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1.
为了探讨铝对中枢神经系统多巴胺能神经元的影响,采用地高辛标记酪氨酸羟化酶(TH)反义RNA探针原位杂交组织化学方法,观察了氯化铝对大鼠黑质THmRNA表达的影响。结果发现,大鼠服用氯化铝3个月后,黑质背侧部多巴胺神经元THmRNA杂交信号弱于对照组,表明氯化铝对大鼠黑质THmRNA表达有抑制作用。  相似文献   

2.
目的构建人生长激素释放激素变异受体1型(GHRHR-SVT1)基因绿色荧光蛋白真核表达载体1(pEG—FP—N1)并在鼠成纤维细胞株NIH-3T3细胞中的表达。方法GHRHR-SVT1基因是由人胃癌细胞株AGS中扩增出的DNA片段,克隆入真核表达载体pEGFP-N1中,用高效转染试剂将pEGFP-N1/GHRHR—SVT1转染NIH-3T3细胞,NIH-3T3细胞分3组:非转染组(对照组),只加转染试剂不加质粒;转染空质粒组(pEGFP-N1组),加转染试剂与空质粒;重组质粒转染组(pEGFP-N1/GHRHR-SVT1组),加转染试剂与重组质粒。采用四甲基偶氮唑盐(MTT)法和流式细胞仪检测各组转染后各组NIH-3T3细胞的生长情况。结果(1)pGEFP—N1/GHRHR-SVT1经限制性内切酶XhoⅠ和BarnHⅠ双酶切产生约1080bp和4700bp2条带。(2)构建的GHRHR-SVT1cDNA序列与GHRHR—SVT1原始序列(AF-282259)进行对比,第54位碱基突变(A突变为T),为无义突变。(3)重组质粒转染组NIH-3T3细胞增殖率明显高于对照组(P〈0.01)。(4)重组质粒转染组NIH-3T3细胞增殖指数明显高于对照组(P〈0.01)。结论在适当浓度GHRH(10μmol/L)的培养基中,GHRHR—SVT1能够促进NIH-3T3细胞增殖,并为肿瘤的治疗提供新的思路。  相似文献   

3.
胡蕴玉  谭祖键 《中华医学杂志》2001,81(17):1070-1073
目的 通过基因转染方法,使成纤维细胞NIH-3T3细胞具有合成及分泌骨形态发生蛋白(BMP)、体内诱导新骨生成的能力。方法 定向克隆法将BMP3基因构建于PcDNA3表达载体,脂质体介导法转染NIH-3T3细胞,G418筛选获得稳定转化体,Northern印迹法检测转染细胞有无BMP3基因表达,免疫组织化学检测转染细胞内的BMP3蛋白合成,钙钴法染色不同时相的转染细胞碱性磷酸酶并作图像分析。将转染细胞注入裸鼠肌袋,观察注射后第4周诱导新骨生成情况。结果 转染细胞体外培养6周Northern印迹法检测到有明显的BMP3基因表达,免疫组织化学染色见转染细胞培养筛选5-8周后细胞浆内有染成黄色的BMP3蛋白质颗粒出现,图像分析蛋白质生成在转染后第6周达高峰。钙钴法碱性磷酸酶染色及图像分析示转染细胞内碱性磷酸酶明显高于同期对照组,两组间有显著性差异(P<0.01)。被转染NIH-3T3细胞裸鼠肌袋诱导成骨实验见注射后4周注射部位有大量软骨细胞出现,并伴有骨小梁生成。结论 通过基因转染方法可以使NIH-3T3细胞具有合成及分泌内源性BMP的能力,合成及分泌的BMP具有良好的生物活性,肌袋实验具有诱导成骨作用。但是,由于基因治疗的某些不可控性因素,其安全性仍有待于进一步研究。  相似文献   

4.
目的 构建MAGE-1基因重组真核表达质粒并在细胞系NIH-3T3中高效表达。方法用RT-PCR方法从人肝细胞肝癌(HCC)组织全RNA中扩增出MAGE-1基因cDNA序列,克隆至真核表达载体pcDNA3.1(+),构建pcDNA3.1-MAGE-1重组质粒.重组质耗用脂质体转染NIH-3T3细胞,经RT-PCR和Western-blot鉴定转化细胞中MAGE-1的表达。结果RT-PCR获得长度为927bp的MAGE-1基因,经T载体和pcDNA3.1(+)真核表达载体克隆、酶切鉴定及序列分析后,证实重组质粒构建正确,并存转化细胞中检测到了MAGE-1的表达。结论 真核表达质粒pcDNA3.1-MAGE-1构建成功,并建立了稳定表达人MAGE-1的NIH-3T3细胞株。  相似文献   

5.
目的 将Aβ阻断肽(ABAD-DP) cDNA插入人硫氧化还原蛋白(hTRX)的活化位点,克隆融合基因TRX1-ABAD-DP-TRX2 (T-A-T) cDNA,为基因治疗阿尔茨海默病奠定基础.方法 通过PCR获得目的片段TRX1、TRX2、ABAD-DP,进一步将上述3目的片段按既定顺序插入腺相关病毒穿梭质粒pSSHG-CMV的多克隆位点,从而获得融合基因TRX1 -ABAD-DP-TRX2.经PEI介导Hela细胞包装重组腺相关病毒rAAV/T-A-T.感染NIH-3T3细胞,通过荧光免疫组织化学染色检测融合基因的表达及其与Aβ42的定位.结果 酶切后得到TRX1 -ABAD-DP-TRX2融合基因片段的大小约为435 bp,与理论值一致.荧光免疫组织化学染色检测到融合基因的表达,且共定位组T-A-T和Aβ42均位于NIH-3T3细胞胞质内.结论 成功克隆及表达了Aβ阻断肽适配子TRX1-ABAD-DP-TRX2融合基因,为进一步将阻断肽用于基因治疗阿尔茨海默病提供了基础.  相似文献   

6.
pEgr-sHemopexin重组质粒的构建及体外辐射诱导表达   总被引:1,自引:0,他引:1  
目的:克隆小鼠分泌型Hemopexin (sPEX) 编码区的cDNA序列,构建含Egr-1启动子的pEgr-sPEX真核表达载体,检测辐射诱导重组质粒在B16F10细胞中的表达。 方法:用RT-PCR从NIH3T3细胞中扩增出sPEX,经测序证实后,构建pEgr-sPEX重组质粒,以脂质体转染B16F10细胞,用Western blotting方法检测B16F10细胞上清中辐射诱导PEX的表达。 结果:测序表明扩增的sPEX cDNA序列与GenBank中登录的序列完全一致,sPEX cDNA正确插入表达载体pcDNA3.1-Egr-1,转染入B16F10细胞内PEX基因在体外辐射诱导下成功获得表达。结论:成功地克隆了小鼠sPEX基因,并在体外证实了pEgr-sPEX具有辐射诱导表达特性。  相似文献   

7.
组织因子对人类大肠癌培养细胞侵袭能力的影响   总被引:5,自引:1,他引:4  
目的:分析组织因子表达对人类大肠癌细胞(HT—29细胞)体外侵袭能力的影响。方法:利用构建有正义/反义组织因子cDNA(TF cDNA)的真核表达载体pcDNA3.1/Zeo,以脂质体法转染HT—29细胞,经验证转染成功的HT—29细胞采用Western印迹分析检测组织因子的蛋白表达水平并进行基质胶(Matrigel)侵袭实验观察细胞体外侵袭能力的变化。结果:转染了正义TFcDNA的HT—29细胞的组织因子表达水平及侵袭能力较未转染的HT—29细胞升高;转染了反义TFcDNA的HT—29细胞的组织因子表达水平及侵袭能力较未转染的HT—29细胞相比降低。结论:组织因子可以增加人类大肠癌细胞的体外侵袭能力。  相似文献   

8.
目的 用RNA干扰方法建立稳定的热休克蛋白90α(HSP90α)抑制表达细胞株,研究热休克蛋白90α抑制表达对细胞应激反应的影响。方法 将人Hsp90αRNA干扰基因片段连接入pSilencer2.1-U6 neo载体,重组质粒pSilencerHSP90经亚克隆、纯化、测序鉴定后,用电穿孔法转染到小鼠成纤维细胞系NIH-3T3细胞内。经G418筛选、克隆分离培养,用免疫荧光、免疫印迹鉴定阳性克隆。用高温作用(44℃,40min)模拟氧化应激环境,以NIH-3T3细胞为对照,流式细胞仪测定DNA受损细胞数,分析在细胞应激状态下对细胞膜和DNA的损害及HSP90低表达对细胞应激反应的影响。结果 转染pSilencerHSP90的NIH-3T3细胞Hsp90免疫荧光染色减弱,主要分布于胞浆;与对照相比,44℃,40min时DNA的损害加重(9.2%vs18.5%,P〈0.01)。结论 建立稳定低表达HSP90αNIH-3T3细胞株;热休克蛋白90表达与其应激保护作用密切相关。  相似文献   

9.
目的:探讨体外培养大鼠海马神经元癫痫模型中神经元丢失的分子机制。方法:制备大鼠海马神经元癫痫样放电细胞模型,以膜片钳全细胞记录对模型放电进行检测,采用RT-PCR法克隆大鼠全长caspase3 cDNA,采用原位杂交和流武细胞术检测模型中caspase3基因表达和神经元凋亡情况。结果:成功克隆获得大鼠全长caspase3 cDNA,其序列与文献报道一致。模型组海马神经元癫痫样放电3h后caspase 3基因表达开始增多,6h后凋亡细胞开始明显增加,两者均较对照组明显增加。结论:癫痫样放电可能启动神经元caspase3表达,继而介导神经元凋亡。  相似文献   

10.
目的构建一种含人肽基脯氨酰顺反异构酶Pin1的质粒,以绿色荧光蛋白作为报告基因,观察其在宫颈癌HeLa细胞中的表达。方法从人宫颈癌HeLa细胞中,以RT-PCR方法扩增出人Pin1基因全长cDNA序列(CDS),插入增强绿色荧光蛋白表达质粒pEGFP-C1,脂质体法转染HeLa细胞,荧光显微镜下观测绿色荧光蛋白的表达,免疫印迹法检测GFP-Pin1蛋白的表达。结果RT-PCR扩增出人Pin1基因;构建pEGFP-C1-Pin1重组质粒,体外成功转染入宫颈癌HeLa细胞,在荧光显微镜下可见强绿色荧光蛋白的表达,免疫印迹法证实存在GFP-Pin1蛋白高表达;经G418筛选获得单细胞克隆。结论重组质粒pEGFP-Pin1体外转染HeLa细胞后,目的基因能够在细胞内有效表达,检测方法简便可靠,为利用转染该目的基因的细胞进行下一步研究提供了实验基础。  相似文献   

11.
Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P< 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P< 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.  相似文献   

12.
Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P < 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (>0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P <0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P < 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.  相似文献   

13.
Objective: To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods: SD rats served as both donors and recipients. 4℃ sodium lactate Ringer's was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava (IVC) inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results: Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion: This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.  相似文献   

14.
Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.  相似文献   

15.
Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.  相似文献   

16.
A clinical guideline for the therapeutic interventions of integrative medicine may be defined as a written document which states a series of recommendations on therapeutic interventions of integrative medicine for a special disease or condition. The guideline may provide assistance to medical professionals in making clinical decisions aimed at improving the clinical outcome of patients and reducing the costs of medical care(~'4~. Recommendations issued by a guideline should be based on the best available evidence in both Western and Chinese medicine. For fulfilling this purpose, the development of clinical guidelines for therapeutic interventions in the field of integrative medicine should follow scientific principles and undergo a rigorous processes.  相似文献   

17.
Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.  相似文献   

18.
Journal of Nanjing Medical University (English Edition) JNMU, sponsored by Nanjing Medical University, was established in 1987. It is a bimonthly comprehensive English medical journal published locally and abroad.Since 2007, Journal of Nanjing Medical University (English Edition )was granted Elsevier the full publishing and distribution rights worldwide for the Electronic Edition, excluding the People's Republic of China.  相似文献   

19.
目的:评价使用安心颗粒对急诊经皮冠状动脉介入术(PPCI)术后生活质量的影响.方法:将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组(术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次)和对照组(仅接受基础药物治疗).所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表(MIDAS)、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果:入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异(P>0.05);出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低(P<0.05);组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低(P<0.05).两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血(P>0.05).两组发生轻度血小板减少症的患者数比较无差异(P>0.05).结论:PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.  相似文献   

20.
Objective To measure Derpl and Blot5 allergen levels in asthmatics' homes in Hongkong.Methods Seventy houses were enrolled for a mite indoor environment study. Dust samples were obtained from two sites of each patients' house: bed and floor. Derpl and Blot5 levels were quantified by a two-site monoclonal antibody-based ELISA technique.Results The levels of Derpl allergens found in bed (geometric mean (GM) 3.43 μg/g of dust; 95%CI, 1.89-4.96 μg/g)and on the floor (GM 1.12 μg/g of dust; 95%CI, 0.71-1.53 μg/g) indicated significant differences (P=0.005). However, the levels of Blot5 allergens found in bed (GM 19.00 μg/g of dust; 95%CI, 0.89-38.90 μg/g) and on the floor (GM 6.14 μg/g of dust; 95%CI, 0.40-11.90 μg/g) showed no statistically significant difference. In addition, in regards to the exposure index for Derpl and Blot5 allergens found in bed and on the floor, 17.6% in bed and 8.6% on the floor had levels of Blot5 ≥ 10 μg/g of dust, higher than those obtained for Derp1 (7.2% and 0% in bed and on the floor respectively, P< 0.05); higher percentages in bed and on the floor (25.0% and 35.7%) were observed for levels of Blot5 =0 μg/g of dust as compared with Derpl in bed and on the floor (4.3% and 14.5% respectively, P< 0.05).Conclusions Derpl and Blot5 are the major allergens found in this regional study, Blot5 is a more potent allergen in Hongkong, probably reflecting the high level of exposure to Blomia tropicalis (Bt). Bt and Dermatophagoides pteronyssinus (Dp) allergens should be included for precise diagnosis and effective immuno-therapeutic treatment of mite allergy in Hongkong.  相似文献   

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