携带hIL-2cDNA的逆转录病毒载体的构建及其在人肝癌细胞中的特异表达研究

将人白细胞介素２（ｈＩＬ-２）ｃＤＮＡ克隆到逆转录病毒载体ＭＮＳＭ的ＰＬ位点　，分别构建了转录受ＳＶ４０早期启动子和人甲胎蛋白增强子调控的重组逆转录病毒载体ＭＮＳＩ和ＭＮＳＩＡ。用脂质体转染法将ＭＮＳＩ和ＭＮＳＩＡ分别转导ＰＡ３１７包装细胞，测质粒转染率为（５～２０）×１０~(－３)克隆／μｇＤＮＡ·１０~６细胞，病毒感染率为（５．４～４５０）×１０~４ＣＦＵ／ｍｌ。重组病毒在４μｇ／ｍｌ ｐｏｌｙｂｒｅｎｅ存在条件下感染人肝癌细胞、肾癌细胞和黑色素瘤细胞，Ｎｅｏ~Ｒ克隆经Ｓｏｕｔｈｅｒｎｂｌｏｔ分析证明ｈＩＬ－２ｃＤＮＡ转入人肿瘤细胞并整合，　Ｒ　ＮＡ斑点杂交及ＩＬ－２活性表达分析证明，人甲胎蛋白增强子可促进异源启动子启动ｈＩＬ－２ｃＤ－ＮＡ在合成甲胎蛋白的人肝癌细胞中高效特异转录和表达。该研究对肝癌特异性免疫增强基因治疗有重要意义。     

突变和修饰IFN—α 2b基因的5‘和3’端对其在E.coli表达的调控作用

目的 对人ＩＦα２ｂ基因进行突变和修饰,从翻译水平上调控ＩＦα２ｂ基因在大肠杆菌中的表达。方法 采用ＰＣＲ技术,人工合成寡核苷酸引物,对人ＩＦＮ－α２ｂ基因进行了改造;在不改变氨基酸的前提下,将５‘端编码区的四个Ｇ、Ｃ位点突变为Ａ,Ｔ;去除３’端非编码区,将突变和修饰后的基因片段分别克隆入原核表达载体ｐＢＶ３２１;在大肠杆菌中进行表达,对表达产物进行活性效价测定。结果 ３‘端删除非编码区,表达水平     

单纯疱疹病毒Ⅰ型SM44株胸苷激酶基因的克隆及序列测定

为了分离、克隆单纯疱疹病毒Ⅰ型（ＨＳＶ－１）ＳＭ４４株胸苷激酶基因（ＴＫ）,采用原代兔婴肾细胞培养ＨＳＶ－Ｉ ＳＭ４４株病毒,提取病毒核酸作为模板,设计了ＨＳＶ－１ ＴＫ基因的上、下游引物,在引物的５‘端分别引入ＢａｍＨ１和ＥｃｏＲ１限制性内切酶位点,通过ＰＣＲ方法扩增出ＴＫ基因,经ＢａｍＨ Ｉ／ＥｃｏＲ １双酶切与ＰＵＣ１９质粒载体相连接,构建重组体转化受体菌ＪＭ１０９,通过筛选和酶切鉴定,获得     

传染性法氏囊病病毒HZ96 VP2 cDNA的结构分析及在大肠杆 …

以随机引物反转录传染性法氏囊病病毒（ＩＢＤＶ）杭州分离株ＨＺ９６基因组合成第１链ｃＤＮＡ。以第１链ｃＤＮＡ为模板,ＰＣＲ扩增ＨＺ９６ ＶＰ２ ｃＤＮＡ;Ｓａｎｇｅｒ双脱氧法测序ＨＺ９６ＶＰ２ｃＤＮＡ,并对其基因结构氨基酸序列进行计算分析;构建非融合表达质粒,在大肠杆菌中表达ＶＰ２蛋白。结果表明,克隆的ＨＺ９６ＶＰ２ｃＤＮＡ全长为１４３１个核苷酸对（ｂｐ）,含起始密码子ＡＴＧ和终止密码子ＴＡＡ,编码     

含稳定整合pMCLacI／Neo质粒的NIH3T3细胞体外突变研究？…

目的 建立一个用于比较研究哺乳动物细胞内表达基因和沉默基因的突变机理的实验模型。方法 以脂质体转染法将线性化的ｐＭＣＬａｃＩ／Ｎｅｏ质粒导入ＮＩＨ３Ｔ３细胞,用Ｇ４１８筛选,造反一个药物抗性细胞克隆进行扩增,用基因组Ｓｏｕｔｈｅｒｎ杂交,ＲＴ－ＰＣＲ及ＲＴ－ＰＣＲ Ｓｏｕｔｈｅｒｎ杂交进行了分子鉴定。结果 （１）在此细胞克隆的基因组中整合有ｐＭＣＬａｃＩ／Ｎｅｏ质粒;（２）该质粒上的两个ｌａｃＩ靶     

H—2K^b基因导入小鼠造血细胞的研究

本研究应用分子克隆技术，用脂质体（ｌｉｐｏｆｅｃｔｉｎ）将ＭＨＣ－Ｉ类基因（即小鼠Ｈ－２Ｋ＾ｂ基因）导入包装细胞ＰＡ３１７。用培养的病毒上清进一步感染ＢＡＬＢ／Ｃ小鼠的造血细胞，且形成了抗Ｇ４１８的ＣＦＵ－ＧＭ。通过多聚酶链式反应（ＰＣＲ）和反转录－多聚酶链反应（ＲＴ－ＰＣＲ），表明病毒上清感染后小鼠造血细胞及其形成的ＣＦＵ－ＧＭ中均整合了ＭＨＣ－Ｉ类基因，且已转录为ｍＲＮＡ，流式细胞仪（ＦＡＣＳ     

大鼠组织中三磷酸肌醇受体亚型基因的表达

目的观察大鼠心肌、小脑和培养的主动脉血管平滑肌细胞（ＶＳＭＣ）中三磷酸肌醇受体（ＩＰ２Ｒ）亚型的基因表达。方法用异硫氰酸胍－酸酚氯仿一步法抽提组织总ＲＮＡ,用特异性引物进行逆转／聚合酶链式反应（ＲＴ—ＰＣＲ）,β－ａｃｔｉｎ为内标半定量检测ＩＰ３Ｒ亚型ｍＲＮＡ的表达。将Ⅱ型ＰＣＲ产物重组、克隆并测序。结果发现ＩＰ３Ｒ三个亚型在心肌、小脑和ＶＳＭＣ中均有表达,小脑组织中表达水平较高。ＰＣＲ扩增片段分别为：Ｉ型ＩＰ３Ｒ,５２５ｂｐ（小脑）或４０５ｂｐ（心肌ＶＳＭＣ）;Ⅱ型ＩＰ３Ｒ,４０５ｂｐ;Ⅲ型ＩＰ３Ｒ,１６９ｂｐ。结论序列分析发现克隆的Ⅱ型ＩＰ３Ｒ ｃＤＮＡ序列与设计的目的 ｃＤＮＡ一致,证实了本文 ＲＴ－ＰＣＲ的特异性和准确性,提示本文所建立的ＲＴ－ＰＣＲ方法能可靠地研究动物组织中ＩＰ３Ｒ不同亚型的基因表达。     

HER2启动子在卵巢癌细胞系中控制报告基因特异性表达

目的 构建一种能在卵巢癌肿瘤组织中进行肿瘤特异性表达的逆转录病毒载体,增强肿瘤基因治疗的靶向性。方法 ＰＣＲ法获得ＨＥＲ２基因的启动子片段并将其克隆至逆转录病毒载体Ｐ＾ＬＸＳＮ中,再将突变型绿色荧光蛋白（ｍｔＧＦＰ）基因克隆至该启动子下游,将此载体转染ＳＫ－ＯＶ３细胞和ＭＣＦ－７细胞。结果 转染３天后可见部分ＳＫ－ＯＶ３细胞呈现绿色荧光,而ＭＣＦ－７中无此现象。结论 ＨＥＲ－２基因启动子可控制报告     

软骨发育不全FGFR3基因突变的研究

目的了解中国人软骨发育不全患者成纤维细胞生长因子受体３（ｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒｒｅｃｅｐｔｏｒ３，ＦＧＦＲ３）基因变异情况。方法应用ＰＣＲ－ＳＳＣＰ和限制性内切酶酶切方法，分析辽宁地区７例软骨发育不全（ａｃｈｏｎｄｒｏｐｌａｓｉａ，ＡＣＨ）患者外周血ＤＮＡ标本中ＦＧＦＲ３基因第１０外显子区域。结果７例患者均检测到相同的Ｇ３８０Ｒ点突变。结论表明Ｇ３８０Ｒ为中国人ＡＣＨ患者常见突变。应用ＰＣＲ－ＳＳＣＰ和限制性内切酶酶切的方法检测ＦＧＦＲ３基因突变是产前诊断和早期诊断ＡＣＨ患者的简便、快速、可靠的手段     

可溶性人干细胞因子基因导入真核细胞及其表达

本文构建了编码可溶性人ＳＣＦ基因的逆转录病毒载体ｐＬＸＳＮ－ＳＣＦ，应用Ｌｉｐｏｆｅｃｔｉｎ基因转移法将重组质粒导入Ψ２和ＰＡ３１７病毒包装细胞，经Ｇ４１８选择性培养基筛选，获得产重组病毒的包装细胞ＰＡ３１７－ＳＣＦ，其病毒效价为２．４×１０５～８．５×１０５ＣＦＵ／ｍｌ，继而感染人造血干祖细胞和ＮＩＨ３Ｔ３细胞。应用ＰＣＲ、ＡＰＡＡＰ免疫组化染色和化学发光一直接酶标法检测上述细胞中人ＳＣＦ基因的转染和表达，结果表明逆转录病毒载体介导转染的ｒｈ～ＳＣＦ基因在真核细胞中获得有效的表达。并将转有外源基因的人骨髓细胞进行体外液体长期培养（ＬＴＣ），观察造血干祖细胞增殖分化期间基因的表达情况。     

血清SF、sTfR、sTfR/SF在诊断难治性贫血与慢性再生障碍性贫血中的价值

目的探讨可溶性转铁蛋白受体(sTfR)、血清中铁蛋白(SF)及它们的比值(sTfR/SF)在鉴别诊断难治性贫血(RA)与慢性再生障碍性贫血(CAA)中的意义.方法分别对34例难治性贫血与37例慢性再生障碍性贫血和30例正常对照组应用免疫比浊法和化学发光法测定sTfR和SF并计算其比值.结果正常人、RA和CAA患者三组之间SF有显著性差异(P《0.05),sTfR有显著性差异(P《0.05).sTfR/SF:三组间及两两比较均有极显著的差异(P《0.001),CAA》正常人》RA.结论同时测定血清SF、sTfR尤其是计算sTfR/SF值在RA与CAA的诊断和鉴别诊断中可提供重要的实验依据.     

肝细胞生长因子研究进展

查阅近二十年国外有关HGF/SF文献,基本弄清了HGF/SF的分子结构及组织分布,说明了HGF/SF的调节和cmet受体及两者的关系,揭示了HGF/SF的生物活性、HGF/SF和肿瘤扩散以及与肝再生的关系.因此,HGF/SF在肝再生中起主要作用;是肾再生的关键因子;可促进伤口愈合;通过增加恶性细胞的侵袭性,HGF/SF可能作为肿瘤转移的重要因素之一.     

Surface Modifications of Silk by Cold SF6 Plasma Treatment

Silk samples, treated in an appositely set‐up radio frequency SF₆ plasma reactor under different operation conditions, were characterized by XPS, EPR, DSC, XRD, ATR analyses and water contact angle measurements, also as a function of storage time after the treatment. Efficient attachment of fluorine atoms on the polymer surface occurred during the plasma treatment, imparting water repellence to it. Also the percentage of surface oxygen increased after the treatment, partially as a consequence of radical scavenging by molecular oxygen and the formation of peroxides. An increase in silk crystallinity during the treatment seems to contribute to ensuring durable water repellence properties.     

Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells

SF2, an activity necessary for 5' splice site cleavage and lariat formation during pre-mRNA splicing in vitro, has been purified to near homogeneity from HeLa cells. The purest fraction contains only two related polypeptides of 33 kD. This fraction is sufficient to complement an S100 fraction, which contains the remaining splicing factors, to splice several pre-mRNAs. The optimal amount of SF2 required for efficient splicing depends on the pre-mRNA substrate. SF2 is distinct from the hnRNP A1 and U1 snRNP a polypeptides, which are similar in size. Endogenous hnRNA copurifies with SF2, but this activity does not appear to have an essential RNA component. SF2 appear to be necessary for the assembly or stabilization of the earliest specific prespliceosome complex, although in the absence of other components, it can bind RNA in a nonspecific manner. SF2 copurifies with an activity that promotes the annealing of complementary RNAs. Thus, SF2 may promote specific RNA-RNA interactions between snRNAs and pre-mRNA, between complementary snRNA regions, and/or involving intramolecular pre-mRNA helices. Other purified proteins with RNA annealing activity cannot substitute for SF2 in the splicing reaction.     

Diverse and potent activities of HGF/SF in skin wound repair

Genetic studies in the mouse have highlighted essential roles for several growth factors in skin repair and have offered a rationale for their use in therapy. The present study shows that the plasminogen-related growth factor HGF/SF (hepatocyte growth factor/scatter factor) promotes wound repair in homozygous diabetic db/db mice by recruiting neutrophils, monocytes, and mast cells to the wound; by promoting the migration of endothelial cells to the injured area; and by enhancing keratinocyte migration and proliferation. As a result, granulation tissue formation, wound angiogenesis, and re-epithelialization are all increased. The results demonstrate that HGF/SF affects and sustains all key cellular processes responsible for wound repair and point to a unique potential of this molecule for the therapy of chronic skin wounds.     

Reduced xenon diffusion for quantitative lung study--the role of SF(6)

The large diffusion coefficients of gases result in significant spin motion during the application of gradient pulses that typically last a few milliseconds in most NMR experiments. In restricted environments, such as the lung, this rapid gas diffusion can lead to violations of the narrow pulse approximation, a basic assumption of the standard Stejskal-Tanner NMR method of diffusion measurement. We therefore investigated the effect of a common, biologically inert buffer gas, sulfur hexafluoride (SF(6)), on (129)Xe NMR and diffusion. We found that the contribution of SF(6) to (129)Xe T(1) relaxation in a 1:1 xenon/oxygen mixture is negligible up to 2 bar of SF(6) at standard temperature. We also measured the contribution of SF(6) gas to (129)Xe T(2) relaxation, and found it to scale inversely with pressure, with this contribution approximately equal to 1 s for 1 bar SF(6) pressure and standard temperature. Finally, we found the coefficient of (129)Xe diffusion through SF(6) to be approximately 4.6 x 10(-6) m(2)s(-1) for 1 bar pressure of SF(6) and standard temperature, which is only 1.2 times smaller than the (129)Xe self diffusion coefficient for 1 bar (129)Xe pressure and standard temperature. From these measurements we conclude that SF(6) will not sufficiently reduce (129)Xe diffusion to allow accurate surface-area/volume ratio measurements in human alveoli using time-dependent gas diffusion NMR.     

5'-溴-2'-脱氧尿嘧啶核苷标记人巨细胞病毒感染SD大鼠脑组织神经元模型的建立

目的 用5'-溴-2'-脱氧尿嘧啶核苷(BrdU)标记的人巨细胞病毒(HCMV)进行脑内定位注射,建立SD大鼠脑组织感染模型;研究大鼠脑组织海马部位神经元对HCMV的容许性及HCMV在其内的感染特点. 方法 运用BrdU标记HCMV基因组,并测定病毒滴度.将21只SD大鼠随机分为病毒感染组(n=18)及对照组(n=3),病毒感染组根据感染时间分为感染24、48及96 h组.每个感染时间组再分为2μl及4μl两个剂量组,每个剂量组包括3只动物,均以脑内注射途径将BrdU标记的HCMV直接注入大脑海马部位;对照组大鼠注射不含病毒的细胞培养上清液(4μl).感染大鼠术后分笼饲养,于各感染时间点处理大鼠取脑组织,冰冻切片;对海马周围部位脑组织分别运用抗BrdU及抗微管相关蛋白2(MAP-2)抗体进行免疫荧光双标染色检测标记病毒;抗pp65抗体免疫组化染色及Nissil复染检测病毒蛋白pp65. 结果 病毒感染后24 h,两个剂量组均未在神经元内检测到标记病毒及pp65.病毒感染后48h和96 h,两个剂量组在海马部位神经元中均检测到了标记病毒阳性荧光及pp65的阳性着色;而且2μl组的受感染神经元细胞数较4μl组少,但同-剂量组在镐h和96 h受感染神经元细胞数未见明显变化. 结论 成功建立了BrdU标记HCNV感染SD大鼠脑组织的模型.大鼠脑组织海马部位神经元是HCMV的半容许细胞,HCMV在其内仅表现为潜伏性感染.