磷脂酰乙醇胺N-甲基转移酶2过表达抑制磷脂酶Cγ1磷酸化及转位

目的探讨转染磷脂酰乙醇胺N-甲基转移酶2(PEMT2)基因抑制大鼠肝癌CBRH-7919细胞增殖的机制。方法采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western blot方法,观察转染PEMT2基因对大鼠肝癌CBRH 7919细胞磷脂酶C γ 1(PLC γ 1)磷酸化及在细胞内转位的影响;同时观察转染PEMT2基因对肝细胞生长因子受体(c- Met)自身磷酸化活化的影响。结果转染PEMT2后,质膜结合的PLC γ下降,为对照组的45％。膜结合的磷酸化PLC γ 1约为对照组细胞的27％,同时c-Met磷酸化程度显著下降,约为对照组细胞的32％。结论转染PEMT2基因可抑制细胞PLC γ 1磷酸化及由胞浆向质膜转位,抑制c-Met自身磷酸化活化,从而下调CBRH-7919细胞c-Met／PLC γ 1信号转导途经。     

IL-2和B7-1基因联合修饰瘤苗预防小鼠肝癌形成的研究

目的 研究IL-2和B7-1基因联合修饰的肝癌瘤苗预防肝癌形成的作用。方法 用重组腺病毒为载体,将目的基因导入小鼠Hepal-6肝癌细胞株,MMC处理后制成肝癌瘤苗,免疫同系小鼠,观察抗亲代Hepal-A细胞攻击的免疫保护作用,并检测免疫小鼠的细胞免疫功能。共设6个实验组:IL-2和B7-1双基因修饰瘤苗组出(Hep6-IL2/B7),IL-2基因瘤苗组(Hep6-IL2),B7-1基因瘤苗组(Hen6-B7),BGFP基因瘤苗组,野生瘤苗组和培养液对照组。结果 各组瘤苗的免疫保护作用:上述 6组小鼠的中位生存期分别为 68、59、54、51、48、38 d,其中 Hep6-IL2/B7瘤苗组生存期最长,且瘤体积最小(Z值为3.20～44.10,P<0.05)。经细胞免疫功能检测和细胞毒活性实验显示以Hep6-IL2/B7瘤苗免疫后可诱导显著提高的NK、LAK和CTL活性(分别为29.0％±2.5％、65.0％±2,9％和83.1％±1.5％,与其它实验组比P<0.05)。结论 IL-2和 B7-1双基因肝癌瘤苗可诱导小鼠产生活化的针对亲代癌细胞的特异性CTL细胞,具有比单基因及常规瘤苗更强的防癌的作用,有希望成为预防肝癌复发转移的治疗手段。     

IL—6基因转导大肠癌细胞的表达

目的:IL-6基因导入大肠癌细胞,建立能有效表达IL-6的转导株HT-29IL-6.方法:采用酶切与连接技术构建重组IL-6基因逆转录病毒载体,基因转染包装细胞,C418筛选克隆,常规制备重组病毒液并感染HT-29细胞,筛选抗性细胞,Southern blot和Northem blot分析基因的整合与mRNA转录水平,MTT显色法及ELISA法检测表达产物的量与活性.结果:成功地构建了重组载体pZIPIL-6cDNA,制备了高滴度的重组病毒液(5.1×10~5cft/ml),其感染率达80%以上,建立了HT-29IL-6表达株,杂交结果证实具有目的基因的稳定整合和相应mRNA的有效转录,表达IL-6的量与活性分别为1132.5pg/ml和15OU/ml.结论:逆转录病毒载体介导的IL-6基因通过转染及筛选能稳定整合在大肠癌细胞染色体,并进行有效的转录与表达,为IL-6转基因治疗大肠癌的研究奠定了基础.     

蛋白激酶B短发夹环RNA表达载体的构建及其对血管平滑肌细胞增殖活性的影响

目的构建靶向蛋白激酶B基因的短发夹环RNA表达载体,观察其对血管平滑肌细胞增殖活性的影响。方法设计多个针对大鼠蛋白激酶B基因的短发夹环RNA序列,化学合成方法合成并经pGEM-T载体克隆后双酶切,将cDNA序列插入逆转录病毒载体pLXIN,包装后获得蛋白激酶B的逆转录表达载体,感染血管平滑肌细胞,Northern blot和Western blot法检测蛋白激酶B及其下游底物的表达变化,流式细胞仪检测细胞周期变化,MTT法检测血管平滑肌细胞增殖活性的改变。结果成功构建蛋白激酶B基因的逆转录病毒载体并包装,感染血管平滑肌细胞,证实其能显著抑制蛋白激酶B的mRNA和蛋白产物表达,下游的p70s6k表达相应减少;被感染血管平滑肌细胞的分裂、增殖过程受阻,更多细胞停滞在G0/G1期。结论成功构建蛋白激酶B基因逆转录病毒RNA干扰表达载体,感染血管平滑肌细胞能够明显抑制其分裂、分化和增殖。     

RNA干扰供体大鼠库普弗细胞B7分子表达对受体大鼠淋巴细胞激活的影响

目的:观察RNA干扰供体Lewis大鼠库普弗细胞(KC)B7分子表达对受体BN大鼠淋巴细胞增殖和生成IL-2的影响.方法:分离培养供体Lewis大鼠KC, 设计大鼠B7分子的干扰片段, 构建并鉴定含B7干扰片段的RNA干扰载体Psilencer 3.1H1-Neo-B7, 将RNA干扰载体转染供体大鼠的KC, 转染后采用RT-PCR方法检测KC上B7分子表达的变化. 将转染后的KC分为3组, 对照组(A); 空载体组(B); RNA干扰B7表达组(C). 分离培养受体BN大鼠的淋巴细胞, 将以上各组细胞分别与BN大鼠的淋巴细胞进行共培养, 采用MTT法检测各组淋巴细胞的增殖情况. 采用ELISA方法检测各组培养上清中IL-2的含量.结果: 分离培养的供体Lewis大鼠KC得率为5×107, 活率大于98%. 构建的RNA干扰载体经酶切和测序鉴定正确. RNA干扰KC后其B7的表达降低了22%(P<0.01). 将干扰B7表达的KC与BN大鼠的淋巴细胞进行共培养, 与对照组相比, 受体BN大鼠的淋巴细胞增殖降低了49%(P<0.01), 细胞培养上清中IL-2的分泌量下降了67%(P<0.01).结论:RNA干扰供体Lewis大鼠KC B7分子的表达可明显抑制受体BN大鼠淋巴细胞的增殖和IL-2的产生.     

RNA干扰Ref-1表达对肝癌顺铂敏感性的影响

目的建立表达氧化还原因子-1（Ref-1）小分子干扰RNA的重组逆转录病毒载体pmscv/siRef-1,观察人肝癌细胞株HepG2下调内源性Ref-1后对顺铂的敏感性变化。方法采用基因重组技术构建逆转录病毒载体pmscv/siRef-1。包装逆转录病毒,感染HepG2。RT-PCR及Western blot法检测感染后细胞内Ref-1 mRNA和蛋白表达,MTT法检测HepG2细胞下调Ref-1表达后对顺铂的敏感性变化。结果pmscv/siRef-1能有效被包装并感染靶细胞,感染pmscv/siRef-1病毒的HepG2细胞Ref-1蛋白与mRNA表达量明显降低,细胞对顺铂敏感性显著增加（P〈0.05）。结论成功构建以逆转录病毒为基础的抑制内源性Ref-1表达的RNA干扰载体;RNA干扰Ref-1基因可显著提高HepG2细胞对顺铂的敏感性。     

IL-17和siRNA-IL-17基因逆转录病毒载体的构建及其转染致糖尿病性T细胞中IL-17的表达

目的 构建含有Thy1.1细胞表面抗原(Thy-1.1 cell surface antigen,Thy1.1)基因的携带IL-17或siRNA-IL-17基因的逆转录病毒载体,并观察逆转录病毒对致糖尿病性BDC2.5 T细胞的转染能力.方法 利用基因工程和细胞克隆技术分别将IL-17的cDNA插入MSCV-IRES-Thy1.1(MIT)逆转录病毒载体,将Thy1.1、U6增强子(U6 promoter)基因和IL-17的siRNA cDNA插入逆转录病毒载体pMND-BANSHEE,采用磷酸钙沉淀法将重组载体转染293包装细胞;收集病毒上清,转染预先活化的NOD/BDC小鼠脾细胞,测定致糖尿病性T细胞的IL-17和siRNA-IL-17的表达.结果 流式细胞术检测显示,成功构建携带IL-17和siRNA-IL-17的逆转录病毒载体.携带IL-17或siRNA-IL-17逆转录病毒分别感染原代培养并活化的NOD/BDC脾细胞,在空白对照组、空载体组、阳性对照组、IL-17组和siRNA-IL-17组,Thy1.1~+/Thy1.2~+细胞比例分别为(0.6±0.3)%,(7.2±2.4)%,(6.8±2.6)%,(6.4±2.4)%和(4.6±1.8)%;体外实验携带IL-17基因逆转录病毒转染BDC2.5 T淋巴细胞后,IL-17的表达显著高于siRNA-IL-17转染组和未转染对照组(均P<0.01).体外培养活化实验显示,携带IL-17逆转录病毒转染非活化组和活化组T淋巴细胞,转染后IL-17的表达均显著高于siRNA-IL-17转染组和未转染对照组(均P<0.01),其中IL-17转染活化组的IL-17的表达较非活化组更高(P<0.01);在siRNA-IL-17转染的非活化组和活化组,IL-17的表达显著降低,与未转染对照组相比无显著性差异(均P>0.05).结论 逆转录病毒载体能够快速、稳定地将外源基因IL-17或siRNA-IL-17转移至BDC/NOD T细胞,可作为介导T细胞基因转移的重要工具.     

胰腺癌正反义K-ras基因片段的分离和定向克隆

目的分离及克隆胰腺癌基因组中K-ras基因片段,构建重组正反义K-ras基因逆转录病毒载体并探讨其临床意义.方法设计两对PCR引物,分别在上下游引物中引进BamH1和EcoR1位点,以胰腺癌细胞基因组DNA为模板扩增K-ras基因外显子4B及侧翼序列,并采用重组DNA技术将目的基因分别定向插入逆转录病毒载体LZRSpBMN-Z中.重组克隆通过菌液PCR和重组质粒限制性内切酶酶切鉴定.结果正反义K-ras基因外显子4B及侧翼序列成功地克隆入LZRSpBMN-Z中.结论LZRSpBMN-Z是胰腺癌反义基因治疗中新型候选载体之一.应用PCR方法获取反义K-ras基因方便可行,可用于重组反义K-ras基因逆转录病毒载体的构建.     

HSV1-tk基因逆转录病毒载体构建、病毒包装及稳定细胞株建立

目的构建携带HSV1-tk基因的逆转录病毒载体pDON-AI-HSV1-tk,包装后产生携带相应基因的逆转录病毒并稳定感染T47D乳腺癌细胞。方法用PCR方法扩增HSV1-tk基因,利用基因重组技术构建逆转录病毒载体pDON-AI-HSV1-tk,经PCR及BamHI、SalI双酶切鉴定后与gag-pol和env表达载体用脂质体法共同转染293T包装细胞,产生含有HSV1-tk基因的逆转录病毒并用其感染T47D细胞,经G418筛选获得稳定表达株,命名为tk/T47D。结果构建了携带HSV1-tk基因的逆转录病毒载体pDON-AI-HSV1-tk,并通过PCR及BamHI、SalI双酶切证实。包装产生的逆转录病毒提取病毒基因组后PCR扩增出目的基因HSV1-tk,并成功地建立稳定表达HSV1-tk基因的T47D细胞。结论成功的构建了含有HSV1-tk基因的逆转录病毒载体并包装出携带相应基因的逆转录病毒,建立稳定表达HSV1-tk基因的乳腺癌T47D细胞株。     

p16真核表达载体的构建及其对肝癌细胞的作用

研究p16基因对肝癌细胞的作用;构建pcDNA3.0/p16真核表达质粒转导到大鼠肝癌细胞CBRH-7919中,对其p16基因的表达、细胞的生长抑制及机制进行分析;转染细胞p16蛋白免疫组化阳性,MTY法结果显示,50×103/cm2细胞经培养24h～96h后,每组细胞数均有不同程度的增加,但经pcDNA3.0/p16转染的CBRH-7919细胞数比对照明显减少。与空白对照比,细胞在24h,48h,72h和96h的生长抑制率分别为29％,29％,40％和52％,流式细胞仪检测细胞周期显示经pcDNA3.0/p16转染的CBRH-7919细胞有显著的细胞凋亡现象和G0/G1期阻滞。pcD-NA3.0/p16真核表达质粒转导到大鼠肝癌细胞CBRH-7919中能够抑制细胞的增殖活性,p16基因可能通过诱导肿瘤细胞凋亡及G1期阻滞在肿瘤的基因治疗方面发挥作用。     

Prevention of hepatocellular carcinoma in mice by IL-2 and B7-1 genes co-transfected liver cancer cell vaccines

AIM: To study the immunoprotective effect of liver cancer vaccine with co-transfected IL-2 and B7-1 genes on hepatocarcinogenesis in mice.METHODS: The murine liver cancer cell line Hepal-6 was transfected with IL-2 and/or B7-1 gene via recombinant adenoviral vectors and the liver cancer vaccines were prepared. C57BL/6 mice were immunized with these vaccines and challenged with the parental Hepal-6 cells afterwards.The immunoprotection was investigated and the reactive T cell line was assayed.RESULTS: The immunoprotection of the tumor vaccine was demonstrated. The effect of IL-2 and B7-1 genes cotransfected Hepal-6 liver cancer vaccine (Hep6-IL2/B7vaccine) on the onset of tumor formation was the strongest.When attacked with wild Hepal-6 cells, the median survival period of the mice immunized with Hep6-IL2/B7 vaccine was the longest (68 days, χ2=7.70-11.69, P＜0.05) and the implanted tumor was the smallest (z =3.20-44.10, P＜0.05).The effect of single IL-2 or B7-1 gene-transfected vaccine was next to the IL2/B7 gene co-transfected group, and the mean survival periods were 59 and 54 days, respectively.The mean survival periods of wild or enhanced green fluorescence protein gene modified vaccine immunized group were 51 and 48 days, respectively. The mice in control group all died within 38 days and the implanted tumor was the largest (z=3.20-40.21, P＜0.05). The cellular immunofunction test and cytotoxicity study showed that the natural killer (NK) cell, lymphokine activated killer (LAK) cell and cytotoxic T lymphocyte (CTL) activities were significantly increased in mice immunized with the Hep6-IL2/B7 vaccine, (29.5±2.5%,65.0±2.9%, 83.1±1.5% respectively, compared with other groups, P＜0.05).CONCLUSION: The Hep6-IL2/B7 liver cancer vaccines can induce the mice to produce activated and specific CTL against the parental tumor cells, and demonstrate stronger effect on the hepatocarcinogenesis than single gene modified or the regular tumor vaccine. Therefore, the vaccines may become a novel potential therapy for recurrence and metastasis of HCC.     

Construction of retroviral vectors to induce a strong expression of human class Ⅰ interferon gene in human hepatocellular carcinoma cells in vitro

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人白细胞介素-1受体拮抗剂或白细胞介素-10逆转录病毒载体的构建与体外实验

目的 构建携带人白细胞介素-1受体拮抗剂(hIL-1Ra)或人白细胞介素-10(hIL-10)基因的重组逆转录病毒(rRv)，体外转染免滑膜成纤维样细胞，检测目的基因的表达水平与持续时间。方法 提取人外周血单个核细胞总RNA，反转录聚合酶链反应(RT-PCR)扩增目的基因；酶切、连接目的基因与逆转录病毒载体pLXSN，筛出阳性克隆，经GP2-293细胞包装，收集病毒并鉴定；rRV-hlL-1Ra、rRV-hlI-10分别体外转染兔滑膜成纤维样细胞，RT-PcR测定目的基因mRNA表达水平与时间的关系。免疫组织化学与免疫印迹测定蛋白水平的表达与持续时间。结果 成功构建了携带hIL-1Ra或hIL-10基因的重组逆转录病毒，rRv-hIL-1Ra与rRV-hIL-10均能有效转染体外培养的兔滑膜成纤维样细胞，RT-PcR测定目的基因mRNA高峰均出现于转染后第5天左右。免疫组织化学和免疫印迹均检测到hIL-lRa、hlL-10的表达。经G418筛选后的细胞中，hIL-1Ra的表达在转染后第30天内达高峰，至少持续60d：hIL-10的表达至少持续40d。结论 以重组逆转录病毒为载体可以成功地将hIL-lRa或hIL-10基因导人体外培养的兔滑膜成纤维样细胞并实现稳定表达。     

Retroviral gene transfer into primary hepatocytes: implications for genetic therapy of liver-specific functions.

The liver is an important target for potential gene therapy because of the critical role it plays in intermediary metabolism and synthesis of serum proteins. We report the use of retroviral vectors for transfer of recombinant genes into primary mouse hepatocytes. Hepatocytes were grown in a defined serum-free medium and expressed liver-specific functions for up to 14 days. Hepatocytes were transformed to Genticin (G418) resistance by infection with recombinant retroviruses carrying the Tn5 neomycin-resistance gene. The G418-resistant cells exhibited characteristic hepatocyte morphology and continued to express liver-specific gene function. A retrovirus that expresses neomycin resistance driven by a herpes simplex thymidine kinase promoter produced the most efficient transformation compared with viruses using the retroviral long terminal repeat promoter or the simian virus 40 early-region promoter. These experiments indicate that primary hepatocytes can be successfully cultured and transformed with recombinant genes using retroviral vectors. These results provide a model for future somatic gene replacement therapy in which functional genes can be introduced into hepatocytes by viral-mediated gene transfer.     

Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors containing genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liver-specific functions, including phenylalanine hydroxylase, alpha 1-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.     

Human T lymphocytes transduced by lentiviral vectors in the absence of TCR activation maintain an intact immune competence

Gene transfer into T lymphocytes is currently being tested for the treatment of lymphohematologic disorders. We previously showed that suicide gene transfer into donor lymphocytes infused to treat leukemic relapse after allogeneic hematopoietic stem cell transplantation allowed control of graft-versus-host disease. However, the T-cell receptor (TCR) activation and sustained proliferation required for retroviral vector transduction may impair the half-life and immune competence of transduced cells and reduce graft-versus-leukemia activity. Thus, we tested lentiviral vectors (LVs) and stimulation with cytokines involved in antigen-independent T-cell homeostasis, such as interleukin 7 (IL-7), IL-2, and IL-15. Late-generation LVs transduced efficiently nonproliferating T cells that had progressed from G0 to the G1 phase of the cell cycle on cytokine treatment. Importantly, IL-2 and IL-7, but not IL-15, stimulation preserved physiologic CD4/CD8 and naive-memory ratios in transduced cells with only minor induction of some activation markers. Functional analysis of immune response to cytomegalovirus (CMV) showed that, although CMV-specific T cells were preserved by all conditions of transduction, proliferation and specific killing of autologous cells presenting a CMV epitope were higher for IL-2 and IL-7 than for IL-15. Thus, LV transduction of IL-2 or IL-7 prestimulated cells overcomes the limitations of retroviral vectors and may significantly improve the efficacy of T-cell-based gene therapy.     

Efficient gene delivery to quiescent interleukin-2 (IL-2)-dependent cells by murine leukemia virus-derived vectors harboring IL-2 chimeric envelope glycoproteins.

Interleukin-2 (IL-2) is a cytokine that induces the proliferation of certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused to the amino-terminus of amphotropic murine leukemia virus (MLV) envelope glycoproteins. Retroviral vectors were pseudotyped with both the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope. The chimeric IL-2 glycoproteins were incorporated on retroviral vectors and the IL-2-displaying vector particles could bind specifically to cell surface IL-2 receptors. In addition, the IL-2-displaying vectors could infect proliferating cells through amphotropic receptors irrespective of whether the cells expressed the IL-2 receptor. IL-2-displaying vector particles could also transiently stimulate the cell cycle entry and proliferation of several IL-2-dependent cell lines. Finally, retroviral vectors displaying IL-2 could efficiently transduce G0/G1-arrested cells expressing the IL-2 receptor at a 34-fold higher efficiency compared with vectors with unmodified envelopes. This new strategy, whereby C-type retroviral vector particles display a ligand that activates the cell cycle of the target cells at the time of virus entry, may represent an alternative to lentivirus-derived retroviral vectors for the infection of quiescent cells. In addition, upon infection of an heterogeneous population of nonproliferating cells, MLV-retroviral vectors that display cytokines/growth factors will allow the transgene of interest to be integrated specifically in quiescent cells expressing the corresponding cytokine/growth factor receptor.     

携带hIL-4逆转录病毒重组体的构建及在类风湿关节炎中体外表达的研究

目的 构建携带人白介素4（hIL-4）表达序列的重组逆转录病毒pLXSN—hIL-4并检测目的基因的表达。方法 2004年6月至12月在中国医科大学基础医学院设计带有酶切位点的引物，含有目的基因的DNA进行聚合酶链反应扩增并纯化目的基因片段。pLXSN与目的基因片段hIL-4进行定向克隆连接，筛出阳性克隆并进行鉴定。扩增重组逆转录病毒载体pLXSN—hIL-4，并转染体外培养的人滑膜成纤维样细胞。Western blotting法测定目的基因蛋白表达水平。结果 成功构建了携带治疗基因的逆转录病毒重组体：rRV—hIL-4。Western blotting法检测到了hIL-4的表达。结论 成功构建了携带治疗基因的逆转录病毒重组体：rRv—hIL-4。以逆转录病毒为载体可以将hIL-4基因导入体外培养的人滑膜成纤维样细胞，转染后的细胞可以表达hIL-4蛋白。