从质粒pMT-E1a和pIRES-neo中获得目的基因E1a和PolyA并连接入质粒pKS-hTERTp,用Xho Ⅰ和Spe Ⅰ双酶切后连接入线性化的pAd-Apoptin,获得穿梭载体pAd-apoptin-PolyA-hTERTp-E1a.穿梭载体pAd-ApoptinPolyA-hTERTp-E1a和腺病毒骨架质粒(pAd5)共转染HEK293细胞,获得重组腺病毒Ad-Apoptin-hTERTp-E1a,采用RT-PCR和Western blot对重组腺病毒进行鉴定,MTS法检测病毒对A549细胞的抑制作用;绘制重组腺病毒生长曲线,监测重组腺病毒在细胞内的复制能力.结果显示:构建出的重组腺病毒中包含有了目的基因Apoptin和E1a,并且目的基因在细胞中正确的表达;MTS结果显示重组腺病毒对A549具有抑制作用,且抑制作用具有一定的时效及剂效关系;重组腺病毒具有在A549细胞中正常复制的能力.结果表明:成功的构建出了具有特异性杀伤和特异性复制能力的双重特异性重组腺病毒Ad-Apoptin-hTERTp-E1a.

本研究构建1株能够稳定表达荧光素酶的人肺癌细胞,并进行生物学特性鉴定及体内成瘤情况验证.将携带有萤火虫荧光素酶基因的pGL4.50质粒转入A549细胞中,利用G418一直加压筛选出能够表达荧光素酶的阳性细胞克隆,并对阳性细胞克隆进行稳定性和体外活体成像检测.测定A549-1uc细胞和A549细胞的生长曲线、迁移、侵袭及细胞周期,确定转染前后细胞生物学特性变化.为检测A549-1uc细胞在体内成瘤和发光情况,建立裸鼠皮下荷瘤模型并应用小动物活体成像系统观察.结果表明,通过G418一直加压筛选和荧光素酶活性检测,筛选出2株荧光素酶活性高的克隆Clone20和Clone28,并连续传至40代,每5代检测1次荧光素酶活性,最终保留荧光素酶活性和稳定性最高的Clone28,Clone28通过体外活体成像检测显示生物发光值与细胞数目呈正相关的线性关系(R2=0.994 8).A549-1uc细胞和A549细胞具有相似的生长特性、迁移和侵袭能力以及细胞周期.成功建立了裸鼠皮下荷瘤模型,其发光强度与肿瘤体积呈正相关的线性关系(R2=0.971 5).本研究稳定表达荧光素酶的人肺癌细胞的构建并成功建立裸鼠皮下荷瘤模型,可通过活体生物成像系统动态监测肿瘤的变化.

The effect of the combination of a recombinant adenovirus (ATV) expressing a specific apoptin protein and cisplatin on human lung cancer cells (A549 cells) was determined. The inhibitory effects of ATV and cisplatin, ATV alone, or cisplatin alone on the migration and invasion of A549 cells were evaluated in vitro using cell proliferation, wound healing, Transwell migration and Matrigel invasion assays. The tumor inhibition effect on A549 cells in vivo was assessed by observing the tumor growth and survival rate of nude mice with subcutaneous tumor xenografts grown from implanted A549 cells after treatment with ATV, cisplatin, or ATV combined with cisplatin. The proliferation (P<0.01), migration (P<0.01), and invasion (P<0.01) on A549 cells was suppressed significantly by ATV, cisplatin, and ATV and cisplatin, in a dose- and time-dependent manner. The inhibition of tumor growth in transplanted nude mice in the ATV combined with cisplatin group was significantly higher than that displayed in the other groups, and the survival rate of the combined treatment group was significantly higher than that of the group treated with cisplatin alone. The results indicated that the combined application of ATV and cisplatin could reduce toxicity and showed a synergistic effect in reducing tumor growth and increasing survival. Thus, there is a potential research value in treating tumors using the combination of ATV and cisplatin, which provides a foundation for future preclinical studies on this antitumor treatment.

Objective:To investigate the effect of apoptin-loaded oncolytic adenovirus ATV infection on the autophagy and apoptosis of human cervical carcinoma HeLa cells.Methods:Apoptin-loaded oncolytic adenovirus ATV and the control virus Ad-MOCK (both were constructed previously) were transfected into HeLa cells.The effect of ATV infection on proliferation of HeLa cells was measured through WST-1 assay;The effect of ATV infection on apoptosis and cell cycle of HeLa cells was detected by flow cytometry;Western blotting was used to detect the effect of ATV infection on the expression of autophagy-related proteins (LC3,P62 and mTOR) in HeLa cells;MDC staining was used to detect the effect of ATV infection on autophagy of HeLa cells.Results:ATV infection inhibited HeLa cell proliferation in a time-dependent manner;and the inhibitory rate reached 59.26％ with ATV 100MOI at 72 h,which was significantly higher than that of the control group (P＜0.01);The apoptotic rate of HeLa cells in ATV group was significantly higher than that in control group[(38.995±4.009)％ vs (14.680±1.174)％,P＜0.01] 48 h after infection.With the infection of ATV,flow cytometry analysis showed that the cell cycle of HeLa was blocked at S phase,which was most significant at 48 h and significantly higher than that in control group [(58.490±2.447)％ vs (43.235±4.419)％,P＜0.05].Western blotting revealed that after ATV infection,LC3 expression was gradually increase at 6,12 h,with a highest level at 12 h(0.368±0.010,P＜0.01)and a lowest level at 24 h (0.106 ± 0.023,P＜0.01),P62 protein showed the highest expression level at 24 h (6.004 ± 1.423,P＜0.01),while mTOR gradually reduced (0.042±0.010,48 h,P＜0.01)with the extension of infection time;under fluorescent microscope,monodansylcadaverin (MDC) positive staining of autophagosomes was observed in the peripheral region of the nucleus of HeLa cells;the number of autophagosomes of ATV group was significantly increased as the ATV infection time prolonged;compared with control group,the number of autophagosomes of ATV group significantly increased at 6 h and 12 h [(28.000±2.828) vs (8.500±2.121),(37.000±4.243) vs (14.000±1.414),P＜0.01],but relatively reduced at 24 h [(12.000±2.828) vs (17.000± 1.414),P＜O.0 1].Conclusion:Apoptin-loaded oncolytic adenovirus ATV can promote the autophagy and apoptosis of human cervical cancer HeLa cells and then specifically kill tumor cells.

Objective To examine the synergistic effect of a dual cancer-specific oncolytic adenovirus (ATV) and cisplatinum on the A549 human lung cancer cell line.Methods A549 cells were infected with ATV,and annexin V was detected.Inhibition of tumor cells by ATV was measured.Cell proliferation in vitro was assessed by infecting A549 cells with the oncolytic adenovirus ATV and treating those cells with cisplatinum,by infecting those cells with the oncolytic adenovirus ATV alone,or by treating those cells with cisplatinum alone.An MTS assay was performed.A wound scratch assay,Transwell migration assay,and Transwell invasion assay were used to detect the migration and invasion of A549 cells that were transfected with constructed adenoviral vectors.Nude mice bearing subcutaneously implanted tumors formed by A549 cells were treated with ATV alone or in combination with cisplatin.Tumor growth was monitored,and the inhibition of tumors by ATV combined with cisplatin was determined in vitro and in vivo.Results The dual cancer-specific oncolytic adenovirus ATV killed A549 cells by inducing apoptosis,and this action was dose-and time-dependent.When A549 cells were treated with ATV combined with cisplatin,they had markedly diminished capacity for migration and invasion compared to cells that were treated with ATV or cisplatin alone.The use of ATV combined with cisplatin inhibited the growth of subcutaneously implanted tumors.Conclusion ATV can induce apoptosis of A549 cells.The combination of ATV and cisplatinum has a synergistic attenuating effect and can enhance inhibition of A549 cell growth and it can inhibit migration and invasion by those cells.

构建含有埃博拉出血热病毒(Ebola hemorrhagic fever virus,EHFV)小片段分泌蛋白S基因、马尔堡出血热病毒(Marburg hemorrhagic fever virus,MHFV)GP基因、辛诺柏病毒(Sin Nombre virus,SNV)G2糖蛋白基因及非洲欧尼恩病毒(Africa O'nyong nyong virus,O’NNV)E2蛋白基因的重组痘苗病毒.设计含EGFP筛选标记和4种外源基因的穿梭质粒,并利用同源重组技术、荧光筛选技术和Cre/LoxP敲除系统,最终获得四联重组痘苗病毒.使用PCR方法鉴定重组痘苗病毒及其遗传稳定性;用透射电镜观察病毒形态.结果显示:所构建的多联重组痘苗病毒rVTT-J-4+具有良好的遗传稳定性,并且具备痘苗病毒的完整形态.结果表明:成功构建了含有4种外源基因的重组痘苗病毒rVTT-J-4,为后续多联疫苗的研究奠定了基础.

为建立Cox.A16型手足口病乳鼠动物模型并进行免疫、致病特性研究.将临床分离的Cox.A16病毒株经蚀斑纯化,乳鼠驯化,最终获得1株能致死11日龄乳鼠的Cox.A16毒株,命名为TS10/08 (GenBank Accession NO.JX068829,Cox.A16-TS).Cox.A16-TS感染11日龄C57BL/6J乳鼠后,观测临床疾病得分、体质量变化、死亡率并测定病毒载量、免疫分子、组织病理损伤等病毒、免疫、病理指标.结果表明,Cox.A16-TS毒株感染11日龄C57BL/6J乳鼠,其病毒毒力为50 LD50/mL,感染后不同时期肌肉病毒载量均高于其他组织中,至4d达到高峰,后不断下降.至感染后6d发病达高峰时做病理检测,相对于脑组织,肌肉中有更严重的淋巴细胞浸润,引起更严重的炎性分子升高.血清中MCP-1,MIP-1alpha,MIP-1beta和CSF3动态变化,并在不同时间形成峰值.本试验初步建立了Cox.A16型手足口病乳鼠动物模型,为药物筛选、疫苗研发和免疫机理研究奠定了基础.

Objective To obtain capsid p27 of SIV for detecting.Methods The p27 gene of SIV was synthesized and inserted into pMAL-p5x vector to construct pMAL-p5x-p27 recombinant plasmid.And then the recombinant plasmid was transformed into E.coli BL21 to express protein and the express product was purified by Amylose Resin maltose-binding protein affinity column.Results SDS-PAGE analysis indicated that pMAL-p5x-p27 can be expressed in E.coli BL21 and the protein molecular weight was about 70×103;The final p27 protein purification could reach 90%.Conclusion p27 of SIV was successfully expressed in E.coli,which lays a foudation for developing the methods of detecting SIV antibody.

Objective:To explore the influence of ozagrel sodium on NO and eNOS in myocardial tissue of myocardial ischemia rat.Methods:The electrocardiogram normal and health Wistar rats were randomly divided into control group(0.15 mL saline),model group(0.15 mL saline),low dose group(8 g/kg ozagrel sodium),middle dose group(12 g/kg ozagrel sodium),high dose group(16 g/kg ozagrel sodium) and positive control group(81.0 mg/kg nitroglycerin).They had continuous intragastric administration for 7 day.from the third day,in addition to the normal control group,the other groups had isoproterenol subcutaneous injection at 5 mg/kg·d 1 hour after administration for 5 days continuously,with interval time as 24 h.The normal control group received normal saline by subcutaneous injection.NO,eNOS of myocardial tissue were determined 2 hour after the injection of isoprenaline.Results:Compared with the control group,eNOS activity and total NOS activity were significantly increased,NO concentration was decreased significantly in model group(P0.05);Compared with model group,eNOS activity and total NOS activity were decreased,and NO concentration was increased in low-dosage group(P0.05).eNOS activity and total NOS activity were significantly decreased,and NO concentration was significantly increased in middle dose group,high dose group,and positive control group(P0.05).Decrease in NOS activity and NOS activity was more significant in middle dose group and high dose group,and increase in NO concentration was not significant.Conclusions:Ozagrel sodium can effectively increase NO concentration in myocardial tissue,inhibit activity coefficient of eNOS and NOS,scavenge free radicals,and reduce myocardial injury.

This study was aimed to prepare the polyclonal antibody against the soluble proliferation-inducing ligand (sAPRIL) antigen and to investigate its effects in suppressing sAPRIL mediated lymphocyte proliferation. Mutated recombinant sAPRIL protein, which lacks biological activity but maintains immunogenicity, was used as antigen to immunize humanized SCID mice. Sera were obtained at 6 weeks after immunization. Indirect ELISA and Western blot were used to detect the antibody titer and specificity. The inhibition of polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL was assessed by the MTT assay. The results showed that the mutant of sAPRIL could induce the production of polyclonal antibodies against human sAPRIL. Western blot and indirect ELISA analyses indicated that the anti-serum had higher specificity with a titer of 1:640. Functional analysis revealed that these polyclonal antibodies significantly inhibited the proliferation of Raji and Jurkat cell stimulated by sAPRIL (p < 0.05). It is concluded the polyclonal antibody against human sAPRIL is successfully prepared, which can inhibit the proliferation of Raji and Jurkat cells stimulated by sAPRIL in vitro.

Objective To develop a proper animal models representing human diabetes,the efficiency and doses of alloxan in male and female animals was investigated.Methods Alloxan were administered to beagle dogs and mice with different dosage.Blood glucose was tested after treatment at the 3rd,7th,14th,and 21th day,respectively.Results The different effects of alloxan on the blood glucose concentration was showed in female and male animals.After identical doses of alloxan injected,the blood glucose concentration in female animals increased quickly and its peak was higher than that in male animals.About 70% female dogs were successfully induced to become diabetic models with 40 mg/kg alloxan,corresponding only 40% for male dogs(P0.01).The most optimum dose of alloxan for male dogs was 50 mg/kg,but 3 female dogs(30%) injected with this doses were dead because of higher blood glucose concentration.Similar phenomenons were found in mice and the most optimum dose of alloxan was 200 mg/kg and 250 mg/kg for female and male respectively.Conclusion Sensitivity of alloxan to female is higher than that to male.For male animals,the dose of alloxan used to duplicate diabetic animal models would be higher about 20% than that for female animals.

目的 检测狂犬病毒NP蛋白的免疫原性.方法 利用RT-PCR扩增狂犬病毒核蛋白(NP)基因,测序后将其克隆到PVAX1真核表达载体上;将PVAX1-NP转染Vero细胞,进行SDS-PAGE及Western-blot分析;以重组质粒PVAX1-NP对小鼠进行免疫试验.结果 经酶切分析、鉴定和测序验证,得到重组表达质粒PVAX1-NP,NP蛋白在Vero细胞中获得了表达,且表达产物具有免疫学活性;免疫小鼠抗体水平显著升高.结论 狂犬病毒NP蛋白具有免疫原性.