三种癌细胞对Ad-E1A的敏感程度研究

目的：研究腺病毒介导的E1A基因（Ad-E1A）体外对Hep-2人喉癌细胞、A375人黑色素瘤细胞、SMMC-7721人肝癌细胞的生长抑制作用及敏感程度。方法：RT-PCR鉴定E1A基因的转录;MTT法检测细胞的生长抑制作用;Hoechst染色法检测细胞核形态改变;流式细胞术（FCM）检测细胞周期和细胞凋亡。结果：Ad-E1A在三种癌细胞内均有效表达,在其感染72h和96h后对Hep-2、A375、SMMC-7721细胞的生长抑制率达分别为18.65%和29.95%;33.02%和45.36%;36.32%和54.11%;其中对SMMC-7721细胞的作用最强,FCM 检测发现其凋亡率为36.92%。Hoechst染色表明Ad-E1A诱导肿瘤细胞凋亡,使其呈现典型的核固缩、断裂并出现凋亡小体等核形态改变。结论：Ad-E1A对Hep-2、A375、SMMC-7721三种癌细胞均有生长抑制作用,尤以对SMMC-7721细胞作用最强、A375细胞次之, Hep-2细胞的敏感性相对较低;此外,Ad-E1A还能诱导肿瘤细胞凋亡。     

Ad-ING4诱导人前列腺癌细胞凋亡的实验研究

目的研究腺病毒介导的ING4基因(简称Ad-ING4)体外对人前列腺癌细胞PC-3的生长抑制作用。方法将携有绿色荧光蛋白(GFP)的腺病毒空载体Ad(简称Ad-GFP)及Ad-ING4分别感染PC-3细胞,RT-PCR检测ING4基因的转录;荧光显微镜观察Ad-ING4对PC-3细胞的细胞毒作用;用MTT比色法绘制细胞生长曲线,计算生长抑制率;流式细胞仪(FCM)检测Ad-ING4对细胞凋亡的影响;Hoechst 33258核荧光染色检测细胞凋亡的核形态学变化。结果 RT-PCR显示Ad-ING4能在PC-3细胞内转录;MTT结果提示Ad-ING4感染72 h和96 h后对细胞生长抑制率达39.29%和50.00%;FCM检测Ad-ING4感染细胞72 h后凋亡率为76.19%;核荧光染色结果进一步证明Ad-ING4对PC-3细胞可呈现典型的细胞凋亡核形态学改变。结论 Ad-ING4能够明显抑制PC-3细胞的生长,并诱导细胞凋亡。     

Therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

Objectivc To observe the therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy60 Co γ-rays irradiation in beagles,and to provide experimental evidences for the clinical treatment of extremely severe myeloid acute radiation sickness(ARS).Methods 16 beagles were given 4.5 Gy60 Co γ-rays total body irradiation,and then randomly assigned into irradiation control group,supportive care group and cytokines group.In addition to supportive care,recombinant human granulocyte colony-stimulating factor (rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2(rhIL-2)were administered subcutaneouly to dogs in cytokines group.Peripheral blood hemogram was examined once every two days.Bone marrow and peripheral blood were collected to proceed colony cultivation 4 d pre-irradiation and 1 and 45 d post-irradiation.Conventional histopathological sections of sternum were prepared to observe the histomorphology changes. Results After irradiation,the population of all kinds of cells in peripheral blood declined sharply.WBC nadir Was elevated(1.04×109/L,but 0.28×109/L and 0.68×109/L for the irradiation control group and the supportive care group separately),the duration of thrombocytopenia was shortened (24 days,but 33 days for the supportive care groug) and red blood cell counts were maintained in the range of normal values after cytokincs treatment in combination.The colony forming efficiency of haemopoietic stem cells(HSCs)in bone marrow and peripheral blood decreased obviously 1 d post irradiation,but recovered to the level of that before irradiation 45 d post irradiation after supportive care and cytokines treatment.Hematopoietic cells disappeared in bone marrow of animals in irradiation control group,but hematopoietic functions were recovered after cytokines were administrated.Conclusions RhG-CSF.rhIL-11 and rhIL-2 used in combination could elevate WBC nadir,accelerate the recovery of leukocytes,platelets and red blood cells and promote the proliferation,differentiation and maturity of HSPCs left in the body after 4.5 Gy γ-rays total body irradiation,eventually restore the hematopoietic function.Hence,combination of rhG-CSF,rhIL-11 and rhIL-2 could serve as better therapeutic strategy to treat extremely severe myeloid ARS.     

Studies on mechanism of treatment of granulocyte colony-stimulating factor,recombinant human interleukin-11 and recombinant human interleukin-2 on hematopoietic injuries induced by 4.5 Gy γ-rays irradiation in beagles

Objective To investigate the mechanism of treatment of granulocyte colony-stimulating factor(rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2 (rhIL-2)on hematopoietic injuries induced by 4.5 Gy60 Coγ-ray irradiation in beagles,and to provide experimental evidence for the clinical treatment of extremely severe myeloid acute radiation sickness (ARS).Methods Sixteen beagle dogs were given 4.5 Gy60 Co γ-ray total body irradiation(TBI),then randomly assigned into irradiation control group,supportive care group or cytokines+supportive care (abbreviated as cytokines)group.In addition to supportive care,rhG-CSF,rhlL-11 and rhIL-2 were administered subcutaneously to treat dogs in cytokines group.The percentage of CD34+cells,cell cycle and apoptosis of nucleated cells in peripheral blood were examined by Flow cytometry.Results After 4.5 Gy 60 Co γ-ray irradiation,the CD34+cells in peripheral blood declined obviously(61.3%and 52.1% of baseline for irradiation control and supportive care group separately).The cell proportion of nucleated cells in Go/G1 phase was increased notably(99.27% and 99.49% respectively).The rate of apoptosis(26.93% and 21.29% separately)and necrosis(3.27% and 4.14%,respectively)of nucleated cells were elevated significantly when compared with values before irradiation(P<0.05) 1 d post irradiation.When beagles were treated with cytokines and supportive care,the CD34+cells in peripheral blood were markedly increased(135.6% of baseline).The effect of G0/G1 phase blockage of nucleated cells became more serious(99.71%).The rate of apoptosis(5.66%)and necrosis(1.60%)of nucleated cells were significantly lower than that of irradiation control and supportive care groups 1 d after exposure.Conclusions Cytokines maybe mobilize CD34+cells in bone marrow to peripheral blood,indce cell cycle block at G0/G1 phase and reduce apoptosis,and eventually cure hematopoieticinjuries induced by irradiation.     

食管癌组织和外周血miRNA检测技术及其应用的研究进展

食管癌是人类常见的八大肿瘤之一,全世界每年约有30万人死于食管癌,其中70%发生在我国。食管癌起病隐匿,大部分患者就诊时已是中晚期,此时实施外科手术治疗的5年存活率仅为20%～30%。如何提高食管癌的早期诊断,尽早治疗对提高食管癌患者生存率具有重要意义。目前用于食管癌筛查的首选方法是胃镜组织活检,但是组织活检可能会导致肿瘤扩散,且胃镜检查患者痛苦,不能被无症状者接受,     

再生丝素膜对鼠胚真皮层成纤维细胞生长的影响

目的 研究再生丝素膜对鼠胚真皮层成纤维细胞生长的影响。方法 采用静置贴壁培养法在再生丝素膜上体外培养鼠胚真皮层成纤维细胞，并用活细胞计数法及MTT比色法观察再生丝素膜对细胞的形态、功能和生长的影响。结果 再生丝素膜对鼠胚真皮层成纤维细胞的贴壁能力和形态及其代谢功能未见明显改变，也未见明显毒性，并能支持细胞正常生长，呈现正常的生长繁殖曲线。结论 再生丝素膜对鼠胚真皮层成纤维细胞具有良好的细胞相容性。     

NKCF检测在疾病诊断中的应用

报告用MIT比色法观察了恶性肿瘤、SLE、哮喘等疾病患者的NKCF活性,并与经PHA刺激的淋巴细胞~3H—TdR 掺入量(cpm)和刺激指数(SI)对比。结果;(1)三类恶性肿瘤患者的NKCF 活性比正常值明显降低,而他们的淋巴细胞cpm 和SI 则与正常值无显著差异;(2)SLE 组NKCF 活性显著下降,而淋巴细胞cpm 和SI 有所上升;(3)哮喘患者的NKCF 活性无明显改变,而淋巴细胞cpm 和SI 升高。结果表明,NKCF 活性检测对恶性肿瘤和SLE 有辅助诊断价值。     

基因工程IFNα_(2a)对肿瘤细胞生长的抑制作用

采用α－干扰素抑制各种肺癌细胞生长的MTT法。结果：不同品牌的10000,1000,100IU／ml的IFNα2a对人脑胶质瘤细胞（SHG—44）、人骨肉瘤细胞（HOS－8603）、人白血病细胞（HL－60）、红白血病细胞（K562）和淋巴瘤细胞（Raji）的生长抑制率分别为20％,10％,5％左右。结论：进口和国产的不同品牌IFNα2a。对上述5种肿瘤细胞株均有较明显的生长抑制效应,且抑制率相近。     

人Epsilon干扰素在CHO细胞中的表达及生物学活性的研究

目的 为了研究新发现的人epsilon干扰素（hIFN-ε）的生物学活性，我们通过RT-PCR克隆了hIFN-ε基因，并构建pcDNA3．1／myc-his（-）A-hIFN-ε（以下简称pcDNA3-1A-hIFN-ε）的真核表达载体，在CHO细胞中表达，并研究真核细胞重组表达的rhIFN-ε的生物学活性。方法 用TNF-α刺激人宫颈癌HeLa细胞，抽提总RNA，经RT-PCR获得全长cDNA，与pcDNA3．1／myc-his（-）A真核表达载体连接，构建重组体pcDNA3．1A-hIFN-ε并在CHO细胞中进行表达，采用微量细胞病变抑制试验研究表达产物中的rhIFN-ε对多种病毒的抗病毒活性；MTT法检测其对A375、HeLa和A549细胞的生长影响，并通过在Wish、HeLa、A375细胞中诱导MxA抗病毒蛋白的产生研究hIFN-ε的抗病毒机制。结果 经PCR和限制性酶切鉴定以及DNA测序，结果表明已经成功构建了重组体pcDNA3．1A-hIFN-ε，并在CHO细胞中稳定表达了rhIFN-ε蛋白，该蛋白具有抗HSV-I、PolioV、Ad3和VSV病毒的作用，能够抑制HeLa、A375、A549细胞的生长，刺激Wish、HeLa、A375细胞产生MxA蛋白。结论 本研究成功地构建了pcDNA3．1A-hIFN-ε真核表达载体并在CHO细胞中获得稳定表达，证明了ddFN．￡蛋白具有抗病毒和抗增殖活性，其抗病毒效应的分子机理可能与诱导细胞产生MxA抗病毒蛋白有关，为今后研究rhIFN-ε的生物学功能和基因重组药物研制及其开展基因治疗奠定了基础。     

脐血LAK与CIK细胞杀伤活性研究

寻求一种可用于过继免疫疗法的高效免疫活性细胞。方法用多种细胞因子诱生ＣＩＫ细胞,用ＭＴＴ法测定ＬＡＫ和ＣＩＫ细胞杀伤活性。结果在峰值期ＣＩＫ细胞杀伤活必均显著高于ＬＡＫ细胞。