂浓度对Tea8113细胞抑制率的影响。结果5-AIA—PDT作用后,Tea8113细胞生长受到抑制,孵育时间和浓度效应关系显著（P〈0．05）。药物最佳作用浓度为1mmoL／l,最佳孵育时间为6h。结论5-氨基酮戊酸一光动力疗法能有效杀伤Tea8113细胞,光敏剂孵育时间、光敏剂浓度是影响疗效的重要因素。     

山楂酸对肺癌95D细胞增殖、凋亡的影响

目的探讨山楂酸对肺癌细胞95D增殖及凋亡产生的影响。方法将山楂酸作用于人的肺癌细胞系95D,通过四甲基偶氮唑蓝(MTT)法检测山楂酸对细胞增殖的影响;通过流式细胞仪Annexin V/PI双染法检测细胞凋亡;通过荧光显微镜检测细胞核的形态变化;采用Western blot方法检测凋亡相关蛋白的影响。结果随着山楂酸浓度增大,95D细胞的增殖率明显降低,早期及晚期凋亡率逐渐增加,与对照组比较差异有统计学意义;荧光电子显微镜观察山楂酸作用后的细胞,可见染色体边集、核碎裂等典型的凋亡改变,而对照组没有相应的变化;Western blot显示,随着山楂酸浓度的增加,Bcl-2蛋白表达逐渐降低,Bax蛋白表达逐渐升高。结论山楂酸可以抑制肺癌95D细胞增殖,且具有浓度依赖性,山楂酸抑制肺癌95D细胞增殖是通过诱导细胞凋亡实现的。     

A Delivery System for Oral Administration of Proteins/Peptides Through Bile Acid Transport Channels

Proteins and peptides are poorly absorbed via oral administration because of the gastrointestinal tract environment and lysosomal digestion after apical endocytosis. A delivery system, consisting of a deoxycholic acid–conjugated nanometer-sized carrier, may enhance the absorption of proteins in the intestine via the bile acid pathway. Deoxycholic acid is first conjugated to chitosan. Liposomes are then prepared and loaded with the model drug insulin. Finally, the conjugates are bound to the liposome surface to form deoxycholic acid and chitosan conjugate–modified liposomes (DC-LIPs). This study demonstrates that DC-LIPs can promote the intestinal absorption of insulin via the apical sodium-dependent bile acid transporter, based on observing fluorescently stained tissue slices of the rat small intestine and a Caco-2 cell uptake experiment. Images of intestinal slices revealed that excellent absorption of DC-LIPs is achieved via apical sodium-dependent bile acid transporter, and a flow cytometry experiment proved that DC-LIPs are a highly efficient delivery carrier. Caco-2 cells were also used to study the lysosome escape ability of DC-LIPs. We learned from confocal microscopy photographs that DC-LIPs can protect their contents from being destroyed by the lysosome. Finally, according to pharmacokinetic analyses, insulin-loaded DC-LIPs show a significant hypoglycemic effect with an oral bioavailability of 16.1% in rats with type I diabetes.