人IL—15cDNA的克隆及真核表达载体的构建

目的 构建人白细胞介素－１５（ＩＬ－１５）ｃＤＮＡ真核表达载体,以期在哺乳类细胞中获得高效、稳定表达。方法 从外周血粘附性单核细胞（ＰＢＭＣ）中提取总ＲＮＡ,经ＲＴ－ＰＣＲ方法获取了人ＩＬ－１５ｃＤＮＡ,并与ｐｃＤＮＡ３．１质粒的ＥｃｏＲＩ和ＸｂａＩ位点定向连接,构建受控于人巨细胞病毒（ＣＭＶ）启动子的重组真核表达载体ｐｃＤＮＡ３１－ＩＬ－１５。结果 经ＰＣＲ扩增鉴定和ＤＮＡ序列分析,证明ＩＬ－１５已经正确插入克隆载体且序列正确。     

产组织型纤溶酶原激活剂CHO工程细胞无血清培基的研究

在对ＤＭＥＭ：Ｆ１２（１：１）进行优化的基础上,以细胞生长和组织型纤溶酶原激活剂（ｔ－ＰＡ）表达为评价指标,筛选无血清培养基添加成分,建立了由优化ＤＭＥＭ：Ｆ１２（１：１）,胰岛素,ＰｌｕｒｏｎｉｃＦ－６８维生素Ｃ,乙醇胺和微量元素混合液组成的适于产ｔ－ＰＡＣＨＯ工程细胞４Ｂ３生长和ｔ－ＰＡ表达效果均优于相同条件下用含血清培基培养的效果。     

IL-15表达质粒能增强HIV-1 DNA疫苗诱导的细胞免疫应答

本研究旨在观察白细胞介素１５（ＩＬ－１５）表达质粒能否增强１型人类免疫缺陷症病毒（ ＨＩＶ－ １） ＤＮＡ疫苗诱导的细胞免疫应答及ＩＬ－ １５与 ＩＬ－ ２或 ＩＬ－１２表达质粒是否具有协同作用。 作者分另将ＨＩＶ－１ＤＮＡ 疫苗（ｐＣＭＶ１６０ⅢＢ和 ｐｃＲＥＶ各 ２μｇ）、ＨＩＶ－１ＤＮＡ疫苗和ＩＬ－１５表达质粒 ｐＣＡＧＧＳ－ＩＬ１５（ １、 １０、５０μｇ）、ＨＩＶ－１ＤＮＡ疫苗和 ｐＣＡＧＧＳ－ＩＬ１５同 ＩＬ－２表达质粒 ｐＢＣＭＧＮｅｏ－ｍＩＬ２（１０μｇ）或ＩＬ－１２表达质粒ｐＣＡＧＧＳ－ＩＬ１…     

新型降钙素的基因合成与克隆表达

借助计算机辅助设计了一种新的人降钙素类似物（新型降钙素）。根据密码子偏爱性原理及基因操作原则了它的基因,运用化学法和酶法相结合的技术合成了该基因,并克隆到质粒ｐＵＣ１９中,ＤＮＡ测序验证正确。该基因被克隆到融合型表达本ｐＧＥＸ－２Ｔ中,重组融合型表达载体ｎＣＴ／ｐＧＥＸ－２Ｔ转化Ｅ．ｃｏｌｉＢＬ２１,增减表达,ＳＤＳ－ＰＡＧＥ分析可见Ｍｒ约３１０００的融合蛋白带,融合蛋白占细胞内总蛋白３４％。Ｗｅ     

基因工程丙酰螺旋霉素

以碳霉素４＂异戊酰基转移酶基因（ＣａｒＥ）为探针，从麦迪霉素产生菌基因文库中，经菌落杂交获得阳性菌落ｐＣＮ１０Ｆ５。经分子杂交证明ｐＣＮ１０Ｆ５ＤＮＡ的ＢａｍＨＩ－ＢａｍＨＩ８．０ｋｂ片段与ＣａｒＥ基因同源。将ｐＣＮ１０Ｆ５ ＢａｍＨＩ８．０ｋｂ片段分别克隆到ｐＩＪ６８０、ｐＷＨＭ３，ｐＷＨＭ６０１质粒载体上，获得重组质粒ｐ６Ｆ５，ｐＷＦ５和ＰＧＦ５。含上述重组质粒的螺旋霉素产生菌克隆菌株均产生与     

冠心病经皮冠状动脉腔内成形术后血浆内皮素和前列环素的变化及意义

报告２１例冠脉造影及１９例经皮冠状动脉腔内成形术（ＰＩＣＡ）患者血浆内皮素（ＥＴ）和前列环素的代谢产物６－酮－ＧＰＦ１α的检测结果。单纯冠脉造影的患者血浆ＥＴ及６－酮－ＰＧＦ１α在手术前后无明显的变化（Ｐ＞０．０５）,而ＰＴＣＡ后２４小时血浆ＥＴ和６－酮－ＰＧＦ１α较术前明显升高（Ｐ＜０．０５）,术后１周又复降至接近术前水平（Ｐ＞０．０５）。单支血管和多支血管的ＰＴＣＡ,血浆ＥＴ和６－酮－ＰＧＦ１α浓度变化比较差异均无统计学意义（Ｐ＞０．０５）。所以,在ＰＴＣＡ ２４小时后应注意防止血小板聚集和血栓形成,术后１周内最好服用预防冠脉痉挛药物。     

突变的人铜、锌超氧化物歧化酶基因的克隆、测序及表达

目的：通过基因工程的方法将ｒｈＣｕ,Ｚｎ－ＳＯＤ ｃＤＮＡ基因改造以得到更加稳定的酶。方法：以本实验室构建的 ｒｈＣｕ,Ｚｎ－ＳＯＤ ｃＤＮＡ为模板,利用含有突变核苷酸的引物进行 ＰＣＲ扩增,将正确测定的含 ｒｈＣｕ, Ｚｎ－ＳＯＤ突变（ｒｈＣｕ,Ｚｎ－ＭＳＯＤ）基因的重组质粒重组到表达载体ＰＥＴ－２２ｂ（＋）中,重组质粒在大肠杆菌ＢＬ２１（ＤＥ３）中表达ｒｈＣｕ,Ｚｎ－ＭＳＯＤ。结果：表达产物占菌体总蛋白的３８％,具有特异性ＳＯＤ酶活性。结论：从基因突变的角度改善酶的性能不仅具有理论意义,又有一定的实用价值。     

致肾盂肾炎大肠杆菌papA免疫保护作用的初步研究

目的：对致肾盂肾炎大肠杆菌ｐ菌毛的结构基因ｐａｐＡ的免疫保护作用进行研究。方法：用ＰＣＲ方法扩增ｐａｐＡ基因,并将５５４ｂｐ扩增产物克隆入真核表达载体ｐｃＤＮＡ３,构建重组质粒ｐＣＴ３７作为ＤＮＡ疫苗,免疫ＢＡＬＢ／ｃ小鼠。结果：全菌ＥＬＩＳＡ测定显示疫苗接种组小鼠血清效价明显高于对照组,分别为１：５７５０和１：４０００（Ｐ〈０．０１）。尿液和肾脏菌落计数在接种组均明显低于对照组（Ｐ〈０．０５）,     

逆转录病毒介导诱导型一氧化氮合酶基因转移载体的构建

目的：构建逆转录病毒介导的诱导型一氧化氮合酶（ｉＮＯＳ）基因转移载体。方法：应用ＤＮＡ重组和ＰＣＲ扩增技术。结果：通过二步克隆，从质粒ｐＫＳｉＮＯＳ分离编码巨噬细胞ｉＮＯＳ的生长ｃＤＮＡ，亚克隆于中间载体ｐＳＰ７２，调整插入片段二端酶切位点；进而构建含ｉＮＯＳ基因、巨细胞病毒启动子和ｎｅｏ抗生基因的逆转录病毒载体ｐＬＮＣＸｉＮＯＳ。限制性酶切分析和ＰＣＲ鉴定证实，重组体ｉＮＯＳ插入片段的大小和方向     

海洛因依赖者外周血多形核白细胞的化学发光研究

本文应用化学发光（ＣＬ）方法体外检测了３９例海洛因依赖者多形核白细胞（ＰＭＮ）的化学发光的变化，结果表明：海洛因滥用者治疗前ＰＭＮ的ＣＬ值，未被ＯＺ刺激积分面积为２２．１±ｓ８．０２（×１０４ｃｐｓ／１０６ＷＢＣ），经脱毒治疗８－１２ｄ后为１５．３６±ｓ５．６１（×１０４ｃｐｓ／１０６ＷＢＣ），较脱毒前有所降低（Ｐ＜０．０５）；经ＯＺ刺激后积分面积为３１５．６±ｓ９４．５（×１０４ｃｐｓ／１０６ＷＢＣ），脱毒８－１２ｄ后明显降低为１７６．２±ｓ９３．９（×１０４ｃｐｓ／１０６ＷＢＣ）（Ｐ＜０．０１）。提示：经脱毒治疗后外周血ＰＭＮ的自由基有明显变化。同时观察了ＰＭＮ－ＣＬ在男女性别之间，与毒品滥用方式（烫吸与注射）、时间（＞１８月与＜１８月）及滥用量（＞１．０ｇ与＜１．０ｇ）的关系，未发现其有明显差异。     

顺铂对人肺腺癌细胞作用与ERCC1、Bcl-2表达的相关性研究

目的:探讨ERCC1、Bcl-2表达与顺铂作用的关系,进而推测ERCC1、Bcl-2在顺铂耐药中的作用.方法:选择人肺腺癌细胞A549及其耐DDP细胞株A549/DDP作为研究对象,采用MTT法检测A549/DDP细胞耐药指数;免疫细胞化学方法、RT-PCR方法检测不同浓度、不同作用时间干预后细胞中ERCC1、Bcl-2的表达.结果:10μg/mL DDP作用12 h,A549细胞中ER-CC1、Bcl-2 mRNA表达开始增高,随着作用时间的延长,ERCC1、Bcl-2 mRNA的表达逐渐增高,在72 h表达最强.浓度为5μg/mL的DDP即可刺激A549细胞中ERCC1、Bcl-2 mRNA表达水平上升,随着DDP浓度的增加,其表达水平逐渐上升,ERCC1民mRNA表达水平在20μg/mL组达到高峰.与亲本细胞相比,A549/DDP中ERCC1、Bcl-2表达明显增高.结论:随着顺铂作用时间的延长和浓度的增加,A549细胞中ERCC1、Bcl-2 mRNA和蛋白的表达量增加,推测ERCC1、Bcl-2可能参与了顺铂继发耐药的形成.     

弓形虫SAG1基因在HeLa细胞表达的研究

目的 :研究弓形虫 SAG1基因在 He L a细胞中的表达 ,为研制弓形虫疫苗奠定基础。方法 :采用磷酸钙 -DNA共沉淀转化法将重组质粒 pc DNA3- SAG1导入 He L a细胞 ,用 G418进行选择培养 ,筛选出表达阳性的细胞克隆 ,再通过培养和加压 ,建立稳定分泌 SAG1抗原的阳性 He L a细胞克隆株 ,分离表达产物 ,进行 SDS- PAGE、Western blot分析。结果 :采用钙沉淀法成功地将重组质粒 SAG1导入 He L a细胞 ,通过 G418选择和加压培养 ,获得稳定分泌 SAG1抗原的阳性 He L a细胞 ,表达的蛋白经 SDS- PAGE、Western blot分析 ,显示其分子量 30 k Da蛋白 ,与理论值相符。结论 :成功构建重组质粒 pc DNA3- SAG1,建立稳定分泌 SAG1抗原的阳性 He L a细胞克隆株     

人Prp8蛋白基因片段的克隆

目的：构建人Prp8基因全长真核表达质粒。方法：从HeLa细胞中提取总体RNA,两步法合成cDNA,利用逆转录聚合酶链反应（RT-PCR）法,扩增出人Prp8基因的四段序列,首先克隆至pTZ57R/T载体,再定向克隆至真核表达载体pcDNA3.1（＋）,构建pcDNA3.1（＋）-Prp8重组质粒。结果：RT-PCR法获得Prp8基因的4段序列,长度分别为2025、1090、1966、1963bp,分别与pTZ57R/T载体连接后,选择合适的酶切位点再连接成全长序列,然后将全长序列和pcDNA3.1（＋）真核表达载体连接、转化、酶切鉴定及序列分析后,证实pcDNA3.1（＋）-Prp8重组质粒构建成功。结论：成功克隆了人Prp8的编码基因,并构建了表达载体pcDNA3.1（＋）-Prp8。     

短发夹RNA对前列腺癌细胞中PIM-1基因沉默效应的研究

目的:探讨脂质体介导的短发夹RNA(shRNA)对前列腺癌细胞中PIM-1基因表达的影响.方法:构建3种针对PIM-1 mRNA不同靶点的shRNA重组质粒表达载体,并转染前列腺癌PC-3细胞.分别采用逆转录-聚合酶链反应(RT-PCR)及蛋白印迹法(Westernblot)检测转染后细胞中PIM-1 mRNA及蛋白的表达情况.结果:转染48 h后,3种PIM-1基因靶向性shRNA表达质粒均可抑制PC-3细胞中PIM-1 mRNA和蛋白的表达,其中靶向PIM-1 mRNA第707-725位和1080-1098位序列的重组质粒干扰效果更显著.结论:本研究所构建的PIM-1 shRNA表达质粒可在体外高效特异地抑制前列腺癌细胞中PIM-1的表达,为下一步研究奠定了基础.     

Assessment of the astrogliotic responses of three human astrocytoma cell lines to ethanol, trimethyltin chloride and acrylamide

The astrogliotic responses of the CCF-STTG1, U251-MG, and U373-MG human astrocytoma lines were determined after exposure to ethanol, trimethyltin chloride (TMTC), and acrylamide over 4, 16, and 24h. Basal glial fibrillary acidic protein (GFAP) expression in the U-251MG and U373-MG cells was 10-fold greater than the CCF-STGG1 line. Ethanol treatment over 24h, but not at 4 and 16h, resulted in significant increases in GFAP in all three glioma lines at sub-cytotoxic levels; the GFAP responses in the CCF-STTG1 line were the most sensitive, as concentrations of 0.1 and 1mM led to increases in GFAP expression compared with control of 56.8+/-15.7 and 58.9+/-11.5%, respectively (P<0.05). Treatment with TMTC (1 microM) over 4h showed elevated GFAP expression in the U251-MG cell line to 28.0+/-15.7% above control levels (P<0.01), but not in the other U373-MG or CCF-STTG1 cells. At 4h, MTT turnover was markedly increased compared with control, particularly in the U373-MG line at concentrations as low as 1 microM (17.1+/-2.3%; P<0.01). TMTC exposure over 16 and 24h resulted in reduction in GFAP expression in all three lines at concentrations; at 24h incubation, the reduction was >50% (P<0.01). There were no changes in GFAP expression or MTT turnover in response to acrylamide except at the highest concentration ranges of 10-100 mM. This study underlines the significance of period of exposure, as well as toxin concentration in astrocytoma cellular response to toxic pressure.     

Influence of sodium butyrate on hepatocellular carcinoma (hepG2) and glioblastoma (C6) cell lines in vitro

Hepatocellular carcinoma and glioblastoma are both very aggressive forms of human neoplasms. The most effective treatment includes the combination of surgery, chemotherapy and radiation. Sodium butyrate (NaB) demonstrates a high efficiency and low toxicity. It inhibits proliferation and cell cycle of the cancer cells. The aim of this study was to investigate the effect of sodium butyrate on HepG2 and C6 cell line viability. Hepatocellular cancer (HepG2) and glioblastoma cell line (C6) were cultured in DMEM/Ham's F12 medium with 10% FBS and antibiotics. Different NaB concentrations (0-10 mM) were tested. The control consisted of cells without tested substance. The incubation times were 24 and 48 h. Cell viability was studied using Trypan Blue exclusion test. Inhibitory influence of sodium butyrate on cell viability in both examined cell lines was confirmed. Strong correlation between NaB concentration and cell viability after 24 h was noticed (correlation coefficient was 0.94 and 0.98 for C6 and HepG2, respectively). IC50 values after 24 h were 8.44 mM and 6.17 mM for C6 and HepG2, respectively. The strongest effect was observed after 48 h of incubation with NaB. IC50 values were 3.44 mM and 1.47 mM for C6 and HepG2 (correlation coefficients after 48 h were 0.91 and 0.631 for C6 and HepG2, respectively). C6 line was more resistant to NaB than HepG2. Both cell lines were sensitive to NaB treatment, which gives the promise that NaB can be used against broader spectrum of neoplasms in the future.     

In vitro sensitivity of normal human mesothelial and malignant mesothelioma cell lines to four new chemotherapeutic agents

In this study, we used four human mesothelioma cell lines (M14K, M24K, M25K and M38K), one transformed human mesothelial cell line (MeT-5A) and one primary mesothelial culture (UPL) to test for in vitro sensitivity to docetaxel, paclitaxel, SN-38 [an active metabolite of irinotecan (CPT-11)] and gemcitabine, as single agents. Subconfluent cell cultures were treated with 2x10(-9), 5x10(-9), 10(-8), 2x10(-8) and 5x10(-8) M concentrations of each drug for 48 h. The sensitivity was measured in terms of cell viability using the Trypan blue exclusion method. All four drugs were potent inhibitors of mesothelioma cell growth, but cell lines from different patients diverged in their sensitivity to the individual agents. In most cases docetaxel, paclitaxel and SN-38 were more potent killers of mesothelioma cells than gemcitabine. The induction of DNA damage was investigated using the Comet assay; cells from two cell lines (M14K and M25K) were treated with subtoxic 10(-8) M concentrations of each drug for 4, 24 and 48 h. Each of the agents caused a slight increase in DNA single-strand breaks at a concentration of 10(-8) M.     

低氧诱导因子-1α和角质细胞生长因子双基因重组减毒沙门菌菌株的构建及其在肠上皮细胞中的表达

目的构建同时携带低氧诱导因子-1α（HIF-1α）和角质细胞生长因子（KGF）真核表达载体的减毒沙门菌菌株（Ty21a-pIRES-HIF,IRES-KGF,TPHK）,观察其在防治肠黏膜损伤方面潜在的应用前景。方法采用RT-PCR法从低氧处理A549细胞扩增HIF-1αcDNA后连接到载体pIRES-SEQ的NheI和MluI酶切位点,构建单基因重组质粒pIRES-HIF。然后以质粒pIRES2-EGFP-KGF为模板扩增KGF基因并克隆到重组质粒pIRES-HIF的XbaI和NotI酶切位点上,获得双基因重组质粒plRES-HIF-IRES-KGF。采用电穿孔法将pIRES-HIF-IRES-KGF质粒转人减毒沙门菌Ty21a中,通过筛选获得TPHK。该菌株转染肠上皮细胞IEC-6后48h,用ELISA法检测上清中HIFId和KGF的表达水平。并采用MTT方法分析不同剂量表达产物对IEC-6细胞的作用。结果通过对构建质粒克隆进行测序及酶切,证实TPHK构建成功。TPHK转染正常肠上皮细胞IEC-6后,48h取上清用ELISA法检测HIF和KGF的表达,结果表明6×10^5个细胞可表达（16．03±1．47）ng的HIF蛋白和（17．77±1．83）ng的KGF蛋白。MTT结果表明表达上清有明显刺激正常肠上皮细胞IEC-6增殖的活性（P〈0．05）,加入20％表达上清时刺激活性达高峰。结论成功构建了TPHK,其可有效转染肠上皮细胞IEC-6,表达上清可显著促进IEC-6细胞增殖。提示TPHK具有潜在的在肠黏膜损伤局部应用的前景。     

Cigarette smoke condensate induces cytochromes P450 and aldo-keto reductases in oral cancer cells

Our objective is to identify molecular factors which contribute to the increased risk of smokers for oral squamous cell carcinoma (OSCC). In the present study, we investigated the effects of cigarette smoke condensate (CSC) on gene expression profiles in different human oral cell phenotypes: normal epidermal keratinocytes (NHEK), oral dysplasia cell lines (Leuk1 and Leuk2), and a primary oral carcinoma cell line (101A). We determined differential gene expression patterns in CSC-exposed versus non-exposed cells using high-density microarray RNA expression profiling and validation by quantitative real-time RT-PCR. A set of 35 genes was specifically up- or down-regulated following CSC treatment (25microg/ml for 24h) by at least 2-fold in any one cell type. Notably, five genes of the cytochrome P450 (CYP1A1, CYP1B1) and aldo-keto reductase (AKR1C1, AKR1C3, AKR1B10) families were highly increased in expression, some of them 15- to 30-fold. The timing and extent of induction for these genes differed among the four cell phenotypes. A potential biological interaction network for the CSC response in oral cells was derived from these data, proposing novel putative response pathways. These CSC-responsive genes presumably participate in the prevention or repair of carcinogen-induced DNA damage in tobacco-related oral carcinogenesis, and may potentially be exploited for determining the severity of exposure and for correcting mutagenic damage in exposed tissues of the oral cavity.     

Enhancement of excision repair of cisplatin-DNA adducts by cell-free extract from a cisplatin-resistant rat cell line.

To characterize the enhanced repair synthesis of defined DNA lesions, oligodeoxyribonucleotides were synthesized and inserted into plasmid DNA. The inserted plasmid DNA was treated with cis-diamminedichloroplatinum(II) (cisplatin) and subjected to in vitro DNA repair assay with soluble extract from the rat liver cell line Ac2F. All cisplatin adducts tested stimulated DNA repair synthesis. Moreover, two cisplatin-resistant cell lines, Ac2F-CR4 and Ac2F-CR10, were established by stepwise exposure of Ac2F cells to this drug. The DNA repair synthesis was enhanced 3- to 4-fold in the extract from cisplatin-resistant Ac2F cells relative to that from Ac2F cells. Such repair synthesis was suppressed by the specific DNA polymerase inhibitor aphidicolin. The results of the present study suggested that the enhanced repair activity induced by a cisplatin adduct can be detected by in vitro DNA repair assay with soluble cell extract.