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1.
目的克隆、构建绿色荧光蛋白(GFP)-AWP1(associatedwithproteinkinaseCrelatedkinase1,AWP1)表达载体,观察AWP1在293细胞中表达和定位。方法采用逆转录PCR(RT-PCR)法从人ECV304内皮细胞中扩增AWP1cDNA编码区,并将其重组于GFP表达载体pEGFP-C2中。经酶切、序列鉴定分析后,将该重组质粒通过DOTAP脂质体介导,转染293细胞。荧光显微镜观察AWP1在细胞内的表达和分布。结果GFP-AWP1融合基因表达载体经酶切鉴定和测序分析确认构建成功,并在293细胞中获得了高效表达。荧光显微镜下,在不携带外源基因的空载体pEGFP-C2转染的对照组293细胞,绿色荧光均匀分布于整个细胞中;在重组质粒pEGFP-C2/AWP1转染的293细胞,绿色荧光弥散分布于细胞质内。结论成功构建GFP-AWP1融合基因表达载体并表达于293细胞胞质中。  相似文献   

2.
赵宁  邓新燕  单于  郭中敏 《热带医学杂志》2012,12(12):1430-1433,1441,1552
目的 构建表达增强型绿色荧光蛋白(EGFP)、rtTA双基因的重组逆转录病毒载体pLXSN-EGFP-rtTA,并在PA317细胞中表达.方法 以质粒pEGFP-C1为模板进行PCR反应获得EGFP基因片段,定向插入逆转录病毒载体pLXSN,筛选出正确的重组逆转录病毒载体pLXSN-EGFP.PCR方法从pTet-on质粒中扩增出rtTA基因,酶切纯化后克隆于逆转 录病毒载体pLXSN-EGFP中,限制性内切酶酶切分析及PCR鉴定筛选出正确的重组载体pLXSN-EGFP-rtTA,脂质体介导转染PA317细胞,PCR方法检测PA317细胞内EGFP、rtTA基因,RT-PCR方法检测PA317细胞内rtTA基因的mRNA表达,荧光显微镜观察EGFP基因的表达情况.以NIH3T3细胞为靶细胞测定重组逆转录病毒滴度.结果 成功构建了表达EGFP、rtTA双基因的重组逆转录病毒载体,转染PA317细胞后,RT-PCR方法可检测到rtTA基因的mRNA表达,荧光显微镜下可观察到EGFP基因表达所产生的绿色荧光,说明连接有EGFP和rtTA双基因的逆转录病毒载体在PA317细胞中可正常表达,转染pLXSN-EGFP-rtTA载体的PA317细胞可产生4.8x 104 CFU/ml病毒滴度的重组病毒.结论 成功构建表达EGFP、rtTA双基因的重组逆转录病毒载体,该载体可在PA317细胞内正常表达,获得较高滴度的病毒上清.  相似文献   

3.
目的 探讨构建人巨细胞病毒(HCMV)截短UL54(UL54S)基因融合增强型绿色荧光蛋白(EGFP)表达质粒pUL54S-EGFP转染人胚肾293细胞,瞬时表达UL54S和EGFP的可行性.方法 采用PCR法扩增UL54s基因并将其克隆入真核表达载体pEGFP-N1中,采用脂质体法转染AD293细胞后通过荧光显微镜和流式细胞仪分析EGFP的表达情况.结果 重组质粒pUL54S-EGFP转染AD293细胞12h后即可见到荧光蛋白的表达,至48h表达达高峰;转染48h pUL54S-EGFP组平均转染率为47%.平均荧光强度为547.结论 重组质粒pUL54S-EGFP可在人胚肾293细胞内瞬时表达UL54S和EGFP融合蛋白,EGFP的表达可报告UL54基因的表达,质粒pUL54S-EGFP构建成功.  相似文献   

4.
目的:构建人H2B基因与GFP融合的绿色荧光蛋白真核表达质粒,并在HEK293细胞中表达.方法:通过RT-PCR从HEK293细胞总RNA中扩增获得H2B基因的部分cDNA序列,与载体pEGFP-C1连接构建H2B基因与GFP融合表达的真核表达质粒pEGFP/H2B,经酶切和测序鉴定;应用脂质体转染技术将重组质粒转染HEK293细胞观察融合蛋白的表达.结果:成功构建H2B基因与GFP融合的绿色荧光蛋白真核表达载体,与对照组中转染空载体pEGFP-C1的HEK293细胞中的弥漫荧光相比,转染重组质粒的细胞中可观察到绿色荧光蛋白集中表达在细胞核内,分裂期细胞中可见与染色体形态一致的绿色荧光.结论:该载体的成功构建为研究染色体的分离机制建立了有效的观察体系.  相似文献   

5.
目的 构建携EGFP的人低氧诱导因子(HIF-1α)真核表达载体,为研究人HIF-1α基因对冠心病的血管新生作用奠定基础。方法 采用分子克隆技术,KpnⅠ、XbaⅠ双酶切pcDNA3.1/V5-Hin—HIF-1仅获得HIF-1仅cDNA,克隆到质粒pcDNA3.1(+),得到质粒pcDNA3.1(+)-HIF-1α,以KpnⅠ,ApaⅠ双酶切质粒pcDNA3.1(+)-HIF-1仅获得HIF-1α cDNA,克隆到质粒pEGFP—c1得到pEGFP-HIF-1α-c1质粒。脂质体法转染HEK293细胞,用荧光显微镜和Western blotting检测EGFP和HIF-1α 在HEK293细胞的表达。均显示融合蛋白在细胞中表达。结果经酶切鉴定及PCR证实重组pEGFP-HIF-1α-c1质粒构建成功。荧光显微镜和Westom blotting显示EGFP和HIF-1α融合蛋白在HEK293细胞中表达。结论 成功构建重组真核表达载体pEGFP—HIF-1α—c1并在HEK293细胞表达,为冠心病的HIF-1基因治疗研究奠定更直观的基础。  相似文献   

6.
目的 利用腺病毒载体介导的RNAi技术,建立糖皮质激素受体基因阻断的人K562细胞模型。方法 将针对糖皮质激素受体发挥RNAi效应的合成寡核苷酸序列连入含有绿色荧光蛋白基因的腺病毒穿梭质粒pRNAT-H1.1/Adeno中,在含有pAdEasy-I病毒骨架的BJ5183大肠菌内进行同源重组;重组体通过脂质体介导转染293T细胞,并在293细胞内包装为具有感染能力的病毒颗粒,通过反复感染扩增病毒以达感染靶细胞的适当滴度,通过绿色荧光表达来监测腺病毒扩增;应用Western blot法检测人K562细胞感染病毒后糖皮质激素受体蛋白的表达。结果 重组腺病毒穿梭质粒psiGR/Adeno和重组腺病毒pAdEasy-siGR的成功构建;转染腺病毒载体的293T细胞表达绿色荧光,随着时间逐渐增强并且出现明显的细胞病变效应,经过3轮扩增,病毒达到合适的滴度;受腺病毒感染的K562细胞内糖皮质激素受体蛋白表达明显降低。结论 可以成功构建针对糖皮质激素受体基因的RNAi腺病毒载体,为进一步研究糖皮质激素受体的生物学功能奠定基础。  相似文献   

7.
目的 克隆、构建绿色荧光蛋白(GFP)-AWPI(associated with protein kinase Crelated kinase I,AWPI)表达载体,观察AWPI在293细胞中表达和定位。方法 采用逆转录PCR(RT—PCR)法从人ECV304内皮细胞中扩增AWPI cDNA编码区.并将其重组于GFP表达载体pEGFP—C2中。经酶切,序列鉴定分析后,将该重组质粒通过DOTAP脂质体介导,转染293细胞。荧光显微镜观察AWPI在细胞内的表达和分布。结果 GFP—AWPI融合基因表达载体经酶切鉴定和测序分析确认构建成功,并在293细胞中获得了高效表达。荧光显微镜下,在不携带外源基因的空载体pEGFP—C2转染的对照组293细胞,绿色荧光均匀分布于整个细胞中;在重组质粒pEGFP—C2/AWPI转染的293细胞,绿色荧光弥散分布于细胞质内。结论 成功构建GFP—AWPI融合基因表达载体并表达于293细胞胞质中。  相似文献   

8.
9.
目的:构建信号调节蛋白α-绿色荧光蛋白(SIRPα-GFP)真核表达载体,转染HEK293T细胞获得稳定表达SIRPα-GFP融合蛋白的细胞系,研究SIRPα-GFP融合蛋白在HEK293T细胞的膜定位。方法:将SIRPα质粒和带有GFP的表达载体分别用限制性内切酶MluⅠ和SgfⅠ进行双酶切,将SIRPα基因克隆到pLenti-GFP载体中,构建pLenti-SIRPα-GFP质粒;将pLenti-SIRPα-GFP质粒转化DH5α大肠杆菌感受态细胞,对重组质粒pLenti-SIRPα-GFP进行DNA测序;采用Lipofectamine 3000转染试剂将pLenti-SIRPα-GFP质粒转染到HEK293T细胞中,通过荧光显微镜观察SIRPα-GFP在稳定转染HEK293T细胞中的表达,采用Western blotting法检测HEK293T细胞中SIRPα-GFP融合蛋白的表达。结果:酶切鉴定和DNA测序,重组质粒pLenti-SIRPα-GFP构建成功。荧光显微镜检测,SIRPα-GFP融合蛋白在HEK293T细胞膜上表达。Western blotting法检测,SIRPα蛋白在pLenti-SIRPα-GFP质粒转染的HEK293T细胞中成功表达。结论:成功构建了pLenti-SIRPα-GFP质粒。通过在HEK293T细胞中转染pLenti-SIRPα-GFP质粒,成功制备了稳定表达SIRPα-GFP的细胞系。SIRPα-GFP蛋白定位于HEK293T细胞的细胞膜上。  相似文献   

10.
人CTLA4Ig和EGFP双基因共表达逆转录病毒载体构建及鉴定   总被引:3,自引:1,他引:2  
目的 构建并鉴定人CTLA4Ig和增强型绿色荧光蛋白(EGFP)双基因共表达逆转录病毒载体。方法 利用基因重组技术将IRES序列与CTLA4Ig和EGFP基因构建到逆转录病毒载体中,脂质体法将重组质粒pCTLA4Ig-IRES2-EGFP转染PA317细胞,在G418选择压力下,通过流式细胞检测筛选得到高效共表达CTLA4Ig和EGFP并能产生高滴度重组逆转录病毒的PA317细胞克隆,MTT法测定CTLA4Ig生物学活性。结果 成功构建出含IRES序列的CTLA4Ig和EGFP双基因共表达逆转录病毒载体,生物学活性测定表明CTLA4Ig生物学活性没有受到影响。结论 IRES序列调控CTLA4Ig与EGFP双基因在细胞中同时独立表达。  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 investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P<0.01);the MAP decreased(P<0.01)and the HR increased drastically(P<0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.  相似文献   

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