人IL-1RⅡ基因腺病毒载体的构建及在子宫内膜异位症细胞中的表达

目的：利用AdEasy XL系统，构建并鉴定IL-1RⅡ基因重组腺病毒载体，并在子宫内膜异位症（EM）细胞中表达。方法-PCR扩增含有IL-1RⅡ全长cDNA的片段，亚克隆到pShuttle—CMV穿梭质粒，经酶切和测序验证无误后，再经电转化与pAdEasy-1质粒在大肠杆菌BJ5183中进行同源重组产生腺病毒载体质粒。经过抗性筛选、酶切鉴定以及再次测序验证无误后得到阳性的重组质粒，经PacⅠ酶切线性化再在293细胞中进行包装扩增，用ELISA检测IL-1RⅡ蛋白的表达。利用Adeasy XL系统的对照载体pShuttle-CMV-LacZ同上操作作为对照。收集的重组腺病毒感染原代培养的EM基质细胞，并以免疫组化法鉴定IL-1RⅡ表达。结果：测序证实连接后IL-1RⅡ序列完全正确；抗性筛选及酶切鉴定均表明重组腺病毒载体构建成功；pShuttle-CMV-LacZ转染293细胞3d后x-gal染色阳性，回收病毒可以重复感染293细胞，ELISA鉴定表达IL-1RⅡ可溶性蛋白，证明病毒包装成功。重组的腺病毒感染EM基质细胞后，免疫组化法证实IL-1RⅡ表达。结论：成功地构建了IL-1RⅡ基因重组腺病毒载体，并且在EM基质细胞中表达，为进一步研究IL-1RⅡ基因在EM中的作用乃至生物治疗都奠定了基础。     

携带HSP70-HBsAg嵌合基因复制缺陷型重组腺病毒的制备

目的 构建表达乙肝病毒表面抗原(HBsAg)和热休克蛋白70(HSPT0)嵌合基因的复制缺陷型重组腺病毒载体.方法扩增结核分枝杆菌HSP70基因片段,亚克隆到穿梭质粒pAdTrack-CMV-HBsAg上,与5型腺病毒骨架质粒pAdeasy-1共同电转化到大肠埃希菌BJ5183内进行同源重组,经卡那霉素抗性筛选和酶切鉴定筛选出携带HBsAg-HSP70嵌合基因的重组腺病毒载体,用脂质体包裹Pac Ⅰ酶切线性化的重组质粒,转染到293细胞内进行重组腺病毒的包装.体外转染真核细胞,通过示踪基因绿色荧光蛋白表达的观察、RT-PCR和ELISA检测目的基因的表达.结果成功获得重组腺病毒质粒pAd-HBsAg-HSP70.重组质粒pAd-HBsAg-HSP70导入293细胞,经包装和二次扩大培养获得了具有感染能力的重组腺病毒颗粒Ad-HBsAg-HSP70,其病毒滴度达2×1012pfu/L,并能在真核细胞中有效表达目的基因.结论成功构建表达HBsAg-HSP70复制缺陷型重组腺病毒载体,为进一步开展HBV基因治疗研究提供实验基础.     

小鼠noggin基因的重组腺病毒构建与鉴定

目的：构建小鼠 noggin 基因的重组腺病毒载体并鉴定。方法：采用基因工程技术。经过2次亚克隆将 noggin 基因片段克隆至穿梭质粒 AdTrack-CMV 上,利用 pAdEasy-1系统进行细菌内同源重组后,脂质体转染 293T 细胞包装、扩增。采用 PCR 方法对重组体腺病毒进行鉴定,利用穿梭质粒中带有绿色荧光蛋白 GFP 报告基因,对病毒滴度和感染效率进行监测。结果：酶切鉴定及 PCR 结果证明 noggin 基因重组腺病毒载体构建成功,病毒滴度达6．3×10~(10)pfu/ml。结论：应用细菌内同源重组法成功构建了含小鼠 noggin 基因的重组腺病毒载体。     

HBsAg腺病毒疫苗载体的构建及293细胞中的包装表达

目的：HBsAg与腺病毒骨架载体质粒构建，在293细胞中包装表达重组腺病毒颗粒，建立HBsAg腺病霉疫苗。方法：以pEcob-6为模板，PCR扩增目的基因HBsAg，腺病毒穿梭载体PAd-track-cmv与HBsAg重组为PAd-track-cmv-HBs穿梭质粒；腺病毒骨架载体质粒PAd-Easy-1再与PAd-track-emv-HBs同源重组为PAd-Easy-1-HBs质粒；用脂质体介导的转染法将PAd-Easy-1-HBs转染到293细胞，包装成重组腺病毒颗粒。酶联免疫吸附法测定细胞上清中HBsAg的表达，建立HBsAg腺病毒疫苗。结果：PAd-Easy-1-HBs转染到293细胞中，合成荧光蛋白，荧光显微镜下细胞发绿色荧光。再次感染293细胞，90％以上的细胞脱落。细胞上清液中也测定出有HBsAg的表达。结论：HBsAg腺病毒疫苗载体构建成功，并能在293细胞中完整包装成重组腺病毒颗粒，为免疫动物实验提供条件。     

含猪链球菌2型溶菌酶释放蛋白基因片段的重组腺病毒构建与鉴定

目的 构建猪链球菌2型(SS2)溶菌酶释放蛋白(MRP)基因片段的重组腺病毒并对其进行鉴定.方法 根据MRP的基因序列,设计合成1对引物,以SS2型基因组为模板,扩增MRP基因片段(467-1351)序列.将PCR产物通过pMD18-T载体克隆后,再连接至腺病毒穿梭载体pShuttle-CMV中构建重组穿梭质粒pShuttle-CMV-MRP,再经PmeI酶切,然后转化至含腺病毒骨架质粒pAdEasy-1的BJ5183-AD-1感受态细胞中,经同源重组获得重组腺病毒质粒pAdeno-CMV-MRP.PacI酶切线性化该重组腺病毒质粒,再转染AD-293细胞进行病毒包装,最后对细胞包装的病毒液进行PCR和Western blot法鉴定.结果 重组腺病毒质粒pAdeno-CMV-MRP转染AD-293细胞8d后出现明显的细胞病变,在培养细胞的上清病毒液中也检测到了MRP基因片段及其表达的蛋白.结论 成功构建了SS2型MRP基因片段的重组腺病毒(rAdeno-MRP).     

HAX1和EGFP共表达重组腺病毒载体的构建、鉴定

目的 构建和鉴定HAX1和EGFP双基因共表达重组腺病毒载体.方法 采用DNA重组技术,将目的 基因HAX1克隆至含有报告基因EGFP的穿梭质粒pAdTrack-CMV中,并转化于大肠埃希菌DH5α;筛选出重组质粒pAdTrack-CMV- HAX1,并在BJ5183细菌中与pAdEasy-1质粒进行同源重组,产生重组腺病毒载体;用lipofectamine将其转染HEK293细胞,包装携带全长HAX1的重组复制缺陷型腺病毒pAd-HAX1-EGFP,酶切和序列测定鉴定;用制备好的Ad-HAX1-EGFP感染HEK293细胞,流式细胞术检测其感染效率,RT-PCR、Western 印迹鉴定外源基因HAX1的表达.BrdU检测感染了Ad-HAX1-EGFP的HEK293细胞增殖情况.结果 pAdTrack-CMV-HAX1重组质粒构建成功.pAdTrack-CMV-HAX1 质粒与pAdEasy-1质粒同源重组后与预期结果相符.构建好的Ad-HAX1-EGFP能有效感染HEK293细胞;外源基因能在239细胞中有效表达.HAX1高表达的HEK293细胞其增殖率得以提高.结论 成功构建了表达HAX1和EGFP共表达的重组腺病毒载体,HAX1能够促进结肠癌细胞HEK293细胞的增殖.     

BMP4-siRNA重组腺病毒的构建及鉴定

目的构建靶向抑制骨形态发生蛋白BMP4基因RNA的siRNA腺病毒,为进一步研究其在乳腺癌骨转移中的作用打下基础。方法以PCR扩增获得BMP4全长片段,插入筛选质粒pSOS载体,同时设计6段靶向BMP4基因RNA的干扰片段,分别经酶切、连接及鉴定后获得pSOS-sirBMP4。脂质体转染293细胞,根据荧光表达量筛选获得干扰效率较高的4个干扰片段,插入穿梭质粒pSES-HUS,经重组、HEK-293细胞包装获得BMP4-siRNA腺病毒。感染乳腺癌细胞系MDA-MB-231,RT-PCR、Western blot检测其干扰效率。MTT法检测其对MDA-MB-231细胞增殖的影响。结果筛选获得4个干扰效率较高的片段,插入穿梭质粒,经重组、包装获得腺病毒pAd-sirBMP4。感染乳腺癌细胞系MDA-MB-231后,明显抑制BMP4的表达并促进MDA-MB-231细胞增殖。结论成功构建具有较高干扰效率的腺病毒pAd-sirBMP4,为进一步研究BMP4在乳腺癌中的作用打下基础。     

重组人肝细胞生长因子-NK4腺病毒的制备及初步鉴定

目的 制备表达人肝细胞生长因子(HGF)-NK4蛋白的复制缺陷型重组腺病毒.方法 酶切pcDNA3-hNK4质粒获得NK4基因编码区序列并克隆至穿梭载体构建重组pAdTrack-CMV-NK4载体,线性化后与pAdEasy-1共转化BJ5183,通过同源重组得到重组pAd-NK4病毒载体.将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒,用病毒悬液感染人肝癌细胞株HepG2.RT-PCR法检测感染肿瘤细胞中NK4 mRNA表达.结果 酶切鉴定得到阳性pAd-NK4重组腺病毒载体,该载体能有效转染HEK293细胞并在细胞内成功包装.在转染2d后能观察到绿色荧光蛋白(GFP)表达.制备的Ad- NK4在体外能有效感染HepG2细胞并获得NK4基因高水平表达.结论 成功构建了NK4基因的重组腺病毒载体并制备重组腺病毒颗粒,为进一步研究NK4基因的功能及应用NK4进行基因治疗提供实验依据.     

应用细菌同源重组法快速构建和制备重组腺病毒

目的：利用大肠杆菌细菌同源重组构建重组腺病毒载体并在293细胞制备高滴度重组病毒。方法：自细胞周期相关激酶真核表达载体pCR31-CCRK中酶切出CCRK基因，亚克隆至带有增强型绿色荧光蛋白（EGFP）表达盒的腺病毒穿梭质粒pAdTrack-CMV中，形成转移质粒pAdTrack-CMV-CCRK，采用电穿孔或化学转化法在大肠杆菌BJ5183内与腺病毒骨架质粒pAdEasy-1同源重组，得到重组腺病毒载体pAd-CCRK。以pAd-CCRK为模板，经DNA测序正确后，用Pac Ⅰ酶切线性化pAd-CCRK，转染293细胞，包装成重组病毒颗粒，荧光显微镜观察转染细胞EGFP的表达，采用PCR的方法对重组腺病毒进行鉴定。将重组病毒上清感染RAW细胞，荧光显微镜下观察感染细胞重组病毒的表达。结果：成功地构建了携带CCRK基因的重组腺病毒载体并制备出高滴度重组病毒，重组病毒能在体外高效表达。结论：应用细菌内同源重组能够快速构建腺病毒载体，可高效制备均一的高滴度重组病毒，为CCRK基因功能的研究奠定了基础.     

cFLIPL和cFLIPS腺病毒载体的构建及其在心肌细胞中的表达

目的 :构建并鉴定cFLIP_L和cFLIP_S重组质粒,并观察其在乳鼠心肌细胞中表达并检测转染效率。方法 :利用基因合成技术获取cFLIP_L和cFLIP_S基因,分别连接pAd-CMV-GFPa1-IRES载体质粒,并转移至穿梭质粒pAdcFLIP_L和pAd-cFLIP_S,经挑选、扩培后进行酶切验证及测序鉴定、包装、扩增、纯化获得一定滴度的pAd-cFLIP_L和pAd-cFLIP_S,之后感染原代乳鼠心肌细胞,观察两者在心肌细胞中的荧光表达并用流式细胞仪检测转染效率。结果 :酶切验证与理论值相符,测序鉴定结果与目的基因序列一致;pAd-cFLIP_L和pAd-cFLIP_S感染人胚肾293A细胞后可见大量绿色荧光蛋白表达和聚集,10 d后出现典型的细胞病变,腺病毒包装成功,并测得滴度均为1×10~9pfu/ml;同时,在荧光显微镜下可见重组腺病毒感染可在乳鼠心肌细胞中稳定表达并发出绿色荧光,流式细胞仪检测结果显示其转染效率为90.30%±3.62%。结论 :成功构建cFLIP_L和cFLIP_S重组腺病毒表达载体且证实其能安全、有效地感染乳鼠心肌细胞。     

Modes of reinforcement and gender and culture of experimenter and subject: effects of "alien" and external reinforcement for Japanese and American men and women

In this study, we examined external and "alien" reinforcement (ER and AR. respectively) as a factor in social learning, and studied the combined effects of culture and reinforcement mode. A female (Experiment 1) and a male (Experiment 2) experimenters conducted experimental sessions. Both men and women, who grew up in the same culture as the experimenter, participated and performed the experimental task. A three-way interaction effect of experimenter gender, culture, and reinforcement mode was found on task performance. And the effect was more pronounced for a Japanese experimenter. A female and a male experimenters conducted Experiments 3 and 4, respectively; however participants this time were men and women who grew up in different cultures than the experimenter. Results indicated that the pattern of the subject gender and reinforcement mode interaction effect, when the experimenter was Japanese with American subjects, was exactly opposite to that when the experimenter was American. These experiments showed that AR was as effective for social learning as ER, and that the cultural backgrounds of experimenter and subject influenced AR and ER effectiveness.     

Rates of oxygen consumption and of anaerobic glycolysis in renal cortex and medulla of adult and new-born rats and guinea-pigs

1. Rates of oxygen uptake and of anaerobic glycolysis were estimated in slices from the renal cortex and medulla (a) of adult rats and guinea-pigs, (b) of new-born (1-, 5- and 21-day-old) rats and of guinea-pigs of 1, 12, 21, 24 and 120 hr age.2. In the adult rat, Q(O2) values for the cortex were 12.55 +/- 0.20 (22) and for the medulla: 8.56 +/- 0.17 (22) mul./hr.mg dry weight, while in the new-born rat (24 hr old) they were 10.99 +/- 0.46 (12) and 9.33 +/- 0.18 (9) mul./hr.mg dry weight respectively.3. Values for Q(CO2) (N2) (anaerobic glycolysis) in the 14 hr old new-born rat were in the renal cortex 9.65 +/- 0.35 (5) and in the medulla 7.39 +/- 0.43 (5) mul./hr.mg dry weight; while in the adult they were 2.25 +/- 0.08 (16) and 5.76 +/- 0.14 (16) mul./hr.mg dry weight, respectively.4. In the adult guinea-pig values for Q(CO2) (N2) were of the same order as in the adult rat, though the rate of O(2) uptake was for the cortex 8.12 +/- 0.22 (12) and for the medulla 5.02 +/- 0.23 (11) mul./hr.mg dry weight.5. Though the Q(O2) values in the renal cortex and medulla were smaller in the 1 hr old new-born guinea-pig, they were already increasing in the 12 hr old neonate.6. The results are discussed in the light of enzyme changes occurring during the process of maturation of the nephron as indicated by histochemical observations.     

Timing and intensity of early fevers and the development of allergies and asthma

BACKGROUND: Early childhood fevers appear to protect against later allergies and asthma. What is not known is the time in which fevers exert this effect and whether the degree of temperature increase is important. OBJECTIVE: We sought to examine the relationship between the time and degree of early fevers and later allergies and asthma. METHODS: Eight hundred thirty-five children from southeast Michigan were enrolled at birth. Clinic records from their first 2 years were abstracted for episodes of fever. At age 6 to 7 years, children underwent allergy testing. We examined fevers occurring within 6-month intervals in the first 2 years of life and outcomes at age 6 to 7 years. The primary outcome measures were allergic sensitization, asthma, asthma with allergic sensitization, and asthma without allergic sensitization. RESULTS: In the unadjusted analysis each episode of fever between 7 and 12 months of age was associated with a lower odds of allergic sensitization (odds ratio [OR], 0.71; 95% CI, 0.54-0.93) and asthma with allergic sensitization (OR, 0.43; 95% CI, 0.21-0.90) at age 6 to 7 years. Likewise, every 1 degrees C increase in the maximum temperature between 7 and 12 months was associated with a lower odds of allergic sensitization (OR, 0.77; 95% CI, 0.61-0.96) and asthma with allergic sensitization (OR, 0.62; 95% CI, 0.40-0.94). After adjusting for potential confounders, each episode of fever between 7 and 12 months was associated with a lower likelihood of asthma with allergic sensitization (adjusted OR, 0.33; 95% CI, 0.11-0.94) at age 6 to 7 years. CONCLUSIONS: Both the timing and intensity of childhood fevers appear to be important factors in the development of allergies and asthma.     

纳米水平分子成像与诊疗一体化研究进展与挑战

分子成像能以非侵入性的方式重现活体细胞的生理功能和生物学过程,提高疾病的早期和特异性诊断水平。纳米颗粒/材料具有物理性质可控性高、易于表面修饰、血液循环时间长和可功能化等优点,在疾病诊断与治疗中显示出巨大潜力。但如何阐明纳米材料多功能间的内在联系、解决其代谢及安全性等关键机制难题、实现纳米颗粒/材料多功能性到临床多功能...