人睾丸基因TDRG1重组真核载体的构建及其表达

目的 构建人睾丸基因TDRGl的真核表达质粒,并研究其表达.方法 取人新鲜正常睾丸组织,提取总RNA,采用逆转录.聚合酶联链反应(RT-PCR)扩增TDRG1基因编码序列;将该基因克隆到真核表达载体pYD5中,构建真核细胞表达载体pYD5-TDRG1,用限制性内切酶酶切分析,DNA序列分析鉴定重组质粒;将测序正确的重组质粒pYD5-TDRG1在脂质体介导下转染293细胞,间接免疫荧光法和Western Blot法鉴定目的 蛋白质的表达.结果 RT-PCR扩增出TDRGl基因编码序列,目的 插入片段长约303bp:产物行限制性内切酶酶切后连接到真核表达载体pYD5,重组质粒pYD5-TDRG1经酶切及DNA测序鉴定构建成功:该质粒转染293细胞48h后在荧光显微镜下可观察到绿色荧光,阳性细胞率96.42%;行Western blot分析检测到约39-8kD的目的 蛋白表达.结论 成功构建了人类睾丸基因TDRG1的真核表达载体pYD5-TDRG1,TDRG1基因在293细胞内成功表达.     

WWOX基因真核表达载体的构建及其在SMMC-7721中的表达

目的克隆人WWOX基因及构建WWOX基因真核表达载体并研究其表达。方法取人新鲜正常肝组织，提取总RNA，采用逆转录-聚合酶联链反应（RT—PCR）扩增WWOX基因，将该基因定向克隆到真核表达载体pEGFP—N1中，构建真核细胞表达载体pEGFP—N1-WWOX，用限制性内切酶酶切分析，DNA序列分析鉴定重组质粒；将测序正确的WWOX基因通过脂质体介导下转染肝癌细胞SMMC-7721。结果重组质粒pEGFP—N1-WWOX经HindⅢ和BamHI双酶切后，获得4700bp片断和1234bp插入片断，序列分析表明插入的片段与Gene Bank发布的序列一致；荧光显微镜下可见转染的SMMC-7721细胞有绿色荧光蛋白的表达。结论成功构建了真核表达载体pEGFP—N1-WWOX，WWOX基因在SMMC-7721细胞内成功表达。     

急性早幼粒性白血病基因重组逆转录病毒载体构建及表达

目的构建重组人早幼粒白血病基因(PML)的逆转录病毒载体及稳定表达PML的膀胱肿瘤细胞株。方法应用DNA重组技术,将PML基因亚克隆后连接在逆转录病毒载体pLXSN上,经酶切鉴定正确后,磷酸钙沉淀法转染到PA317包装细胞包装病毒,并计算重组病毒的滴度。聚合酶链反应(PCR)鉴定重组PML逆转录病毒能够成功感染靶细胞。激光共聚焦和Westernblot鉴定PML蛋白的表达。结果经酶切分析证实有大约2.1kb大小的片段插入到逆转录病毒载体中。PCR结果显示感染重组PML逆转录病毒的细胞可以扩增出304bp大小的片段。激光共聚焦显微镜显示,感染重组PML病毒的膀胱肿瘤细胞胞核内有散在的斑点样的绿色荧光亮点。Westernblot也发现感染PML组有大约90×103的特异性的蛋白条带。结论我们成功构建重组人PML基因的逆转录病毒载体并且建立了稳定表达PML的膀胱肿瘤细胞株。     

真核表达载体pcDNA3.1-β淀粉样前体蛋白裂解酶的构建及其在COS-7细胞中瞬时表达

目的 构建β淀粉样前体蛋白裂解酶（β-site amyloid precursor protein cleaving enzyme，BACE）基因的真核表达载体，为修复阿尔茨海默病（Alzheimer’s disease，AD）损伤神经元奠定基础。方法采用RT-PCR方法，从人的成神经细胞瘤的总cDNA中，扩增出1．5kb的BACE cDNA片段，再用BamHI和XhoI双酶切后定向克隆到真核细胞表达载体pcDNA3.1中，用限制性内切酶酶切分析和DNA序列分析鉴定重组质粒；以免疫细胞化学法检测BACE基因的表达情况。结果人BACE基因的cDNA已克隆到真核细胞表达载体pcDNA3.1质粒中；经脂质体转染COS-7细胞后，并由潮霉素进行筛选，可见转染细胞胞浆中和胞膜上有较高量的BACE蛋白表达。结论成功构建了pcDNA3．1-BACE的真核表达载体，为研究BACE基因在AD发病机制中的作用及抑制BACE，为治疗AD奠定一定的实验基础。     

大鼠神经营养因子3的体外表达与分析

目的：为了有效地解决脊髓损伤后轴突再生不良的问题，作者构建大鼠源性神经营养因子(neurotrophin-3，NT3)真核表达载体，并探讨其在真核细胞中的表达。方法：以大鼠肝细胞染色体为模板，采用PCR方法扩增出NT3的编码基因，将所得的基因重组于：PcDNA3．1( )真核表达载体，筛选正确的克隆，转染COS7细胞。结果：重组克隆酶切鉴定正确，序列测定与文献报道完全一致。转染COS7细胞后可检测到。NT3mRNA的表达，Western Blot在COS7细胞裂解液中检测到13．6KD的蛋白表达。结论：基因重组的PcDNA3．1( )-NT3载体能有效地将目的基因带入真核细胞并表达，为进一步进行脊髓损伤的修复实验作了准备。     

DcR3反义RNA表达载体的构建及表达

目的：构建DcR3反义RNA表达载体PVAXI-as-DeR3，并研究其对肝癌细胞中DcR3蛋白表达的影响。方法：从肝癌组织中提取总RNA，RT-PCR法克隆出DcR3基因片段，将其与PMD18-T载体连接，经BamHⅠ、XholⅠ双酶切TA—DeR3及PVAXⅠ（-）真核表达载体后，使用T4DNA连接酶连接，构建反义DcR3表达载体。转染肝癌细胞株SMMC7721。Western blot法检测DeR3蛋白表达。结果：用RT-PCR方法从肝癌组织中扩增出DcR3片段，并经测序证实。重组质粒经酶切鉴定正确，转染肝癌细胞株SMMC7721后，经Western blot证实DcR3蛋白表达较对照组下降。结论：成功构建反义RNA表达载体PVAXⅠ-as-DcR3，构建的反义表达载体PVAXⅠ—as—DcR3具有阻断DcR3基因表达的效应。     

SV40LT抗原基因逆转录病毒载体的构建及其在肝细胞中的表达

目的　构建含SV 40LT抗原基因的逆转录病毒载体并转染鼠肝细胞 ,检测SV 40LT抗原基因在肝细胞中的表达情况 ,为肝细胞移植研究打下基础。方法　利用体外基因重组技术构建含SV 40LT抗原基因的逆转录病毒载体并酶切、测序鉴定 ,脂质体介导转染PA3 17细胞 ,G 418抗性筛选阳性克隆 ,通过NIH 3T3细胞测定病毒滴度 ;分离、纯化大鼠肝细胞后 ,将含SV 40LT抗原基因的假病毒颗粒感染肝细胞 ,用PCR及免疫组化法检测转染肝细胞中SV40LT抗原基因的表达情况。结果　( 1)酶切分析、测序证明重组逆转录病毒载体含有SV 40LT抗原基因 ;( 2 )病毒滴度为 1.3× 10 6 cfu/ml ;( 3 )转染后的肝细胞含有SV40LT抗原基因 ,其表达在转染后 2 4h明显高于 96h(P <0 .0 5 )。结论　成功构建含SV40LT抗原基因的逆转录病毒载体 ,转染后的肝细胞表达有目的基因 ,有望作为肝细胞体外培养的可用技术     

人VEGF-C基因荧光真核表达载体的构建和表达

目的:构建含有绿色荧光蛋白基因的人血管内皮生长因子C基因的真核表达载体,检测该基因在真核细胞中的表达,研究VEGF-C基因在淋巴管生成中的作用.方法:含有绿色荧光蛋白基因的质粒pEGFP-1通过末端平滑化,酶切获得5'(粘端)BamHI-EGFP-XbaI(平端)3'片断,已经构建的VEGF-C表达载体pcDNA3.1/VEGF-C通过末端平滑化,酶切获得5'-(粘端)BamHI-pcDNA3.1-目的基因-KpnI(平端)3',两片段连接构建含有绿色荧光蛋白的真核表达载体,琼脂糖凝胶电泳并测序检测插入片段的正确性.将该载体通过脂质体转染到真核细胞CHO中,观察绿色荧光蛋白在CHO细胞中的表达情况.通过G418筛选阳性转染细胞,提取总RNA,行RT-PCR,PCR产物琼脂糖凝胶电泳观察VEGF-C基因在CHO细胞中的表达情况.结果:琼脂糖凝胶电泳证实了插入EGFP片段的大小,测序结果证实了插入片段的正确性,CHO细胞中看到绿色荧光蛋白的表达,RT-PCR产物中含有VEGF-C cDNA.结论:成功构建了含有绿色荧光蛋白基因的VEGF-C真核表达载体并检测到在真核细胞CHO中的表达.     

人血管生成素1真核表达载体的构建及在兔骨髓间充质干细胞的表达

目的 探索构建人血管生成素1(human angiopoietin 1，hAng-1)真核表达载体，转染体外培养兔骨髓间充质干细胞(marrow mesenchymal stemcells，MSCs)修复骨缺损的可能性。方法 基因重组和限制性内切酶酶切构建真核表达载体pcDNA3-hAng 1，经脂质体DOTAP介导质粒转染兔MSCs，G418筛选阳性克隆，RTPCR及免疫印迹杂交检测hAng-1 mRNA及蛋白表达。结果 重组真核表达载体pcDNA3-hAng-1经限制性内切酶Xho-I和BamH-I酶切后，电泳显示1．4kb hAng-1目的片段和5．4kb pcDNA3载体片段，转染兔MSCs后，RT-PCR检测出目的基因mRNA，免疫印迹杂交检出hAng-1的蛋白表达。结论 构建的真核表达载体pcDNA3-hAng-1能在转染的兔MSCs中表达，可以为组织工程骨修复奠定基础。     

真核表达载体 pcDNA3.1-h转化生长因子-β1的构建

目的：为研究软骨细胞基因转染的可行性创造条件,从而为基因修饰的软骨组织工程研究奠定基础。方法：应用基因重组技术和限制性内切酶酶切构建并鉴定pcDNA3.1-TGF-β1真核表达载体,经Fugene6介导质粒转染3T3细胞后应用逆转录－聚合酶链反应（RT－PCR）检测hTGF-β1 mRNA在真核细胞中的表达。结果：重组真核表达载体pcDNA3.1-TGF-β1经限制性内切酶Xho I和Hind Ⅲ酶切,电泳后显示1．35kb的hTGF-β1目的片段和5．40kb的pcDNA3.1载体片段,证明重组质粒连接正确;经RT－PCR检测了hTGF-β1在转录水平mRNA的表达情况,表明质粒转染3T3细胞后hTGF-β1mRNA的表达明显增强。结论：重组真核表达载体构建正确,并能在真核细胞3T3中表达hTGF-β1 mRNA,为其在软骨组织工程中的基因转染奠定了基础。     

CD1D基因与B7-1基因共转染胰腺癌细胞及其抗癌作用的实验研究

目的:观察共转染小鼠B7-1和CD1D 基因对小鼠胰腺癌细胞的免疫应答。方法:将表达B7-1和CD1D 基因的逆转录病毒载体导入包装细胞系,然后转染入胰腺癌细胞。用PCR和Western方法鉴定细胞表达;用B7-1和CD1D 阳性的细胞在体内诱导抗肿瘤免疫反应。结果:B7-1和CD1D 阳性的细胞在同基因小鼠体内能诱导抗肿瘤免疫反应并抑制肿瘤生长。结论:B7-1基因和CD1D 基因共转染肿瘤细胞,能抑制肿瘤生长,可能为肿瘤的基因治疗开辟一条新途径。     

人IL-1受体拮抗蛋白重组逆转录病毒载体的构建、鉴定及在骨关节炎软骨细胞中的表达

目的 构建含人IL-1受体拮抗蛋白(IL-1 receptor antagonist, IL-1Ra)逆转录病毒表达载体(PLXRN-IL-1Ra),体外转染人骨关节炎(osteoarthritis, OA)软骨细胞,研究其相关特性.方法 利用细菌内同源重组技术快速构建PLXRN-IL-1Ra逆转录病毒重组质粒,经测序及酶切鉴定正确后转染PT67细胞,包装成为重组PLXRN-IL-1Ra逆转录病毒,并使用小鼠肾成纤维细胞系NIH/3T3对病毒进行滴度测定.实验分为3组未转染组(A组)、PLXRN空质粒转染组(B组)、PLXRN-IL-1Ra转染组(C组),病毒感染人OA软骨细胞后,RT-PCR检测细胞内IL-1Ra基因的转录和表达;ELISA法检测细胞培养上清液中人IL-1Ra蛋白表达.结果 酶切鉴定及基因测序证实重组逆转录病毒质粒中含有人IL-1Ra cDNA,测定包装的病毒滴度为3 × 104 CFU/mL.原代软骨细胞体外培养呈多角形或梭形,甲苯胺蓝染色见细胞内有紫色异染颗粒.RT-PCR结果显示在C组出现311 bp人IL-1Ra mRNA片段,A、B组未见人IL-1Ra mRNA的表达带,GAPDH在各组均有表达.ELISA检测发现C组细胞上清有一定量的人IL-1Ra表达,蛋白浓度为(60.47±15.13)ng/L,A组和B组均无人IL-1Ra表达.结论 构建的IL-1Ra逆转录病毒表达载体成功地感染人OA软骨细胞,并在体外获得稳定表达,为将表达人IL-1Ra基因的人OA软骨细胞用于OA基因治疗提供了实验依据.     

人B7-1基因与GPI信号肽序列融和基因的原核表达载体的构建及表达

目的 构建GPI锚定蛋白基因与B7-1分子融合的原核高效表达载体(GPI-B7-1),观察融合蛋白的抗肿瘤效应.方法 分别从新鲜胎盘和ConA刺激的外周血单核细胞中克隆人胎盘碱性磷酸酶(hPLAP-1)C末端信号肽序列(GPI)和CD80(B7-1)基因.利用PCR技术将GPI和B7-1胞外编码区基因进行融合,并将融合基因亚克隆入原核表达载体(pET-30a)中.重组载体pET-30a-GPI-B7-1转化表达菌株E.coli BL,纯化GPI-B7-1融合蛋白,SDS-PAGE、Western blot分析鉴定其免疫活性.结果 PCR和RT-PCR产物经电泳鉴定,在100～250 bp处可见到133 bp的GPI目的 条带.在750 bp左右可见到792 bp的B7-1胞外编码区基因目的 条带.重组载体pET-30a-GPI-B7-1经PCR检测获得900 bp左右的目的 片段,经EcoRⅠ、SAlⅠ双酶切鉴定,得到5000 bp和900 bp左右大小2个片段,实现了融合蛋白(GPI-B7-1)在大肠杆菌中的高效表达,在非变性条件下纯化该融合蛋白,SDS-PAGE分析显示,表达蛋白的分子量约为38 kDa,Western blotting证实38 kDa处出现一条特异的显色带,该融合蛋白即为目的 蛋白.结论 GPI-B7-1表达载体可在大肠杆菌中高效表达获取融合蛋白,为肿瘤免疫治疗奠定了基础.     

Early osteoblastic differentiation induced by dexamethasone enhances adenoviral gene delivery to marrow stromal cells.

We investigated the implications of induced osteogenic differentiation on gene delivery in multipotent rat marrow stromal cells (MSCs). Prior to genetic manipulation cells were cultured with or without osteogenic supplements (5x10(-8) M dexamethasone, 160 microM l-ascorbic acid 2-phosphate, and 10 mM beta-glycerophosphate). Comparison of liposome, retroviral, and adenoviral vectors demonstrated that all three vectors could mediate gene delivery to primary rat MSCs. When these vectors were applied in the absence or presence of osteogenic supplements, we found that MSCs differentiated prior to transduction with adenovirus type 5 vectors produced a 300% increase in transgene expression compared to MSCs that were not exposed to osteogenic supplements. This differentiation effect appeared specific to adenoviral mediated gene delivery, since there was minimal increase in retroviral gene delivery and no increase in liposome gene delivery when MSCs were treated with osteogenic supplements. In addition, we also determined this increase in transgene production to occur at a higher concentration of dexamethasone (5x10(-8) M) in the culture medium of MSCs prior to adenoviral transduction. We found that this increased transgene production could be extended to the osteogenic protein, human bone morphogenetic protein 2 (hBMP-2). When delivered by an adenoviral vector, hBMP-2 transgene production could be increased from 1.4 ng/10(5) cells/3 days to 4.3 ng/10(5) cells/3 days by culture of MSCs with osteogenic supplements prior to transduction. These results indicate that the utility of MSCs as a therapeutic protein delivery mechanism through genetic manipulation can be enhanced by pre-culture of these cells with dexamethasone.     

Gene therapy of metastatic pancreas cancer with intraperitoneal injections of concentrated retroviral herpes simplex thymidine kinase vector supernatant and ganciclovir.

OBJECTIVE: The objective of this study was to determine the efficacy of intraperitoneal (IP) injections of a new concentrated herpes simplex thymidine kinase (HS-tk) retroviral vector and ganciclovir (GCV) for peritoneal metastases from pancreas cancer. SUMMARY BACKGROUND DATA: Metastatic pancreas cancer is fatal. Gene therapy may provide a novel approach for this disease. Gene therapy with adeno- or retroviral-mediated transfer of the HS-tk gene into tumor cells renders the cells susceptible to GCV. Intratumoral or intracavity injections of retroviral vectors have been ineffective in previous studies. METHODS: Pancreatic cancer B x PC3 cells (3 x 10(7)) were injected into the tail of pancreas in nude mice. Mice received IP injections of a concentrated HS-tk vector (5 x 10(7)) cfu/mliters) or a control vector (G1Na) without the tk gene for 10 days and GCV (100 mg/kg) for 14 days. To determine whether the vector would survive in the milieu of the peritoneal cavity, the authors examined the effects of ascitic fluid on the vector. Pancreas cancer cells were transduced in vitro with HS-tk vector in presence of media or ascitic fluid and treated with GCV. RESULTS: Highly significant reductions in the mass of metastatic peritoneal tumor deposits were found in HS-tk-treated group (124 +/- 27 mg; n = 11) compared with G1Na vector controls (910 +/- 168 mg; n = 8; p < 0.0001). Results of polymerase chain reaction analysis demonstrated integration of the vector in the tumors, and on immunohistochemistry, expression of the TK protein was seen in the number of surviving colonies (representing nontransduced cells) were similar in both groups, suggesting that the vector effectively transduced tumor cells bathed in the ascitic fluid. CONCLUSIONS: Results demonstrate that IP administration of concentrated retroviral HS-tk vectors is effective treatment for pancreas cancer metastatic to the peritoneal cavity; furthermore, the vector is active in the presence of ascitic fluid. Intraperitoneal retroviral HS-tk may provide a novel approach to treatment of metastatic pancreas cancer.     

逆转录病毒载体介导的HyTK基因在人膀胱癌细胞株EJ中的表达

为探索ＨＳＶ１ＴＫ基因对人膀胱癌的治疗作用，作者用一种与潮霉素磷酸转移酶（ｈｐｔ）基因相融和的Ⅰ型单纯疱疹病毒胸苷激酶（ＨＳＶ１ＴＫ）基因（简称ＨｙＴＫ基因）作目的基因，用逆转录病毒载体将ＨｙＴＫ基因导入人膀胱癌细胞株ＥＪ细胞中表达。含ＨｙＴＫ基因的逆转录病毒载体ＬＨｙＴＫＮ，经包装细胞ＰＡ３１７包装后，获得重组逆转录病毒；病毒感染人膀胱癌细胞株ＥＪ细胞，经潮霉素Ｂ筛选培养，获得潮霉素Ｂ的抗性克隆；ＰＣＲ扩增证实：病毒感染的ＥＪ细胞中导入了ＨＳＶ１ＴＫ基因，为开展ＨｙＴＫ基因治疗膀胱癌打下了基础。     

HyTK基因逆转录病毒重组质粒的构建及其在PA317细胞中的高表

目的:为开展逆转录病毒介导的HyTK基因治疗恶性肿瘤奠定实验基础。方法:利用逆转录病毒载体与潮霉素磷酸转移酶和单纯疱疹病毒胸腺嘧啶核苷激酶的融合基因(HyTK)连接构建重组质粒pL(HyTK)SN,应用新一代的非脂质体基因转导系统FuGENETM6将构建的pL(HyTK)SN导入Ψ2单嗜性包装细胞并获得瞬间表达,继而再转染PA317双嗜性细胞。结果:获得高滴度重组病毒,上清逆转录病毒滴度为5.4×106cfu/ml。结论:所构建的HyTK基因可用于治疗恶性肿瘤。     

胞嘧啶脱氨酶基因体外特异性前药转换抗大肠癌作用研究

为使胞嘧啶脱氨酶(CD)基因在大肠癌细胞中特异表达。方法:构建了以CEA基因顺式转录调控序列驱动CD基因的组织特异型重组逆转录病毒载体G1CEACDNa,以脂质体法将重组载体及转录受LTR驱动的CD基因逆转录病毒载体pCD2分别转导入高分泌CEA的大肠癌细胞LoVo中,经G418完全选择后进行前药5-FC敏感试验。结果:转导普通型及组织特异型CD基因的LoVo细胞较亲代细胞对5-FC的前药转换抑瘤效应明显(均为P<0.01);而转导组织特异性CD基因的LoVo细胞较转导普通型CD基因的LoVo细胞对5-FC的敏感性显著增高(P<0.01),其IC_(50)分别为0.1mmol/L和0.5mmol/L。结论:表明CEA TRS可驱动自杀基因在高分泌CEA大肠癌细胞中特异性表达,在一定程度上达到了靶向杀伤肿瘤细胞的目的。     

甲氧西林耐药金黄色葡萄球菌临床分离株青霉素结合蛋白2a的克隆表达及鉴定

目的 应用基因重组技术对编码甲氧西林耐药金黄色葡萄球菌（MRSA）临床分离株青霉素结合蛋白2a（PBP2a）的mecA基因片段进行克隆、表达。方法 从临床样本巾分离鉴定出MRSA，根据基因文库收录的mecA基因编码序列，针对编码PBP2a第25～668位氨基酸的mecA基因片段设计引物，扩增目的基因片段，克隆至pQE30载体，经酶切鉴定、测序后，再转化E．coliM15（pREP4）。采用1mmol／L的异丙硫半乳糖苷（IPTG）进行诱导表达及鉴定。结果重组表达质粒pQE30-mecA构建成功，基因测序结果显示，扩增的mecA基因DNA片段全长为1932bp，其巾有9个碱基突变。IPTG诱导后6h，聚丙烯酰胺凝胶电泳显示，M15（pQE30-mecA）出现一条相对分子质量约74×10^3的蛋白条带，且一部分以可溶性形式存在；M15（pQE30）未出现特异性蛋白条带。经诱导表达和鉴定证实，表达出的可溶性目的蛋白为PBP2a。结论 利用本技术可成功表达MRSA临床分离株中的可溶性PBP2a。     

B7-1 gene transfer into human cancer cells by infection with an adenovirus-B7 (Ad-B7) expression vector

Background: Transfection of the costimulatory molecule B7-1 into some murine tumors can increase antitumor immunity and eradicate tumor growth. The purpose of this work was to construct an adenovirus-B7 (Ad-B7) expression vector and study B7-1 gene transfer into human cancer cells. Methods: The human B7-1 cDNA was ligated into an expression cassette containing the human cytomegalovirus immediate early gene promoter and then inserted into the E1 region of the Ad5 genome by homologous recombination. The resulting Ad-B7 vector was used to infect established cancer cell lines and freshly resected cancers. Resected tumors were disaggregated into single cell suspensions by mechanical mincing and enzymatic digestion. Surface expression of B7-1 after infection was verified by flow cytometry. Results: Expression kinetics in three cell lines showed that infected cells began to express B7-1 within 24 h. The proportion of B7-1⁺ cells continued to increase during the next 48 h, after which expression remained relatively constant during the next 5 days (up to 98% B7-1⁺ cells). Fresh tumor cells from various cancers displayed similar kinetics, but with greater variability in the proportion of cells expressing B7-1 (13% to 95% B7-1⁺ cells). Cancers which were successfully infected included 3 colorectal adenocarcinomas, 2 leiomyosarcomas, 2 lung squamous cell carcinomas, and 1 renal cell carcinoma. Conclusions: The Ad-B7 vector is a rapid and efficient means of gene transfer which does not require host cell proliferation. The ultimate objective is to engineer autologous tumors to express B7-1 and vaccinate cancer patients in an adjuvant or palliative setting. Results of this study were presented at the 48th Annual Cancer Symposium of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.