目的:检测细胞增殖相关基因人特异性蛋白激酶-15(specific protein kinase 15,NYD-SP15)基因在增生牙龈及正常牙龈组织中的表达,初步探讨该基因与牙龈增生发生的关系。方法:应用RT-PCR及免疫组化法检测27例正常牙龈组织(取自外伤冠折后...目的:检测细胞增殖相关基因人特异性蛋白激酶-15(specific protein kinase 15,NYD-SP15)基因在增生牙龈及正常牙龈组织中的表达,初步探讨该基因与牙龈增生发生的关系。方法:应用RT-PCR及免疫组化法检测27例正常牙龈组织(取自外伤冠折后行牙冠延长术中切除的牙龈组织)及40例牙龈增生患者(以增生表现为主的慢性牙龈炎12例、药物性牙龈增生13例、牙龈瘤15例)牙龈切除术中切除的增生牙龈组织标本中NYD-SP15 mRNA及蛋白的表达。结果:增生牙龈组织中NYD-SP15的mRNA水平显著高于正常牙龈组织(P<0.05)。免疫组化结果显示,在正常牙龈组织中该基因高表达于胞浆,在增生牙龈组织中高表达于胞核。结论:NYD-SP15表达量异常以及定位差异在人牙龈增生发生发展过程中起到一定的作用,但该基因高表达与牙龈增生发生的相关机制尚待进一步研究。展开更多
肺表面活性蛋白A(surfactant protein A,SP-A)具有维持肺内稳态、防御病原感染以及协助免疫应答等重要作用。本试验建立了定量检测绵羊SP-A基因及内参基因B2M(beta 2 microglobulin)m RNA表达水平的SYBR Green I实时荧光定量RT-PCR方法...肺表面活性蛋白A(surfactant protein A,SP-A)具有维持肺内稳态、防御病原感染以及协助免疫应答等重要作用。本试验建立了定量检测绵羊SP-A基因及内参基因B2M(beta 2 microglobulin)m RNA表达水平的SYBR Green I实时荧光定量RT-PCR方法,并采用该方法对健康滩羊、小尾寒羊和滩寒杂交羊三个绵羊品种肺脏中SP-A m RNA的表达情况进行了检测。结果显示,SP-A基因和B2M基因的扩增效率分别为94.1%和95.9%;线性范围分别在1×10-1~1×10-6和1×10-1~1×10-7;相关系数分别为0.998和0.999;熔解曲线分别在84.5℃和83.0℃出现一个特异峰;滩羊SP-A m RNA的表达水平略高于小尾寒羊和滩寒杂交羊。本试验建立的绵羊SP-A基因实时荧光定量RT-PCR方法特异性好、检测线性范围广,为绵羊SP-A m RNA表达水平的检测提供了重要方法。展开更多
目的:探讨磷酸酶–张力蛋白基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)在正常脑组织及星形细胞瘤中的表达及其意义。方法:采用免疫组织化学法及RT-PCR方法检测正常脑组织及不同分级星形细胞瘤组织中PTEN蛋...目的:探讨磷酸酶–张力蛋白基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)在正常脑组织及星形细胞瘤中的表达及其意义。方法:采用免疫组织化学法及RT-PCR方法检测正常脑组织及不同分级星形细胞瘤组织中PTEN蛋白及mRNA的表达水平,探讨并分析其与星形细胞瘤组织学分级的关系,Western印迹法检测正常脑组织及星形细胞瘤脑脊液中PTEN蛋白的表达。结果:免疫组织化学方法显示PTEN在正常脑组织中阳性率较高,为90%(9/10),星形细胞瘤中阳性率较低,为47.5%(19/40),且PTEN在不同的组织学分级中,阳性率不同。RT-PCR结果显示PTEN m RNA在正常脑组织中表达较高,其相对表达量为0.861±0.072,显著高于星形细胞瘤组织中的表达(0.127±0.008),差异有统计学意义(P<0.05),并随着组织学级别增加,其表达呈降低趋势。Western印迹法结果显示P TEN蛋白在非星形细胞瘤组脑脊液中的相对表达量为1.549±0.194,高于星形细胞瘤组脑脊液中的相对表达量(0.602±0.058),差异具有统计学意义(P<0.05)。结论:检测PTEN基因蛋白表达有望作为评估人星形细胞瘤生物学行为和预后的参考指标。展开更多
Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the s...Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the spring epidemics of the disease, which determines the crop loss. Estimation of P. striiformis f. sp. tritici winter survival requires processing a large number of samples for sensitive detection of the pathogen in wheat leaf tissue using real-time quantitative reverse transcription PCR （qRT-PCR）. A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for qRT-PCR to distinguish dead and alive fungal hyphae inside leaves. Although several methods have been described in the literatures and commercial kits are available for RNA extraction, these methods are mostly too complicated, expensive and inefficient. Thus, we modified three previously reported RNA extraction methods with common and low-cost reagents （LiCI, SDS and NaCI） to solve the problems and selected the best to obtain high quality and quantity RNA for use in qRT-PCR. In the three improved methods, the NaCI method was proven to be the best for extracting RNAfrom urediniospores of and wheat leaves infected by P. striiformis f. sp. tritici, although the modified LiCI and SDS methods also increased yield of RNA compared to the previous methods. The improved NaCI method has the following advantages： 1） Complete transfer of urediniospores of P. striiformis f. sp. tritici from the mortar and pestle can ensure the initial amount of RNA for the qRT-PCR analysis; 2） the use of low-cost NaCI to replace more expensive Trizol can reduce the cost; 3） the yield and quality of RNA can be increased; 4） the improved method is more suitable for a large number and high quantity of samples from fields. Using the improved NaCI method, the amount of RNA was increased three times from urediniospores of P. striiformis f. sp. tritici compared from the extraction kit. Approximately, 10.11 IJg total RNA of high quality was obtained from 100 mg of infected leaves, which was 8.8, 6.5, 3.4 and 2.1 folds of the amounts obtained from the previous LiCI, SDS, NaCI and traditional Trizol methods, respectively. The method could be used to study the overwintering rates of R striiformis f. sp. tritici over a large region of wheat production for predicting epidemic levels by determining pathogen survival levels after winter. The method can alsobe used in any studies which need a large number of high quality RNA samples.展开更多
文摘目的:研究赖氨酰氧化酶样蛋白-2(lysy loxidase-like 2 protein,LOXL2)及黏附蛋白E-cadherin在胆管癌的表达,探讨其表达情况与胆管癌生物学行为的关系.方法:应用SP法及RT-PCR技术分别检测47例胆管癌、47例配对的癌旁组织、20例正常胆管组织中LOXL2和E-cadherin的表达情况,对结果应用SPSS17.0统计软件进行.数据进行统计学分析.结果:(1)在47例胆管癌组织、癌旁组织、正常胆管组织中L O X L2蛋白的阳性表达率分别为68.1%、10.6%、10.0%(P<0.05);E-cadherin蛋白阳性表达率分别为21.3%、7 4.5%、9 5.0%,差异均具有统计学意义(P<0.0 5);(2)胆管癌组织、癌旁组织、正常胆管组织中LOXL2 m RNA的表达分别为154.63±128.96、5.35±11.85、4.52±10.79(P<0.05);E-c adherinm RNA的表达分别为0.18±0.25、0.95±0.62、1.08±0.33(P<0.05);(3)胆管癌组织中E-cadherin的表达减弱和LOXL2的表达增强均与患者肿瘤的分化程度、分期、淋巴结转移和组织器官转移相关(P<0.05);(4)胆管癌中E-cadherin和LOXL2的表达呈负相关.结论:在胆管癌组织中E-cadherin的表达低于癌旁组织减弱,而LOXL2表达高于癌旁组织;二者的表达呈负相关且与肿瘤的分化程度有明显相关性.
文摘目的:检测细胞增殖相关基因人特异性蛋白激酶-15(specific protein kinase 15,NYD-SP15)基因在增生牙龈及正常牙龈组织中的表达,初步探讨该基因与牙龈增生发生的关系。方法:应用RT-PCR及免疫组化法检测27例正常牙龈组织(取自外伤冠折后行牙冠延长术中切除的牙龈组织)及40例牙龈增生患者(以增生表现为主的慢性牙龈炎12例、药物性牙龈增生13例、牙龈瘤15例)牙龈切除术中切除的增生牙龈组织标本中NYD-SP15 mRNA及蛋白的表达。结果:增生牙龈组织中NYD-SP15的mRNA水平显著高于正常牙龈组织(P<0.05)。免疫组化结果显示,在正常牙龈组织中该基因高表达于胞浆,在增生牙龈组织中高表达于胞核。结论:NYD-SP15表达量异常以及定位差异在人牙龈增生发生发展过程中起到一定的作用,但该基因高表达与牙龈增生发生的相关机制尚待进一步研究。
文摘肺表面活性蛋白A(surfactant protein A,SP-A)具有维持肺内稳态、防御病原感染以及协助免疫应答等重要作用。本试验建立了定量检测绵羊SP-A基因及内参基因B2M(beta 2 microglobulin)m RNA表达水平的SYBR Green I实时荧光定量RT-PCR方法,并采用该方法对健康滩羊、小尾寒羊和滩寒杂交羊三个绵羊品种肺脏中SP-A m RNA的表达情况进行了检测。结果显示,SP-A基因和B2M基因的扩增效率分别为94.1%和95.9%;线性范围分别在1×10-1~1×10-6和1×10-1~1×10-7;相关系数分别为0.998和0.999;熔解曲线分别在84.5℃和83.0℃出现一个特异峰;滩羊SP-A m RNA的表达水平略高于小尾寒羊和滩寒杂交羊。本试验建立的绵羊SP-A基因实时荧光定量RT-PCR方法特异性好、检测线性范围广,为绵羊SP-A m RNA表达水平的检测提供了重要方法。
文摘目的:探讨磷酸酶–张力蛋白基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)在正常脑组织及星形细胞瘤中的表达及其意义。方法:采用免疫组织化学法及RT-PCR方法检测正常脑组织及不同分级星形细胞瘤组织中PTEN蛋白及mRNA的表达水平,探讨并分析其与星形细胞瘤组织学分级的关系,Western印迹法检测正常脑组织及星形细胞瘤脑脊液中PTEN蛋白的表达。结果:免疫组织化学方法显示PTEN在正常脑组织中阳性率较高,为90%(9/10),星形细胞瘤中阳性率较低,为47.5%(19/40),且PTEN在不同的组织学分级中,阳性率不同。RT-PCR结果显示PTEN m RNA在正常脑组织中表达较高,其相对表达量为0.861±0.072,显著高于星形细胞瘤组织中的表达(0.127±0.008),差异有统计学意义(P<0.05),并随着组织学级别增加,其表达呈降低趋势。Western印迹法结果显示P TEN蛋白在非星形细胞瘤组脑脊液中的相对表达量为1.549±0.194,高于星形细胞瘤组脑脊液中的相对表达量(0.602±0.058),差异具有统计学意义(P<0.05)。结论:检测PTEN基因蛋白表达有望作为评估人星形细胞瘤生物学行为和预后的参考指标。
基金supported by the National Key Basic Research Program of China (2013CB127700)the Na-tional Natural Science Foundation of China (31071640 and 31271985)partially supported by the 111 Project from Education Ministry of China (B07049)
文摘Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the spring epidemics of the disease, which determines the crop loss. Estimation of P. striiformis f. sp. tritici winter survival requires processing a large number of samples for sensitive detection of the pathogen in wheat leaf tissue using real-time quantitative reverse transcription PCR （qRT-PCR）. A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for qRT-PCR to distinguish dead and alive fungal hyphae inside leaves. Although several methods have been described in the literatures and commercial kits are available for RNA extraction, these methods are mostly too complicated, expensive and inefficient. Thus, we modified three previously reported RNA extraction methods with common and low-cost reagents （LiCI, SDS and NaCI） to solve the problems and selected the best to obtain high quality and quantity RNA for use in qRT-PCR. In the three improved methods, the NaCI method was proven to be the best for extracting RNAfrom urediniospores of and wheat leaves infected by P. striiformis f. sp. tritici, although the modified LiCI and SDS methods also increased yield of RNA compared to the previous methods. The improved NaCI method has the following advantages： 1） Complete transfer of urediniospores of P. striiformis f. sp. tritici from the mortar and pestle can ensure the initial amount of RNA for the qRT-PCR analysis; 2） the use of low-cost NaCI to replace more expensive Trizol can reduce the cost; 3） the yield and quality of RNA can be increased; 4） the improved method is more suitable for a large number and high quantity of samples from fields. Using the improved NaCI method, the amount of RNA was increased three times from urediniospores of P. striiformis f. sp. tritici compared from the extraction kit. Approximately, 10.11 IJg total RNA of high quality was obtained from 100 mg of infected leaves, which was 8.8, 6.5, 3.4 and 2.1 folds of the amounts obtained from the previous LiCI, SDS, NaCI and traditional Trizol methods, respectively. The method could be used to study the overwintering rates of R striiformis f. sp. tritici over a large region of wheat production for predicting epidemic levels by determining pathogen survival levels after winter. The method can alsobe used in any studies which need a large number of high quality RNA samples.
