CLONING PROMOTER OF HUMAN SATB1 GENE AND EFFECT OF ATRA AND CoCl2 ON ITS ACTIVITY

Objective To explore the structure and activity of SATB1 promoter in different cells, ATRA and CoCl2 effect on its activity. Methods Using luciferase system to assay the promoter activity of human SATB1 gene, three luciferase reporter vectors were constructed which driven by different regions of 5＇ untranslated sequence from human SATB1 gene , called pGL3-SP2946-luc , pGL3-SP1718-luc and pGL3-SP751-luc , and transfected into Jurkat T, K562, U937 and Hela cells transiently using lipofectinamine, the expression activity was detected at different dosage of A TRA and CoCl2treatment for different time course. Results The reporter gene expression from SATB1 promoter were high activity in U937 cell, moderate in Jurkat T cell, low activity in K562 cell and showed no obvious activity in Hela cell, the reporter gene expression from pGL3-SP751-1uc kept on the higher lever in Jurkat T, K562 and U937 cells than the other two vectors. We also found that the repressive effect of CoCI2 on SATBI＇s mRNA expression and the relative luciferase expression from pGL3-SP751-luc in U937 cell was down-regu- lated obviously by ATRA and CoCI2 in the concentration- and time-dependent manners. Conclusion SATB1 pro- rooter drives gene expression with cell-specificity and its core promoter region maybe exist in the - 751 - - 9bp of 5＇ untranslated region of human SATB1 gene. Combined with the experiment result we found before that SATB1 was down-regulated by ATRA in U937, the results imply that STAB1 maybe is down-regulated by ATRA and CoCl2 through its promoter in the differentiation of myeloid cell line-U937.     

CLONING PROMOTER OF HUMAN SATBl GENE AND EFFECT OF ATRA AND CoCl_2 ON ITS ACTIVITY

Objective To explore the structure and activity of SATBI promoter in different cells, ATRA and CoCl2 effect on its activity. Methods Using luciferase system to assay the promoter activity of human SATB1 gene, three luciferase reporter vectors were constructed which driven by different regions of 5' untranslated sequence from human SATB1 gene, called pGL3-SP2946-luc, pGL3-SP1718-luc and pGL3-SP751-luc, and transfeted into Jurkat T,K562, U937 and Hela cells transiently using lipofectinamine, the expression activity was detected at different dosage of ATRA and CoCl2 treatment for different time course. Results The reporter gene expression from SATB1 promoter were high activity in U937 cell, moderate in Jurkat T cell, low activity in K562 cell and showed no obvious activity in Hela cell, the reporter gene expression from pGL3-SP751-luc kept on the higher lever in Jurkat T,K562 and U937 cells than the other two vectors. We also found that the repressive effect of CoCl2 on SATBI' s mRNA expression and the relative luciferase expression from pGL3-SP751~luc in U937 cell was down-regulated obviously by ATRA and CoCl2 in the concentration- and time-dependent manners. Conclusion SATB1 promoter drives gene expression with cell-specificity and its core promoter region maybe exist in the - 751 ~ - 9bp of 5' untranslated region of human SATBI gene. Combined with the experiment result we found before that SATB1 was down-regulated by ATRA in U937, the results imply that STAB1 maybe is down-regulated by ATRA and CoCl2 through its promoter in the differentiation of myeloid cell line-U937.     

DF3启动子调控的hTERT基因RNAi载体的构建及靶向干扰效果的鉴定

目的：构建带有肿瘤特异性DF3启动子、针对hTERT基因的RNA干扰表达载体pGenesil-10-miR30-DF3-hTERT,探讨该表达载体的肿瘤靶向性基因抑制作用。方法：分别用针对hTERT基因的RNA干扰寡核苷酸序列及DF3启动子取代质粒pGenesil-10-Micro30中原有的miR30序列及CMV启动子,构建pGenesil-10-miR30-DF3-hTERT载体并进行酶切及测序鉴定。将该载体分别转染人乳腺癌MCF-7细胞、肝癌HepG2细胞及造血干细胞,ELISA法检测hTERT蛋白的表达情况。结果：重组质粒经酶切及测序鉴定证实符合设计要求,构建成功。ELISA法检测结果显示,实验组MCF-7细胞及HepG2细胞的hTERT蛋白下降显著（P〈0.05）,其中以MCF-7细胞下降更为明显;造血干细胞实验组则无明显变化（P〉0.05）。结论：DF3启动子调控的hTERT基因的RNA干扰表达载体能有效抑制DF3阳性肿瘤细胞中hTERT的表达,以乳腺癌细胞效果最为显著,而对端粒酶阳性的正常细胞不产生影响。     

乙型肝炎病毒C基因启动子区域T1762 A1764变异的临床及干扰素应答

目的研究乙型肝炎病毒(HBV)C基因启动子(CP)区域T1762A1764变异与慢性乙型肝炎病情严重性及干扰素应答的关系.方法通过聚合酶链反应(PCR)及其产物直接测序,检测4例无症状慢性HBV携带者(AsC)、27例慢性乙肝病人血清的HBVCP序列,并定量检测病人血清的HBVDNA水平.结果CPT1762A1764变异在慢性乙肝病人的发生率为51.9%(14/27),更多的是在病情较重(肝损害中度以上)的病例中出现(P＜0.05),且该变异株具有生存优势.干扰素治疗3个月后,感染该优势变异株的慢性乙肝病人血清HBVDNA拷贝数下降36.6±25.1倍,显著高于非变异株病人(2.5±2.1倍);HBeAg阴转率(75%)及HBVDNA(斑点法)阴转率(77.8%)也显著高于对照组(16.7%,16.7%);对4例变异干扰素组病人3个月后再次测序,有3例其变异株的生存优势被野生株(WT)替代.结论HBVCPT1762A1764变异株可能对干扰素较为敏感.     

急性脑梗死病人纤溶酶原激活物抑制物活性及其基因启动子区域4G/5G基因多态性的研究

①目的　探讨纤溶酶原激活物抑制物 1 (PAI 1 )活性及其启动子区 4G/ 5G基因多态性在急性脑梗死(ACI)发病过程中的作用。②方法　分别采取ACI组和正常对照组外周血 ,酚抽提法提取DNA ,然后应用聚合酶链反应限制性片段长度多态技术 (PCR RFLP)检测PAI 1基因 4G/ 5G多态性 ;发色底物法测定每个标本血浆PAI 1活性。③结果　ACI组PAI 1活性明显高于正常对照组 (t=2 .731 ,P <0 .0 1 ) ,但ACI组各基因型血浆PAI 1活性水平无显著性差异 (F =2 .2 4 ,P >0 .0 5 ) ;ACI组 5G5G纯合子基因型比正常对照组明显增多 (χ2 =5 .88,P <0 .0 5 ) ,两组 4G5G杂合子基因型和 4G4G纯合子基因型比较无显著性差异 (χ2 =1 .1 6、0 .30 ,P >0 .0 5 ) ;ACI组 5G等位基因分布频率 (0 .4 0 )明显高于正常对照组 (0 .2 7) (χ2 =4 .2 5 ,P <0 .0 5 )。④结论　 5G5G基因型个体可能是我国汉族人ACI的易患者 ,可考虑将PAI 1基因 4G/ 5G基因多态性检查用于ACI高危人群的筛查工作     

全反式维甲酸对兔颈动脉内皮损伤后内膜增殖和VEGF及VCAM-1表达的影响

目的 观察全反式维甲酸(atRA)对兔颈动脉内皮损伤后内膜增殖和血管内皮生长因子(VEGF)、血管细胞黏附分子-1(VCAM-1)表达的影响.方法 新西兰雄性大白兔36只,随机分为6组:治疗A、B组,对照A、B组,假手术A、B组.治疗组和对照组给予高脂饮食并行颈动脉内膜空气干燥术,假手术组手术暴露颈动脉而不损伤内膜,治疗组给予atRA连续灌胃.分别于术后7、28 d处死A、B组动物,取病变血管进行形态学观察.免疫组织化学方法检测病变血管VEGF、VCAM-1表达水平.结果 假手术组动脉内膜光滑,无粥样硬化病变及VCAM-1表达.对照组术后7 d动脉内膜层出现泡沫细胞,平滑肌细胞增殖,VEGF、VCAM-1表达明显增加,28 d时血管内膜明显增厚,有粥样斑块形成.治疗组动脉内膜增生较轻,28 d时内膜面积低于对照组(t=4.770,P<0.001),动脉粥样硬化病变轻于对照组(H=4.89,P<0.05),VEGF、VCAM-1表达低于对照组(t=3.280～4.288,P<0.01、0.05).结论 atRA可抑制兔颈动脉内皮损伤后新生内膜增殖,下调VEGF、VCAM-1表达是其作用机制之一.     

细脚拟青霉对小鼠血清及肝,脾,肾组织酸性磷酸酶活力的影响

本文观察了细脚拟青霉菌丝体水煎剂对正常及环磷酰胺所致免疫抑制小鼠血清及肝、脾、肾组织中酸性磷酸酶活力的影响作用。结果表明：细脚拟青霉水煎剂给小鼠灌胃２１天后，小鼠血清中酸性磷酸酶活力无显著改变，正常小鼠脾组织酸性磷酸酶活力显著提高，对环磷酰胺所致小鼠肝、脾、肾组织酸性磷酸酶活力下降具有非常显著的抑制作用。提示：细脚拟青霉对免疫抑制小鼠脏器组织内的酸性磷酸酶活力具有调节作用。     

HIF-1在肿瘤的凋亡和增殖中的作用

低氧是大多数肿瘤的特征性微环境改变。HIF-1是由α和β两个亚单位组成的低氧转录激活因子。HIF-1调节多种靶基因，近年来HIF-1与细胞凋亡和增殖的关系受到广泛关注。本文针对肿瘤低氧和HIF-1对细胞凋亡的影响作一综述。     

人类PU.1 cDNA的克隆及表达产物的生物活性

目的：重叠聚合酶链反应（Ｏｖｅｒｌａｐｐｉｎｇ Ｐｏｌｙｍｅｒａｓｅ ＣｈａｎｉＲｅａｃｔｉｏｎ ＰＣＲ）法是在上下游引物之间设计一对碱基互站的嵌套引物,第一轮ＰＣＲ分别扩增５’端和３’端片段,第二轮ＰＣＲ以第一轮ＰＣＲ的５’端和３’端片段混合物为模板,用上下游引物扩增出全长片段。该文用逆转录－Ｏｖｅｒｌａｐｐｉｎｇ ＰＣＲ法快速高效地从前骨髓干细胞中扩增到人类ＰＵ．１全长ｃＤＮＡ为４４２ｂｐ,酶切和测序证实完全与前人报道一致。构建的真核表达质粒ｐｃＤＮＡ３．１－ｈＰＵ．１可以被特异的抗体识别,体外细胞株转梁证实具有特异的激活启动子活性的功能。     

叶酸及维生素B_(12)伍用抗精神病药物治疗精神障碍的疗效观察

本文报告了100例精神障碍患者,随机分为两组,Ⅰ组单用抗精神病药物治疗,Ⅱ组合并用叶酸及维生素 B_(12)治疗。两组在治疗前后均测血清叶酸及维生素 B_(12)浓度,其中Ⅱ组疗效较Ⅰ组明显。这些资料提示对精神障碍患者在常用抗精神病药物之外加用叶酸及维生素 B_(12)可能有益。     

褪黑素对大鼠脑缺血时MPO的活性和ICAM-1表达的影响

目的研究褪黑素(melatonin,MT)对大鼠脑缺血损伤区髓过氧化物酶(myeloperoxidase,MPO)的活性及细胞间黏附分子-1(intercellular adhesion molecule-1,ICAM-1)表达的影响。方法线栓法造成大鼠大脑中动脉栓塞制成脑缺血模型,分为正常对照组、缺血组、MT治疗组,MT治疗组缺血前30 min给予MT(20 mg/kg)腹腔注射。术后在相应时间点取缺血侧脑组织,用分光光度计检测MPO的活性,用免疫组织化学法检测ICAM-1蛋白的表达。结果与正常对照组比较,缺血组各时间点MPO的活性及ICAM-1蛋白的表达均升高,而与缺血组比较MT治疗组各时间点MPO的活性及ICAM-1蛋白的表达均降低。结论MT可能通过降低MPO的活性抑制白细胞浸润和降低ICAM-1蛋白表达对缺血脑组织产生保护作用。     

食管鳞状细胞癌中SDF-1的表达及DC抗肿瘤活性的影响

的探讨食管鳞癌中基质细胞衍生因子-1的表达与树突状细胞密度的相互关系及它们与患者临床病理特征的关系。方法运用免疫组化和原位杂交方法检测49例食管鳞癌组织中基质细胞衍生因子-1蛋白和mRNA、S-100蛋白标记的树突状细胞的表达,根据染色面积和染色强弱判断它们表达的水平。结果（1）肿瘤细胞分化和浸润深度与基质细胞衍生因子-1的表达呈正相关（rs分别为0．498、0．313,P〈0．05）;而与S-100蛋白阳性树突状细胞的密度呈负相关（rs分别为-0．501、-0．466,P〈0．05）。（2）基质细胞衍生因子-1与树突状细胞密度间存在负相关性（FS=-0．619,P〈0．05）。结论（1）基质细胞衍生因子-1蛋白的表达与食管鳞癌的临床病理特征存在明显相关性。（2）食管鳞癌中基质细胞衍生因子-1的表达可影响鳞癌组织中树突状细胞的密度,可能影响宿主的抗肿瘤能力及肿瘤的浸润转移。     

VKORC1-1639A/G基因多态性对机械心脏瓣膜置换术后妊娠期华法林抗凝效果的影响

目的 探讨维生素K环氧化物还原酶复合体亚单位1基因（VKORC1）多态性对机械心脏瓣膜置换术后妊娠期华法林抗凝效果的影响。方法 收集56例机械心脏瓣膜置换术后妊娠者,妊娠期全程口服华法林,应用聚合酶链反应、限制性片段长度多态性方法检测VKORC1基因多态性,同时记录服用华法林初始抗凝效果、维持剂量,以及妊娠结局情况。结果 VKORC1-1639AA型频率最高（78.57%）,VKORC1-1639AG型频率为21.43%,未发现VKORC1-1639GG型;VKORC1-1639AA型华法林初始达标剂量、时间及维持剂量均少于VKORC1-1639AG型（P〈0.05）;VKORC1-1639AA型妊娠结局优于VKORC1-1639AG型。结论 VKORC1-1639AA型对华法林敏感度更高;建议VKORC1-1639AA型妊娠期可考虑口服华法林,VKORC1-1639AG型建议联合用药。     

病程对1型糖尿病血脂、血尿酸水平及并发MS的影响

目的探讨病程对1型糖尿病病人血脂、血尿酸水平及并发代谢综合征（MS）的影响。方法随机选取144例1型糖尿病病人,依据病程长短分为A组（病程〈5年）、B组（病程5～15年）、C组（病程〉15年）,分析比较3组病人体质量指数（BMI）、血总胆固醇（TC）、三酰甘油（TG）、血尿酸（UA）水平及并发MS情况。结果C组病人BMI明显高于A组,差异有显著性（F=3.449,q=3.656,P〈0.05）;C组病人血TC、TG、UA水平及MS发生率明显高于B组,B组明显高于A组,差异有显著性（F=5.60～90.25,q=3.122～18.988,P〈0.01;χ^2=8.52,P〈0.05）。结论随着病程延长,1型糖尿病病人BMI、血TC、TG、UA水平逐渐升高,MS发生率增加。     

IN VIVO 1H MAGNETIC RESONANCE SPECTROSCOPY IN EVALUATION OF HEPATOCELLULAR CARCINOMA AND ITS EARLY RESPONSE TO TRANSCATHETER ARTERIAL CHEMOEMBOLIZATION

MAGNETIC resonance spectroscopy (MRS) isa new modality using nuclear magnetic reso-nance and chemical shift effect·It is the uni-que noninvasive technique to quantitatively study metabolicand biochemical concentrationin vivo·The spectrum con-sists with …     

DNA错配修复蛋白hMLH1在大肠癌组织中的表达及其与大肠癌分化的关系

目的:了解错配修复蛋白hMLH1在大肠癌组织中的表达及其与大肠癌分化的关系。方法:应用SP免疫组织化学法对65例大肠癌组织病理切片进行免疫组化分析。结果:大肠癌旁正常组织hMLH1的表达阳性率为80%,明显高于癌组织hMLH1的表达阳性率46.2%(P<0.01);高分化、中分化和低分化大肠癌组织中hMLH1的表达率分别为68.2%、44.1%和22.2%,高分化大肠癌与低分化大肠癌之间hMLH1的表达存在显著性差异(0.010.05)。结论:hMLH1表达水平与大肠癌的发生及分化相关,大肠癌分化程度越低,hMLH1表达水平越低。     

晚期乳腺癌患者ERCC1、RRM1表达与吉西他滨联合顺铂化疗疗效

目的 探讨ERCC1、RRM1在晚期乳腺癌中的表达与吉西他滨联合顺铂化疗疗效关系.方法 经病理组织学诊断的Ⅲb-Ⅳ期晚期乳腺癌患者30例,通过免疫组化法检测乳腺癌组织中ERCC1、RRM1的表达水平,预测吉西他滨联合顺铂在晚期乳腺癌中的化疗疗效.结果 晚期乳腺癌病理组织中ERCC1阳性率为30.0％ (9/30),低表达与高表达化疗后有效率分别为85.7％、33.3％,两组差异有统计学意义(P＜0.01);RRM1阳性率为33.3％ (10/30),低表达与高表达化疗有效率分别为65.0％、40.0％,两组差异有统计学意义(P＜0.01).结论 ERCC1和RRM1低表达的患者选用吉西他滨联合顺铂方案可提高化疗疗效.提示,ERCC1、RRM1可作为晚期乳腺癌选用吉西他滨联合顺铂方案化疗敏感性的预测指标.     

p27KIP1和CyclinE表达与喉鳞状细胞癌生物学行为的关系

目的:探讨p27^KIP1和CyclinE基因蛋白与喉鳞状细胞癌（LSCC）生物学行为的关系。方法：采用免疫组化法检测60例LSCC、40例喉不典型增生病变和20例喉正常粘膜中p27^KIP1和CyclinE基因蛋白的表达。结果：p27^KIP1和CyclinE阳性表达主要定位于细胞核；在LSCC、喉不典型增生病变和喉正常粘膜中p27^KIP1阳性表达率分别为：58．3％（35／60）、67．5％（27／40）和90．0％（18／20）（P＜0．05）；CyclinE阳性表达率分别为：45．0％（27／60），20．0％（8／40），5．0％（1／20）（P＜0．001）。LSCC中颈淋巴结转移组和非淋巴结转移组，淋巴结转移阳性组p27^KIP1阳性表达率明显低于阴性组（P＜0．01）；淋马结转移组CyclinE阳性表达率明显高于非淋巴结转移组（P＜0．01）。p27^KIP1和CyclinE表达显著相关性（P＜0．01）。结论：p27^KIP1和CyclinE异常表达可能是喉癌发生中早期分子事件，对喉癌发生发展起重要作用。     

EFFECT OF INTERLEUKIN-1β ON GROWTH HORMONE GENE EXPRESSION AND ITS POSSIBLE MOLECULAR MECHANISM IN RAT MtT/S SOMATOTROPH CELLS

Objective To elucidate the effect of interleukin-1β （IL- 1β） on human growth hormone （hGH） gene expression in a rat somatotropic pituitary cell line MtT/S.
Methods Stably transfected MtT/S cells were firstly established by transfecting 484-Lucl plasmid which contained hGH gene promoter --484 to ＋30 bp and luciferase reporter gene. The effect of IL-1β on hGH gene expression was determined by assaying the luciferase activities. RT-PCR method was also used to determine whether IL-1 recepor mRNA was expressed in MtT/S cells.
Results The 10^3 U/mL IL-1β stimulated secretion and synthesis of GH, and promoted the 5＇-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.38 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase （MAPKK/MEK） inhibitor PD98059 （40 μmol/L） and p38 mitogen-activated protein kinase （MAPK） inhibitor SB203580 （5 μmol/L） completely blocked the stimulatory effect of IL-1μ, and phosphatidylinositol-3-kinase （PI3-K） inhibitor LY294002 partly abolished the effect of IL-1μ. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit- 1 nor inhibition of Pit- 1 expression affected induction of hGH promoter activity by IL-1μ. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-1β, and results showed that the stimulatory effect of IL-1β was abolished following deletion of the --196 to -- 132 bp fragment.
Conclusions IL-1β promotes GH secretion and synthesis in rat MtT/S somatotroph cells. The stimulatory effect of IL-1β on hGH gene promoter appears to require the activation of MEK, p38 MAPK, PI3-K, and a fragment of promoter sequence that spans the -196 to -132 bp of the gene, but it may be unlinked with Pit-1 protein.