To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector...To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofe-ctamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. Results COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. Conclusion We successfully established a hBD3-expressing cell line.展开更多
为了探究公羊睾丸和附睾不同区段β防御素124(beta-defensins 124,BD124)的表达特征与精液品质之间的关系,该研究选取20只健康杜泊羊×湖羊杂交种公羊,采集新鲜精液测定精子运动学参数,根据精子活力(sperm motility,SM)将公羊分为...为了探究公羊睾丸和附睾不同区段β防御素124(beta-defensins 124,BD124)的表达特征与精液品质之间的关系,该研究选取20只健康杜泊羊×湖羊杂交种公羊,采集新鲜精液测定精子运动学参数,根据精子活力(sperm motility,SM)将公羊分为高品质组(SM≥60%)和低品质组(SM≤15%),每组各选取3只采集睾丸和附睾不同区段组织,通过免疫组织化学法和Western blot检测BD124定位及表达。结果表明:(1)BD124在附睾头(epididymis head,EH1~EH2)平均光密度值和相对灰度值极显著高于附睾体(epididymis body,EB1~EB3)和附睾尾(epididymis tail,ET1~ET2)(P<0.01),高品质组输出小管(efferent ducts,ED)、EH2、EB1、EB3和ET2区段BD124平均光密度值和相对灰度值均极显著高于低品质组(P<0.01);(2)高品质组精子活率(sperm viability,SV)、A级精子比例(percentage of spermatozoa of the A class,A percent)、平均路径速度(average path velocity,VAP)、直线速度(straight line velocity,VSL)、曲线速度(curvilinear velocity,VCL)和平均侧摆幅值(amplitude of lateral head displacement,ALH)均极显著高于低品质组(P<0.01);(3)相关性分析显示EB1、ET2区段BD124相对灰度值和平均光密度值与SV呈极显著正相关(P<0.01),EB1、ET2区段BD124相对灰度值和平均光密度值与D级精子比例呈极显著负相关(P<0.01),ED、EH2、EB1、EB3、ET2区段BD124的相对灰度值和平均光密度值与A级精子比例、VAP、VSL、VCL、ALH呈极显著正相关(P<0.01)。由此可知,EB1和ET2区段BD124表达与公羊精液品质呈明显正相关关系。展开更多
目的探索肺结核患者血清甲壳质酶蛋白40(chitinase protein 40,YKL-40)、β-防御素-2(human beta defensin 2,HBD-2)水平及其对肺结核的诊断价值。方法选取2021年6月—2023年6月山东省立医院收治的86例肺结核患者为肺结核组,根据痰涂片...目的探索肺结核患者血清甲壳质酶蛋白40(chitinase protein 40,YKL-40)、β-防御素-2(human beta defensin 2,HBD-2)水平及其对肺结核的诊断价值。方法选取2021年6月—2023年6月山东省立医院收治的86例肺结核患者为肺结核组,根据痰涂片结果分为活动性肺结核组(n=50)和非活动性肺结核组(n=36),另选取86例来自我院体检中心的健康受试者作为对照组。检测所有受试者血清中YKL-40、HBD-2水平并分析肺结核患者血清中YKL-40与HBD-2的相关性;采用受试者工作特征(receiver operating characteristic,ROC)曲线分析血清中YKL-40、HBD-2水平对肺结核及活动性肺结核的诊断价值。结果肺结核组血清YKL-40、HBD-2水平高于对照组(P均<0.05);活动性肺结核组血清YKL-40、HBD-2水平高于非活动性肺结核组(P均<0.05)。肺结核患者血清中YKL-40与HBD-2的水平呈正相关(r=0.601)。YKL-40、HBD-2及2者联合诊断肺结核的曲线下面积(area under the curve,AUC)分别为0.895、0.922、0.962,2者联合诊断优于单独诊断;YKL-40、HBD-2及2者联合诊断活动性肺结核的AUC分别为0.891、0.881、0.959,2者联合诊断优于单独诊断。结论肺结核患者血清YKL-40、HBD-2水平显著升高,且随病情加重而升高,2者对肺结核及活动性肺结核的具有一定的诊断价值。展开更多
β-防御素体外试验显示具广谱抗菌活性 ,本研究应用转基因方法评估其体内抗菌作用。构建大鼠 β-防御素 - 2 (r BD2 )重组质粒 p BK- CMV- r BD2和 p CDNA- 3.1- Myc- His(+) - r BD2 ,通过脂质体包裹的方法将重组质粒经气管滴入 ,检测...β-防御素体外试验显示具广谱抗菌活性 ,本研究应用转基因方法评估其体内抗菌作用。构建大鼠 β-防御素 - 2 (r BD2 )重组质粒 p BK- CMV- r BD2和 p CDNA- 3.1- Myc- His(+) - r BD2 ,通过脂质体包裹的方法将重组质粒经气管滴入 ,检测其在气管和肺组织表达情况 ,并应用肺组织匀浆上清菌落计数法检察对绿脓杆菌的肺清除率。结果显示在基因转染大鼠的气管和肺组织内均检测到 r BD2 - His融合蛋白基因 m RNA和蛋白的表达 ,说明脂质体包裹的重组 β-防御素 - 2 p BK- CMV- r BD2质粒可有效转染气道上皮组织。肺细菌清除率实验显示构建重组质粒 p BK-CMV- r BD2气道转染与对照相比可显著提高肺组织对绿脓杆菌的清除率 (n(8,p(0 .0 1)。本研究表明 β-防御素基因气道转染可提高呼吸道天然抗感染防御功能 ,可能在呼吸道感染的防治实践中具有潜在应用价值。展开更多
文摘To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofe-ctamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. Results COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. Conclusion We successfully established a hBD3-expressing cell line.
文摘为了探究公羊睾丸和附睾不同区段β防御素124(beta-defensins 124,BD124)的表达特征与精液品质之间的关系,该研究选取20只健康杜泊羊×湖羊杂交种公羊,采集新鲜精液测定精子运动学参数,根据精子活力(sperm motility,SM)将公羊分为高品质组(SM≥60%)和低品质组(SM≤15%),每组各选取3只采集睾丸和附睾不同区段组织,通过免疫组织化学法和Western blot检测BD124定位及表达。结果表明:(1)BD124在附睾头(epididymis head,EH1~EH2)平均光密度值和相对灰度值极显著高于附睾体(epididymis body,EB1~EB3)和附睾尾(epididymis tail,ET1~ET2)(P<0.01),高品质组输出小管(efferent ducts,ED)、EH2、EB1、EB3和ET2区段BD124平均光密度值和相对灰度值均极显著高于低品质组(P<0.01);(2)高品质组精子活率(sperm viability,SV)、A级精子比例(percentage of spermatozoa of the A class,A percent)、平均路径速度(average path velocity,VAP)、直线速度(straight line velocity,VSL)、曲线速度(curvilinear velocity,VCL)和平均侧摆幅值(amplitude of lateral head displacement,ALH)均极显著高于低品质组(P<0.01);(3)相关性分析显示EB1、ET2区段BD124相对灰度值和平均光密度值与SV呈极显著正相关(P<0.01),EB1、ET2区段BD124相对灰度值和平均光密度值与D级精子比例呈极显著负相关(P<0.01),ED、EH2、EB1、EB3、ET2区段BD124的相对灰度值和平均光密度值与A级精子比例、VAP、VSL、VCL、ALH呈极显著正相关(P<0.01)。由此可知,EB1和ET2区段BD124表达与公羊精液品质呈明显正相关关系。
文摘目的探索肺结核患者血清甲壳质酶蛋白40(chitinase protein 40,YKL-40)、β-防御素-2(human beta defensin 2,HBD-2)水平及其对肺结核的诊断价值。方法选取2021年6月—2023年6月山东省立医院收治的86例肺结核患者为肺结核组,根据痰涂片结果分为活动性肺结核组(n=50)和非活动性肺结核组(n=36),另选取86例来自我院体检中心的健康受试者作为对照组。检测所有受试者血清中YKL-40、HBD-2水平并分析肺结核患者血清中YKL-40与HBD-2的相关性;采用受试者工作特征(receiver operating characteristic,ROC)曲线分析血清中YKL-40、HBD-2水平对肺结核及活动性肺结核的诊断价值。结果肺结核组血清YKL-40、HBD-2水平高于对照组(P均<0.05);活动性肺结核组血清YKL-40、HBD-2水平高于非活动性肺结核组(P均<0.05)。肺结核患者血清中YKL-40与HBD-2的水平呈正相关(r=0.601)。YKL-40、HBD-2及2者联合诊断肺结核的曲线下面积(area under the curve,AUC)分别为0.895、0.922、0.962,2者联合诊断优于单独诊断;YKL-40、HBD-2及2者联合诊断活动性肺结核的AUC分别为0.891、0.881、0.959,2者联合诊断优于单独诊断。结论肺结核患者血清YKL-40、HBD-2水平显著升高,且随病情加重而升高,2者对肺结核及活动性肺结核的具有一定的诊断价值。
文摘β-防御素体外试验显示具广谱抗菌活性 ,本研究应用转基因方法评估其体内抗菌作用。构建大鼠 β-防御素 - 2 (r BD2 )重组质粒 p BK- CMV- r BD2和 p CDNA- 3.1- Myc- His(+) - r BD2 ,通过脂质体包裹的方法将重组质粒经气管滴入 ,检测其在气管和肺组织表达情况 ,并应用肺组织匀浆上清菌落计数法检察对绿脓杆菌的肺清除率。结果显示在基因转染大鼠的气管和肺组织内均检测到 r BD2 - His融合蛋白基因 m RNA和蛋白的表达 ,说明脂质体包裹的重组 β-防御素 - 2 p BK- CMV- r BD2质粒可有效转染气道上皮组织。肺细菌清除率实验显示构建重组质粒 p BK-CMV- r BD2气道转染与对照相比可显著提高肺组织对绿脓杆菌的清除率 (n(8,p(0 .0 1)。本研究表明 β-防御素基因气道转染可提高呼吸道天然抗感染防御功能 ,可能在呼吸道感染的防治实践中具有潜在应用价值。
