肝癌细胞系DcR3的表达及意义

目的 探讨肝癌细胞系诱捕受体3(DcR3)的表达变化及意义. 方法 分别取人肝癌细胞HepG2、HepB3、SMMC-7721、bel-7402、bel-7405及癌旁肝细胞QSG-7701和正常肝细胞HL-7702,RT-PCR和EnVision法分别检测其DcR3 mRNA和蛋白;MTT法检测加入DcR3中和性抗体后的肝癌细胞存活率. 结果 DcR3在5种肝癌细胞中均有mRNA和蛋白的表达,QSG-7701及HL-7702均无表达.加入DcR3中和性抗体后肝癌细胞存活率下降. 结论 肝癌细胞高表达DcR3,DcR3中和性抗体可抑制肝癌细胞的生长.     

甘露糖受体在肝细胞癌中的表达及意义

目的:观察甘露糖受体(mannose receptor,M R)在肝细胞癌组织/肝癌细胞系中的表达,探讨其与肝细胞癌发生、发展及恶性程度的关系.方法:应用免疫组织化学检测50例肝细胞癌组织及癌旁组织、10例正常肝组织中MR的表达,免疫荧光法、Western blot法检测肝癌细胞系、肝细胞系中MR的表达.结果:免疫组织化学染色:肝细胞癌组织中M R的表达水平明显高于癌旁组织及正常肝组织(86%vs 76%,30%,P0.05).免疫荧光:MR在肝癌细胞系BEL-7402、Hep G2和人肝细胞系HL-7702中均有表达.在肝癌细胞系BEL-7402、Hep G2上有很强的MR表达,在肝细胞系HL-7702上MR的表达分布明显较少.Western blot法:MR在肝癌细胞系Hep G2、BEL-7402和人肝细胞系HL-7702上均有表达,肝癌细胞BEL-7402中MR的表达水平明显高于肝癌细胞Hep G2(P0.01)、肝细胞HL-7702(P0.01).结论:MR在肝细胞癌组织/肝癌细胞系中高表达提示可能与肝细胞癌的发生、发展及恶性程度密切相关.     

5-羟色胺通过上调2B受体抑制放线菌素D诱导肝细胞凋亡初步实验研究

目的探讨5-羟色胺(5-HT)在肝细胞凋亡中的作用及机制。方法以不同浓度梯度放线菌素D(AcD)分别诱导HepG2和7702细胞凋亡,采用AnnexinV-FITC/PI流式细胞术和TUNEL方法检测细胞凋亡,MTT法检测细胞存活率。AcD预处理HepG2细胞后分别加入5-HT、5-HT 2A受体激动剂DOI、5-HT 2B受体激动剂M110、5-HT 2B受体拮抗剂SB204+5-HT,AnnexinV-FITC/PI流式细胞术和MTT法分别检测细胞凋亡率和存活率,蛋白质印迹法检测蛋白水解酶3(caspase3)、丝氨酸/苏氨酸蛋白激酶(AKT)及磷酸化丝氨酸/苏氨酸蛋白激酶(p-AKT)的表达。结果 AcD以浓度依赖性方式分别诱导HepG2细胞和7702细胞的凋亡,50 ng/mL AcD处理两种细胞凋亡率均高于无血清对照组(HePG2细胞t=1.71,7702细胞t=1.98,均P0.05),与单用AcD组相比,5-HT使HepG2细胞凋亡率从31.96%±2.13%降至10.24%±2.59%(t=1.62,P0.05),5-HT 2B受体激动剂使其降至8.41%±0.96%(t=1.75,P0.05),反之,5-HT 2B受体拮抗剂能拮抗5-HT的抗凋亡作用,其凋亡率为25.92%±1.33%(t=1.45,P0.05)。5-HT及5-HT 2B受体激动剂均能够减少caspase3活化,增加AKT的磷酸化,使细胞凋亡减少。结论 5-HT通过上调5-HT 2B受体促进AKT磷酸化,从而抑制AcD诱导的肝细胞凋亡。     

酮色林对HepG2细胞Sp1表达的影响

Sp1是Sp蛋白家族中的一员．是序列特异性的DNA结合蛋白，可与启动子中富含GC序列结合以凋控细胞和病毒基因的转录过程．Sp1在肿瘤中表达明显增高。有报道5-羟色胺（5-HT）及其受体存在于肝细胞癌（HCC）上．提示HCC可能存在自分泌5-HT的机制。本实验柃测酮色林（5HT2A受体阻断剂）干预HepG2细胞后Sp1mRNA的表达．探讨Sp1在肝癌发生、发展中的作用。     

人乳头状瘤病毒16型伪病毒载体对肝癌细胞系HepG2细胞的杀伤研究

目的 构建基于人乳头状瘤病毒（HPV）16型的伪病毒载体,并检测其对肝癌细胞系HepG2细胞的杀伤活性。方法利用昆虫什状病毒表达系统表达病毒蛋白,在体外将病毒蛋白包装白喉毒（DT）A链表达型质粒,形成伪病毒。透射电镜观察病毒样颗粒（VLP）的结构,并转染肝癌细胞系HepG2。乳酸脱氢酶释放法检测对肝癌细胞系HepG2的杀伤能力。结果透射电镜观察显示病毒蛋白可自我组装成VLP,在转染肝癌细胞系HepG2后,乳酸脱氢酶释放法成功检测到伪病毒埘肝癌细胞系HepG2的杀伤作用。结论基于HPV16的新型伪病毒载体能成功转染肝癌细胞系HepG2,并产生杀伤活性,为肝癌的基因治疗提供了一种可选择的方法。     

B7-H1在HBV感染导致肝细胞癌中的调控作用

目的探讨B7-H1（B7-homolog1）在乙型肝炎病毒（HBV）感染导致的肝细胞癌（HCC）中的作用机制。方法通过慢病毒lentivirus．B7．H1转染人正常肝细胞系L02、人肝癌细胞系HepG2、HepG2．2．15后,应用MTT法观察细胞活性,流式细胞仪检测细胞的凋亡情况,Westernblot技术检测蛋白B7-H1、Survivin蛋白的变化情况。结果抑制B7-H1表达水平后,通过MTT检测表明细胞系HepG2．2．15细胞活性比HepG2细胞、L02细胞活性降低,流式细胞仪检测凋亡增加,同时Westernblot显示Survivin蛋白的表达减少。结论通过慢病毒转染抑$1JB7．H1的表达可以促进细胞系HepG2．2．15凋亡,B7-H1可能是通过调控Survivin的变化影响HBV感染的肿瘤细胞的增殖与凋亡。     

几种肺癌细胞系中FHIT基因和蛋白表达的研究

目的研究不同人肺癌细胞系FHIT基因和蛋向的表达，为探讨外源FHIT基因对不同FHIT状态人肺癌细胞恶性表型的影响筛选受体细胞。方法提取B01D（人肺巨细胞癌细胞系）、LTEP-A2（人肺腺癌细胞系）、LTEP-Sm（人小细胞肺癌细胞系）和A549（人肺腺癌细胞系）细胞总RNA，定量后逆转录合成cDNA第一链，分别用5131、3D1和5U2、3D2两对引物进行巢式PCR扩增FHIT基因外显子1～10。1．5％琼脂糖凝胶电泳检测4种细胞株的FHIT基因表达。将扩增的PCR产物纯化后进行DNA测序。用FHIT单克隆抗体进行免疫组化染色检测4种细胞FHIT蛋白表达。结果801D和LTEP-Sm细胞有正常FHIT基因转录本，LTEP-A2细胞有一条正常FHIT基因转录本和一条异常转录本，正常转录本测序显示与FHIT cDNA同源，无缺失及重排。801D和LTEP-Sm细胞FHIT蛋向表达呈强阳性。A2细胞FHIT蛋白表达减弱。A549缺乏FHIT基因转录本和蛋白表达。结论4种细胞系代表3种FHIT基因状态，为进一步研究外源FHIT基因对不同FHIT状态人肺癌细胞恶性表型的影响提供了合适的受体细胞。     

ku80蛋白表达水平与肝癌细胞侵袭迁移能力的相关性

目的观察ku80蛋白表达水平与肝癌细胞侵袭迁移能力的相关性。方法使用肝癌细胞系MHCC97-H、 MHCC97-L、HepG2及正常人肝细胞HL-7702为研究对象,分别采用RT-PCR、Western印迹方法检测ku80 mRNA及蛋白水平;体外划痕实验、Transwell侵袭实验观察4株细胞迁移、侵袭能力;免疫荧光检测ku80亚细胞定位;线性相关分析ku80及其mRNA与肝癌细胞系迁移、侵袭能力的相关性。结果 ku80 mRNA及蛋白在4株细胞系中均有表达,且在3株肝癌细胞中的表达明显高于正常肝细胞系HL-7702(均P0.05),其中MHCC97-H细胞表达水平最高(P0.01);体外划痕实验、Transwell侵袭实验显示3株肝癌细胞系的迁移、侵袭能力明显高于HL-7702细胞系(均PO.05),其中MHCC97-H的迁移侵袭能力最强(P0.01)。ku80蛋白定位于细胞核。线性相关性分析显示ku80蛋白及mRNA的表达水平与肝癌细胞系的迁移、侵袭能力呈正相关。结论 ku80蛋白主要在细胞核表达,其表达水平与肝癌细胞系的迁移、侵袭能力呈正相关。     

Egr-1基因表达及其在射线诱导的肝癌细胞凋亡中的作用

目的:研究Egr-1基因的表达在放射诱导的细胞凋亡中的作用．方法:选择肝癌细胞系HepG2,SMMC-7721和正常肝细胞系HL-7702培养;培养细胞接受4Gy X射线照射;收获受照前和受照后1,2,4, 6,12和24 h的细胞,采用荧光定量PCR(FQ- PCR)检测0,1,2和4 h Egr-1基因的表达,采用流式细胞术(FCM)检测0,6,12和24 h细胞周期和细胞凋亡．结果:随HepG2,SMMC-7721和HL-7702在4GY X射线照射后1 h即诱导了Egr-1基因表达增高,4 h均未达峰值,分别为ΔEgrHepG2(12．9629±1．0649)、ΔEgr7702(0．0096±0．0008)和ΔEgr7721(0．0017±0．0003),HepG2显著高于HL-7702和SMMC-7721(P<0．01)．照射后6 h射线诱导的3株细胞凋亡均不明显,但在12 h均诱导了明显的细胞凋亡,而且HepG2(41．16％)和HL-7702(27．45％)已达峰值; SMMC-7721诱导的细胞凋亡水平较低,24 h仅为24．94％,且未达峰值．在射线诱导的细胞周期变化中,HepG2和SMMC-7721 S期的变化与细胞凋亡变化在6-12 h走势相反．结论:在HepG2,SMMC-7721和HL-7702细胞中,射线通过诱导Egr-1基因表达而诱导了细胞周期和细胞凋亡的变化;射线诱导的Egr-1基因表达水平可能与射线诱导的细胞凋亡成正相关;S期肿瘤细胞可能易发生射线诱导的细胞凋亡．     

干细胞相关基因Oct-4在人肿瘤细胞系中的表达及其意义

目的 探讨干细胞相关基因Oct-4在多种人肿瘤细胞系中的表达.方法 体外培养人胰腺癌、宫颈癌、肝癌、乳腺癌、结肠癌细胞系,应用RT-PCR和Western blot法分别检测Oct-4 mRNA和蛋白在各种人肿瘤细胞系中的表达.结果 Oct-4 mRNA和蛋白在胰腺癌细胞系SW1990、PANC1、BxPC3、PC3,宫颈癌细胞系HELA,肝癌细胞系HepG2,乳腺癌细胞系MCF-7和结肠癌细胞系CaCo2中均有表达.结论 干细胞相关基因Oct-4在肿瘤细胞中的表达,一方面说明恶性肿瘤与干细胞具有密切的关系,另一方面也说明Oct-4基因在这些肿瘤的发生中起到重要作用.     

人肝癌细胞系7721丙型肝炎病毒体外感染模型的建立

目的　建立接近体内自然感染状态并能稳定支持丙型肝炎病毒（ＨＣＶ） 体外长期复制的感染细胞模型。方法　将人肝癌细胞系７７２１ 与慢性丙型肝炎患者血清共温育８ 小时后，分别以逆转录聚合酶链反应、免疫组化、原位杂交检测细胞和／ 或培养上清中的ＨＣＶ ＲＮＡ、ＨＣＶ 抗原表达及ＨＣＶＲＮＡ 在感染细胞内的定位。结果　感染血清和细胞共同温育后的第２ ～６６ 天，从细胞内和培养上清中均可间断地检测出ＨＣＶ 正、负链ＲＮＡ，即使其间细胞传代６ 次，ＨＣＶ ＮＳ３ 、ＮＳ５ 抗原在细胞内能稳定表达，原位杂交显示ＨＣＶ ＲＮＡ 阳性物质多位于细胞浆。结论　７７２１ 细胞不但对ＨＣＶ 易感，而且可以稳定地支持ＨＣＶ 的体外长期复制，此模型可以用于ＨＣＶ 感染、复制机制的研究、抗病毒药物的评价及保护性抗体／ 疫苗的初步筛选。     

肝癌干细胞逃避5-氟尿嘧啶单药杀伤可能是造成化疗失败的重要原因

目的通过观察5-氟尿嘧啶(5-FU)处理人肝癌细胞系BEL-7402和SMMC-7721,分析细胞生物学特性的变化。方法采用Cell Counting Kit-8法检测5-FU作用前后肝癌细胞系BEL-7402和SMMC-7721增殖能力的变化;采用流式细胞法检测细胞周期组成变化和细胞群体中肿瘤干细胞标志物EpCAM、ABCG2和ICAM-1阳性细胞的比例变化。结果 10μg.ml-15-FU作用48h后,BEL-7402和SMMC-7721细胞增殖均明显受抑制(P<0.05);两细胞系实验组静息期(G0/G1期)细胞比例较对照组均明显升高(P<0.05);两细胞系中肿瘤干细胞标志物阳性细胞比例均明显升高(P<0.05)。结论肝癌细胞系BEL-7402及SMMC-7721中的肿瘤干细胞能够逃避一定剂量5-FU的杀伤作用。肝癌干细胞逃避5-FU单药杀伤可能是造成临床化疗失败的重要原因。     

Differential functional effects of two 5-HT4 receptor isoforms in adult cardiomyocytes

Serotonin 5-HT4 receptors are present in human atrial myocytes and have been proposed to contribute to the generation of atrial fibrillation. However, 5-HT4 receptors have so far been only found in human and pig atria and are absent from the heart of small laboratory animals, such as rat, guinea pig, rabbit and frog, which limits the experimental settings for studying their functional properties. In this study, we developed an adenovirus expression system to examine the properties of two human 5-HT4 receptor splice variants, h5-HT4(b) and h5-HT4(d), expressed in adult cardiomyocytes devoid of native 5-HT4 receptors. When expressed in the HL-1 murine cell line of atrial origin, both receptors caused specific binding of the 5-HT4 selective antagonist GR113808 and activated adenylyl cyclase in the presence of serotonin (5-HT, 1 microM). When expressed in freshly isolated adult rat ventricular cardiomyocytes, a stimulation of the L-type Ca2+ current (ICa,L) by 5-HT (100 nM) was revealed. Both effects were blocked by GR113808. In HL-1 cells, the h5-HT4(d) receptor was found to be more efficiently coupled to adenylyl cyclase than the h5-HT4(b). Pertussis toxin treatment (250 ng/ml for 5 h) potentiated the stimulatory effect of 5-HT on ICa,L in rat myocytes expressing the h5-HT4(b) but not the h5-HT4(d) receptor, indicating a likely coupling of the (b) isoform to both Gs and Gi/o proteins. Adenoviral expression of h5-HT4 receptor isoforms in adult cardiac myocytes provides a valuable means for the exploration of the receptor signaling cascades in normal and pathological situations.     

Cell adhesion and migration of different human colon cell lines and primary tumors

Summary Several cell lines derived from colon tumors (HT-29, WiDr, PT4, PT5) and from human liver metastases (LM3) and primary human colon tumor cells (PTR) were compared with regard to their ability to migrate and to attach to different substrates (collagen G, laminin, fibronectin). Cells from the WiDr cell lines migrated actively, whereas the other cell lines, and LM3 and the PTs showed almost no migratory activity. The attachment efficiency was best in all cell lines assayed when tested on collagen followed by laminin and fibronectin as substrates. The HT-29 cells showed the strongest adhesion to all substrates, while the adhesiveness of PT4, PT5, and LM3 was reduced. The WiDr cells which migrated best showed the lowest adhesion to the substrates.     

Absence or decreased levels of the hMLH1 protein in human gastric carcinoma cell lines: implication of hMLH1 in alkylation tolerance

Defective hMLH1 function has been increasingly associated with acquired cellular resistance to DNA alkylation damage in human colorectal and endometrial cancer cells. To investigate the relationship between the DNA alkylation tolerance and the hMLH1 status in human gastric carcinoma cells, we determined the cellular response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), the mutational changes, and the expression of hMLH1 in 11 human gastric carcinoma cell lines. Of 11 cell lines, 4 (SNU-5, -16, -620, and -719) were sensitive, whereas 7 (SNU-1, -216, -484, -520, -601, -638, and -668) were resistant to the cytotoxic effect of MNNG. As determined by Western analysis, it was evident that all the MNNG-resistant cell lines except one (SNU-601) produced very low or undetectable levels of hMLH1 protein compared to the MNNG-sensitive cell lines. A homozygous non-sense mutation that resulted in truncated protein was found in one MNNG-resistant cell line (SNU-1). Therefore, to determine whether the sensitivity of cells to MNNG can be restored by exogenous expression of hMLH1 protein, wild-type hMLH1 cDNA was introduced into the MNNG-resistant cells (SNU-1). The cytotoxicity test showed that expression of exogenous wild-type hMLH1 protein caused an increase in sensitivity to the cytotoxic effect of MNNG. This restoration was confirmed by an increase in the cell population containing less than the G1 amount of DNA (cell death) in the wild-type hMLH1-transfected cells, as determined by flow cytometry analysis. Together our results suggest that (1) the absence or decreased level of wild-type hMLH1 protein may be a frequent event in the human gastric carcinoma cell lines, (2) such alterations in the hMLH1 protein are closely associated with the MNNG tolerance in the human gastric carcinoma cell lines, and (3) the hMLH1 protein participates not only in the repair of DNA mismatches but also in the mechanism of escape from the cytotoxic effects of DNA alkylation damage. Received: 26 January 1998 / Accepted: 18 March 1998     

Induction of differentiation in the human leukemia cell line SPI-802: morphological, immunological, and isoenzymatic changes

The phorbolester 12-0-tetradecanoylphorbol 13-acetate (TPA) was used for the induction of differentiation in cells of the human leukemia cell line SPI-802. The cellular morphology, surface marker antigen expression, and isoenzyme profiles of four enzymes (carboxylic esterase, acid phosphatase, hexosaminidase, and lactate dehydrogenase) served as parameters for monitoring the induced phenotypical changes. TPA led to distinct alterations of the morphology and significantly affected the growth rate with cessation of cell proliferation. No major increase in the number of nitro blue tetrazolium-positive cells or aggregation of cells, phagocytosis of latex beads, adherence to plastic surface, or development of pseudopodia were observed. As TPA-treated SPI-802 cells remained negative for these markers of the monocyte-macrophage complex, it can be concluded that the cells did not differentiate into monocytes and macrophages. The immunological marker profile based on testing of 55 monoclonal antibodies, terminal deoxynucleotidyl transferase and two erythrocyte rosette tests indicated a differentiation of SPI-802 cells along the granulocytic cell lineage. This was confirmed by isoenzyme analysis, especially that of carboxylic esterase. An isoenzyme specific for monocytes and macrophages was not detected. In earlier studies it was found that SPI-802 cells produce hemoglobin upon exposure to TPA or hemin. This latter observation and the present results suggested a comparison with the two erythroleukemia cell lines K-562 and HEL. SPI-802 cells appear to have the potential to differentiate along several cell lineages.     

Adenylate cyclase activity in human pancreatic adenocarcinoma cell lines

Summary Conclusion BxPC-3, Hs 766T, Capan-2, Panc-1, and Capan-1 cells possess receptors for VIP and β-adrenergic agonists that are functionally coupled to adenylate cyclase. In this respect, they resemble pancreatic duct cells. However, we speculate that the process of neoplastic transformation has either downregulated the expression of secretin receptors or led to a defect in the receptor itself, placing a question mark over the usefulness of these adenocarcinoma cell lines as models of the pancreatic ductal epithelium. Background Because of the importance of ducts in pancreatic disease, we wished to establish which duct cells receptors are functional on adenocarcinoma cell lines. Methods We investigated the expression of agonist-stimulated adenylate cyclase activity in six human pancreatic adenocarcinoma cell lines. Known stimulants of pancreatic ductal secretion, VIP, PHI, secretin, β-adrenergic, and dopamine, were tested. Results For responsive cell lines, VIP was the most effective stimulant followed by adrenaline, isoprenaline, PHI, and secretin. Dopamine was without effect. Since high concentrations of PHI and secretin were required to stimulate cyclase activity, their effect is probably mediated by VIP receptors. Based on the degree of stimulation observed with the individual agonists, Hs 766T and BxPC-3 were the most responsive cell lines, followed by Capan-2 and Capan-1, and finally Panc-1. MIAPaCa-2 cells did not respond to any of the agonists tested.     

阳离子脂质体介导的虫荧光素酶基因表达

目的 研究阳离子脂质体Lipofectin介导虫荧光素酶基因在不同细胞株中的表达。方法 质粒pDR2luc在大肠杆菌DH5a中扩增后用碱裂解法提取,并经Sepharose 2B凝胶过滤柱层析,通过凝胶电泳及紫外分光光度计分析纯度、定量,以Lipofectin分别转染人肝癌细胞株HepG2、SMMC7721、人肾癌细胞株GRC及非洲绿猴肾细胞COS7,然后以液体闪烁计数仪单光子计数法测定荧光素酶活性。结果 荧光素酶活性在4种细胞株中均有表达,且均有显著性差异(均P<0.05)。结论 阳离子脂质体Lipofectin可用于多种真核细胞基因转染。     

Co-expression of CD4 and CD8 associated with elevated interleukin-4 in a cord T cell line derived by cocultivating normal cord leukocytes and an HTLV-II-producing simian leukocyte cell line (Si-IIA)

A new interleukin-2(IL-2)-dependent T cell line, designated CS-IIA, was established by cocultivating normal human cord leukocytes and a lethally X-irradiated HTLV-II-producing simian leukocyte cell line (Si-IIA). CS-IIA showed CD4 dominance during the early culture. However, after addition of IL-2, CS-IIA predominantly co-expressed CD4 and CD8 (69.5%) and also expressed the surface markers CD1^–, CD3⁺, CD19^–, CD25⁺ and HLA-DR⁺. A significantly elevated level of IL-4 (1697 pg/ml) was observed in the culture supernatant from CS-IIA. In addition, the conversion of phenotype from some CD4⁺CD8⁺ cells to CD4⁺CD8^– was demonstrated by the neutralization assay using anti-IL-4 antibody. CS-IIA had a normal human karyotype and was free from Epstein-Barr virus nuclear antigen and immunoreactive with sera of HTLV-I- or HTLV-II-infected patients and anti-HTLV-1, p19 or p24 mAb. The provirus genome of HTLV-II was detected in this cell line by the polymerase chain reaction combined with a digoxigenin-enzyme-linked immunosorbent assay. However, electron microscopy of CS-IIA cells revealed no C-type virus particles in the extracellular space. These results indicate that HTLV-II can be transmitted from an HTLV-II-infected simian leukocyte cell line to human cord T lymphocytes and suggest that co-expression of CD4 and CD8 on T cells may be induced by the high level of IL-4, which can mediate CD8 induction on CD4⁺ T cell clones.