EGFR小分子多肽配体修饰提高阳离子脂质体对肿瘤细胞的转染效率

目的:用针对表皮生长因子受体(epidermal growth factor receptor,EGFR)的小分子多肽配体D4修饰PEG化的阳离子脂质体,观察其提高质粒DNA和siRNA对肿瘤细胞转染效率的作用。方法:将D4连接在DSPE-PEG2000的末端以修饰PEG化的阳离子脂质体,检测该载体系统对高表达EGFR的人非小细胞肺癌细胞株H1299中质粒DNA转染效率的影响,Sirius照度仪检测质粒DNA转染后H1299细胞荧光素酶的表达,荧光显微镜观察转染FAM-siRNA后H1299细胞的荧光强度。结果:制备的脂质体/质粒DNA复合物随着电荷比的提高,复合物粒径逐渐减小,复合物Zeta电位逐渐升高。在质粒DNA的转染中,与无修饰的非靶向脂质体相比,D4修饰的脂质体可以显著提高H1299细胞中荧光素酶的表达(P<0.05或P<0.01);D4修饰的脂质体在各个电荷比处对H1299细胞的转染效率显著高于无修饰的非靶向脂质体(P<0.05或P<0.01)。在FAM-siRNA的转染中,荧光显微镜下可以观察到D4修饰的脂质体组有更高水平的FAM荧光强度。结论:D4修饰的阳离子脂质体提高高表达EGFR肿瘤细胞中质粒DNA和siRNA的转染效率。     

基于HPV16的新型伪病毒肿瘤疫苗对Hela细胞的杀伤作用研究

目的：构建基于人乳头状瘤病毒（humanpapillomavirus，HPV）16型的肿瘤疫苗，并检测其对Hela肿瘤细胞的杀伤活性。方法：利用昆虫杆状病毒表达系统表达病毒蛋白，在体外变性与复性过程中将病毒蛋白包装绿色荧蛋白EGFP和白喉毒素（Diphtheriatoxin，DT）A链（DT—A）的双表达质粒，形成伪病毒疫苗。透射电镜观察病毒样颗粒（virus-likeparticle，VIJP）的结构，并转染Hela肿瘤细胞，荧光显微镜和流式细胞仪检测其转染效率和对Hela肿瘤细胞的杀伤能力。结果：透射电镜观察证实病毒蛋白可自我组装成VLP，在转染Hela肿瘤细胞后，荧光显微镜和流式细胞仪成功检测到荧光的表达和对Hela肿瘤细胞的杀伤作用。结论：基于HPV16的新型伪病毒肿瘤疫苗能成功转染Hela肿瘤细胞并产生杀伤活性，为肿瘤的基因治疗提供了依据。     

转染MUC1=基因树突状细胞活化的淋巴细胞对BIU-87细胞的体外杀伤作用(英文)

目的观察转染 MUCl 基因的树突状细胞(DCs)活化的淋巴细胞对 BIU-87细胞的体外杀伤作用,确定 MUCl-DCs 作为膀胱肿瘤免疫治疗疫苗的可能性。方法应用脂质体将人 MUCl 基因转染体外培养的外周血来源的 DCs,并活化 T 细胞,以流式细胞术和 MTT 法检测其转染率和免疫活性,以不同效靶细胞的比例对 BIU-87细胞和膀胱正常上皮细胞进行体外杀伤实验,MTT 法测定杀伤率。结果转染 MUCl 基因后的 DCs 活化的 T 细胞对 BIU-87细胞和正常膀胱上皮细胞均有杀伤作用,但是,对 BIU-87的杀伤作用明显增强(P<0.05);转染后的 DCs 活化的 T 细胞对 BIU-87细胞的杀伤作用明显大于未转染 DCs 活化的 T 细胞(P<0.01)。结论转染 MUCl 基因的 DCs 活化的淋巴细胞对 BIU-87细胞有明显的杀伤作用,MUCl 基因可作为免疫治疗膀胱肿瘤的攻击靶点。     

阳离子脂质体介导小鼠白细胞介素2基因治疗头颈鳞癌的抗肿瘤作用研究

白细胞介素2(IL-2)是肿瘤兔疫治疗中最有效的细胞因子之一。运用转基因技术将IL-2基因转入肿瘤细胞并表达,可克服全身应用IL-2蛋白质制剂所带来的副作用。国内外一些研究认为合适的脂质体和核酸物质的质量比是形成复合物理化特性的决定性因素,并影响其转染效率。转染肿瘤因肿瘤细胞的类型、脂质体结构和电荷不同等因素影响而效果各异。我们利用多价阳离子脂质体LipofectAMINE携带小鼠IL-2基因的真核表达质粒转染头颈鳞癌SCCⅦ细胞株及其移植瘤,旨在探讨脂质体转染特性,寻求最佳脂质复合物(Lipoplexes)转染比例,为进一步基因治疗提供最佳转导途径。     

放射诱导HSV-TK基因肺癌靶向性表达的研究

目的:构建由Egr-1启动子驱动HSV-TK基因表达的重组质粒,通过放射诱导调控HSV-TK基因的肿瘤靶向表达,以提高肺癌基因治疗的选择性和有效性。方法:利用基因重组方法构建tgEgr-HyTK表达载体;脂质体介导转染肺癌A549、SHG44细胞系,给以不同剂量γ射线照射,观察照射前后各时相点肺癌细胞HSV-TK表达情况;以及不同浓度前药GCV作用下肺癌细胞相对存活率的变化。结果:γ射线可诱导HSV-TK基因在被转染肺癌细胞表达显著增强,呈剂量依赖性;照射后3h HSV-TK基因表达增强,于第8小时达峰值,第36小时恢复至照射前水平。经放射诱导后,tgEgr-HyTK转染肺癌细胞对GCV的敏感性明显升高(P<0.05);进一步发现,HSV-TK/GCV系统对放射线有较明显的增敏作用。放射和HSV-TK/GCV在肿瘤杀伤作用上存在明显协同效应。结论:利用Egr-1启动子调控HSV-TK基因的肿瘤靶向性表达,是一种高效、特异的肺癌基因治疗新策略。     

脂质体RNA负载慢性粒细胞白血病来源树突状细胞的性能比较

目的研究人慢性粒细胞白血病(CML)总RNA经过脂质体负载后体外转染自体慢性粒细胞白血病 树突状细胞（CML-DC）,并诱导特异性细胞毒T淋巴细胞（CTL）免疫反应。方法CML患者骨髓单个核细胞（CML-BMMNC）在rhIL-4、rhGM-CSF、rhTNF-α细胞因子联合培养诱导出CML-DC。采用Trizol法提取CML-BMMNC的总RNA;反复冻融法制备CML BMMNC抗原。于CML-DC培养第5天,加入脂质体转染的CML总RNA、CML总RNA、CML肿瘤冻融抗原及不加抗原。倒置显微镜观察DC形态学变化;流式细胞仪器检测免疫表型变化;染色体G显带技术检测其染色体核型及逆转录聚合酶链反应（RT-PCR）检测Bcr-abl融合基因的表达;用MTT法检测CTL作用。结果CML BMMNC诱导成CML-DC, CD_1α、CD₈₃表型表达较诱导前明显上调,形态学有典型的DC形态,且均存在Bcr-abl基因带和Ph₁染色体,提示CML-DC为白血病源性。总RNA经脂质体转染CML-DC、CML肿瘤冻融抗原负载CML-DC、总RNA不经脂质体转染CML-DC、未负载抗原CML-DC分别致敏的CTL及IL-2培养的T淋巴细胞在效：靶比为20∶1时的杀伤效率依次降低。结论CML BMMNC来源的 DC既具有CML白血病源性,又具有DC细胞的特性,能诱导特异性CTL杀伤作用;总RNA经脂质体转染的CML-DC诱导的CTL杀伤性对CML细胞的杀伤作用最强。     

新城疫病毒HN基因对肝癌细胞SMMC7721的细胞毒性研究

目的:探讨新城疫病毒HN基因对肝癌细胞SMNC7721的杀伤作用及其机制.方法:以脂质体介导方法在体外转染含新城疫病毒HN基因的质粒pVHN于细胞SMMC7721 24 h后,采用MTT法检测细胞活性;3,5-二羟基甲苯测定其唾液酸含量的变化;丫啶橙/溴化乙锭(AO/EB)染色,荧光显微镜观察细胞形态学改变;以及FCM检测病毒HN基因和HLA-A,B,C的表达.结果:体外转染pVHN能显著地降低细胞表面唾液酸含量(P＜0.05),有效地杀伤肿瘤细胞SMMC7721;荧光显微镜观察可见典型的细胞死亡形态学改变;实验组细胞与对照组比较HN表达差异显著,HLA-A,B,C表达上调.结论:HN基因在体外能够显著降低肿瘤细胞表面唾液酸含量,同时,使其高表达HN抗原,上调HLA-A,B,C表达,从而,可能增强了肿瘤细胞的抗原性和免疫识别,诱导了细胞SMMC7721死亡.     

转染MUC1基因树突状细胞活化的淋巴细胞对BIU-87细胞的体外杀伤作用

目的观察转染MUC1基因的树突状细胞(DCs)活化的淋巴细胞对BIU-87细胞的体外杀伤作用,确定MUC1-DCs作为膀胱肿瘤免疫治疗疫苗的可能性。方法应用脂质体将人MUC1基因转染体外培养的外周血来源的DCs,并活化T细胞,以流式细胞术和MTT法检测其转染率和免疫活性,以不同效靶细胞的比例对BIU-87细胞和膀胱正常上皮细胞进行体外杀伤实验,MTT法测定杀伤率。结果转染MUC1基因后的DCs活化的T细胞对BIU-87细胞和正常膀胱上皮细胞均有杀伤作用,但是,对BIU-87的杀伤作用明显增强(P<0.05);转染后的DCs活化的T细胞对BIU-87细胞的杀伤作用明显大于未转染DCs活化的T细胞(P<0.01)。结论转染MUC1基因的DCs活化的淋巴细胞对BIU-87细胞有明显的杀伤作用,MUC1基因可作为免疫治疗膀胱肿瘤的攻击靶点。     

体外构建靶向脂质体靶向杀伤肿瘤血管内皮模型

目的 :于体外构建靶向脂质体标杀肿瘤血管内皮的小鼠模型并进行验证。方法 :利用肿瘤细胞分泌的IFN γ刺激内皮细胞表达MHCⅡ ,以连有抗MHCⅡ抗体的免疫载药脂质体靶向杀伤内皮细胞。内皮细胞的MHCⅡ阳性率由细胞流式仪监测。免疫脂质体识别靶细胞的能力由结合实验验证。细胞毒实验采用的是MTT检测法。结果 :肿瘤细胞分泌的IFN γ可刺激内皮细胞表达MHCⅡ。内皮细胞SVEC与转染IFN γ基因的神经母细胞瘤C130 0 (Muγ)的上清培养 2d后 ,约有 2 3%的上皮细胞表达MHCⅡ。连有抗MHCⅡ抗体的脂质体可与表达MHCⅡ抗原的内皮细胞特异结合。结合率可达非特异性脂质体的 3倍。载药的抗MHCⅡ脂质体可特异杀伤MHCⅡ阳性内皮细胞。结论 :免疫靶向脂质体可于体外特异杀伤因肿瘤分泌的细胞因子的作用而呈递抗原的内皮细胞。     

pGL3-hTERT-tk/GCV对胃癌细胞的促凋亡作用

目的:探讨重组质粒pGL3hTERTtk/GCV对胃癌细胞的促调亡作用。方法：以基因工程方法构建重组质粒pGL3hTERTtk和相应的荧光报告质粒pGL3hTERTtkLuc+;脂质体LipofectamineTM2000瞬时转染胃癌细胞系SGC7901并用GCV干预,荧光显微镜观察细胞形态变化和转染效率,TUNEL标记和流式细胞术观察转染后胃癌细胞的凋亡;以上实验均以正常肝细胞L02为对照。结果：经鉴定,重组质粒pGL3hTERTtk中tk片段的长度为1 100 bp。荧光素酶标记的阳性、阴性对照及治疗报告质粒pGL3hTERTtkLuc+均能有效转染高表达端粒酶活性的胃癌细胞SGC7901,转染效率为(8.2±114)%。重组质粒转染胃癌细胞后与GCV共育4 d,细胞的凋亡率为(60.0±1.56)%;被pGL3hTERTtk转染的肿瘤细胞细胞周期发生了变化,处于细胞周期早期的细胞大量凋亡,早期凋亡率为（47.1±1.35）%。〖HT5W〗结论：〖HT5"SS〗pGL3hTERTtk/GCV对胃癌细胞有强烈的杀伤作用,但不影响正常细胞的生长,有潜在临床应用前景。     

利用GFP逆转录病毒标记肿瘤与血管内皮细胞

目的 制备携带GFP基因的逆转录病毒,以简便、快速标记活细胞。方法 将编码GFP的cDNA片段插入逆转录病毒载体pLNCX构建重组逆转录病毒载体pLNCX-GFP,借助脂质体转染单嗜性及双嗜性逆转录病毒包装细胞,G418筛选抗性克隆,然后利用荧光显微镜选出GFP表达最高的克隆。取含有毒颗粒的上清液感染NIH3T3及多种肿瘤或血管内皮细胞。结果 重组逆转录病毒载体pLNCX-GFP转染包装细胞后,包装细胞可表达GFP并产生GFP逆转录病毒。尽管GFP逆转录病毒对动物及人的肿瘤细胞或血管内皮细胞的感染能力各不相同,但由于逆转录病毒能整合进入宿主细胞基因组DNA内,经短期G418筛选后容易获得持续、稳定、高水平表达GFP的阳性细胞。表达GFP的肿瘤细胞仍可在同系动物体内生长并持续表达GFP。携带GFP基因的逆转录病毒能简便、快速介导稳定的基因转移,遗传标记多种细胞,比表达质粒等更优越。     

Interleukin 4 potentiates the antiproliferative effects of tumor necrosis factor on various tumor cell lines.

In response to a given stimulus, usually a number of cytokines are secreted simultaneously by the immune system. Whether these cytokines are meant to function as a single agent or in combination with others is not understood. Tumor necrosis factor (TNF) has been shown to exhibit antiproliferative effects against a wide variety of tumor cell lines in vitro. In the present report, we investigated the effects of a T-cell-derived cytokine, interleukin 4 (IL-4), on the antiproliferative effects of TNF against different tumor cell lines. The growth characteristics of human breast cancer cells (MDA-MB-330) were minimally affected when the cells were exposed to either TNF or IL-4 alone. However, together these 2 cytokines inhibited cell growth in a dose-dependent manner. The enhancement of the cytotoxic effects of TNF by IL-4 were not just limited to breast tumor cells, but were also observed with human epidermoid carcinoma cells (A-431) and human histiocytic lymphoma cells (U-937). The enhancement of the cytotoxic effect of TNF by IL-4 against various tumor cell lines was found comparable with that by gamma-interferon (IFN-gamma). Interestingly, for certain tumor cell types, IL-4 alone was found to enhance cell proliferation. IL-4 had no effect on the growth-stimulatory activity of TNF on normal human foreskin fibroblasts. Pre-exposure of U-937 cells to IFN-gamma led to a greater than 2-fold induction in TNF receptors, but no modulation of TNF receptors by IL-4 was observed. Moreover, the presence of IFN-gamma was found to further potentiate the antiproliferative effects of TNF and IL-4. These results clearly suggest that IL-4 potentiates the antiproliferative responses of TNF by a mechanism different from that of IFN-gamma. Although it is well known that IL-4 can modulate the production of TNF from macrophages, this is the first report to suggest that IL-4 can also modulate TNF-dependent antiproliferative responses.     

Human tumor cell strains both unable to repair O6-methylguanine and hypersensitive to killing by human {alpha} and {beta} interferons

Human brain tumor cell strains were previously found by othersto be sensitive to growth inhibition by human inter-feron-ß(HuIFN-ß). We noticed that the sensitive strains weresome that we had found deficient in the repair of O⁶-methyl-guanine(O⁶MeG), a characteristic of 20% of the human tumor cell strainswe have studied. We confirmed this sensitivity to HuIFN-ß,and have further shown that human brain tumor cells which repairO⁶MeG are resistant to the growth inhibitory effects of HuIFN-ß.In addition, treatment with HuIFN- or HuIFN-ß resultedin more killing (reproductive inactivation) of six human tumorcell strains deficient in repairing O⁶-methylguanine in DNAthan did such treatment of six strains of cells proficient insuch repair. Further, we found two human lines, altered to becomeO⁶MeG repair deficient after establishment of the primary tumorcell culture, that were resistant to interferon. IFN treatmentproduced no DNA damage detectable by either chemical or biologicalassays. It is suggested that the genes responsible for resistanceto IFN treatment and to agents that produce O⁶MeG are oftencoordinately shut down.     

Antitumor efficacy of a human interleukin-12 expression plasmid demonstrated in a human peripheral blood leukocyte/human lung tumor xenograft SCID mouse model

Genes encoding the p35 and p40 subunits of human interleukin-12 (IL-12) and the bacterial aminoglycoside phosphotransferase were cloned into a mammalian expression plasmid. The resultant plasmid, pCMVIL-12neo, was used to transfect human lung tumor cell lines in vitro. Stably transfected subclones were generated and found to secrete human IL-12 for at least 10 days following a lethal dose of gamma-radiation. The ability of the IL-12--producing tumor cells to promote an antitumor response in vivo was evaluated in SCID mice co-engrafted subcutaneously with human peripheral blood lymphocytes (PBLs) and viable human lung tumor cells (SCID-Winn assay). Using this model system, it was established that IL-12 released locally into tumors by irradiated IL-12--transfected cells activated the human PBL and promoted their ability to suppress tumor development in a dose-dependent fashion. PBL subset depletion studies revealed that the antitumor effect promoted by the IL-12--modified cells was dependent on the presence of human CD8(+) T cells and, to a lesser extent, human CD56(+) natural killer cells within the xenograft. We conclude that (a) irradiated human lung tumor cells genetically modified with pCMVIL-12neo secrete bioactive human IL-12 at concentrations sufficient to promote a human lymphocyte-mediated antitumor response in the microenvironment of the xenograft, and (b) that the SCID-Winn assay provides a useful model for the preclinical evaluation of cytokine-based human immunotherapy protocols.     

The presence of p53 transformation-related protein in Ab-MuLV transformed cells is required for their development into lethal tumors in mice

p53, a cellular-encoded protein, is synthesized at elevated levels in a wide range of tumor cells. Ab-MuLV-transformed cells expressing both the viral-encoded p120 oncogene and the cellular-encoded p53 display a lethal tumor phenotype in syngeneic mice. L12 is an exceptional Ab-MuLV-transformed cell line that expresses the p120 oncogene and lacks the p53 cellular protein. Injection of L12 cells into syngeneic mice is followed by the development of local tumors that are subsequently rejected. Prolonged treatment of L12 cells with TPA, a tumor cell promoter, gave rise to L12T cells that synthesize the p53 protein and exhibit a lethal tumor phenotype. Comparison of one-dimensional proteolytic partial peptide map of p53 obtained from L12T to that obtained from other Ab-MuLV-transformed cell lines confirmed their identity. These results suggest a correlation between the cellular expression of p53 in Ab-MuLV-transformed cells and their capacity to develop into lethal tumors in syngeneic mice.     

Inhibition of potentially lethal radiation damage repair in normal and neoplastic human cells by 3-aminobenzamide: an inhibitor of poly(ADP-ribosylation)

The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, on potentially lethal damage repair (PLDR) was investigated in normal human fibroblasts and four human tumor cell lines from tumors with varying degrees of radiocurability. The tumor lines selected were: Ewing's sarcoma, a bone tumor considered radiocurable and, human lung adenocarcinoma, osteosarcoma, and melanoma, three tumors considered nonradiocurable. PLDR was measured by comparing cell survival when cells were irradiated in a density-inhibited state and replated at appropriate cell numbers at specified times following irradiation to cell survival when cells were replated immediately following irradiation. 3AB was added to cultures 2 hr prior to irradiation and removed at the time of replating. Different test radiation doses were used for the various cell lines to obtain equivalent levels of cell survival. In the absence of inhibitor, PLDR was similar in all cell lines tested. In the presence of 8 mM 3AB, differential inhibition of PLDR was observed. PLDR was almost completely inhibited in Ewing's sarcoma cells and partially inhibited in normal fibroblast cells and osteosarcoma cells. No inhibition of PLDR was observed in the lung adenocarcinoma or melanoma cells. Except for the osteosarcoma cells, inhibition of PLDR by 3AB correlated well with radiocurability.     

Repair of fractionated radiation in plateau phase cultures of human tumor cells and human multicellular tumor spheroids

We have measured potentially lethal damage repair (PLDR) after fractionated radiation, delivered at 24-h intervals in density-inhibited plateau phase cultures of four human tumor cell lines derived from tumors of differing radiocurability (two melanoma and two breast). The repair of potentially lethal damage conferred significant radioresistance on the human melanoma cells but not on the breast carcinoma cells. We examined the effects of fractionated radiation on human tumor cells adapted to become multicellular tumor spheroids (MTS). MTS derived from a human neuroblastoma were "cured" by a total fractionated radiation dose about 50% of that required for MTS derived from a human melanoma.     

肝癌患者A-LAK细胞与苯乙酸协同抗瘤的观察

目的：构建重组人FN多肽的真核表达载体,研究体内表达对免疫细胞的趋化作用及抑制肿瘤的生长的作用。方法：采用重组DNA技术构建表达质粒;体内外进行基因转染;用Western blot方法鉴定表达产物;肌肉组织切片与染色观察体内基因转染后的趋化作用;小鼠实体瘤模型研究基因转染抑制肿瘤生长的作用。结果：将人FN cDNA5'端非编码区及信号肽编码区、CH50多 编码区cDNA、人FN cDNA的3'端非编码区重组连接并插入pcDNA3.1质粒,构建出pCH510。以pCH510转染小鼠NIH3T3细胞,可以表在产生CH50多肽。肌肉内注射转染pCH510可对免疫细胞产生趋化作用,抑制实体肿瘤的生长。结论：质粒pCH510可在细胞中及小鼠体内表达;体内表达可对免疫细胞产生趋化作用,并可抑制实体瘤生长。     

Thrombin induces apoptosis in human tumor cells

Thrombin is a serine protease that is produced during the coagulation process and plays an essential role for hemostasis, thrombosis and wound healing. It is a potent activator of platelets, induces proliferation of a wide variety of normal and malignant human cells, and enhances their invasiveness and metastatic potential. We studied the effect of thrombin on the proliferation of a wide variety of human tumor cells and report here that, at low concentrations, thrombin induces proliferation of these cells. However, at higher concentrations, thrombin inhibited their proliferation. We show that this inhibition of cell proliferation was due to apoptosis of the tumor cells. The thrombin-mediated apoptosis was inhibited significantly by its specific inhibitor, hirudin. Furthermore, no consistent pattern of induction and/or modulation of p53, p21 and bcl-2 was observed in the thrombin-mediated apoptosis. To our knowledge, this is the first report to describe the pro-apoptotic effects of thrombin on human tumor cells and may have implications for chemotherapy in cancer patients and for the pathogenesis of AIDS as well.     

Growth hormone releasing hormone plasmid supplementation, a potential treatment for cancer cachexia, does not increase tumor growth in nude mice

Growth hormone releasing hormone (GHRH) is known to have multiple anabolic effects and immune-stimulatory effects. Previous studies suggest that treatment with anabolic hormones also has the potential to mitigate the deleterious effects of cancer cachexia in animals. We studied the effects of plasmid-mediated GHRH supplementation on tumor growth and the role of antitumor immune cells with two different human tumor cell lines, NCI-H358 human bronchioalveolar carcinoma and MDA-MB-468 human breast adenocarcinoma, subcutaneously implanted in nude mice. GHRH supplementation by delivery of human GHRH from a muscle-specific GHRH expression plasmid did not increase tumor progression in tumor-bearing nude mice. Male animals implanted with the NCI-H358 tumor cell line and treated with the GHRH-expressing plasmid exhibited a 40% decrease in the size of the tumors (P<.02), a 48% increase in white blood cells (P<.025) and a 300% increase in monocyte count (P<.0001), as well as an increase in the frequency of activated CD3+ and CD4+ cells in the tumors, compared to tumors of control animals. No adverse effects were observed in animals that received the GHRH-plasmid treatment. The present study shows that physiological stimulation of the GHRH-GH-IGF-I axis in mice with cancer does not promote tumor growth and may provide a viable treatment for cancer cachexia in humans.