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
目的分析人β防御素1(HBD1)单核苷酸多样性(SNP)与老年肺癌患者胸腔镜下肺癌根治术术后呼吸机相关肺炎(VAP)易感性及转归的相关性。方法选取2022年1月至2024年12月空军军医大学唐都医院收治的胸腔镜下肺癌根治术术后需呼吸机辅助治疗的...目的分析人β防御素1(HBD1)单核苷酸多样性(SNP)与老年肺癌患者胸腔镜下肺癌根治术术后呼吸机相关肺炎(VAP)易感性及转归的相关性。方法选取2022年1月至2024年12月空军军医大学唐都医院收治的胸腔镜下肺癌根治术术后需呼吸机辅助治疗的266例老年肺癌患者,根据是否并发VAP分为VAP组(41例)和对照组(225例),并根据VAP组患者预后不同分为死亡组(13例)和存活组(28例)。分析VAP患者病原菌及耐药性,采用聚合酶链反应(PCR)法测定HBD1基因第一外显子5′非编码区(-52A/G、-44C/G、-20A/G)SNP的基因型,采用Logistic回归分析SNP位点与老年肺癌患者胸腔下肺癌根治术后并发VAP和预后的关系。结果41例VAP患者共分离出病原菌85株,其中革兰阴性菌占48.24%,革兰阳性菌占20.00%,真菌占31.76%。革兰阴性菌鲍曼不动杆菌对氨曲南、头孢唑啉、头孢曲松、头孢他啶、磺胺甲噁唑及氨苄西林耐药率均为100%,肺炎克雷伯菌对氨曲南、头孢唑啉、头孢曲松、头孢吡肟、头孢他啶、磺胺甲噁唑、氨苄西林耐药率均为100%,铜绿假单胞菌对头孢唑啉、头孢曲松、磺胺甲噁唑、氨苄西林/舒巴坦耐药率均为100%,大肠埃希菌对氨曲南、环丙沙星、头孢唑啉、头孢曲松、头孢吡肟、头孢他啶、左氧氟沙星、磺胺甲噁唑、氨苄西林及氨苄西林/舒巴坦耐药率均为100%。VAP组HBD1基因5′非编码区-44 SNP GG基因型及G等位基因频率高于对照组(P<0.05);两组CC、CG基因型及其相应的C等位基因频率比较,差异无统计学意义(P>0.05);多因素Logistic回归分析显示,HBD1基因5′非编码区-44 SNP位点GG基因型(OR=2.855,95%CI:1.347~6.083)患VAP的风险显著升高,而CC基因型(OR=0.440,95%CI:0.230~0.845)患VAP的风险显著降低(P<0.05)。VAP死亡组HBD1基因5′非编码区-44 SNP GG基因型及G等位基因频率高于存活组(P<0.05);两组CC、CG基因型及其相应的C等位基因频率比较,差异均无统计学意义(P>0.05);多因素Logistic回归分析显示,HBD1基因5′非编码区-44 SNP位点GG基因型(OR=4.392,95%CI:1.673~11.524)的死亡风险高,CC基因型(OR=0.291,95%CI:0.122~0.690)的死亡风险低(P<0.05)。结论老年肺癌患者胸腔镜下肺癌根治术后VAP致病菌以革兰阴性菌为主,耐药性较高,临床应根据药敏试验合理应用抗菌药物,且HBD1基因-44C/G位点SNP与VAP易感性和预后具有一定的相关性,G等位基因可能会增加老年肺癌患者胸腔镜下肺癌根治术后VAP的易感性和死亡风险。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.
文摘目的分析人β防御素1(HBD1)单核苷酸多样性(SNP)与老年肺癌患者胸腔镜下肺癌根治术术后呼吸机相关肺炎(VAP)易感性及转归的相关性。方法选取2022年1月至2024年12月空军军医大学唐都医院收治的胸腔镜下肺癌根治术术后需呼吸机辅助治疗的266例老年肺癌患者,根据是否并发VAP分为VAP组(41例)和对照组(225例),并根据VAP组患者预后不同分为死亡组(13例)和存活组(28例)。分析VAP患者病原菌及耐药性,采用聚合酶链反应(PCR)法测定HBD1基因第一外显子5′非编码区(-52A/G、-44C/G、-20A/G)SNP的基因型,采用Logistic回归分析SNP位点与老年肺癌患者胸腔下肺癌根治术后并发VAP和预后的关系。结果41例VAP患者共分离出病原菌85株,其中革兰阴性菌占48.24%,革兰阳性菌占20.00%,真菌占31.76%。革兰阴性菌鲍曼不动杆菌对氨曲南、头孢唑啉、头孢曲松、头孢他啶、磺胺甲噁唑及氨苄西林耐药率均为100%,肺炎克雷伯菌对氨曲南、头孢唑啉、头孢曲松、头孢吡肟、头孢他啶、磺胺甲噁唑、氨苄西林耐药率均为100%,铜绿假单胞菌对头孢唑啉、头孢曲松、磺胺甲噁唑、氨苄西林/舒巴坦耐药率均为100%,大肠埃希菌对氨曲南、环丙沙星、头孢唑啉、头孢曲松、头孢吡肟、头孢他啶、左氧氟沙星、磺胺甲噁唑、氨苄西林及氨苄西林/舒巴坦耐药率均为100%。VAP组HBD1基因5′非编码区-44 SNP GG基因型及G等位基因频率高于对照组(P<0.05);两组CC、CG基因型及其相应的C等位基因频率比较,差异无统计学意义(P>0.05);多因素Logistic回归分析显示,HBD1基因5′非编码区-44 SNP位点GG基因型(OR=2.855,95%CI:1.347~6.083)患VAP的风险显著升高,而CC基因型(OR=0.440,95%CI:0.230~0.845)患VAP的风险显著降低(P<0.05)。VAP死亡组HBD1基因5′非编码区-44 SNP GG基因型及G等位基因频率高于存活组(P<0.05);两组CC、CG基因型及其相应的C等位基因频率比较,差异均无统计学意义(P>0.05);多因素Logistic回归分析显示,HBD1基因5′非编码区-44 SNP位点GG基因型(OR=4.392,95%CI:1.673~11.524)的死亡风险高,CC基因型(OR=0.291,95%CI:0.122~0.690)的死亡风险低(P<0.05)。结论老年肺癌患者胸腔镜下肺癌根治术后VAP致病菌以革兰阴性菌为主,耐药性较高,临床应根据药敏试验合理应用抗菌药物,且HBD1基因-44C/G位点SNP与VAP易感性和预后具有一定的相关性,G等位基因可能会增加老年肺癌患者胸腔镜下肺癌根治术后VAP的易感性和死亡风险。
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。
