Ectopic Over-expression of Oncogene Pim-2 Induce Malignant Transformation of Nontumorous Human Liver Cell Line L02

In order to prove that ectopic over-expression of Pim-2 could induce malignant transformation of human liver cell line L02, three groups of cells were set up including human liver cell line L02 (L02), L02 cells transfected with Pim-2 gene (L02/Pim-2) and L02 cells transfected with empty-vector (L02/Vector). Pim-2 expression levels were detected. The morphology, proliferation level, apoptosis rate and migration ability of the cells were detected respectively. Then the cells were subcutaneously inoculated into athymic mice and the microstructures of the neoplasm were observed. Compared with the controls, Pim-2 expression levels were significantly higher in L02/Pim-2 cells (P<0.05), and their morphology had obvious malignant changes. They also showed a significantly increased proliferation rate (P<0.05) and migration capacity (P<0.05), as well as a significantly decreased apoptosis rate (P<0.05). Only the athymic mice inoculated with L02/Pim-2 cells could generate neoplasm, and the morphology of the neoplasm coincided with that of the hepatoma. The results manifest that ectopic Pim-2 gene could be stably expressed in L02/Pim-2 cells. Both the morphological and biological changes of L02/Pim-2 cells demonstrate the trend of malignant transformation. L02/Pim-2 cells could generate hepatoma in athymic mice. In conclusion, Pim-2 could induce malignant transformation of human liver cell line L02.     

肾综合征出血热病毒感染正常人肝脏组织细胞培养研究

将４个型的９株肾综合征出血热病毒分别感染体外培养的正常人胚肝单层细胞，结果９株均能感染肝细胞，而且繁殖滴度较高，分别在６－１１ｄ达到高峰，其中以从肝分离的Ｌ２０株滴度为高而且维持时间较长。说明了肾综合征出血热病毒对肝脏有直接作用的可能。     

Hepatitis B Virus Vector Carries a Foreign Gene into Liver Cells In Vitro

Hepatitis B viruses (HBV) specifically target the liver, where they efficiently infect quiescent hepatocytes. Thus, HBV virus has potential to be used as vectors for liver-directed gene transfer. We constructed a new HBV-based vector system. It is composed of transfer vector for transferring a foreign gene, green fluorescence protein (GFP) gene, and a helper vector. When the transfer vector and the helper vector were cotransfected into HepG2 cells, the recombinant HBV (rHBV) particles were generated by trans-complementation between two vectors. The rHBV particles carrying the foreign gene were identified by the Southern blot assay. To test gene delivery and the transduction of the rHBV, we infected primary human hepatocytes and immortalized, HepG2 cells with rHBV in vitro. The results using fluorescence microscopy confirmed that the inserted GFP gene was successfully transferred and expressed both in primary human hepatocytes and HepG2 cells.     

甲基丙烯酸环氧丙酯致人胚肺成纤维细胞恶性转化的研究

本实验对经典的细胞转化实验进行了改进，应用甲基丙烯酸环氧丙酯（简称ＧＭＡ）多次作用，建立了人胚肺成纤维细胞转化系统。观察了人胚肺成纤维细胞恶性转化过程中细胞形态和原癌基因ｃ－ｍｙｃ的改变。结果表明：在ＧＭＡ的多次作用下，细胞的恶性程度逐渐增高，形态上由于正常到开始排列紊乱，最终形成大量细胞致密堆集的转化灶。经多次染毒后形成的转化细胞在含３％小牛血清的培养基中生长旺盛，其原癌基因ｃ－ｍｙｃ的ＭＲＮＡ     

慢性乙型肝炎患者外周血单核细胞与血清HBV DNA定量对比研究

目的　探讨慢性乙肝患者外周血单个核细胞 (PBMC)内HBV -DNA的出现与血清HBV -DNA浓度之间关系 .方法　应用荧光定量PCR技术检测 5 7例慢性乙肝患者血清和PBMC中HBV -DNA含量、对血清中不同的病毒浓度进行分组比较分析 .结果　① 5 7例PBMC内HBV DNA总检测出率为 4 2 .1% (2 4 / 5 7) ,两者检测结果一致占 88.6 % .②根据血清内HBV -DNA浓度分成三组 ,三组PBMC内HBV -DNA浓度及阳性率比较p <0 .0 1,存在显著差异 ;③血清HBV -DNA阴性而PBMCHBV -DNA阳性只有 1例 (1.7% ) ,其浓度为 1.5 0× 10 8/L .结论　①血清HBV -DNA浓度与PBMC内HBV -DNA的出现及HBV -DNA浓度存在明显相关性 .②临床上检测PMBC中HBV -DNA是对血清HBV -DNA的一个重要补充 ;③PBMC内HBV -DNA检测对观察病毒在非血清内的状态、间接反映肝细胞病毒复制情况以及进一步指导抗病毒治疗有一定意义     

人乳头瘤病毒16型E6E7 ORF转化作用的研究

构建了含人乳头瘤病毒１６型（ＨＰＶ１６）－Ｅ６Ｅ７ＯＲＦｓ（ｎｔ８３－８５５）片段和ＨＰＶ１６长控制区（ＬＣＲ）加Ｅ６Ｅ７ＯＲＦｓ片段（ｎｔ７００７－７９０４／０－８７９）的逆转录病毒载体ｐＨ２１和ｐＨ１８质粒，利用Ｌｉｐｏｆｅｃｔｉｎ分别将它们导入病毒包装细胞ｐＡ３１７中，经过筛选获得Ｇ４１８抗性的病毒包装细胞，产生的重组病毒Ｈ２１和Ｈ１８感染的ＮＩＨ３Ｔ３细胞都具有恶性细胞的形态学特征，并能在裸鼠体内形成肿瘤。Ｓｏｕｔｈｅｒｎ杂交结果证明，上述两基因片段都整合到细胞基因组中。本实验结果说明ＨＰＶ１６－Ｅ６Ｅ７基因片段是ＨＰＶ１６转化ＮＩＨ３Ｔ３细胞的关键早期区，其自身ＬＣＲ区在该转化过程中没有显示出重要作用。     

Human Fetal Liver Cells Induce Colonies in Spleen of Lethally Irradiated Mice

Murine hematopoietic tissues contain cells which, upon injection into lethally irradiated mice, produce nodules on the surface of their spleen (colony-forming unit—spleen; CFU-S). The exact hierarchical level of the hematopoietic progenitors which give rise to CFU-S is not fully established; however, cell populations highly enriched for repopulating stem cells appear to contain a high percentage of CFU-S. The experiments reported here involved the injection of human fetal liver cells into mice, under conditions similar to those of the CFU-S test. These data demonstrate that human fetal liver cells are able to induce spleen colonies (tentatively called human CFU-S) when injected into lethally irradiated mice. The number of CFU-S was increased by prior purification of human fetal liver cells. When mice were injected with human fetal liver cells inactivated by irradiation, no human CFU-S were observed. Positive staining of cells found in spleen colonies, using monoclonal antibodies specific for various human determinants, indicated the human origin of part of them. The presence of human cells within the colonies was further confirmed by in situ hybridization using a probe specific for human DNA. A mean of 30–40% of analyzed colonies was thus shown to contain some patches of human cells. These data confirm that human hematopoietic cells are able to seed, proliferate, and differentiate in a murine microenvironment.     

GM1体外诱导人脐带血间充质干细胞分化为神经样细胞研究

目的 研究单唾液酸四己糖神经节苷脂通过体外诱导人脐带间充质干细胞分化为神经样细胞并探索其最佳诱导浓度.方法 取足月妊娠剖宫产健康胎儿的脐带,剔除脐动脉和脐静脉,用酶消化法获得MSCs,贴壁培养,采用流式细胞技术行细胞表型检测.扩增后,取第4代细胞,分为A、B、C、D、E5组,前4组为实验组,E组为对照组,实验组各组GM1浓度分别为:A组50μg/mL、B组100μg/mL、C组150μg/mL、D组200μg/mL,诱导液用L-DMEM培养基配制,对照组仅使用L-DMEM培养基.观察5组诱导人脐带血间质干细胞向神经样细胞分化的作用,每60min观察细胞诱导前后的形态,在第360min时采用免疫细胞染色法检测胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、神经丝蛋白(neurofilaments protein H,NF-H)以及神经元特异性标记物微管结合蛋白-2(microtubule associated protein 2,MAP-2)的表达情况,并计算阳性细胞率.结果 ①大部分原代细胞在接种9h后贴壁,呈多边形、菱形,之后变为长梭形,细胞开始以漩涡、放射状生长.②P3、P5和P10代细胞表面的分子标记经流式细胞术检测发现,均表达CD105、CD90和CD73,却不表达HLA-DR、CD19、CD11b、CD45以及CD34.③诱导至第360分钟后,实验组各组细胞均有神经样细胞出现,表现为细胞胞体收缩成椭圆形,伸出较长突起,以双极细胞居多.免疫细胞化学检测显示实验组各组NF-H、MAP-2表达阳性,胶质纤维酸性蛋白GFAP表达为阴性,C组(150μg/mL)细胞NF-H、MAP-2阳性表达率最高.对照组细胞诱导前后变化不明显.结论 GM1可以诱人脐带间充质干细胞向神经样细胞分化,150μg/mL为最合适诱导剂量.     

转化人胚腱细胞在PLA和PLGA表面上的粘附特性研究

为了定量研究转化人胚腱细胞在聚乳酸和聚乳酸与羟基乙酸共聚物８５／１５表面上的粘附特性,我们采用微管吸吮技术测定了单个ＴＨＥＴＣ与ＰＬＡ膜和ＰＬＧＡ８５／１５膜表面的粘附力学特性。结果显示,ＴＨＥＴＣ与ＰＬＡ膜和ＰＬＧＡ８５／１５膜表面的粘附率和粘附力明显大于膜表面裱衬牛血清白蛋白实验组,同时小于膜表面裱衬聚赖氨酸实验组,而且ＰＬＧＡ８５／１５膜更易于ＴＨＥＴＣ粘附。表明ＢＳＡ能抑制ＴＨＥＴＣ在聚全     

替格瑞洛对oxLDL诱导的人脐静脉内皮细胞ICAM-1表达的影响

目的 探究不同浓度的替格瑞洛对氧化型低密度脂蛋白诱导的人脐静脉内皮细胞在蛋白及RNA水平表达细胞间黏附分子-1的影响。方法 离体培养人脐静脉内皮细胞,平均接种于6孔培养板,待细胞生长至融合状态时取三孔加入ox-LDL（50 μg/ml）,再分别加入不同浓度的替格瑞洛（0、20、40 μmol/L）。刺激干预24 h后采用Western Blot法测定ICAM-1的蛋白表达水平。重复实验,采用Real-time PCR法测定ICAM-1的RNA表达水平。结果 ①oxLDL能明显上调ICAM-1的表达;②Western Blot蛋白定量结果显示,oxLDL诱导组ICAM-1的蛋白表达明显高于对应的非诱导组（P＜0.05）;未予替格瑞洛干预组ICAM-1的蛋白表达高于替格瑞洛低浓度组（P＜0.05）;替格瑞洛低浓度组ICAM-1的蛋白表达高于替格瑞洛高浓度组（P＜0.05）;③Real-time PCR结果与Western Blot蛋白定量结果一致。结论 oxLDL能在RNA水平提高ICAM-1表达,替格瑞洛能在RNA水平抑制ICAM-1的表达,并与浓度正相关。     

Transplantation of Human Embryonic Myoblasts and Bone Marrow Stromal Cells into Skeletal Muscle of C57BL/10J-mdx Mice

We studied expression of dystrophin in skeletal muscles of C57BL/10J-mdx mice after transplantation of human embryonic and fetal myoblasts and bone marrow stromal cells. Dystrophin-positive areas corresponding to the location of transplanted cell were detected in muscles of all recipient mice after transplantation of different cell cultures, but the distribution of dystrophin characteristic of normal muscle fibers was detected only after transplantation of embryonic myoblasts. Dystrophin distribution in muscle fibers after transplantation of fetal myoblasts and bone marrow stromal cells was atypical.     

第3～5周人胚肝的细胞特征和生长因子及受体表达的研究

用第 3～ 5周人胚 ,石蜡切片 ,免疫组化染色 ,光镜下观察人胚肝的细胞特征和HGF、IGF -I、TGFβ1等生长因子及其受体、PCNA、AFP、CK19等的表达。结果发现第 3周末肝芽形成 ,第 4周肝索开始形成 ,第 3～ 4周人胚肝由同一类具有幼稚细胞形态学特征的细胞构成。这些细胞为AFP、c Met阳性反应。第 5周时肝索细胞的数量增加 ,开始出现PCNA的表达 ,仍仅为同一类细胞。第 5周肝索细胞呈IGF -I、TGFβ1及其受体免疫反应阳性 ,HGF阴性 ,其周围的心肌细胞及间充质细胞为HGF阳性反应。结果提示第 3～ 5周 ,组成肝芽和肝索的细胞属于肝干细胞 ,其形态和因子表达的差异说明肝干细胞可能处于不同的发育阶段 ,AFP、c Met可以作为此阶段肝干细胞的标记物 ,HGF、IGF -I、TGFβ1及其受体可能参与对早期人胚肝发育的调节。     

PC-1基因表达诱导NIH3T3细胞恶性转化

目的 通过建立稳定表达外源PC-1基因的小鼠成纤维细胞株,初步探讨PC-1基因表达对肿瘤发生、发展的影响。方法 通过脂质体介导的方法,将真核表达载体pcDNA3、1(-)／myc-his-pc-1稳定转染NIH3T3细胞,之后利用PCR、逆转录PCR(RT-PCR)技术,确定外源PC-1基因在靶细胞染色体上的整合及在转录水平的表达。通过细胞形态学分析、MTT实验、细胞周期分析、软琼脂集落形成和裸鼠成瘤实验,观察PC-1基因表达对NIH3T3生物学特性的影响。结果建立了稳定转染PC-1基因的NIH3T3细胞株。PC-1基因表达的小鼠成纤维细胞NIH3T3生长速度加快,在软琼脂上生长并形成集落,接种裸鼠后可成瘤(6／6)。结论 PC-1基因在NIH3T3细胞中稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。     

维生素B_1、B_6对高频音引起小鼠肝糖原含量变化的影响

小鼠纯音暴露(3kHz,115dB)60min时,肝糖原含量显著高于对照组(P<0.01),事先腹腔注入Vit B_1、B_6肝糖原含量显著低于单纯接受纯音组(均为P<0.01),而与对照组无差异。说明单纯纯音可使小鼠肝糖含量升高;Vit B_1、B_6均有明显对抗纯音所致的肝糖原含量升高作用。     

Epstein-Barr病毒BARF1基因协同TPA诱发猴肾上皮细胞恶性转化的研究

目的 探讨Epstein-Barr病毒（EBV）BARF1基因单独或协同佛波醇乙酯（TPA）诱导猴肾上皮细胞恶性转化的作用。方法 用猴肾永生化上皮细胞PT1和PT7单独或进一步加促癌物TPA注射裸鼠或Scid小鼠背部皮下,观察成瘤情况。结果 EB病毒BARF1永生化细胞PT1客PT7在裸鼠不能形成肿瘤,但在免疫力严重缺陷的Scid小鼠能形成肿瘤,成瘤率为66．7％－100％,成瘤时间较长为45－60d。加TPA后裸鼠及Scid小鼠均能形成肿瘤,裸鼠的成瘤时间为20－22d,Scid小鼠的成瘤时间为5－7d。用聚合酶链反应（PCR）可从肿瘤组织中扩增出BARF1基因。结论 EB病毒BARF1基因是致癌基因,甚至无需促癌物等辅助条件,也能诱发猴肾上皮细胞恶性转化。     

肺炎克雷伯杆菌分泌因子及活菌诱导人肺上皮细胞表达分泌IL-8的研究

目的 :探讨肺炎克雷伯杆菌 (Klebsiellapneumoniae ,Kp)分泌因子及活菌诱导肺上皮细胞株表达和分泌IL 8的状况。方法 :用临床分离株Kp0 3 1 1 6、Kp0 3 1 83的细菌培养上清或活菌刺激肺上皮细胞株A5 49和SPC A 1 ,酶联免疫吸附实验 (ELISA)检测细胞IL 8表达量。结果 :①两株Kp培养上清分别刺激A5 49或SPC A 1后 ,IL 8表达量均有显著增高 (P <0 .0 1 ) ,且随上清刺激浓度增加而上升。②两株Kp及DH5 (活菌分别与SPC A 1共孵育 2h ,用庆大霉素杀死胞外菌 ,继续孵育 2 4h后两株Kp诱导细胞IL 8表达量均显著高于DH5α(P <0 .0 1 ) ,且随细菌数 /细胞数比例增大而上升。结论 :Kp分泌因子及活菌都能够诱导肺上皮细胞IL 8表达 ,而活菌上调IL 8的效应较培育上清分泌因子更显著 ,提示肺上皮细胞在Kp感染刺激的肺部炎症反应中起重要作用。     

Oxidative Stress in HEp-2 Human Laryngeal Carcinoma Cells Induced by Combination of Vitamins B12b and C

Incubation of human laryngeal epidermoid carcinoma HEp-2 cells with hydroxocobalamin (vitamin B12b) and ascorbic acid (vitamin C) for 1 h initiated oxidative stress accompanied by damage to mitochondria and increase in intracellular oxidative activity. Studies of the kinetics of these processes showed that the increase in intracellular H2O2 activity and mitochondrial damage are more likely a result, but not the cause of cell apoptosis during the first hour of their incubation with vitamins B12b and C.     

药物流产终止乙肝病毒感染患者10~14周妊娠的 安全性及有效性分析

目的 观察药物流产终止乙型肝炎病毒感染患者10~14周妊娠的安全性及有效性。方法 选取我院2013年1月~2017年12月120例乙型肝炎患者作为观察组,其中大三阳46例,小三阳74例,肝功能均正常。选取同期非肝病药物流产患者60例作为对照组,两组患者均采用药物终止妊娠。比较两组患者流产成功率、转氨酶水平和药物副反应。结果 观察组流产成功率86.67%,对照组的流产成功率为 88.33%,两组比较,差异无统计学意义（P＞0.05）;药物流产前后两组患者转氨酶比较,差异无统计学意义（P＞0.05）;药物副反应包括胃肠道反应、过敏反应、阴道出血,两组比较,差异均无统计学意义（P＞0.05）。结论 两组患者完全流产率无差异,无严重副反应及重症肝炎的发生,说明乙型肝炎患者孕10~14周行药物流产是安全有效的,作为非侵入性终止妊娠的方法值得推广应用。     

Infection of Human Dendritic Cells by Dengue Virus Activates and Primes T Cells Towards Th0-Like Phenotype Producing Both Th1 and Th2 Cytokines

Dengue viruses (DV) infection is an important public health issue all over the world. Although the pathogenesis remains unclear, the overwhelmingly triggered immune responses have been consistently observed. Recently, we and other researchers demonstrated that the natural hosts for DV are dendritic cells (DC), the primary sentinels of immune system. In light of the significance of T cells in dengue virus pathogenesis, here, we examine the possible consequences of DC-T cell interaction that is supposed to be happening in lymphoid tissues after infection. We showed that DV-infected DC induced the interacting T cells to proliferate, to produce interleukin-2 as well as to express activation markers on cell surface. Compared to mock-infected DC, the infection of DC by DV also induced T cells to produce interleukin-4, interleukin-10 and interferon-gamma, a cytokine pattern suggesting Th0 phenotype. Such an effect was either totally abolished or greatly reduced when DV were pre-inactivated with heat or ultraviolet before infection. In addition, we demonstrated that such a Th0 phenotype shift of T cells was affected neither by different dosages of viruses that infected DC nor by different durations of DC-T cell interaction. Our results provide a basic support for clinical observations and may be of help in understanding the pathogenesis of DV infection.