目的:利用噬菌体随机肽库筛选与人肺泡Ⅱ型上皮A549细胞存在特异性结合的多肽,为实现对肺泡Ⅱ型上皮细胞的靶向干预和开发针对急性肺损伤等呼吸系统疾病治疗的导向药物载体提供实验依据。方法:采用全细胞筛选的方式,通过对人肺泡Ⅱ型上...目的:利用噬菌体随机肽库筛选与人肺泡Ⅱ型上皮A549细胞存在特异性结合的多肽,为实现对肺泡Ⅱ型上皮细胞的靶向干预和开发针对急性肺损伤等呼吸系统疾病治疗的导向药物载体提供实验依据。方法:采用全细胞筛选的方式,通过对人肺泡Ⅱ型上皮A549细胞的4轮筛选和与人前列腺癌上皮PC-3细胞的差减筛选,从噬菌体随机肽库中筛选出与人肺泡Ⅱ型上皮A549细胞存在特异性结合的多肽,选择丰度较高的一条命名为NTG,用其构建含有增强绿色荧光蛋白(EGFP)的原核表达载体,转化感受态细菌,层析法获得重组蛋白His-NTG-EGFP,测定其分子质量。向A549细胞和PC-3细胞分别加入含有重组蛋白His-NTG-EGFP或His-EGFP的培养基,荧光显微镜下观察。结果:从第1轮至第4轮,淘选所得的噬菌体回收率逐步提高,经过PC-3细胞差减筛选,少量的与A549细胞存在特异性结合的噬菌体得到了回收。重组表达并纯化得到的His-NTG-EGFP,测定其分子质量为33 k D,与预期相符。荧光显微镜下观察发现,经融合蛋白His-NTG-EGFP处理后的A549细胞表面有绿色荧光分布,经融合蛋白His-NTG-EGFP处理后的PC-3细胞以及经对照蛋白处理的A549细胞和PC-3细胞表面均未出现绿色荧光,说明这一融合蛋白可以特异性结合A549细胞。结论:人肺泡Ⅱ型上皮A549细胞特异性靶向肽被成功获得。展开更多
There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) in Arabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters ...There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) in Arabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5' region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants by Agrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5' coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5' coding region of PAH or PAD was 60-100-fold higher than that without the corresponding 5' region. However, the effect of 5' coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference) . The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by the 5' region of PAI genes have the function of PTP.展开更多
AIM：To investigate the effect of tissue factor targeting peptide（TF-TP）on retinal pigment epithelium（RPE）cells tight junctions.METHODS：Cell counting kit-8（CCK-8）was used to measure the proliferation of ARPE-19...AIM：To investigate the effect of tissue factor targeting peptide（TF-TP）on retinal pigment epithelium（RPE）cells tight junctions.METHODS：Cell counting kit-8（CCK-8）was used to measure the proliferation of ARPE-19 cells.Expression of tight junction,ZO-1 in ARPE-19 cells was measured by Western blot and immunofluorescent staining.Western blot was also used to detect the expression of tissue factor（TF）.CEC Transmigration Assay was used to measure the migration of ARPE-19 cells.The transport of fluorescent markers [fluorescein isothiocyanate dextrans of 4,10,20（FD4,FD10,FD20） ]and the transepithelial electrical resistance（TEER）were used to measure in ARPE-19 cell RESULTS：CCK-8 assay showed that 5μmol/L TF-TP can inhibit ARPE-19 cells abnormally proliferation stimulated by lipopolysaccharide（LPS;P〈0.05）.LPS increased the transport of fluorescent markers（FD4,FD10,FD20）and decreased TEER levels in ARPE-19 cells,respectively,which were prevented by 5μmol/L TF-TP pretreatment（P〈0.05）. Furthermore,LPS significantly up-regulated the expression of TF and downregulated the expression of ZO-1（P〈0.05）in ARPE-19 cell which was inhibited by the TF-TP（P〈0.05）.In addition,TF-TP inhibited the abnormal migration induced by LPS in ARPE-19 cell（P〈0.05）.CONCLUSION：Our findings suggest that TF-TP suppressed proliferation and migration of ARPE-19 cells induced by LPS,and maintained the RPE tight junctions through inhibition of TF expression and increased expression of ZO-1.展开更多
文摘目的:利用噬菌体随机肽库筛选与人肺泡Ⅱ型上皮A549细胞存在特异性结合的多肽,为实现对肺泡Ⅱ型上皮细胞的靶向干预和开发针对急性肺损伤等呼吸系统疾病治疗的导向药物载体提供实验依据。方法:采用全细胞筛选的方式,通过对人肺泡Ⅱ型上皮A549细胞的4轮筛选和与人前列腺癌上皮PC-3细胞的差减筛选,从噬菌体随机肽库中筛选出与人肺泡Ⅱ型上皮A549细胞存在特异性结合的多肽,选择丰度较高的一条命名为NTG,用其构建含有增强绿色荧光蛋白(EGFP)的原核表达载体,转化感受态细菌,层析法获得重组蛋白His-NTG-EGFP,测定其分子质量。向A549细胞和PC-3细胞分别加入含有重组蛋白His-NTG-EGFP或His-EGFP的培养基,荧光显微镜下观察。结果:从第1轮至第4轮,淘选所得的噬菌体回收率逐步提高,经过PC-3细胞差减筛选,少量的与A549细胞存在特异性结合的噬菌体得到了回收。重组表达并纯化得到的His-NTG-EGFP,测定其分子质量为33 k D,与预期相符。荧光显微镜下观察发现,经融合蛋白His-NTG-EGFP处理后的A549细胞表面有绿色荧光分布,经融合蛋白His-NTG-EGFP处理后的PC-3细胞以及经对照蛋白处理的A549细胞和PC-3细胞表面均未出现绿色荧光,说明这一融合蛋白可以特异性结合A549细胞。结论:人肺泡Ⅱ型上皮A549细胞特异性靶向肽被成功获得。
基金Project supported by the National Distinguished Young Scholar Grant
文摘There are three non-allelic isogenes encoding phosphoribosylanthranilate isomerase (PAI) in Arabidopsis thaliana. The expression plasmids were constructed by fusion of the GUS reporter gene to the three PAI promoters with or without the 5' region encoding PAI N-terminal polypeptides and transferred into Arabidopsis plants by Agrobacterium tumefaciens. Analysis of GUS activity revealed that the PAI 5' coding region was necessary for high expression of GUS activity. GUS activity in transgenic plants transformed with the expression plasmids containing the 5' coding region of PAH or PAD was 60-100-fold higher than that without the corresponding 5' region. However, the effect of 5' coding region of PAI2 gene on the GUS activity was very small (only about 1 time difference) . The GUS histochemical staining showed a similar result as revealed by GUS activity assay. It was expressed in the mesophyll cells and guard cells, but not in the epidermic cells, indicating that the N-terminal polypeptides encoded by the 5' region of PAI genes have the function of PTP.
基金Supported by Science and Technology Project of Guangzhou City(No.2014J4100035)the Project of the Third Affiliated Hospital of Guangzhou Medical University(No.2013Y06)
文摘AIM：To investigate the effect of tissue factor targeting peptide（TF-TP）on retinal pigment epithelium（RPE）cells tight junctions.METHODS：Cell counting kit-8（CCK-8）was used to measure the proliferation of ARPE-19 cells.Expression of tight junction,ZO-1 in ARPE-19 cells was measured by Western blot and immunofluorescent staining.Western blot was also used to detect the expression of tissue factor（TF）.CEC Transmigration Assay was used to measure the migration of ARPE-19 cells.The transport of fluorescent markers [fluorescein isothiocyanate dextrans of 4,10,20（FD4,FD10,FD20） ]and the transepithelial electrical resistance（TEER）were used to measure in ARPE-19 cell RESULTS：CCK-8 assay showed that 5μmol/L TF-TP can inhibit ARPE-19 cells abnormally proliferation stimulated by lipopolysaccharide（LPS;P〈0.05）.LPS increased the transport of fluorescent markers（FD4,FD10,FD20）and decreased TEER levels in ARPE-19 cells,respectively,which were prevented by 5μmol/L TF-TP pretreatment（P〈0.05）. Furthermore,LPS significantly up-regulated the expression of TF and downregulated the expression of ZO-1（P〈0.05）in ARPE-19 cell which was inhibited by the TF-TP（P〈0.05）.In addition,TF-TP inhibited the abnormal migration induced by LPS in ARPE-19 cell（P〈0.05）.CONCLUSION：Our findings suggest that TF-TP suppressed proliferation and migration of ARPE-19 cells induced by LPS,and maintained the RPE tight junctions through inhibition of TF expression and increased expression of ZO-1.
