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目的构建人类HER2基因的真核表达载体,并对其促进乳腺癌细胞增殖效果及靶向药物敏感性进行验证。方法采用PCR技术扩增出HER2基因,将其插入到pXJ-40-myc载体中,酶切和测序验证后,将其转染到乳腺癌ZR75-1细胞中,Western blot法检测其表达情况;CCK8法测定细胞生长曲线;加入靶向药物曲妥珠单克隆抗体后,观察转染细胞对药物的反应。结果酶切和测序结果证实表达质粒构建成功;Western blot法结果显示,myc-HER2蛋白在转染细胞中成功表达;转染myc-HER2的乳腺癌细胞较空载体细胞生长较快;加入曲妥珠单克隆抗体后,转染myc-HER2的细胞生长明显受到抑制。结论成功构建了带myc标签的HER2基因真核表达载体,为进一步研究曲妥珠单抗的耐药奠定了实验基础。 相似文献
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目的 构建HBV融合抗原真核表达质粒pIRES-neo-HBAg,并验证其在293T细胞中的表达.方法 以含有HBV融合抗原的质粒pVAX1-HBV为模板,PCR扩增该融合基因.PCR产物经纯化后,将其克隆至载体pMD18-T中,构建pMD18-T-HBV质粒,经酶切和测序鉴定后,将其定向克隆入真核表达载体pIRES-neo,获得真核表达质粒pIRES-neo-HBAg.将该重组表达质粒瞬时转染人293T细胞,采用Western blot法、流式细胞术和免疫荧光细胞化学技术验证HBV融合抗原的表达.结果 成功构建了HBV融合抗原真核表达质粒pIRES-neo-HBAg,Western blot法、流式细胞术和免疫荧光细胞化学技术结果显示融合抗原能够在293T细胞中表达.结论 成功构建了HBV融合抗原真核表达质粒pIRES-neo-HBAg. 相似文献
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《解剖科学进展》2017,(3)
目的构建小鼠Prohibitin基因真核表达质粒,在293T细胞中进行表达鉴定,为研究小鼠Prohibitin基因功能奠定基础。方法以小鼠Prohibitin基因cDNA序列为模板,设计特定引物,应用PCR的方法扩增其编码序列全长,并将其克隆到真核表达载体pEFP-C2中,将获得的重组表达质粒转染293T细胞,应用RTPCR、Western blot方法检测其表达。结果成功构建了pEGFP-Prohibitin真核表达质粒,将其成功转染293T细胞,pEGFP-Prohibitin转染组Prohibitin蛋白和mRNA在293T细胞的表达量比转染空载转染组明显增加。结论成功构建了真核表达质粒pEGFP-Prohibitin,并在真核细胞表达良好,为研究小鼠Prohibitin的基因功能奠定了基础。 相似文献
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目的:构建大鼠CD36真核表达载体,并在293T细胞中表达.方法:应用RT-PCR技术,从大鼠肺泡巨噬细胞NR8383细胞提取的总RNA中,获得CD36基因编码序列片段,克隆至真核表达载体pEGFP-N1中,对重组质粒进行酶切和测序鉴定后,以脂质体介导法转染至293T细胞,通过荧光显微镜和Western blot检测其在293T细胞中的表达.结果:酶切和测序证明重组真核表达载体pEGFP-N1-CD36构建成功,荧光显微镜及Western blot确认目的基因序列在293T细胞中过表达.结论:成功构建大鼠CD36基因的重组真核表达载体pEGFP-N1-CD36,并在293T细胞中过表达. 相似文献
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目的:构建人pescadillo(PES1)基因siRNA的真核表达载体,观察其对PES1表达的影响。方法:利用RNA干扰(RNAi)技术,设计并合成了2条针对PES1基因的siRNA,将其克隆到siRNA表达载体pSliencer2.1-U6neo上。分别用以下3种方法检测RNAi的抑制效果:(1)将RNAi重组质粒和带FLAG标签的PES1共转染293T人胚肾细胞,用FLAG抗体进行Western blot;(2)将RNAi重组质粒和带GFP标签的PES1共转染293T细胞,用荧光显微镜观察GFP的亮度;(3)在293T细胞中仅转染小干扰RNA(siRNA)重组质粒,用PES1抗体进行Western blot分析。结果:测序证明,成功构建了PES1siRNA真核表达载体。通过Western blot实验证明,构建的siRNA能有效抑制外源性及内源性PES1基因表达;荧光显微镜检查发现siRNA能明显减弱细胞中荧光强度,抑制PES1的表达。结论:成功地构建了PES1siRNA的真核表达载体,利用3种不同方法均证明该siRNA能有效地抑制PES1基因的表达。 相似文献
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目的:设计并构建ErbB2的小干扰RNA,检测其对ErbB2蛋白表达的干扰效果,并检测其对乳腺癌细胞ZR75-1生长的影响。方法:设计2条针对ErbB2的siRNA,并克隆到siRNA表达载体pSliencer 2.1-U6 neo上。经酶切和测序证明构建成功后,将其与pcDNA3-FLAG-ErbB2共转染于293T细胞,以及单转siRNA于乳腺癌SKBR3和ZR75-1细胞,通过Western blot分别检测siRNA对外源和内源ErbB2的干扰效果。通过结晶紫实验研究siRNA对ZR75-1生长的影响。结果:Western blot证明构建的两条ErbB2 siRNA均能有效抑制外源和内源ErbB2蛋白的表达,并抑制乳腺癌ZR75-1细胞的生长。结论:构建的siRNA能有效地抑制ErbB2蛋白的表达并抑制ZR75-1细胞的生长。 相似文献
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目的构建表达人Sprouty 2(SPRY2)基因的重组表达载体pDC315/hSPRY2,转染人胚肾(HEK)293T细胞表达目的蛋白,初步探讨其对HEK293T细胞增殖与存活的影响。方法构建hSPRY2重组腺病毒表达载体,体外转染HEK293T细胞,以Western blot法检测目的蛋白的表达,以CCK-8法检测SPRY2对HEK293T细胞增殖或去血清条件下细胞存活的影响。结果成功构建重组表达载体pDC315/hSPRY2,在转染的HEK293T细胞中检测到目的蛋白hSPRY2的表达,转染pDC315/hSPRY2组HEK293T细胞的增殖率及存活率均明显高于对照组即转染pDC315/EGFP组(P0.05)。结论成功构建了hSPRY2重组表达载体,过表达hSPRY2可促进HEK293T细胞的增殖与存活。 相似文献
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目的构建表达人Sprouty 2(SPRY2)基因的重组表达载体pDC315/hSPRY2,转染人胚肾(HEK)293T细胞表达目的蛋白,初步探讨其对HEK293T细胞增殖与存活的影响。方法构建hSPRY2重组腺病毒表达载体,体外转染HEK293T细胞,以Western blot法检测目的蛋白的表达,以CCK-8法检测SPRY2对HEK293T细胞增殖或去血清条件下细胞存活的影响。结果成功构建重组表达载体pDC315/hSPRY2,在转染的HEK293T细胞中检测到目的蛋白hSPRY2的表达,转染pDC315/hSPRY2组HEK293T细胞的增殖率及存活率均明显高于对照组即转染pDC315/EGFP组(P<0.05)。结论成功构建了hSPRY2重组表达载体,过表达hSPRY2可促进HEK293T细胞的增殖与存活。 相似文献
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真核表达载体pEGFP-claudin-1的构建及其在293T细胞中的表达 总被引:1,自引:0,他引:1
目的:构建真核表达载体pEGFP-claudin-1,并在293T细胞中进行表达.方法:用反转录聚合酶链反应(RT-PCR)方法扩增claudin-1开放读码框(ORF)基因,将其插入到pEGFP-C3载体的Xho Ⅰ和BamH Ⅰ酶切位点,构建真核表达载体pEGFP-claudin-1,酶切鉴定并测序.通过脂质体法转染293T细胞,进行荧光检测和Western blot分析.结果:构建了含有claudin-1 ORF的真核表达质粒pEGFP-claudin-1,转染293T细胞后,经荧光可见细胞膜有EGFP-claudin-1 融合蛋白的表达,Western blot检测发现有相对分子质量(Mr)49 000的蛋白条带.结论:成功地构建真核表达载体pEGFP-claudin-1,并在293T细胞中表达,为研究claudin-1的功能奠定了基础. 相似文献
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目的:构建密码子优化的HPV16衣壳基因真核共表达载体pcDNA3.1-L1-IRES-L2.方法:用PCR技术从988载体中获得L1-IRES-L2片段,将该片段克隆到pCR -XL-TOPO 载体,然后定向亚克隆到pcDNA3.1( )真核表达载体中,从而构建真核共表达载体pcDNA3.1-L1-IRES-L2;通过水动力转染技术(hydrodynamics-based transfection)和脂质体细胞转染法(liposome-mediated transfection of cells),检测衣壳基因的体内、外转录情况;重组质粒转染后293T细胞后观察其形态变化,用Western blot方法检测293T细胞中L1衣壳蛋白的表达.结果:酶切和测序结果表明真核共表达载体pcD-NA3.1-L1-IRES-L2构建正确.重组质粒中的L1和L2基因在小鼠肝脏、293T细胞中均发生转录.重组质粒转染293T细胞后出现CPE(cytopathic effect)现象,表明衣壳基因在细胞中已表达.Western blot方法检测发现L1蛋白在293T细胞中表达.结论:成功地构建了pcDNA3.1-L1-IRES-L2共表达真核载体,为进一步研究HPV16感染机制奠定基础. 相似文献
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Riann M. Palmieri-Smith Mark Villwock Brian Downie Garin Hecht Ron Zernicke 《Journal of Athletic Training》2013,48(2):186-191
Context:
Quadriceps dysfunction is a common consequence of knee joint injury and disease, yet its causes remain elusive.Objective:
To determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion affect the magnitude of quadriceps dysfunction.Design:
Crossover study.Setting:
University research laboratory.Patients or Other Participants:
Fourteen (8 men, 6 women; age = 23.6 ± 4.8 years, height = 170.3 ± 9.16 cm, mass = 72.9 ± 11.84 kg) healthy volunteers.Intervention(s):
All participants were tested under 4 randomized conditions: normal knee, effused knee, painful knee, and effused and painful knee.Main Outcome Measure(s):
Quadriceps strength (Nm/kg) and activation (central activation ratio) were assessed after each condition was induced.Results:
Quadriceps strength and activation were highest under the normal knee condition and differed from the 3 experimental knee conditions (P < .05). No differences were noted among the 3 experimental knee conditions for either variable (P > .05).Conclusions:
Both pain and effusion led to quadriceps dysfunction, but the interaction of the 2 stimuli did not increase the magnitude of the strength or activation deficits. Therefore, pain and effusion can be considered equally potent in eliciting quadriceps inhibition. Given that pain and effusion accompany numerous knee conditions, the prevalence of quadriceps dysfunction is likely high.Key Words: arthrogenic muscle inhibition, central activation failure, voluntary activation, musclesKey Points
- Knee pain and effusion resulted in arthrogenic muscle inhibition and weakness of the quadriceps.
- The simultaneous presence of pain and effusion did not increase the magnitude of quadriceps dysfunction.
- To reduce arthrogenic muscle inhibition and improve muscle strength, clinicians should employ interventions that target removing both pain and effusion.
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即早基因c-fos与脑血管病及学习记忆 总被引:5,自引:1,他引:5
即早基因c-fos是广泛存在于原核细胞和真核细胞的高度保守基因.在正常情况下,c-fos基因参与细胞生长、分化、信息传递、学习和记忆等生理过程,而在病理情况下c-fos基因表达及调控变化与多种疾病的发生和发展有关.C-fos在中枢神经系统的某些部位可有基础水平的表达,但表达很低,当受到如脑缺血、脑出血、痫性发作、应激等刺激后,其在数十分钟内做出反应,在对外界刺激-转录耦联的信忠传递过程中起着核内第三信使的重要作用. 相似文献
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Schmidt PJ 《The New England journal of medicine》2002,346(8):617-620
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OBJECTIVE: The purpose of this article is to review the role of behavioral research in disease prevention and control, with a particular emphasis on lifestyle- and behavior-related cancer and chronic disease risk factors--specifically, relationships among diet and nutrition and weight and physical activity with adult cancer, and tracking developmental origins of these health-promoting and health-compromising behaviors from childhood into adulthood. METHOD: After reviewing the background of the field of cancer prevention and control and establishing plausibility for the role of child health behavior in adult cancer risk, studies selected from the pediatric published literature are reviewed. Articles were retrieved, selected, and summarized to illustrate that results from separate but related fields of study are combinable to yield insights into the prevention and control of cancer and other chronic diseases in adulthood through the conduct of nonintervention and intervention research with children in clinical, public health, and other contexts. RESULTS: As illustrated by the evidence presented in this review, there are numerous reasons (biological, psychological, and social), opportunities (school and community, health care, and family settings), and approaches (nonintervention and intervention) to understand and impact behavior change in children's diet and nutrition and weight and physical activity. CONCLUSIONS: Further development and evaluation of behavioral science intervention protocols conducted with children are necessary to understand the efficacy of these approaches and their public health impact on proximal and distal cancer, cancer-related, and chronic disease outcomes before diffusion. It is clear that more attention should be paid to early life and early developmental phases in cancer prevention. 相似文献