人端粒酶蛋白催化亚单位基因转染对人胚胎成纤维细胞增殖的影响

目的探讨外源性人端粒酶蛋白催化亚单位(hTERT)基因转染对人胚胎成纤维细胞(hEF)增殖的影响。方法体外培养hEF。应用脂质体转染法将pIRES2增强性绿色荧光蛋白(EGFP)hTERT正义重组质粒及pIRES2EGFP空载质粒分别导入hEF中,相应称为hEFhTERT和hEFEGFP。采用蛋白质印迹法(Westernblot)检测正常hEF、hEFhTERT、hEFEGFP中hTERT蛋白、分化抑制因子Id1、增殖细胞核抗原(PCNA)及Ⅰ、Ⅲ型胶原的表达水平。用噻唑蓝(MTT)法测定3种细胞的生长曲线,用流式细胞仪检测细胞周期并计算增殖指数(PI)。结果(1)Westernblot与hEFEGFP和hEF比较,hEFhTERT中的hTERT蛋白、Id1、PCNA和Ⅰ、Ⅲ型胶原表达水平均较高。(2)MTT法细胞接种后第4—6天,hEFhTERT的吸光度(A)值明显高于hEF和hEFEGFP(P<005),接种后1—6dhEF与hEFEGFP的A值比较,差异均无统计学意义(P>0.05)。(3)细胞周期与hEFEGFP和hEF比较,hEFhTERTG0/G1期细胞较少,S、G2/M期细胞较多;其PI为5747%,高于hEFEGFP(13.13%)和hEF(17.38%)。结论外源性hTERT基因转染可使hEF增殖能力增强。     

模拟缺血再灌注后心肌细胞胰岛素抵抗机制的初步研究

Objective Recent.studies have found a strong association of insulin resistance, which might occur during ischemia reperfusion in vitro in the experimental dogs, with disturbed function of cardiomyocytes. Obvious acute insulin resistance, along with glucose dysmetabolism in the reperfused cardiomyocytes, was furher observed in the study performed with ischemia-reperfused ventric- ular myocytes of rats. We tried to investigate preliminarily the molecular mechanisms of insulin resistance in the cardiomyocytes after ischemia reperfusion. Methods An experimental model of insulin-stimulated ischemia reperfusion (SI/R) was created by isolating cardiomyocytes from adult rats. Glucose uptake of the cardiomyoctyes was evaluated with isotope-labeling technique. Glucose trans- porter 4 (GLUT4) translocation induced by insulin was investigated with Western blot analysis, and the intracellular level of free Ca2+ ([Ca2+]I) was measured quantitatively with Ca2+ indicator Fura-2. Results Insulin can stimulated glucose uptake by cardiomyo- cytes, indicating that these cells were insulin-sensitive. Cardiomyocytes were demonstrated notable acute insulin resistmce during reperfusion. Insulin-stimulated GLUT4 translocation in the cardiomyocytes 15 minutes after reperfusion was 72.2% of that in the con- trol group(P<0.05), in which the GLUT4 content in plasma membrane remained unchanged. The finding suggested that a disturbed GLUT4 translocation might happen in the cardiomyocytes during insulin-stimulated ischemia-reperfusion. Calcium overload was identi- fied in the cardiomyocytes with ischemia reperfusion. At 15 minutes of reperfusion, [Ca2+]I was significantly higher in the reperfused cardiomyocytes than that in the control cardiomyocytes[(318.66±23.06)vs(130.70±0.82) nmol/L, P<0.05], and kept at a higher level [(177.79±17.46) nmol/L] at 60 minutes of reperfusion (P<0.05, vs control). Partial correlation analysis revealed a negative correlation of[Ca2+]I with insulin-induced ghcose uptake in the cardiomyoctyes (r = -0.557,P=0.006). Conclusion Disturbed GLUT4 translocation and decreased intrinsic activity may be important molecular mechanisms for the development of insulin resistance in the cardiomyocytes of rat during insulin-simulated ischemia reperfusion,. [Ca2+]I overload may account for the de- creased intrinsic activity d GLUT4.     

CpG-ODN对婴幼儿喘息性支气管炎外周血单个核细胞T-bet表达的影响

目的：探讨CpG-ODN干预对婴幼儿喘息性支气管炎外周血单个核细胞（PBMC）T-bet表达和IFN-γ分泌的影响。方法：对28例喘息性支气管炎（喘支组）患儿于急性期和恢复期分别抽取抗凝静脉血并分离PBMC,以终浓度为10μg/ml的B型CpG-ODN刺激48小时,分别应用RT-PCR和Western blot检测CpG-ODN刺激前后患儿PBMC的T-bet mRNA和蛋白表达情况,以ELISA检测PBMC培养上清IFN-γ的变化,同时设立26例健康婴儿对照组。结果：与正常对照组比较,喘支患儿PBMC表达T-bet mRNA及蛋白明显减少（P〈0.05）,应用CpG-ODN刺激后,两组小儿PBMC表达T-bet均显著上调,但产生IFN-γ无明显增多,对照序列ODN对T-bet表达及IFN-γ的分泌无明显影响。相关分析表明,喘支患儿T-bet mRNA与IFN-γ分泌呈正相关（r=0.57,P〈0.01）,CpG-ODN刺激后,二者相关性明显下降（r=0.19,P〉0.05）。结论：T-bet在喘支患儿PB-MC中表达减少,提示T-bet表达缺陷参与了喘支的免疫病理,B型CpG-ODN作为一种免疫调节剂,可有效上调T-bet的表达,但不能诱导IFN-γ的产生。     

小型钛板坚固内固定在治疗下颌骨折中的应用

目的：探讨小型钛板坚固内固定在治疗下颌骨折中的疗效。方法：62例下颌骨折患者切开复位，根据骨折部位和骨折线情况选用钛板置于下颌骨折线上内固定。结果：62例应用此项技术取得了满意疗效，较好地恢复了咬牙厶功能。结论：下颌骨折小型钛板内固定操作简单，固定可靠，可缩短颌间固定的时间。口内切口面部无疤痕，无面神经损伤的危险，是一种较理想的治疗下颌骨折的内固定方法。     

血浆同型半胱氨酸水平及MTHFR基因多态与血管性痴呆的关系

目的探讨血浆同型半胱氨酸(H cy)水平及M THFR基因多态与血管性痴呆(VD)的关系。方法应用高效液相色谱仪和电化学检测法测定37例VD患者的血浆总H cy水平,并与40例同龄对照组及40例非痴呆脑梗死组比较,运用多聚酶链反应-限制性内切酶片段长度多态性技术(PCR-RFLP)检测M THFR基因多态性,同时测定血浆叶酸及维生素B12水平。结果VD患者血浆总H cy水平显著高于同龄对照组(P<0.001)和非痴呆脑梗死组(P<0.05);M THFR基因型有3种,即纯合子(T/T)型,杂合子(T/C)型,纯合子(C/C)型。3组基因型和等位基因频率相比,差异均无显著性(P>0.05);VD组血浆叶酸及维生素B12水平明显低于同龄对照组(P<0.05)和非痴呆脑梗死组(P<0.05)。结论高同型半胱氨酸血症是VD发病的一个新的危险因素。     

模拟缺血再灌注后心肌细胞胰岛素抵抗机制的初步研究

Objective Recent.studies have found a strong association of insulin resistance, which might occur during ischemia reperfusion in vitro in the experimental dogs, with disturbed function of cardiomyocytes. Obvious acute insulin resistance, along with glucose dysmetabolism in the reperfused cardiomyocytes, was furher observed in the study performed with ischemia-reperfused ventric- ular myocytes of rats. We tried to investigate preliminarily the molecular mechanisms of insulin resistance in the cardiomyocytes after ischemia reperfusion. Methods An experimental model of insulin-stimulated ischemia reperfusion (SI/R) was created by isolating cardiomyocytes from adult rats. Glucose uptake of the cardiomyoctyes was evaluated with isotope-labeling technique. Glucose trans- porter 4 (GLUT4) translocation induced by insulin was investigated with Western blot analysis, and the intracellular level of free Ca2+ ([Ca2+]I) was measured quantitatively with Ca2+ indicator Fura-2. Results Insulin can stimulated glucose uptake by cardiomyo- cytes, indicating that these cells were insulin-sensitive. Cardiomyocytes were demonstrated notable acute insulin resistmce during reperfusion. Insulin-stimulated GLUT4 translocation in the cardiomyocytes 15 minutes after reperfusion was 72.2% of that in the con- trol group(P<0.05), in which the GLUT4 content in plasma membrane remained unchanged. The finding suggested that a disturbed GLUT4 translocation might happen in the cardiomyocytes during insulin-stimulated ischemia-reperfusion. Calcium overload was identi- fied in the cardiomyocytes with ischemia reperfusion. At 15 minutes of reperfusion, [Ca2+]I was significantly higher in the reperfused cardiomyocytes than that in the control cardiomyocytes[(318.66±23.06)vs(130.70±0.82) nmol/L, P<0.05], and kept at a higher level [(177.79±17.46) nmol/L] at 60 minutes of reperfusion (P<0.05, vs control). Partial correlation analysis revealed a negative correlation of[Ca2+]I with insulin-induced ghcose uptake in the cardiomyoctyes (r = -0.557,P=0.006). Conclusion Disturbed GLUT4 translocation and decreased intrinsic activity may be important molecular mechanisms for the development of insulin resistance in the cardiomyocytes of rat during insulin-simulated ischemia reperfusion,. [Ca2+]I overload may account for the de- creased intrinsic activity d GLUT4.     

一低剂量辐射诱导新基因的转录调控和初步功能分析

目的：克隆低剂量辐射诱导基因，研究辐射对其转录调控作用和生物学功能。方法：用mRNA差异显示技术分离辐射诱导表达基因，用RACE技术获取cDNA旁侧序列，Northern杂交分析基因的转录调节，生物信息学分析基因的结构和功能。结果：获得包括3'端在内的辐射诱导新基因LRIGx的cDNA片段，序列同源性比较显示与人染色体20ql 1.2-12一段DNA高度同源（＞99％），Northern杂交结果揭示该基因转录子全长约8．5kb，在0．2Gy照射后2h出现诱导表达，4h后转录子水平为对照细胞的5倍多，也受0．02Gy更低剂量照射诱导表达，2Gy大剂量照射后1h出现短暂诱导表达，但不如低量照射明显，2h后恢复正常水平，生物信息学分析结果显示该基因编码产物含有解旋酶活性保守区，结论：分离鉴定出一低剂量辐射反应基因，其编码蛋白可能参与DNA代谢（如修复）等细胞辐射反应过程。     

免疫吸附预防高致敏肾移植受者超急性排斥反应

目的探讨免疫吸附对高致敏‘肾移植受者超急性排斥反应的预防作用。方法对10例群体反应抗体（PRA）〉40％的‘肾移植受者术前行免疫吸附治疗，观察其超急性排斥反应发生情况及不良反应。结果10例高致敏肾移植受者均未发生超急性排斥反应，仅有2例发生急性排斥反应，并通过免疫吸附及调整免疫抑制剂得到逆转。所有受者随访至今移植‘肾功能良好，未发生排斥反应。结论免疫吸附可以安全、有效地预防高致敏人群‘肾移植术后超急性排斥反应。     

定量组织多普勒对主动脉瓣硬化左室舒张功能的评价

目的:应用定量组织多普勒速度成像(QTVI)技术探测二尖瓣环舒张早、晚期运动速度,评价主动脉瓣硬化(AVS)病人左室舒张功能。方法:选择主动脉瓣硬化无主动脉瓣狭窄54例,主动脉瓣硬化伴主动脉瓣狭窄38例,正常对照组43例。取标准心尖四腔观,采用QTVI测定二尖瓣环侧壁段舒张早期最大运动速率(Em)、舒张晚期最大运动速率(Am);应用脉冲波多普勒测量二尖瓣口舒张早期最大流速曲线(E)和舒张晚期最大流速曲线(A)。结果:主动脉瓣硬化伴主动脉瓣狭窄组Em、Am均低于正常对照组和主动脉瓣硬化无主动脉瓣狭窄组(P<0.05),E、A均高于正常对照组和主动脉瓣硬化无主动脉瓣狭窄组(P<0.05);主动脉瓣硬化无主动脉瓣狭窄组与正常对照组相比,Em降低(P=0.015)、A明显升高(P<0.001);E、Am无显著性差异(P>0.05)。结论:主动脉瓣硬化患者左室舒张功能下降,组织多普勒较血流脉冲频谱多普勒更能敏感、精确地反映主动脉瓣硬化尤其是主动脉瓣硬化伴主动脉瓣狭窄患者的左室舒张功能改变。     

DNA-PKcs反义核酸增强HeLa细胞对辐射及化疗药物顺铂的敏感性

目的构建表达DNA-PKcs反义核酸的真核表达载体，建立DNA—PKcs表达被反义核酸抑制的细胞模型。方法采用RT-PCR扩增DNA—PKcs的cDNA片段，DNA重组技术构建四环素控制表达的真核表达载体；利用Lipofectamine介导基因转染，Western blot分析鉴定反义核酸抑制DNA—PKcs表达的细胞模型；细胞克隆形成法和细胞生长曲线分析细胞对UV和顺铂的敏感性。结果克隆了DNA-PKcs 5’端320bp cDNA片段，重组于tet启动子控制表达质粒pCl^neo-Tet-splice，转染Hela细胞，获得3个稳定转化克隆；Western blot结果表明细胞中DNA-PKcs表达受到反义核酸的抑制，并且细胞对紫外线辐射和顺铂敏感性增加。结论成功建立了DNA-PKcs表达受到反义核酸抑制的细胞模型，抑制DNA-PKcs表达提高了细胞的辐射和顺铂的敏感性。