转染超抗原SEA肝癌细胞的构建及鉴定

目的用基因重组的方法将葡萄球菌肠毒素A（SEA）基因转染至肝癌细胞．方法先从产SEA标准菌株中提取并扩增SEA全长基因，将SEA基因克隆及亚克隆至真核表达载体pLXSN构建pLXSN—SEA重组质粒，再将重组质粒转染至人肝癌细胞系BEL-7402细胞，用RT—PCR和ELISA法鉴定．结果从产SEA标准菌株ATCCl3565中提取并扩增出SEA基因全长片段；SEA基因克隆和亚克隆至真核表达载体pLXSN，经测序证实所得序列与GenBank中的标准序列完全一致；将pLXSN—SEA质粒转染肝癌细胞BEL-7402，经筛选获得稳定表达的抗性单克隆．RT—PCR扩增出约800bD的基因片段，经ELISA分析细胞培养上清中SEA蛋白的含量在皮克的水平．结论成功构建了重组超抗原SEA基因的克隆载体及真核表达载体，将SEA基因转染至肝癌细胞株BEL－7402细胞后癌细胞能够表达并持续分泌SEA蛋白．     

超抗原SEA抗肿瘤研究进展

超抗原是指一些细菌或病毒编码的蛋白分子, 只需极低浓度即可激活大量的淋巴细胞克隆.作为一种强大的T细胞激活剂,超抗原成为肿瘤免疫治疗研究的热点之一.本文对其特性及目前的研究进展作一综述.     

超抗原SEA抗肿瘤研究进展

超抗原是指一些细菌或病毒编码的蛋白分子，只需极低浓度即可激活大量的淋巴细胞克隆．作为一种强大的T细胞激活剂，超抗原成为肿瘤免疫治疗研究的热点之一．本文对其特性及目前的研究进展作一综述．     

超抗原SEA的基因克隆、原核表达与鉴定

目的：构建pRSET-SEA重组表达载体，转化大肠杆菌BL21(DE3)pLysS，诱导表达超抗原葡萄球菌肠毒素A(staphylococcal enterotoxin A，SEA)，进行分离、纯化及western bolt鉴定。方法：采用PCR技术，从产SEA的葡萄球菌标准菌株FRI100基因组DNA中获得SEA全长序列，克隆人pUC19中，进行测序，构建pRSET-SEA表达质粒，转化大肠杆菌BL21(DE3)pLysS，通过异丙基硫代-β-D-半乳糖苷（isopropyl-beta-D-thiogalactopyranoside，IPTG）诱导表达，分离、纯化及western blot鉴定。结果：PCR获得超抗原SEA基因片段，DNA测序结果与文献报道一致；构建了pRSET-SEA表达质粒，并成功地诱导表达出32000u的蛋白；Western blot鉴定所得蛋白能够与SEA单克隆抗体特异性结合。结论：本研究成功地克隆了SEA全长，并进行了原核表达和分离、纯化，获得了SEA蛋白。     

超抗原SEA(D227A)的构建及其免疫活性鉴定

目的 构建SEA(D227A)基因的原核表达载体，并进行表达、分离纯化与免疫活性鉴定。方法 采用PCR技术从产SEA的葡萄球菌标准株中克隆SEA基因，通过下游引物上的点突变，使SEA基因第680位碱基由A突变为C，致使SEA成熟蛋白的第227位氨基酸残基由天冬氨酸(GAT)突变为丙氨酸(GCr)，构建pGEX-SEA(D227A)重组表达质粒，在大肠杆菌DHSa中原核表达，再通过淋巴细胞增殖实验对其免疫活性进行测定。结果 构建的SEA(D227A)基因经测序证实与设计的序列一致；成功地构建pGDX-SEA(D227A)重组表达质粒，转化感受态大肠杆菌DH5α，通过IPTG诱导、GSTrap FF柱分离纯化及凝血酶切除CST后，得到Mr为27000的目的蛋白。淋巴细胞增殖实验显示，100ng/mL重组SEA(D227A)可明显刺激淋巴细胞增殖。结论 成功地构建了SEA(D227A)基因原核表达载体，并在大肠杆菌中得到高效表达，所得重组SEA(D227A)能够刺激淋巴细胞增殖，但作用与野生型SEA相比较温和。该结果为寻找有效、毒副作用低的超抗原提供了线索。     

超抗原-SE抗肿瘤作用的研究进展

葡萄球菌肠毒素(Staphylococcal entero-toxin，SE)是一种外源性超抗原，仅需微量即能高效地刺激T细胞增殖，促使其产生大量的细胞因子及细胞毒作用，提高机体总体细胞免疫水平，达到抗肿瘤的能力。体外细胞水平和动物体内的研究结果表明：SE能够产生强大的杀伤和抑制肿瘤细胞效应，是一种很有前途的新型肿瘤免疫治疗剂。目前已开始应用于肿瘤病人的治疗及对肿瘤病人在进行放化疗时保护机体白细胞降低以及防止其他毒副反应的发生。     

超抗原-SE抗肿瘤作用的研究进展

葡萄球菌肠毒素(Staphylococcal entero-toxin,SE)是一种外源性超抗原,仅需微量即能高效地刺激T细胞增殖,促使其产生大量的细胞因子及细胞毒作用,提高机体总体细胞免疫水平,达到抗肿瘤的能力.体外细胞水平和动物体内的研究结果表明SE能够产生强大的杀伤和抑制肿瘤细胞效应,是一种很有前途的新型肿瘤免疫治疗剂.目前已开始应用于肿瘤病人的治疗及对肿瘤病人在进行放化疗时保护机体白细胞降低以及防止其他毒副反应的发生.     

点突变SEA(D227A)的基因构建、原核表达与鉴定

目的：进行点突变的SEA(D227A)基因原核表达载体的构建，并进行表达、分离纯化与鉴定。方法：采用PCR技术从产SEA的葡萄球菌标准株中克隆SEA基因，通过下游引物上的点突变，使得到的SEA基因752位碱基突变由A突变为c，致使SEA成熟蛋白的第227位氨基酸残基由D(DAT)突变为A(GCF)，构建pRSET：SEA(D227A)重组表达质粒，在大肠杆菌BL21(DE3)pLysS中进行原核表达，再通过Western blot进行鉴定。结果：构建的SEA(D227A)基因经测序证实与设计的序列一致。成功地构建pRSET-SEA(D227A)重组表达质粒，转化感受态大肠杆菌BL21(DE3)pLysS，通过IPTG诱导得到分子量约为32kD的蛋白，Western blot显示其能够与抗SEA的单克隆抗体特异性结合，最后采用Ni^2 -NTA柱进行分离纯化。结论：成功地构建SEA(D227A)基因原核表达载体，并在大肠杆菌中得到高效表达，为寻找有效低毒副作用的超抗原提供了线索。     

肝癌靶向性SEA-CD80基因共表达重组腺病毒载体的构建及表达鉴定

目的:构建肝癌靶向性葡萄球菌肠毒素A(SEA)和CD80基因共表达重组腺病毒载体。方法:首先利用现有的腺病毒穿梭质粒pShuttle和pShuttleCMV,构建新的不带CMV增强子/启动子而带有polyA加尾信号穿梭质粒,命名为pShuttle2。将AFP增强子、启动子、SEA及CD80基因分别从已构建的pKSEP载体和pMD18TBIS载体上,分别亚克隆至pShuttle2中,再与腺病毒骨架质粒pAdEasy1共转化E.coliBJ5183。以获得的重组子转染HEK293细胞后制备重组腺病毒,然后感染高表达AFP的肝癌细胞系Hepa16和不表达AFP的黑色素瘤细胞系B16、成纤维细胞系NIH3T3。采用间接免疫荧光法,激光共聚焦显微镜观察和流式细胞术检测SEA和CD80在细胞膜表面的表达。采用3H掺入法检测膜表达的SEA诱导淋巴细胞增殖的活性。结果:以制备的重组腺病毒感染肿瘤细胞后,SEA和CD80能够靶向性地共表达在高表达AFP的Hepa16细胞膜上,而在不表达AFP的B16、NIH3T3细胞膜上不表达。结论:成功地构建肝癌靶向性SEA和CD80基因共表达重组腺病毒载体,为进一步研究SEA和CD80在肝癌靶向基因治疗中的联合应用及其抗肿瘤免疫机制奠定了基础。     

超抗原SED在大肠杆菌中的表达

目的 构建稳定的SED原核表达系统。方法 采用PCR技术扩增葡萄球菌D型肠毒素（SED）超抗原的成熟蛋白编码区DNA序列，构建SED DNA与6个组氨酸基因融合的表达载体pTrcHis-SED，转入E.coli DH5α，IPTG诱导后，用SDS－PAGE和免疫印迹检测融合蛋白的表达情况。目的蛋白用Ni-NTA金属亲和层析法进行纯化，SDS－PAGE和毛细管电泳检测蛋白纯度。结果 成功构建了原核表达载体pTrcHis-SED。分子量约为28000u的6His-SED融合蛋白可在E.coli DH5α中稳定表达。Ni-NTA金属亲和层析后得到纯度较高（＞95％）的SED融合蛋白，且具有免疫学活性。结论 本研究为SED免疫识别研究奠定了实验基础。     

超抗原SEA活化淋巴细胞抗肿瘤活性的实验研究

纯化SEA在 10 -12 ～ 10 -5g/ml浓度范围对体外培养的BALB/c鼠脾细胞表现了细胞增殖诱导能力 ,并呈剂量依赖关系 ,其中 10 -7～ 10 -5g/mlSEA的作用强于最适量 ( 2 5 μg/ml)PHA。在E/T为 5∶1～ 2 0∶1条件下 ,10 -5g/mlSEA活化 48h的BALB/c鼠脾细胞对YAC 1细胞的杀伤活性高于NK细胞 ,但SEA未能增强BALB/c鼠脾细胞对B16细胞的杀伤活性。 5 μg和5 0 μgSEA体内用药对L615白血病无治疗作用 ,但转输经SEA活化的同系小鼠脾细胞对L615白血病有一定疗效。实验结果提示 ,SEA活化淋巴细胞对NK敏感的淋巴瘤、白血病有杀伤及治疗作用 ,这种作用依赖于较大数量级SEA活化淋巴细胞的存在 ,并且与SEA活化T细胞分泌的细胞因子有关     

Functional Impairment of Dendritic Cells Caused by Murine Hepatocellular Carcinoma

Immunosuppression in tumor-bearing hosts may be a major obstacle in eradicating tumors. This study investigated whether hepatocellular carcinoma suppressed the functions of dendritic cells to escape tumor surveillance. Dendritic cells (DC), propagated from C57BL/10J mice, were cocultured with or without murine hepatoma Hepa1-6 cells to examine the influence of hepatocellular carcinoma on dendritic cells. The results revealed that dendritic cells cocultured with hepatoma cells expressed low levels of costimulatory molecules, and the stimulatory capacity was decreased. The antigen-specific cytotoxic effects of T cells activated by the DC cocultured with hepatoma cells were also decreased. In ex vivo studies, the maturation and function of dendritic cells propagated from tumor-bearing mice were suppressed. The suppressive effect of Hepa1-6 cells on dendritic cells could be partially reversed by neutralizing IL-10. In conclusion, the maturation and stimulatory function of DC are suppressed by hepatocellular carcinoma. IL-10 release may be one of the mechanisms employed by hepatocellular carcinoma to suppress dendritic cells.     

抗人肝癌单克隆抗体HL2的制备及鉴定

用肝癌细胞株QGY-7703、BEL-7402、SMMC-7721以贯序法免疫BALB/c小鼠,获得一株分泌肝癌单抗的杂交窟株HL_2,其染色体数目为97—105。单抗HL_2系IgG_2b亚类。吸收实验及阻断实验证明HL_2抗原与CEA、AFP、HBsAg、HBcAg及HBeAg无免疫同源性。ABC法和ELISA法说明单抗HL_2与四株肝癌细胞、六例肝癌组织均呈明确阳性反应,除与SGC-7901、H_(128)、K_(562)、Hela细胞株及部分胚胎肝脏,胚胎结肠有较弱性反应外,与肝癌旁组织、正常肝脏、其它肿瘤组织和细胞株、二种正常人细胞及其它胚胎组织无反应。显示了单抗HL_2的特异性较好、阳性率较高,是一个有应用前景的单抗。     

A Novel Approach for Cancer Immunotherapy: Tumor Cells with Anchored Superantigen SEA Generate Effective Antitumor Immunity

Murine B16 melanoma cell line is poorly immunogenic and highly aggressive. We recently reported that the transmembrane staphylococcal enterotoxin A (TM-SEA) anchors onto B16 cells and stimulates lymphocyte proliferation. The purpose of the study was to investigate whether vaccination with B16 cells bearing membrane-anchored TM-SEA fusion protein could cause tumor-specific immunity. Mice in the therapeutic vaccination group received B16 tumor inoculations, followed by treatment with B16-TM-SEA vaccine or control vaccines. Mice in the prophylactic vaccination group were given B16-TM-SEA vaccine or control vaccines, followed by challenge with wild type B16 or control EL4 cells. Significant tumor growth inhibition, prolongation of survival, and marked augmentation of NK and CTL activities were observed in mice which received B16-TM-SEA vaccine as compared to controls. Overall, our results suggest that the TM-SEA cellular vaccine is a novel and effective strategy for cancer immunotherapy.     

Tumor Growth of FGF or VEGF Transfected MCF-7 Breast Carcinoma Cells Correlates with Density of Specific Microvessels Independent of the Transfected Angiogenic Factor

We have previously shown that fibroblast growth factor (FGF)-1-, FGF-4-, or vascular endothelial growth factor (VEGF/VPF)-transfected MCF-7 breast carcinoma cells growing as tumors in nude mice are tamoxifen resistant and/or estrogen independent. These transfectants provide opportunity for study of in situ tumor-induced angiogenesis promoted by the individual angiogenic factors under growth-promoting versus growth-inhibiting hormonal conditions. In the present study, vessels in tumors harvested at varying times after tumor cell injection were immunohistochemically highlighted and vessel morphology and topography were scored on a scale of 0 to 4 by blinded observers. In tumors produced by all cell lines under all growth-promoting hormonal conditions, there was significantly increased abundance (P < 0.05) of edge-associated and intratumor microvessels, but not of stromally located microvessels, when compared with tumor nodules harvested under growth-inhibiting conditions, regardless of the identity of the angiogenic factor or the hormonal treatment. Image analysis of bromodeoxyuridine (BrdU)-labeled nuclei of tumors produced by all cell lines under all hormonal conditions harvested at early time points showed that mean labeling indices were highest for hormonal conditions that produced the most robust growth in that particular cell line, implying that a high BrdU labeling index is a predictor of future tumor growth in individual tumors. These results confirm previous studies that established the importance of neovascularization for tumor growth and provide validation for use of these cell lines to study the process of angiogenesis in vivo. Study of gene expression in endothelial cells in edge-associated or intratumor vessels using this model might reveal mechanisms important in tumor-induced angiogenesis in human breast cancer.     

Proteomic-Based Approach for the Identification of Tumor Markers Associated with Hepatocellular Carcinoma

There is increasing evidence for an immune response to cancer in humans, demonstrated in part by the identification of autoantibodies to tumor antigens. The identification of panels of tumor antigens that elicit a humoral response may have utility in cancer screening, diagnosis or in establishing prognosis. Several approaches are currently available for the identification of tumor antigens. We have used a proteomic-based approach for the identification of tumor antigens that induce an antibody response which we have applied to hepatocellular carcinoma, a major type of cancer worldwide. Two-dimensional gel electrophoresis allows simultaneous separation of several thousand individual proteins from tumor tissue or tumor cell lines. Proteins eliciting a humoral response in HCC were identified by 2-D Western blotting using sera from patients with hepatocellular carcinoma, followed by mass spectrometry analysis and database search. The common occurrence of autoantibodies to specific proteins may have utility for HCC screening and diagnosis.     

肝癌细胞侵袭行为与其流变特性相关

以原代肝细胞、肝细胞癌（Ｈｅｐａｔｏｃｅｌｌｕｌａｒ ｃａｒｃｉｎｏｍａ,ＨＣＣ）细胞及对人工基底膜Ｍａｔｒｉｇｅｌ具有侵袭能力的ＨＣＣ细胞（ＨＣＣ－Ｉｎｖ细胞）为研究对象,研究了三种细胞的粘弹特性、与胶原蛋白Ⅰ的粘附力学特性及与肝血窦内皮细胞（Ｌｉｖｅｒ ｓｉｏｎｓｏｉｄａｌ ｅｎｄｏｔｈｅｌｉａｌ ｃｅｌｌ,ＬＥＣ）的粘附力学特性。结果发现,ＨＣＣ细胞的上述流变特性较之于肝细胞发生明显的变化,     

基质刚度对肝癌细胞增殖及糖代谢的影响

目的研究不同基质刚度对肝癌细胞HepG2增殖和糖代谢的影响,探究细胞外基质(extracellular matrix,ECM)刚度对肝癌细胞代谢和生物学行为影响的相关性。方法采用CCK8和细胞计数法检测培养在不同刚度基质上HepG2细胞增殖的变化,利用2-(N-7-硝基-2,1,3-苯并恶二唑-4-氨基)-2-脱氧-D-葡萄糖(2-NBDG)并通过流式细胞术检测基质刚度对HepG2葡萄糖摄取的影响,利用荧光定量RT-PCR检测基质刚度对HepG2葡萄糖转运蛋白1(glucose transporter 1,Glut1)表达的影响,并利用糖酵解抑制剂2-脱氧葡萄糖(2-deoxy-D-glucose,2-DG)阻断糖酵解途径后检测基质刚度对HepG2细胞增殖的影响。结果随基质刚度增加,HepG2细胞增殖能力、葡萄糖摄取和Glut1表达都显著增加。糖酵解途径被阻断后,不同刚度基质上HepG2细胞增殖能力基本相同。结论肝癌细胞所处力学微环境对肝癌细胞的增殖有重要影响;较大的基质刚度可能通过调控糖代谢途径促进肝癌细胞的增殖。研究结果为肝癌的临床治疗以及糖代谢为治疗靶点的药物开发提供相应的实验依据。