去唾液酸糖蛋白-人端粒酶逆转录酶-胸苷激酶/丙氧鸟苷的靶向促HepG2细胞凋亡作用及旁观者效应

目的 观察去唾液酸糖蛋白(AF)-pGL3-人端粒酶逆转录酶(hTERT)-胸苷激酶(TK)对肝癌细胞株HepG2细胞生长及凋亡的影响.方法 培养细胞并构建pGL3-hTERT-TK质粒、AF脂质体复合物后,转染HepG2细胞和L02细胞,通过单光子液闪计数仪,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法和流式细胞仪观察自杀基因对肝癌细胞生长和凋亡的影响,以及其对自杀基因的旁观者效应.结果 在肝癌细胞HepG2中,TK基因可以被hTERT启动子驱动高效的表达,而不影响正常肝细胞L02的生长,AF通过识别去唾液酸糖蛋白受体结合到HepG2细胞表面,其携带的TK基因更易进入肝癌细胞,同时增强自杀基因TK的高效表达,在旁观者效应机制的参与下,肝癌细胞总的凋亡率达85%±3%,而正常肝细胞则仅为16%±2%.结论 AF-pGL3-hTERT-TK可以靶向攻击肝癌细胞,对正常肝细胞几乎无影响,其基因传递系统具备靶向治疗肝癌的潜力.     

汉坦病毒L99株G2糖蛋白基因的克隆及在真核细胞中的瞬时表达

从感染病毒的Vero-E6细胞中提取总RNA,采用RT-PCR和分子克隆技术将扩增到的C2糖蛋白基因插入含有CMV启动子的真核表达质粒pVAX1.通过脂质体介导转染cos-7细胞,用间接免疫荧光法检测瞬时表达的蛋白.结果获得含有编码汉坦病毒L99株包膜糖蛋白G2基因的重组质粒pVAX/C2.在转染cos-7细胞内.用IFA可检测到细胞内有特异性荧光.证实成功构建了汉坦病毒L99株包膜糖蛋白C2基因真核表达载体,并可在cos-7细胞中瞬时表达;为出血热综合征疫苗制备奠定了基础.     

肝脏疾病的导向治疗

肝脏疾病的治疗效果受到很多因素的影响。其中之一就是治疗药物或治疗技术不能有效地到达肝脏部位,或者不能相对集中在肝脏。解决这类问题的办法就是探索肝脏疾病导向治疗的新方法。目前具有应用前景的研究方向,主要是根据肝脏去唾液酸糖蛋白受体（ASGR）特点设计的肝脏疾病导向治疗,以及应用病毒特异性抗体的导向治疗。一、去唾液酸糖蛋白受体导向治疗去唾液酸糖蛋白受体是肝细胞膜上特异性表达的受体类型,可以与半乳糖或半乳糖胺集团结合。因此,将肝脏疾病的治疗药物进行半乳糖化修饰,就能使其通过与ASGR之间的特异性结合,实现…     

去唾液酸糖蛋白受体1在动脉粥样硬化中的研究进展

心血管疾病严重威胁全球人类的生命健康,动脉粥样硬化是其主要的病理学基础。动脉粥样硬化是一种脂质代谢紊乱引发的慢性血管壁炎症过程。去唾液酸糖蛋白受体1作为凝集素家族成员,参与血清糖蛋白的内吞和降解,在多种生理过程中发挥重要作用。有研究表明去唾液酸糖蛋白受体1突变与低密度脂蛋白胆固醇水平等心血管疾病风险相关,提示去唾液酸糖蛋白受体1可能与动脉粥样硬化的发生发展有着紧密的联系。本文就去唾液酸糖蛋白受体1在动脉粥样硬化中的作用进行综述,并对其可能的机制进行总结。     

肝癌细胞BEL7402中神经元特异性烯醇化酶的表达

目的:分别从转录水平、蛋白水平及细胞水平检测人神经元特异性烯醇化酶(NSE)在肝癌细胞BEL7402中的表达情况.方法:利用NSE特异引物,通过逆转录聚合酶链式反应(RT-PCR)从人肝癌细胞BEL7402中扩增人NSE基因的转录产物,采用免疫印迹(Westernblot)、免疫细胞化学(ICC)染色技术检测NSE在肝癌细胞中的表达.结果:通过RT-PCR方法可以从BEL7402中扩增1305bp的NSE产物;Westernblot方法证实BEL7402细胞可以表达Mr50000的NSE蛋白;免疫细胞化学染色显示BEL7402与抗NSE单抗呈阳性反应,这说明从转录水平、蛋白水平及细胞水平均检测到了NSE在肝癌细胞BEL7402中的表达.结论:NSE可在肝癌细胞BEL7402转录和表达.     

汉滩病毒核蛋白部分片段与囊膜糖蛋白G1在昆虫细胞中的融合表达

目的　应用Bac -to -Bac杆状病毒表达系统融合表达汉滩病毒囊膜糖蛋白G1与核蛋白部分片段。方法　构建含有汉滩病毒G1S0 7嵌合基因的杆状病毒表达载体 pFBD -G1S0 7,转化DH10Bac致敏菌 ,利用其含有的细菌Tn7转座系统将嵌合基因重组至穿梭质粒Bacmid上 ,快速筛选出含有G1S0 7嵌合基因的重组杆状病毒 ,在昆虫细胞中表达该融合蛋白 ,利用间接免疫荧光、ELISA和免疫印迹对表达产物进行检测。结果　构建的含G1S0 7嵌合基因之重组杆状病毒可在昆虫细胞中表达出融合蛋白 ,该蛋白可被抗汉滩病毒核蛋白及糖蛋白G1特异性单抗所识别 ,表达产物主要集中在细胞内。结论　在昆虫细胞中表达出具有生物学活性的G1S0 7融合蛋白 ,为进一步研究其免疫学特性奠定了基础     

甘露聚糖、壳聚糖、α-酸性糖蛋白和姜黄素对脂蛋白(a)和去唾液酸脂蛋白(a)代谢影响的对比分析

分析多糖和姜黄素对脂蛋白 (a)和去唾液酸脂蛋白 (a)代谢的影响 ,从刺猬腋下静脉注入甘露聚糖、壳聚糖、α -酸性糖蛋白和姜黄素 ,2min后注射12 5I-脂蛋白 (a)或12 5I-去唾液酸脂蛋白 (a) ,1h后处死动物 ,测定血、肝、肾、脾、胆汁和肾上腺的同位素含量。结果发现 ,脂蛋白 (a)去唾液酸后能大量进入肝脏 ,加速在体内的分解代谢 ,使血中浓度迅速降低。α -酸性糖蛋白抑制组织对脂蛋白 (a)和去唾液酸脂蛋白 (a)的摄入 ,使血中脂蛋白 (a)和去唾液酸脂蛋白 (a)含量显著增高。壳聚糖和姜黄素增加肝脏和肾上腺对脂蛋白 (a)的摄取 ,使血中脂蛋白 (a)含量略降低 ,但对去唾液酸脂蛋白 (a)代谢无明显影响。甘露聚糖增加脾脏对脂蛋白 (a)的摄取 ,减少胆囊中脂蛋白 (a)含量 ,但增加肾脏和胆囊对去唾液酸脂蛋白 (a)的摄取 ,降低肾上腺对去唾液酸脂蛋白 (a)的摄取。结果提示 ,脂蛋白 (a)去唾液酸后能使脂蛋白 (a)分解代谢加快 ,脂蛋白 (a)分子中的唾液酸在结构稳定中起重要的作用。α -酸性糖蛋白抑制脂蛋白 (a)和去唾液酸脂蛋白 (a)代谢 ,而壳聚糖和姜黄素则促进脂蛋白 (a)代谢     

沉默热休克蛋白70-2基因经线粒体依赖通路介导肝癌细胞凋亡

目的 探讨沉默热休克蛋白(HSP)70-2对肝癌细胞生长、凋亡的影响,以及诱导肝癌细胞凋亡的详细作用机制. 方法 Westem blot、免疫细胞化学法检测HSP70-2在4种肝癌细胞和正常肝细胞中的表达.设计合成针对HSP70-2基因的特异性短发夹状RNA(shRNA),构建真核表达载体HSP70-2 shRNA1和HSP70-2 shRNA2.转染肝癌细胞后,四甲基偶氮唑盐法检测细胞增殖,膜联蛋白/碘化丙啶双染法检测细胞凋亡情况,罗丹明染色检测细胞线粒体跨膜电位变化,Western blot检测多聚ADP-核糖聚合酶、caspase-3、caspase-9蛋白切割情况,以及细胞色素C、Bax,Bcl-2蛋白的表达变化. 结果 HSP70-2在4种肝癌细胞中均高表达,在L02细胞中仅有微量表达.HSP70-2 shRNA1、shRNA2均能有效抑制肝癌细胞中HSP70-2的表达.沉默HSP70-2基因显著抑制肝癌细胞生长,诱导肝癌细胞凋亡,转染48 h后,shRNA1和shRNA2诱导HepG2细胞的凋亡指数分别为55.8%±1.8%和57.1%±1.6%,诱导Huh细胞凋亡指数分别为54.9%±1.1%和60.2%±2.6%,与对照组相比,差异均有统计学意义,P值均＜0.01.转染HSP70-2 shRNA 48 h后,肝癌细胞线粒体跨膜电位显著下降,细胞色素C从线粒体释放到胞质中,caspase-3、caspase-9激活,多聚ADP核糖聚合酶被剪切降解,抗凋亡蛋白Bcl-2表达减少,促凋亡蛋白Bax表达明显增加.结论 沉默HsP70-2能通过激活线粒体凋亡通路导致肝癌细胞凋亡.     

幽门螺杆菌γ谷氨酰转肽酶同源基因的表达与活性检测

目的:评价幽门螺杆菌( H pylori)ggt同源基因的结构特征和功能.方法:通过生物信息学软件分析H pylori γ谷氨酰转肽酶(ggt)同源基因产物的结构特征; 提取H pylori DNA, 并以之为模板, 采用PCR法扩增该基因的全长及去信号肽片段; 进行序列分析后, 将全长片段与gfp片段连接, 分别克隆进杆状病毒的转移质粒pFastBac1中, 利用杆状病毒的Bac-to-Bac系统分别获得重组杆状病毒的DNA; 通过PCR方法验证这些片段克隆进杆状病毒; 将重组病毒的DNA以脂质体为媒介转染家蚕细胞,以获得重组杆状病毒; 分别收集这些重组病毒, 并再感染家蚕细胞, 进行蛋白表达实验和活性检测, 以荧光显微镜观察融合蛋白在细胞中的分布.结果:通过PCR方法成功克隆了各种片段; 测序的结果表明, 未发生突变; 基因片段扩增的结果说明, 获得了3种重组的杆状病毒, 且这3种重组病毒均表达了ggt片段, 全长、去信号肽片段及去信号肽片段与gfp融合的片段的表达产物活性分别为3.61, 10.50及9.31 U/L.Western blot的结果证实了去信号肽片段与GFP的融合表达. 通过荧光显微镜观察去信号肽与gfp的融合片段的表达产物在细胞中的定位, 荧光不具有区域的特异性, 充满了整个细胞, 表明该基因产物并非作用于专一的细胞器.结论:H pylori ggt同源基因适合于杆状病毒表达, 表达产物具有GGT活性, 但他对细胞的作用有待进一步研究.     

血管紧张素（1-7）对血管紧张素Ⅱ激活人脐静脉内皮细胞p38MAPK通路的拮抗作用

目的 探讨血管紧张素(1-7)[Ang-(1-7)] 阻断血管紧张素Ⅱ(AngⅡ)致炎作用的可能机制.方法 原代培养人脐静脉内皮细胞,取2～5代用于实验.培养细胞随机分两组:Ⅰ组:对照组,AngⅡ组和AngⅡ+不同浓度Ang (1-7)组;Ⅱ组:对照组,AngⅡ组,Ang (1-7)组,AngⅡ+Ang-(1-7)组,AngⅡ+Ang (1-7)+A-779组,A-779组.用免疫印迹法测定细胞p38MAPK磷酸化表达.培养细胞用RT-PCR法测定Ang(1-7)的特异性受体Mas受体的表达.结果 100 nmol/L Ang(1-7)可以拮抗100 nmol/L AngⅡ诱导的人脐静脉内皮细胞p38MAPK磷酸化表达,且呈剂量依赖性.随着Ang(1-7)剂量的增加p38MAPK磷酸化表达逐渐减弱,在1000 nmol/L Ang(1-7)时即有明显减弱.Ang(1-7)受体特异性拮抗剂A-779可显著抑制Ang(1-7)的此作用.结论 Ang(1-7)呈剂量依赖性拮抗AngⅡ激活人脐静脉内皮细胞p38MAPK通路的作用.     

Improved method increases sensitivity for circulating hepatocellular carcinoma cells

AIM:To improve an asialoglycoprotein receptor(ASGPR)-based enrichment method for detection of circulating tumor cells(CTCs)of hepatocellular carcinoma(HCC).METHODS:Peripheral blood samples were collected from healthy subjects,patients with HCC or various other cancers,and patients with hepatic lesions or hepatitis.CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation.The remaining cells were cytocentrifuged on polylysine-coated slides.Isolated cells were treated by triple immunofluorescence staining with CD45antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1(CPS1),used as liver-specific markers,and costained with DAPI.The cell slide was imaged and stained tumor cells that met preset criteria were counted.Recovery,sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines.Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining,respectively.RESULTS:CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR+selection(P s<0.05).The expression rates of either ASGPR or CPS1were different in various liver cancer cell lines,ranging between 18%and 99%for ASGPR and between 9%and 98%for CPS1.In both human HCC tissues and liver cancer cell lines,there were a few HCC cells that did not stain positive for ASGPR or CPS1.The mixture of monoclonal antibodies against ASGPR and CPS1identified more HCC cells than either antibody alone.However,these antibodies did not detect any tumor cells in blood samples spiked with the human breastcancer cell line MCF-7 and the human renal cancer cell line A498.ASGPR+or/and CPS1+CTCs were detected in 29/32(91%)patients with HCC,but not in patients with any other kind of cancer or any of the other test subjects.Furthermore,the improved method detected a higher CTC count in all patients examined than did the previous method(P=0.001),and consistently achieved 12%-21%higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs.CONCLUSION:Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells.     

Ethanol Feeding Causes Inactivation of Both State 1 and State 2 Rat Hepatic Asialoglycoprotein Receptors

Previous studies have shown that ethanol feeding in rats causes inactivation and redistribution of ˜50% of the total asialoglycoprotein receptors (ASGPRs) in hepatocytes (Tworek et al., J. Biol. Chem. 271:2531, 1996), and that two equal populations of hepatic ASGPRs mediate ligand uptake and processing via two functionally different pathways (Weigel in Glycoconjugates: Composition, Structure and Function , Marcel Dekker, 1992, p. 421). The purpose of this study was to determine if ethanol feeding causes preferential inactivation of only one of these two ASGPR populations, which have been designated state 1 and state 2 ASGPRs. The state 2, but not state 1, ASGPRs are inactivated in isolated hepatocytes by a variety of drugs and inhibitors. State 2 ASGPRs can also be inactivated in permeable cells by ATP treatment and then reactivated by treatment with fatty acyl coenzyme As. In the present study, permeable cell assays for state 2 ASGPR inactivation and reactivation were used to assess whether hepatocytes from ethanol-fed rats contain inactive state 2 ASGPRs. The results show that preferential inactivation of one ASGPR population does not occur after ethanol feeding. That inactive ASGPRs could not be reactivated by treatment with palmitoyl-coenzyme A to a greater extent in ethanol-fed versus control cells indicates there is not a larger pool of inactivated state 2 ASGPRs in treated cells. We conclude that ethanol feeding causes equal inactivation of both state 1 and state 2 ASGPRs. Ethanol feeding may represent the first treatment found to inactivate state 1 ASGPRs.     

Tissue-specific and cell surface expression of human major histocompatibility complex class I heavy (HLA-B7) and light (beta 2-microglobulin) chain genes in transgenic mice

We introduced the human genes HLA-B7 and B2M encoding the heavy (HLA-B7) and light [beta 2-microglobulin (beta 2m)] chains of a human major histocompatibility complex class I antigen into separate lines of transgenic mice. The tissue-specific pattern of HLA-B7 RNA expression was similar to that of endogenous class I H-2 genes, although the HLA-B7 gene was about 10-fold underexpressed in liver. Identical patterns of RNA expression were detected whether the HLA-B7 gene contained 12 or 0.66 kilobase(s) (kb) of 5' flanking sequence. The level of expression was copy number dependent and as efficient as that of H-2 genes; gamma interferon enhanced HLA-B7 RNA expression in parallel to that of H-2. In addition to the mechanism(s) responsible for gamma interferon-enhanced expression, there must be at least one other tissue-specific mechanism controlling the constitutive levels of class I RNA. Tissue-specific human beta 2m RNA expression was similar to that of mouse beta 2m, including high-level expression in liver. Cell surface HLA-B7 increased 10- to 17-fold on T cells and on a subset of thymocytes from HLA-B7/B2M doubly transgenic mice compared to HLA-B7 singly transgenic mice. The pattern of expression of HLA-B7 on thymocytes resembled that of H-2K as opposed to H-2D. These results confirm that coexpression of both human chains is required for efficient surface expression and that HLA-B7 may share a regulatory mechanism with H-2K, which distinguishes it from H-2D.     

Separate analysis of asialoglycoprotein receptors in the right and left hepatic lobes using (99m)Tc-GSA SPECT in patients with acute hepatic damage.

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the sinusoidal surfaces of hepatocytes. However, regional expression and clinical significance of the ASGPR in acute hepatic damage is presently unknown. Our aim was to clarify the clinical significance of the regional expression of ASGPR in human livers with acute hepatitis (AH) and fulminant hepatic failure (FHF). Eighteen volunteers, 42 patients with AH and 10 with FHF were studied using a newly developed, conventional (99m)Tc-GSA SPECT analysis. Using Cantlie's line as a guide, ASGPR expression was analyzed separately in the right and left hepatic lobes, as well as in the whole liver, using novel indices (the liver uptake ratio [LUR] and liver uptake density [LUD], which reflect the amount and density of ASGPRs in the liver, respectively). Mean LUR and LUD values for the whole liver and the right and left lobes decreased in accordance with the severity of acute hepatic damage. In the FHF group, the reduction in LUR and LUD values in the right lobes was more significant than in the left lobes. The LUR and LUD values for the whole liver correlated well with hepatic functional reserve and total bilirubin levels. The right LUR and LUD values in particular correlated well with these parameters. A time-course observation of 13 patients with either AH or FHF revealed that the expression of ASGPRs in the right lobe recovered faster than in the left. We first evaluated the regional expression of AGSPRs by (99m)Tc-GSA SPECT analysis in both AH and FHF patients, which is a clinically useful and reliable indicator for assessing the severity of regional hepatic damage and evaluating regional liver regeneration.     

骨髓间充质干细胞对乙型肝炎病毒的易感性及与无涎糖蛋白受体的关系

目的 评价骨髓间充质干细胞(BMSC)向肝细胞诱导过程中对HBV的易感性及无涎糖蛋白受体(ASGPR)对BMSC感染HBV的作用.方法 体外使用肝细胞生长因子、成纤维细胞生长因子-4和表皮生长因子,将BMSC诱导分化为肝细胞.检测乙型肝炎患者BMSC的HBV感染情况,并对原代及诱导培养后的BMSC进行体外HBV感染实验,检测BMSC感染后的HBsAg、HBcAg表达情况,并检测BMSC诱导前后ASGPR的表达.每个实验采用来自不同的5个标本,分别重复3次,数据统计采用非参数检验.结果 诱导培养第6天开始,BMSC开始表达甲胎蛋白(AFP)、细胞角蛋白18(CK18)和Alb,并随着诱导时间延长,CK18及Alb表达逐渐增多,而AFP则逐渐减少,并具有糖原合成、尿素分泌及Alb合成的肝细胞功能.BMSC在体内及体外都不能被HBV感染,经过向肝细胞诱导之后,仍然不能被感染,ASGPR在BMSC向肝细胞诱导后表达增多,但是与对照组HepG2细胞相比,仍然呈低水平表达.结论 BMSC在体内外能抵抗HBV感染.ASGPR可能是导致HBV不能感染BMSC的重要原因之一.     

Identification of biomarkers for hepatocellular carcinoma by semiquantitative immunocytochemistry

AIM:To investigate the expression of key biomarkers in hepatoma cell lines,tumor cells from patients’blood samples,and tumor tissues.METHODS:We performed the biomarker tests in two steps.First,cells plated on coverslips were used to assess biomarkers,and fluorescence intensities were calculated using the NIH Image J software.The measured values were analyzed using the SPSS19.0 software to make comparisons among eight cell lines.Second,eighty-four individual samples were used to assess the biomarkers’expression.Negative enrichment of the blood samples was performed,and karyocytes were isolated and dropped onto pretreated glass slides for further analysis by immunofluorescence staining.Fluorescence intensities were compared among hepatocellular carcinoma(HCC)patients,chronic HBV-infected patients,and healthy controls following methods similar to those used for cell lines.The relationships between the expression of biomarkers and clinical pathological parameters were analyzed by Spearman rank correlation tests.In addition,we studied the distinct biomarkers’expression with three-dimensional laser confocal microscopy reconstructions,and Kaplan-Meier survival analysis was performed to understand the clinical significance of these biomarkers.RESULTS:Microscopic examination and fluorescence intensity calculations indicated that cytokeratin 8/18/19(CK)expression was significantly higher in six of the seven HCC cell lines examined than in the control cells,and the expression levels of asialoglycoprotein receptor(ASGPR)and glypican-3(GPC3)were higher in all seven HCC cell lines than in the control.Cells obtained from HCC patients’blood samples also displayed significantly higher expression levels of ASGPR,GPC3,and CK than cells from chronic HBV-infected patients or healthy controls;these proteins may be valuable surface biomarkers for identifying HCC circulating tumor cells isolated and enriched from the blood samples.The stem cell-like and epithelial-mesenchymal transition-related biomarkers could be detected on the karyocyte slides.ASGPR and GPC3 were expressed at high levels,and thus three-dimensional reconstructions were used to observe their expression in detail.This analysis indicated that GPC3 was localized in the cytoplasm and membrane,but that ASGPR had a polar localization.Survival analyses showed that expression of GPC3 and ASGPR is associated with a patient’s overall survival(OS).CONCLUSION:ASGPR,GPC3,and CK may be valuable HCC biomarkers for CTC detection;the expression of ASGPR and GPC3 might be helpful for understanding patients’OS.     

Covalent HLA-B27/peptide complex induced by specific recognition of an aziridine mimic of arginine.

The class I major histocompatibility complex (MHC) glycoprotein HLA-B27 binds short peptides containing arginine at peptide position 2 (P2). The HLA-B27/peptide complex is recognized by T cells both as part of the development of the repertoire of T cells in the cellular immune system and during activation of cytotoxic T cells. Based on the three-dimensional structure of HLA-B27, we have synthesized a ligand with an aziridine-containing side chain designed to mimic arginine and to bind covalently in the arginine-specific P2 pocket of HLA-B27. Using tryptic digestion followed by mass spectrometry and amino acid sequencing, the aziridine-containing ligand is shown to alkylate specifically cysteine 67 of HLA-B27. Neither free cysteine in solution nor an exposed cysteine on a class II MHC molecule can be alkylated, showing that specific recognition between the anchor side-chain pocket of an MHC class I protein and the designed ligand (propinquity) is necessary to induce the selective covalent reaction with the MHC class I molecule.     

Direct gene transfer with DNA-liposome complexes in melanoma: expression, biologic activity, and lack of toxicity in humans.

Direct gene transfer offers the potential to introduce DNA encoding therapeutic proteins to treat human disease. Previously, gene transfer in humans has been achieved by a cell-mediated ex vivo approach in which cells from the blood or tissue of patients are genetically modified in the laboratory and subsequently returned to the patient. To determine the feasibility and safety of directly transferring genes into humans, a clinical study was performed. The gene encoding a foreign major histocompatibility complex protein, HLA-B7, was introduced into HLA-B7-negative patients with advanced melanoma by injection of DNA-liposome complexes in an effort to demonstrate gene transfer, document recombinant gene expression, and determine the safety and potential toxicity of this therapy. Six courses of treatment were completed without complications in five HLA-B7-negative patients with stage IV melanoma. Plasmid DNA was detected within biopsies of treated tumor nodules 3-7 days after injection but was not found in the serum at any time by using the polymerase chain reaction. Recombinant HLA-B7 protein was demonstrated in tumor biopsy tissue in all five patients by immunochemistry, and immune responses to HLA-B7 and autologous tumors could be detected. No antibodies to DNA were detected in any patient. One patient demonstrated regression of injected nodules on two independent treatments, which was accompanied by regression at distant sites. These studies demonstrate the feasibility, safety, and therapeutic potential of direct gene transfer in humans.