大鼠GLUT3启动子的克隆及其缺糖调控活性的测定

目的: 构建葡萄糖转运体3(GLUT3)启动子的报告基因,并在正常和缺糖情况下检测其转录活性。方法: 使用生物信息学软件预测了GLUT3 的启动子序列,长度1 292 bp, 包含第1个外显子, 通过PCR及双酶切方法,从大鼠全血基因组DNA中获得GLUT3 基因编码序列, 包含GLUT3 启动子序列,然后克隆到报告基因pGL3-Basic载体上,构建GLUT3 启动子的报告基因;用脂质体转染法将pGL3-GLUT3与胸腺嘧啶脱氧核苷激酶(pRL-TK)共转染入PC12细胞中,以pRL-TK载体作内参照,分别给予正常和缺糖培养,采用双萤光素酶报告系统评估GLUT3 启动子的活性。结果: 经PCR方法扩增出GLUT3 启动子序列,测序与GenBank序列一致;转染后检测显示,该pGL3-GLUT3明显具有转录活性,而且在缺糖24 h情况下其双萤光素酶活性有明显升高。结论: 成功构建出GLUT3 启动子报告基因,pGL3-GLUT3表现出很好的缺糖诱导活性,缺糖24 h是研究pGL3-GLUT3在缺糖情况下转录调控很有意义的时间点。     

Cyclin H基因启动子区-1 SNP对转录活性的影响

目的 探讨CCNH基因启动子区-1SNP位点碱基变异对CCNH基因转录活性的影响.方法 通过PCR和定点突变技术,构建了含CCNH基因基础启动子及-1G突变启动子,用双荧光素酶报告系统观察此SNP对CCNH基因启动子转录活性的影响.结果 在AD293细胞中,-1G突变启动子活性比CCNH基础启动子活性低26%(P<0.05).结论 -1T→G碱基变异可显著降低CCNH基因启动子转录活性.     

人GDDR基因启动子的克隆和报告基因载体构建

目的:克隆人GDDR基因的启动子;构建GDDR启动子的报告基因载体并进行活性分析.方法:设计合成GD-DR启动子引物,采用PCR技术从人基因组DNA中扩增GD-DR启动子;将扩增片段插入T载体并利用酶切与测序进行鉴定;亚克隆该基因至pGL3-Basic荧光报告基因载体;瞬时转染细胞,用双荧光素酶报告基因系统检测报告基因载体的活性.结果:PCR扩增得到人GDDR基因启动子;成功构建pGL3-GDDR-promoter 报告基因载体,测序结果表明启动子序列正确;双荧光素酶报告基因检测系统证实构建的报告基因载体具有启动子活性.结论:成功地构建人GDDR基因启动子的克隆及其报告基因载体的构建,为深入研究GDDR转录表达的调控机制提供基础.     

Cyclin H基因启动子区-1 SNP对转录活性的影响

目的　探讨CCNH基因启动子区- 1SNP位点碱基变异对CCNH基因转录活性的影响。方法 通过PCR和定点突变技术,构建了含CCNH基因基础启动子及- 1G突变启动子,用双荧光素酶报告系统观察此SNP对CCNH基因启动子转录活性的影响。结果　在AD293细胞中, -1G突变启动子活性比CCNH基础启动子活性低26%（P<0.05）。结论 -1T→G碱基变异可显著降低CCNH基因启动子转录活性。     

5HRE和CEAp联合调控的TSST-1激活淋巴细胞特异杀伤CEA阳性肿瘤细胞

目的:研究5拷贝缺氧反应元件(5HRE)增强子和癌胚抗原启动子(CEAp)联合调控的超抗原中毒性休克综合征毒素-1(TSST-1)在缺氧环境下激活淋巴细胞对CEA阳性的结肠癌细胞株LoVo的杀伤作用。方法:用5HRE和CEAp构成基因表达调控元件,构建出以该元件调控跨膜型超抗原TSST-1-linker-CD80TM(TC)基因靶向表达的真核表达载体。用该载体转染CEA阳性的人结肠癌细胞株LoVo及CEA阴性的人宫颈癌细胞株HeLa,利用G418筛选稳定转染的细胞。用RT-PCR检测TC融合基因的表达。分离健康人外周血淋巴细胞(PBL),用稳定转染TC的细胞裂解物进行PBL刺激实验;将PBL与稳定转染的细胞共培养,行淋巴细胞杀伤实验;MTT法检测TSST-1促PBL增殖和PBL对稳定转染细胞的杀伤效应。结果:成功筛选出稳定转染目的基因的单克隆LoVo细胞和HeLa细胞。RT-PCR证实单克隆LoVo细胞中TC在mRNA水平的表达,且缺氧环境中的表达量更高;单克隆HeLa细胞在常氧和缺氧条件下均无TC的表达。MTT法检测发现,缺氧环境下单克隆LoVo细胞表达的TSST-1能有效激活人PBL增殖,PBL剂量依赖性的抑制表达TSST-1的LoVo细胞的生长(P0.05);但HeLa细胞和野生型LoVo细胞不能刺激PBL增殖,PBL对其也无抑制作用。结论:5HRE和CEAp双重调控的超抗原TSST-1在体外缺氧环境下可激活人PBL特异杀伤CEA阳性肿瘤细胞。     

缺氧反应基因及调控机制研究进展

细胞在缺氧情况下,可诱导缺氧反应基因转录增加,维持血氧稳定。缺氧诱导因子-1和包含有启动子、增强子序列的顺序作用元件为转录调控的关键环节,缺氧诱导因子-1可通过顺式作用元件内的缺氧诱导因子-1结合位点与之相结合,二者通过复杂的相互作用来实现转录调控。     

KIR3DL1基因启动子亚克隆及表达调控载体的构建

为研究KIR3DL1基因的表达调控机制,亚克隆KIR3DL1基因的核心启动子序列,并构建KIR3DL1基因启动子-荧光素酶报告载体,采用PCR法从含有KIR3DL1基因转录起始位点5'侧翼区的质粒中扩增KIR3DL1基因核心启动子序列,产物纯化回收后与pGEM-T easy载体连接,测序鉴定正确的重组子经限制性内切酶BglⅡ和NcoⅠ双酶切后,插入同样经BglⅡ和NcoⅠ双酶切的用于基因表达调控研究的pGL3-Basic报告载体,最终获得了长度为254bp的KIR3DL1基因启动子克隆,构建了调控荧光素酶报告基因的真核表达载体.通过酶切及基因测序的方法证实所构建的重组子是正确的,说明KIR3DL1基因启动子表达调控载体的构建是成功的.     

SOD2启动子的克隆及转录活性的检测

目的： 克隆小鼠锰超氧化物歧化酶基因(MnSOD, SOD2) 5非编码区启动子序列，通过报告基因技术检测在静息或脂多糖（LPS）、亚砷酸钠(NaAsO2)等刺激下该段启动子的转录活性。方法: 提取小鼠肝组织基因组DNA，PCR扩增小鼠SOD2启动子序列(-1 554～+48)；采用基因重组技术构建由SOD2启动子驱动的红色荧光蛋白报告基因载体，将该载体瞬时转染小鼠胚胎成纤维细胞(MEF)，荧光显微镜下观察静息或NaAsO2、LPS、佛波酯(PMA)刺激下红色荧光蛋白表达。结果: 正确扩增出小鼠SOD2启动子(-1 554～+48)片段；成功构建其红色荧光蛋白报告基因载体，证实该质粒转染MEF细胞后静息状态下仅可见少量而微弱的红色荧光，经NaAsO2、LPS、PMA刺激后，红色荧光强度和亮度明显增加。结论: 小鼠SOD2启动子(-1 554～+48)在静息状态下即具有转录活性，炎性、氧化应激刺激后SOD2表达增强；该启动子的成功克隆和其报告基因载体的构建，为研究SOD2的基因表达调控机制提供了重要基础和工具。     

缺氧、复氧条件下低氧反应元件（HRE）对心肌细胞转染hVEGF165基因表达的调控作用

目的：探讨缺氧、复氧条件下，低氧反应元件(HRE)作为氧条件基因表达控制开关，对心肌细胞转染rAAV-HRE9-hVEGF165基因表达的调控作用。方法:分离新生SD大鼠心肌细胞,采用无血清培养,将在HEK293T细胞进行包装后获得的腺相关病毒转染培养的心肌细胞。实验共分为8组：Ⅰ组（空白对照组）：常氧培养 24 h（氧浓度21％）；Ⅱ组（缺氧对照组）：常氧培养16 h，缺氧8 h（氧浓度1％）；Ⅲ组（转基因对照组）：常氧培养 24 h；Ⅳ组（转基因缺氧1组）：转基因后缺氧8 h；Ⅴ组（复氧1组）：常氧培养16 h，缺氧8 h、复氧4 h；Ⅵ组（复氧2组）：常氧培养16 h，缺氧8 h、复氧8 h；Ⅶ组（复氧3组）：常氧培养16 h，缺氧8 h、复氧12 h；Ⅷ组（转基因缺氧2组）：转基因后常氧培养16 h，缺氧20 h。ELISA法测定培养液VEGF蛋白含量；细胞免疫荧光染色及RT-PCR分别检测细胞内VEGF蛋白及VEGF165 mRNA的表达。结果:95%的培养心肌细胞可见自律搏动，cTn-I染色阳性率为86%；病毒转染率约为87%。Ⅳ、Ⅴ、Ⅷ组培养液中VEGF蛋白含量显著高于其它各组（P<0.01），细胞免疫荧光VEGF蛋白染色呈阳性；RT-PCR测定显示，Ⅳ、Ⅴ及Ⅷ组可见484 bp目的条带。结论：rAAV-HRE9-hVEGF165可成功地转染原代培养心肌细胞，在缺氧环境下，受HRE的调控，VEGF165 mRNA及VEGF165蛋白可有效表达，而在常氧状态下，目的基因的表达及蛋白合成即行中止。     

纤维连结蛋白启动子的克隆及转录活性的研究

目的：克隆纤维连结蛋白（FN） 启动子和检测其转录活性，并初步研究增加SV40增强子对其转录活性的影响。方法:从正常人gDNA中克隆FN启动子，驱动氯霉素乙酰转移酶报告基因表达，构建成有SV40增强子和无SV40增强子两个重组体报告载体。利用脂质体介导的转染技术导入HT1080细胞，检测各重组体的瞬时表达，确定克隆FN启动子转录活性和SV40增强子对其转录活性的影响。 结果:克隆的FN启动子在HT1080细胞中活性比SV40启动子稍强，在加入SV40增强子时活性提高了约6倍。 结论:成功的克隆FN启动子，在HT1080细胞中有活性，通过增加SV40增强子能大大提高其活性，为运用FN启动子和特异性靶细胞治疗各种遗传性疾病提供了一定基础。     

Differences in ventilatory responses to hypoxia and hypercapnia between normal and judo athletes with moderate obesity

Summary Hypercapnic and hypoxic ventilatory sensitivities were compared in twenty-one judoists and 24 control subjects with similar degrees of moderate obesity. Data from ten non-obese control subjects were also included as a reference. Mean body weight (BW) and % of ideal body weight in the judoists and the obese and non-obese controls were 100±14.8, 94.4±5.3 and 63.4±6.1 (mean±SD) kg, and 142.3±16.7, 142.2±12.9 and 98.4±10.7%, respectively. Mean body fat in the judoists was 16.2±13.9%, being 25.3±7.7% in the obese control group, the difference being significant (p<0.01).Hypercapnic sensitivities in terms of the CO₂ ventilatory response slope (S) and its normalized value for 70 kg BW (S_N) of the obese controls were higher than the judoists. These findings were also verified by the CO₂-occlusion pressure responses. S and S_N in the obese controls were significantly correlated with BW and % body fat. However, no positive correlation was found between BW and S or S_N in the judoists as well as between lean body mass and S or S_N in the obese control. Hypoxic sensitivity in terms of the ventilation hyperbola slope (A) and its normalized value (A_N) in the obese control was significantly higher than the non-obese control, but the difference from the judoists was not significant. A and A_N were found to increase with increasing % body fat in both judoists and obese controls. We conclude that respiratory compensation to mechanical limitation due to fat accumulation may be one of the possible factors to enhance hypercapnic as well as hypoxic ventilatory chemosensitivities in moderate obesity.     

缺氧预适应抑制缺氧/复氧诱导心肌细胞凋亡的作用及机制

目的：研究缺氧预适应（HP）对缺氧复氧（H/R）诱导新生大鼠心肌细胞凋亡的影响及其作用机制。方法： 体外培养新生大鼠心肌细胞，分3组：正常对照组、HP+ H/R组和H/R组，吖啶橙（AO）染色法观察心肌细胞凋亡形态学特征，流式细胞术检测心肌细胞凋亡率，比色法检测心肌细胞caspase-3的活性，免疫组织化学法结合计算机图像分析检测心肌细胞Bcl-2蛋白的表达。结果： 心肌细胞H/R损伤后，AO染色可见典型凋亡细胞，流式细胞仪检测其凋亡率为(29.7±5.4)%，HP可显著降低心肌细胞凋亡率至(7.8±1.3)%（P＜0.01）。H/R组心肌细胞caspase-3的相对活性为5.9±0.8，HP+H/R组心肌细胞caspase-3的相对活性为2.6±0.5，显著低于H/R组（P＜0.01）。Bcl-2在正常心肌细胞即有表达，其阳性染色吸光度值为119.4±7.1，H/R组为99.6±5.0，显著低于正常组（P＜0.01），HP+H/R组为126.5±6.2，显著高于H/R组（P＜0.01）。结论： HP可通过增加心肌细胞Bcl-2的表达、降低caspase-3活性而抑制H/R诱导的心肌细胞凋亡，发挥心肌细胞保护作用。     

人胰岛素样生长因子Ⅱ基因P1、P3启动子克隆及意义

目的:人胰岛素样生长因子Ⅱ(IGF-Ⅱ)基因P1、P3启动子的克隆。方法:根据GenBank数据库提供的IGF-Ⅱ基因DNA全序列及有关文献, 应用巢式PCR技术从L-02正常人胎肝细胞系中扩增分离出P1、P3启动子片段, 并采用TOPO TA Cloning kit将PCR产物克隆入pCR2.1-TOPO T载体。结果:经琼脂糖凝胶电泳及直接测序鉴定, 克隆的IGF-Ⅱ基因P1、P3启动子片段碱基序列与GenBank数据库一致。结论:成功克隆了IGF-II基因P1、P3启动子。     

雄激素应答元件陷阱DNA抑制PSA基因启动子的作用并诱导LNCaP细胞凋亡(英)

目的：研究雄激素应答元件陷阱DNA（ARE decoy）对前列腺特异抗原（PSA）基因启动子的抑制作用及其对前列腺癌细胞LNCaP细胞生长活性的影响，为前列腺癌的基因治疗寻求新的策略。 方法:联合运用报告基因和陷阱DNA策略，构建含PSA基因5’侧启动子区640 bp DNA的荧光素酶表达载体pGL3-PSA， ARE陷阱DNA共转染前列腺癌细胞株PC3-M。应用双荧光素酶测定系统，检测荧光素酶的表达活性。然后，应用2 mg/L ARE decoy转染LNCaP细胞，通过相差显微镜观察细胞超微结构变化，MTT比色法检测细胞生长活性，DNA ladder和流式细胞技术（FCM）检测细胞凋亡以研究ARE decoy DNA对前列腺癌细胞LNCaP细胞生长活性的影响。同时提取LNCaP细胞核蛋白，应用电泳迁移率变动分析(EMSA)检测ARE decoy DNA与雄激素受体的特异结合。结果:ARE decoy DNA显著抑制报告基因荧光素酶的表达，抑制率可达95%。EMSA显示ARE decoy DNA能特异与核蛋白中雄激素受体结合。LNCaP细胞转染ARE decoy DNA后，镜下观察部分细胞出现凋亡形态学的改变，细胞体外生长受到抑制，染色体DNA凝胶电泳可见明显梯形条带。转染48h的凋亡率为22.4%。 结论:实验表明ARE decoy DNA能竞争结合雄激素受体(AR)，阻断AR的作用而诱导LNCaP细胞凋亡，有可能为前列腺肿瘤的治疗提供新的策略。     

Comparison of the basal and glucocorticoid-inducible activities of the upstream regulatory regions of HPV18 and HPV31 in multiple epithelial cell lines

Steroid hormone receptors have been shown to bind to response elements in the upstream regulatory region (URR) of human papillomavirus (HPV) in a ligand-dependent manner to affect viral promoter activity. To better understand how the enhancer activity of the URR differs between high risk HPV types, we chose to compare the basal and glucocorticoid-dependent activities of the URRs of HPV18 and HPV31. We found that the URR of HPV18 is a stronger enhancer than the URR of HPV31 in six different cell lines of epithelial origin. Furthermore, the activity of the URR of HPV31 was not inducible by the synthetic glucocorticoid dexamethasone (dex) in any cell line tested, while the URR of HPV18 was dex-inducible in the majority of these lines. These studies indicate significant differences between the URRs of high risk HPV types.     

Effect of hypoxia on expiratory muscle activity in fetal sheep

The fetal respiratory response to acute hypoxia is characterized by depression, often to apnea. This study examined the effect of hypoxia on the electromyogram (EMG) of the thyroarytenoid (TA) muscle. Under anesthesia catheters were placed in the fetal sheep carotid artery, fourth cerebral ventricle, trachea and amniotic fluid and wires sewn into the diaphragm and TA muscle. During normoxic episodes of slow fetal breathing (<40 breaths per min) TA EMG activity was phasic beginning immediately after diaphragmatic EMG bursts and ending well before the next burst. This timing is consistent with the post-inspiratory (post-I) phase of the respiratory cycle. Lowering fetal arterial Pa O₂ from ∼20 mmHg to ∼13 mmHg resulted in arrest of diaphragm EMG and tonic TA activity. Instillation of the (R,S)- -amino-3- hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) ionotrophic glutamate receptor antagonist 2,3-dihydro-6-nitro-7-sulphamoyl-benzo(f) quinoxaline (NBQX) into the cerebrospinal fluid (CSF) of the fourth ventricle abolished tracheal pressure deflections and diaphragmatic EMG activity. Tonic TA activity, however, could still be evoked by hypoxia. These results indicate that fetal post-I motoneurons are not inhibited by moderate hypoxia and that their tonic activity may be due to a loss of inhibitory input.     

Changes in ventilatory response to hypoxia in the rat during growth and aging

The influence of aging on the ventilatory response to hypoxia was studied in the halothane-anesthetized male Wistar rats of various ages (1.5–20 months). The magnitude of increase in ventilation (normalized for body weight) during hypoxia in isocapnic conditions was attenuated in parallel with advancing age. However, ventilation in hyperoxia, normoxia or mild hypoxia did not differ among various age groups when the ventilatory volume was normalized for O₂ consumption. Furthermore, threshold end-tidal PO ₂ for ventilatory depression in deeper hypoxia became progressively lower with advancing age. The results suggest that the age change in ventilatory response to hypoxia depends largely upon the progressive reduction in basal O₂ requirement (consumption) with age.