RT-PCR克隆人IL-6、IL-10、IL-13和SCF等四种细胞因子膜外区cDNA

在逆转录病毒介导下将TNF基因转染入小鼠LAK细胞中．结果发现．转染TNF基因的LAK细胞比正常LAK细胞和转染对照基因的LAK细胞分泌更高水平的TNF．其体外生长能力和杀伤活性均显著升高．抗TNF单抗可显著抑制其体外杀伤活性．表明其杀伤活性升高是由基因转染后所表达的TNF介导的。将较低数量的TNF基因转染的LAK细胞与IL-2联合腹腔内注射后，对腹水型肝癌小鼠有显著的治疗效果．     

联合B7-1分子、IL-2及LAK细胞治疗肝癌的实验研究

目的：研究共刺激信号在肿瘤免疫中的作用。方法：构建含人B7-1基因的真核表达载体PRCCMV-B7-1，脂质体介导DNA转移法将人B7-1基因转入人肝癌细胞株SMMC7721，建立新的细胞株SMMC-B7-1，PCR及逆转录PCR（RT-PCR），流式细胞仪检测，四唑卤（MTT）比色试验体外检测白细胞介素-2（IL-2）激活的LAK细胞（LAK-C），对两种肝癌细胞的杀伤活性，结果：PCR，RT-PCR法证实SMMC-B7-1细胞稳定表达B7基因，LAK细胞对SMMC-B7-1细胞的杀伤活性明显高于SMMC7721，结果有统计学意义。结论：B7基因可以体外转染人肝癌细胞，并表达有活性的B7分子，明显增强IL-2激活的LAK细胞的肿瘤杀伤作用。为进一步开展临床应用奠定基础。     

联合IL-2基因和B7-1基因转染G422细胞体外生物学特性

目的　通过研究联合基因转染后胶质母细胞瘤G42 2细胞生物学特性的变化 ,探讨脑胶质瘤细胞因子基因治疗的新途径。方法　通过重组腺病毒作载体 ,将IL 2基因和B7 1基因转染至小鼠G42 2细胞 ,通过CTLL 2细胞增殖的MTT法检测IL 2的分泌水平 ,FACS检测B7 1和ICAM 1的表达 ,MTT法检测转基因后G42 2细胞的增殖活性 ,双层琼脂糖培养法检测其克隆形成能力。结果　IL 2基因转染 4小时后即可检测到IL 2的分泌 ,2 4小时达高峰 ( 4 83U/ml) ,一周时表达明显下降 ,B7 1高表达 ,ICAM 1表达增加 ,增殖能力和克隆形成能力无变化。结论　重组腺病毒作载体 ,IL 2基因及B7 1基因共转染G42 2细胞 ,能得到目的基因的高效表达 ,同时促进了粘附分子ICAM 1的表达 ,体外增殖活性不受影响。     

IL-24对B16细胞生长抑制作用的体内外实验研究

目的:构建白细胞介素24(Interleukin24,IL24)真核表达质粒,研究其体内外表达对肿瘤细胞的生长抑制作用。方法:采用重组DNA技术构建IL24真核表达质粒pEGFPIL24。用脂质体法将重组质粒及空载体体外转染B16细胞,再经激光扫描共聚焦显微镜(LSM)观察其表达,用MTT法检测B16细胞的体外增殖能力,用流式细胞仪检测细胞周期。小鼠实体瘤模型研究基因转染对肿瘤的体内生长抑制作用。结果:pEGFPIL24转染B16细胞后,LSM可观察到IL24在细胞中的表达。转染IL24基因的B16细胞体外增殖能力明显受到抑制,细胞被阻滞在G2/M期。与对照组相比,IL24基因治疗组肿瘤在体内的生长受到明显抑制,P<0.05。结论:IL24基因转染的肿瘤细胞体外生长受抑。于荷瘤小鼠的瘤内注射pEGFPIL24可抑制肿瘤生长,提示IL24具有明显的体内外抗肿瘤作用。     

肿瘤生物治疗新方法的研究

目前肿瘤基因治疗的研究已经取得了一定的进展,已从实验室基础研究阶段发展到临床应用阶段。在临床应用研究上大致可分为免疫基因治疗;耐药基因治疗;自杀基因治疗;肿瘤抑制基因治疗及基因标记示踪等。“九五”期间,我国把“肿瘤生物治疗等新方法研究”列为攻关课题。该项目的研究目标是以腺病毒为载体,将目的基因TNF－a、IL－2及匕等基因导人肝癌及肺癌细胞,及将目的基因TNFa、ILZ基因导人A－LAK细胞,有效地扩增导人目的基因的A－LAK细胞。通过体内外实验研究确定其临床应用的可行性及安全性。在此基础上,争取国家药政部门…     

Rb基因对肺腺癌细胞株生长的抑制作用

目的：探讨重组腺病毒载体的转染效率及其介导外Ｒｂ基因进肿瘤细胞的效果，观察外源Ｒｂ基因在肿瘤细胞内的表达及其对捉瘤细胞生长的抑制作用。为肿瘤的基因治疗提供实验依据。方法：构建Ｒｂ基因重组腺病毒载体。体外转染人肺腺癌细胞标ＧＬＣ－８２。应用免疫组化和聚合酶链反应等技术，观察Ｒｂ基因在细胞内的状态及表达情况。应用细胞计数及流式细胞仪检测肿瘤细胞的生长情况。结果：ＧＬＣ－８３细胞内存在基因组Ｒｂ基因，但     

IκBα基因转染抑制人肺癌细胞株H460成瘤性的实验研究

背景与目的核因子κB(NFκB)参与多种肿瘤的发生发展,抑制其活性可能抑制肿瘤细胞生长。本研究旨在观察抑制NFκB对人肺癌细胞株H460生长的影响,探讨其抑制或杀伤肿瘤的分子机制。方法体外实验:用表达抑制性κB(IκBα)或LacZ的腺病毒(AdIκBα或AdLacZ)转染H460细胞,测定其生长和凋亡情况,并检测细胞培养上清液中caspase3和TNFα表达。体内实验:用预先转染AdIκBα/AdLacZ的H460细胞建立肿瘤模型,以观察基因转染对成瘤性的影响;对未转染的H460肿瘤小鼠,于瘤内注射AdIκBα/AdLacZ/PBS,观察小鼠肿瘤的生长。留取小鼠肿瘤标本进行免疫组化染色,检测p65、p53和VEGF表达。结果①转染AdIκBα可抑制肿瘤细胞生长,促进凋亡发生;②成瘤性抑制实验显示接种30MOIAdIκBα转染的H460细胞的小鼠中77.8%在6周内无肿瘤形成,而30MOIAdLacZ转染组仅12.5%(P=0.012);③对已建立的H460肿瘤,瘤内注射30MOIAdIκBα可明显延缓肿瘤生长(P<0.05);④免疫组化染色提示各实验组的p53表达无显著性差异,而VEGF表达在30MOIAdIκBα转染组明显减少,转染AdIκBα抑制了H460细胞的p65活性。结论AdIκBα转染阻滞人肺癌细胞H460的NFκB活性,抑制肿瘤细胞的成瘤性,并延缓肿瘤生长。体内外实验显示AdIκBα转染不仅通过诱导凋亡而控制细胞     

重组腺病毒介导的人野生型p53、GM—CSF和B7—1基因在肝癌细胞中的表达

目的：观察腺病毒载体对肝癌细胞的转染效率及其介导的人野生型p53，GM－CSF和B7－1基因在肝癌细胞中的表达。方法：不同MOI的Ad-GFP感染肝癌细胞，48h后计算感染效率；BB－102以50MOI感染肝癌细胞，48h后免疫组化法及western blot检测p53表达，ELISA检测GM0CSF的含量，FACS测定B7－1的表达。结果MOI为50pfu/细胞时对肝癌细胞的转染效率达80％以上，目的基因均可在肝癌细胞中高效表达。结论：腺病毒载体对肝癌细胞具有较高的转染效率，目的基因均可在肝癌细胞中高效表达，为进一步研究BB－102在肝癌治疗中的应用奠定了基础。     

复制缺陷型重组腺病毒介导mIFN-β基因治疗小鼠黑色素瘤的研究

目的:探讨人类复制缺陷型重组腺病毒载体介导mIFN-β基因治疗小鼠黑色素瘤的可行性和效果。方法:利用人类复制缺陷型重组腺病毒将目的基因mIFN-β经体外、体内两种途径导入小鼠黑色素瘤细胞(B16细胞)中,观察体外转mIFN-β基因的B16细胞的体内致瘤性和瘤苗作用,及通过腺病毒载体介导的mIFN-β对体内局部肿瘤治疗和抗肿瘤转移的作用。结果:1)携带目的基因的重组腺病毒感染B16细胞能获得较高的转移效率和目的基因的有效表达;2)将mIFN-β基因导入B16细胞对B16细胞的体外增殖能力无明显影响,但能显著提高细胞表面MHC-I类抗原的表达;3)将转mIFN-β基因的B16细胞接种小鼠,其体内致瘤性明显降低,且对对侧野生型B16细胞的致瘤性有抑制作用;4)瘤体内注射和经尾静脉注射途径给予AdCMVmIFN-β,对局部肿瘤和转移瘤有治疗作用。结论:利用人类复制缺陷型腺病毒载体介导mIFN-β基因治疗小鼠黑色素瘤是可行的,并具有较好疗效,提示用腺病毒载体携带有效的目的基因来开发瘤苗和治疗肿瘤具有良好的临床应用前景。     

用表达IL-2的重组腺病毒瘤体内注射诱导肿瘤消退及免疫反应

IL-2能通过增强CTL细胞的杀伤活性、诱导LAK细胞及激活TIL等具有抗肿瘤作用。而腺病毒以其扩增快、转染范围广,以及不整合入感染细胞的基因组等优点适于临床应用。本文作者将携带IL-2基因的重组腺病毒AdCA IL-2转入肿瘤细胞后种入小鼠体内或直接在肿瘤部位进行瘤体内注射,检测了IL-2的表达,并观察了对肿瘤生长的影响。     

肝细胞癌中PGC-1α基因的表达下调及其意义

目的 探讨PGC-1α在肝细胞癌(HCC)组织中的表达及其在肝细胞癌发生发展中的调节作用.方法 采用定量逆转录聚合酶链反应(RT-PCR)分析HCC组织和正常肝组织中PGC-1α基因的表达,比较癌组织与正常组织的表达差异.采用siRNA干扰技术体外抑制人正常肝细胞株L02中PGC-1α基因的表达,并在光镜下观察细胞形态,电镜下观察细胞超微结构的改变.应用人全基因组Oligo DNA芯片分析比较肝组织和肝细胞基因的表达变化.结果 HCC组织中PGC-1α基因的表达明显下调,仅为正常肝组织的25.0%.抑制PGC-1α基因的表达可使人正常肝细胞株L02呈现出类似肝癌细胞的形态和超微结构变化,即细胞变小、变圆,胞质减少、核增大,细胞数量减少,成簇分布,线粒体膜呈髓鞘样变性等.基因芯片分析显示,抑制PGC-1α基因的表达可使人正常肝细胞株L02呈现出类似肝癌组织的基因变化趋势,即细胞黏附基因(TNFAIP6、ITGAX、ICAM1、FN1、EMR1、ITGA6、PARVA)、成瘤基因(PMAIP1、CRKL、FHIT、IRF1、PLK)和促进细胞增殖基因(STAT1、CCL3L1、IL6、WARS、PCK1、MUC2、PRKR、IFITM1)表达上调,肿瘤抑制基因(FHIT、PRKR、IL24、DLEU1、RCN2)表达下调.结论 PGC-1α表达下调能诱导人正常肝细胞株L02的肿瘤性转化,PGC-1α在肝癌发生发展中具有重要的调节作用.     

RNAi抑制PI3Kp85α蛋白活性对人乳腺癌细胞系MCF-7生长抑制的体外研究

目的:探讨敲低PI3Kp85α表达对人乳腺癌细胞系MCF-7细胞生长的影响和机制.方法:用靶向PI3Kp85α的siRNA转染人乳腺癌细胞系MCF-7,使用Real-time PCR法鉴定转染PI3Kp85α表达水平;MTT法评价PI3Kp85α siRNA对乳腺癌细胞系MCF-7生长的影响;流式细胞术检测转染后细胞周期分布和凋亡;采用免疫荧光染色及Western blot方法观察IA型PI3K/AKT通路主要成员的表达.结果:Real-timePCR结果显示PI3Kp85α siR-NA转染导致PI3Kp85α表达下调;MTT结果显示PI3Kp85α siRNA转染抑制肿瘤细胞生长;流式细胞术检测可见PI3Kp85α siRNA转染组细胞周期存在G_0/G_1期阻滞而且凋亡率显著高于对照组与空载体组(F=19.255,P=0.002).结论:应用PI3Kp85α siRNA转染人乳腺癌细胞系MCF-7细胞,可抑制其增殖和诱导细胞凋亡,因此PI3Kp85α可以作为人乳腺癌基因治疗的候选靶点.     

Induction of tumor regression in experimental model of human head and neck cancer by human A-LAK cells and IL-2

In a nude mouse model of human squamous-cell carcinoma of the head and neck (SCCHN), locoregional therapy with interleukin 2 and human lymphokine-activated killer (LAK) cells resulted in a significant inhibition of growth of 3-day established tumors. The same model was used for therapy of 7-day established tumors with highly enriched populations of human adherent (A)-LAK (CD3- CD56+) cells and IL-2. Peritumoral transfer of 10 x 10(6) A-LAK cells, whose in vitro cytotoxicity against a SCCHN cell line (PCI-I) was not significantly different from that of LAK cells, resulted in complete regression of all 3-day or 7-day human SCCHN in nude mice. An initial inflammatory-type reaction, which appeared within hours of the first peritumoral cell transfer, was accompanied by infiltration initially by granulocytes and plasma cells, and later by mononuclear cells into the tumor stroma. A-LAK cells labelled with a fluorescent dye prior to injection appeared in the tumor stroma within 24 hr and were localized around or in the basal epithelial tumor layer by 48 hr. Histologic sections revealed an increasing epithelial disorganization and progressively decreasing basal epithelial layer, which were proportional to the increasing number of A-LAK cells transferred. Within 4 weeks, the tumors were reduced to amorphous keratinic remnants surrounded by the connective tissue containing abundant mononuclear cells. Local administration of human A-LAK cells and IL-2 to SCCHN tumors growing in nude mice led to accelerated tumor differentiation, keratinization and regression.     

Calcineurin promotes proliferation, migration, and invasion of small cell lung cancer

A novel role for calcineurin (Cn) has been reported recently regarding the oncogenic potential in pancreatic and colorectal cancer. The aim of this study was to investigate the putative causal role calcineurin could play in the development of lung cancer with bone metastases. We found that CnAα, an isoform of calcineurin, was significantly overexpressed in lung cancer tissues with bone metastasis as compared to tumors with non-bone metastases as investigated by RT-PCR. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasmic staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis. Western blots of nuclear proteins from lung cancer tissues indicated that CnAα was highly expressed in lung cancer tissues with bone metastases, but not in those with non-bone metastases. In vitro, it was demonstrated that the CnAα gene obviously promoted cell proliferation and inhibited cell apotosis. The CnAα gene affected the cell cycle and promoted G1?S transition in SBC-3 cells. Transfection with the CnAα gene promoted cell migration and invasion. These results indicated that CnAα may affect the biological behavior of the human small cell lung cancer cell line SBC-3 in vitro and may be a candidate tumor promotor gene for developing bone metastases.     

过表达microRNA-7对人肺癌95D细胞凋亡的影响

目的 探讨过表达microRNA-7对人肺癌95D细胞体内外凋亡的作用。方法 将miR-7真核表达载体（命名为p-miR-7）体外瞬时转染人肺癌95D细胞,利用TUNEL法观察95D细胞凋亡的变化;免疫荧光技术检测各组细胞磷酸化Caspase3和Caspase9蛋白的表达;建立人肺癌裸鼠模型,肿瘤局部注射p-miR-7后利用TUNEL法检测肿瘤局部细胞凋亡变化,同时,免疫组织化学法检测凋亡相关的磷酸化Caspase3和Caspase9蛋白的表达变化。结果 体外转染p-miR-7后可导致人肺癌细胞的凋亡（P<0.05）。同时,细胞中磷酸化Caspase3和Caspase9蛋白水平显著增加（P<0.05）;肿瘤局部注射p-miR-7后,肿瘤局部miR-7表达水平及细胞凋亡也明显增强（P<0.05）,磷酸化Caspase3和Caspase9蛋白水平也显著增加。结论 过表达miR-7可以导致肿瘤细胞凋亡,这与凋亡相关蛋白Caspase3和Caspase9磷酸化水平增加有关。     

Comparison of adherent lymphokine-activated killer (a-lak) cells generated by IL-2 and IL-7 - cellular modifications induced by IL-7

At present, the clinical application of plastic-adherent-lymphokine-activated killer (A-LAK) cells shows limited success in the immunotherapy of patients with advanced cancer because of a low responder rate, severe side effects and failures in yielding sufficient numbers of cells for adoptive transfers. Since interleukin-7 (IL-7) is able to induce LAK activity independently of IL-2, we investigated the ability of IL-7 to improve the yield and the properties of A-LAK cells. A-LAK cells from 7 healthy donors generated in the presence of IL-2, IL-7 or combinations of IL-2 plus IL-7 (each 1000 U/ml) were compared with regard to plastic adherence, expansion rate, immunophenotype, cytokine secretion and cytotoxicity against malignant melanoma cells and non-malignant target cells. Our results demonstrate that A-LAK cells generated by a simultaneous stimulation of IL-2 plus IL-7 displayed a significantly higher expansion rate (10.7-fold vs. 9.0-fold), but showed no difference in the cytolytic activity compared to A-LAK cells generated by IL-2 alone. A-LAK cells generated by IL-7 alone demonstrated a low expansion rate (1.1-fold vs. 8.8-fold), and decreased in other properties like plastic adherence, CD56(+)/CD3(+) cell-ratio and cytolytic activity compared to A-LAK cells generated by IL-2 alone. A-LAK cells generated by IL-7 or a sequential stimulation of IL-2 and IL-7, on the other hand, exhibited a more selective cytotoxicity for malignant melanoma cells compared to the non-malignant keratinocyte target cell line (HaCaT) and normal fibroblasts. A sequential replacement of LL-2 by IL-7 might help to reduce the severe side effects of IL-2. In vivo experiments are necessary to evaluate the potential value of IL-7 in adoptive immunotherapy.     

紫草素衍生物SYUNZ-7的抗肿瘤作用及其机制的初步研究

背景与目的:实验证明天然紫草素类化合物及其衍生物具有不同程度的细胞毒作用和抗肿瘤作用。本实验研究紫草素萘茜类衍生物[2或3,11-双苯硫基-6-异己萘茜]代号为SYUNZ-7的体内外抗肿瘤作用,并探讨其作用机制。方法:应用MTT法检测SYUNZ-7对多种肿瘤细胞的体外抗增殖作用,并计算IC50值;用小鼠移植肿瘤模型和人鼻咽癌裸鼠移植瘤模型进行SYUNZ-7的体内抗瘤实验;流式细胞术检测细胞凋亡和细胞周期的变化;免疫组化法检测SYUNZ-7对血管生成的影响。结果:SYUNZ-7对人肺癌细胞GLC-82、人鼻咽癌细胞CNE2、人口底癌细胞KB、人胃癌细胞MGC-803和人肝癌细胞HepG2的IC50值分别为(2.18±0.04)μg/ml、(4.17±0.09)μg/ml、(5.41±0.10)μg/ml、(6.41±0.14)μg/ml和(9.99±0.21)μg/ml。SYUNZ-7对GLC-82细胞的体外抗增殖作用最强。小鼠艾氏腹水癌EAC(实体型)抗瘤实验结果显示,在1mg/kg、2mg/kg、4mg/kg和8mg/kg的剂量下SYUNZ-7的抑瘤率分别为(40.5±0.1)%、(50.9±2.3)%、(61.3±1.8)%和(65.6±7.4)%(P<0.01)。1mg/kg、2mg/kg和4mg/kg的SYUNZ-7对CNE2细胞裸小鼠移植瘤的抑瘤率分别为24.7%、38.3%和41.2%(P<0.05)。流式细胞术检测结果显示,SYUNZ-7能浓度依赖性和时间依赖性诱导CNE2细胞的凋亡;随着作用时间的延长或处理浓度的增加,SYUNZ-7可以阻滞CNE2细胞由S期向G2/M期转化。免疫组化结果显示:SYUNZ-7能够呈浓度依赖性抑制CNE2细胞裸小鼠移植瘤的血管生成。结论:紫草素萘茜类衍生物SYUNZ-7具有较强的体内外抗瘤作用,其抗瘤机制与诱导细胞凋亡、阻滞细胞周期和抑制血管生成有关。     

Tumor-derived transforming growth factor-β1 and interleukin-6 are chemotactic for lymphokine-activated killer cells

Adherent lymphokine-activated killer (A-LAK) cells are purified IL-2 activated natural killer (NK) cells with potent anti-tumor cytotoxic activity. They have been used in the adoptive immunotherapy of metastatic canters. However, it has been shown that intravenously transferred LAK cells have a poor horning capacity to tumor sites. For the present study, the effects of tumor-derived factors on the in vitro migratory capacity of A-LAK cells was investigated. In a micropore migration assay the conditioned medium from 3LL Lewis lung carcinoma cell cultures was found to exert a strong chemotactic, but not chemokinetic effect on A-LAK cells. This effect was partially inhibited by neutralizing antibodies against the cytokines TGF-β1 and IL-6. A combination of the 2 antibodies completely suppressed the chemotactic activity of tumor-cell-conditioned medium. Purified TGF-β1 and recombinant IL-6 were chemotactic for A-LAK cells. Biological activities of both cytokines were detectable in the tumor-cell-conditioned medium. The in vivo relevance of these findings, with respect to tissue infiltration of NK cells and LAK cells in inflammation or cancer, remains to be elucidated.     

单链抗体阿霉素结合物对人肺腺癌细胞体外生长的抑制作用

目的观察抗肺腺癌单链抗体(ScFv)2A7-1-阿霉素(ADM)结合物对人肺腺癌细胞体外生长的抑制作用。方法从抗肺癌可溶性ScFv抗体分泌克隆2A7-1E清提取可溶性抗体,浓缩、亲和层析纯化,以分光光度计测定浓度。戊二醛法连接2A7-1、ADM制备结合物,分光光度计测定结合物中所含2A7-1和ADM的浓度,计算结合比例。集落形成试验观察该结合物对肺癌细胞体外生长的抑制作用。结果制备的2A7-1-ADM结合物中,ADM和2A7-1的克分子比为3：1。连接后的2A7-1-ADM免疫结合物仍可特异结合肺腺癌A2细胞,其对肺癌细胞的抑制作用是单纯使用ADM的4倍。结论2A7-1-ADM免疫结合物对肺癌细胞的生长抑制作用明显强于单独使用ADM,具有潜在的临床应用价值。