[ Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [ Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus, two...[ Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [ Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus, two pairs of pdmers were designed to amplify the HN and F genes from GX1 strain of goose paramyxovirus isolated from diseased goose in Guangxi Zhuang Autonomous Region; the amplified products were ligated into pMD18-T vector and sequenced. [ Result ] HN and F genes of this strain tested were 1 716 and 1 662 bp in full nucleotide length, respectively; both showed the homologues of about 97.3% with GPV- SF02 strain, of 80.3% -97.5% with strains LaSota, F48E9 and JS, of just 84.8% with Miyadera strain. [ Conclusion] The results show that isolated strain BX1 matches to virulent APMV-1 strain, belonging to genotype Ⅶ of APMV-1 strain.展开更多
AIM： To investigate the biological function of F protein by yeast two-hybrid system. METHODS： We constructed F protein bait plasmid by cloning the gene of F protein into pGBKT7, then recombinant plasmid DNA was tran...AIM： To investigate the biological function of F protein by yeast two-hybrid system. METHODS： We constructed F protein bait plasmid by cloning the gene of F protein into pGBKT7, then recombinant plasmid DNA was transformed into yeast AH109 （a type）. The transformed yeast AH109 was mated with yeast Y187 （α type） containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-HisAde） containing X-α-gal for selection and screening. After extracting and sequencing plasmids from positive （blue） colonies, we underwent sequence analysis by bioinformatics. RESULTS： Thirty-six colonies were selected and sequenced. Among them, 11 colonies were zymogen granule protein, 5 colonies were zinc finger protein, 4 colonies were zinc-α-2-glycoprotein, 1 colony was sialyltransferase, 1 colony was complement control protein factor I, 1 colony was vitronectin, and 2 colonies were new genes with unknown function. CONCLUSION： The yeast two-hybrid system is an effective method for identifying hepatocyte proteins interacting with F protein of hepatitis C virus. F protein may bind to different proteins. 2005 The WJG Press and Elsevier Inc. All rights reserved展开更多
AIM： To clone human liver special F protein and to express it in a prokaryotic system. METHODS： Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse prim...AIM： To clone human liver special F protein and to express it in a prokaryotic system. METHODS： Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this, cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein＇s cDNA was subdoned into the expression vector pET-15b and transformed into E. coli BL21 （DE3） pLyss. Isopropy-β-D-thiogalactoside （IPTG） was then used to induce expression of the target protein. RESULTS： The cDNA clone of human liver special F protein （1134bp） was successfully produced, with the cDNA sequence being published in Gene-bank： DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted. CONCLUSION： F protein expresses cDNA clone in a prokaryotic system, which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein.展开更多
[Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prekaryotic cells as well...[Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prekaryotic cells as well as to study the reactogenieity of the expressed products. [ Method ] RT-PCR amplification was used to obtain H and F protein genes; TA cloning and subclonlng techniques were used to construct the cloning plasmids（pMD-18T-H and pMD-18T-F） and recombinant expression plasmids（pET28a-H and pET28a-F） ; SDS-PAGE and Western-blotting were adopted to verify whether the target proteins were successfully expressed. [ Result] The recombinant expression plasmids pET28a-H and pET28a-F containing H and F protein genes of Canine distemper virus isolated from a mink were successfully constructed, and both the expressed H and F proteins with respectively relative molecular mass of 31 400 and 38 200 produced positive reac- tion with the CDV standard positive serum. [ Conclusion] The H and F proteins expressed in prokaryotic cells were the same with the natural ones in terms of reac- togenicity, which can be utilized for diagnosis of a CDV＇s infection or for an epidemiological investigation. Meanwhile, they also provide a basis for developing ge- netically engineered subunit vaccines.展开更多
目的:探讨血清抗着丝粒蛋白F抗体(anti-centromere protein F antibody,anti-CENPF)在乳腺癌中的临床价值。方法:收集100例初诊乳腺癌(breast cancer,BC)患者、40例乳腺良性疾病(non breast cancer,non-BC)患者和40名健康体检者(healthy...目的:探讨血清抗着丝粒蛋白F抗体(anti-centromere protein F antibody,anti-CENPF)在乳腺癌中的临床价值。方法:收集100例初诊乳腺癌(breast cancer,BC)患者、40例乳腺良性疾病(non breast cancer,non-BC)患者和40名健康体检者(healthy control,HC)血清,采用酶联免疫吸附试验检测血清中的anti-CENPF水平,同时化学发光法测定血清糖类抗原153(carbohydrate antigen 153,CA153)的水平。结果:BC组血清anti-CENPF水平高于non-BC组和HC组,差异有统计学意义(P<0.05)。anti-CENPF和CA153诊断乳腺癌中的曲线下面积(area under the curve,AUC)分别为0.714和0.672,两者联合检测的AUC为0.739。有淋巴结转移、远处转移或者人表皮生长因子受体2(human epidermal growth factor receptor-2,HER-2)阴性的乳腺癌患者血清anti-CENPF浓度明显升高(P<0.05)。不同肿瘤大小、临床分期、分子分型乳腺癌患者血清anti-CENPF水平比较,差异有统计学意义(P<0.05)。组间比较显示Ⅳ期和Ⅲ期血清anti-CENPF浓度高于Ⅰ期和Ⅱ期,HER-2过表达型、Luminal B型血清anti-CENPF水平低于三阴性乳腺癌(triple negative breast cancer,TNBC)患者。雌激素受体(receptors estrogen,ER)阳性的乳腺癌患者中,HER-2阴性组的anti-CENPF浓度高于HER-2阳性组,差异有统计学意义(P=0.026)。结论:血清antiCENPF在乳腺癌的诊断、临床分期及分子分型中发挥了重要作用,其水平可能与乳腺癌预后呈负相关,有望成为乳腺癌潜在的疾病标志物。展开更多
目的:探讨着丝粒蛋白F(centromere protein F,CENPF)在腺样囊性癌(adenoid cystic carcinoma,ACC)发生发展中的作用和分子机制。方法:通过小干扰RNA转染敲低ACC细胞中的CENPF。通过细胞计数试剂盒(cell counting kit-8,CCK-8)、划痕实验...目的:探讨着丝粒蛋白F(centromere protein F,CENPF)在腺样囊性癌(adenoid cystic carcinoma,ACC)发生发展中的作用和分子机制。方法:通过小干扰RNA转染敲低ACC细胞中的CENPF。通过细胞计数试剂盒(cell counting kit-8,CCK-8)、划痕实验和Transwell测定评估敲低CENPF对ACC细胞增殖、迁移、侵袭的影响;通过蛋白免疫印迹实验分析改变CENPF表达后ACC细胞中磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/丝氨酸/苏氨酸蛋白激酶(serine/threonine protein kinase,AKT)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路相关蛋白表达变化,通过CCK-8、划痕实验和Transwell测定评估敲低CENPF联合PI3K抑制剂BKM-120对ACC细胞增殖、迁移、侵袭的影响。结果:敲低ACC-M中CENPF表达后,CCK-8实验证明其增殖能力减弱;划痕实验表明其迁移能力减弱,Transwell实验表明其侵袭能力减弱,PI3K/AKT通路关键蛋白p-PI3K、p-AKT和p-mTOR蛋白表达水平下降。在下调CENPF的ACC-M中加入PI3K抑制剂,PI3K/AKT通路关键蛋白表达水平进一步下降。此外,加入PI3K抑制剂后,CENPF下调的ACC-M增殖、迁移、侵袭能力较单纯下调CENPF的ACC-M进一步减弱。结论:CENPF通过调控PI3K/AKT信号通路参与ACC进展。展开更多
Objective To determine if DNA excision repair enzymes oxoguanine glycosylase 1 （OGG1） and xeroderma pigmentosum group F protein （XPF） are involved in the pathogenesis of Parkinson＇s disease （PD） in a cell model...Objective To determine if DNA excision repair enzymes oxoguanine glycosylase 1 （OGG1） and xeroderma pigmentosum group F protein （XPF） are involved in the pathogenesis of Parkinson＇s disease （PD） in a cell model. Methods PC12 cells were treated with 1-Methyl-4-phenylpyridine ion （MPP＋） for various periods of time to induce oxidative DNA damage. MTT assay was used to determine cell viability. Immunocytochemistry with antibody against 8-hydroxy-2＇- deoxyguanosine （8-oxodG） was used to evaluate oxidative DNA damage. Immunoblotting was used to detect the protein levels of OGG1 and XPF. Results MPP＋ treatment （1 mmol/L） for 18 h and 24 h reduced cell viability to 78.6% and 70.3% of the control, respectively, in a time-dependent way. MPP＋ increased the immunoreactivity of 8-oxodG in the cytoplasm at 3 h and in the nucleus at 24 h of treatment. With the treatment of MPP＋, the expression of OGG1 was significantly increased at 1 h, reaching a peak at 3 h, and then it was decreased at 24 h, as compared to that with vehicle treatment. The same effect was exerted on XPF level, except that the XPF level reached a peak at 18 h of MPP＋ treatment. Moreover, the maximally-increased protein level of OGG1 by MPP＋ was approximately 2-fold higher than that of XPF. Conclusion MPP＋ treatment could time- dependently induce increases in OGG1 and XPF expressions in PC12 cells. Also, this study indicates that the base and nucleotide excision repair pathways may be compensatorily activated in the early stage of pathogenesis in the cells after MPP＋ treatment.展开更多
Objective Fusion protein is a subunit of the human respiratory syncytial virus(HRSV)and a potential vaccine candidate.Thus,a study on the genetic characteristics of F protein was considered important for further inves...Objective Fusion protein is a subunit of the human respiratory syncytial virus(HRSV)and a potential vaccine candidate.Thus,a study on the genetic characteristics of F protein was considered important for further investigations in this field.The aim of this study was to determine the prevalence and genetic diversity of the F gene of HRSV infections in hospitalized pediatric patients in Beijing with acute lower respiratory tract infections and to compare the circulating genotypes that are currently found worldwide.Methods HRSV particles were amplified by RT-PCR and the PCR products were purified for sequencing.Further analysis was carried out by Bioedit and MEGA 3.0 biological software programs.Results Seventy-six samples(23.1%)were positive for HRSV.The percentage of cases in patients younger than 1year was 84.21%.Among the six Beijing isolates,four belonged to subgroup A,whose respective F genes shared97.0%-97.4%nucleotide sequence identity and 92.1%-93.0%amino acid sequence identity.The other two isolates belonged to subgroup B.Here,97.3%and 98.2%sequence identity were found at nucleotide and amino acid levels,respectively.Conclusions Phylogenetic analysis of nucleotide sequences revealed that those four isolates within subgroup A were monophyletic and closely related to each other,but those two within subgroup B distributed in two distinct clusters.Subgroup A and B strains co-circulated,indicating that two different transmission chains occurred in Beijing from 2003-2004.展开更多
目的·研究F-box蛋白38(F-box only protein 38,FBXO38)对眼部黑色素瘤增殖的作用以及潜在的调控通路。方法·使用FBXO38短发夹RNA(short hairpin RNA,shRNA)和FBXO38过表达质粒构建FBXO38敲低以及过表达的人皮肤黑色素瘤A375...目的·研究F-box蛋白38(F-box only protein 38,FBXO38)对眼部黑色素瘤增殖的作用以及潜在的调控通路。方法·使用FBXO38短发夹RNA(short hairpin RNA,shRNA)和FBXO38过表达质粒构建FBXO38敲低以及过表达的人皮肤黑色素瘤A375和葡萄膜黑色素瘤OMM2.3细胞系,并通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和Western blotting在转录和蛋白水平验证FBXO38的敲低和过表达效率。通过克隆形成实验、BrdU免疫荧光染色和CCK8细胞增殖实验,探究FBXO38对黑色素瘤细胞增殖的影响。使用肿瘤基因组图谱计划数据库(The Cancer Genome Atlas,TCGA),分析FBXO38高表达和低表达组中的差异表达基因,并进行京都基因与基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集,揭示与FBXO38相关的信号通路。进一步通过CCK8细胞增殖实验检测信号通路抑制剂对不同FBXO38表达量细胞的抑制率。同时通过qRT-PCR和Western blotting,验证在敲低FBXO38之后该通路是否激活。结果·qRT-PCR和Western blotting验证A375和OMM2.3细胞系中的FBXO38的mRNA及蛋白质表达水平,发现与对照组相比敲低组的FBXO38表达水平下降,与野生型相比过表达组的FBXO38的表达水平提高(P<0.05)。克隆形成实验、BrdU免疫荧光染色和CCK8细胞增殖实验显示,敲低FBXO38显著增强A375和OMM2.3细胞的增殖能力(P<0.05),反之过表达FBXO38抑制A375和OMM2.3细胞增殖(P<0.05)。KEGG通路富集分析显示,在皮肤黑色素瘤和葡萄膜黑色素瘤中,FBXO38的表达影响磷脂酰肌醇3激酶/蛋白激酶B(phosphoinositide 3-kinase/protein kinase B,PI3K-Akt)通路激活。与对照组相比,PI3K抑制剂LY294002和mTOR1抑制剂Everolimus对FBXO38敲低组的抑制率显著提升(P<0.05),对FBXO38过表达组的抑制率则显著下降(P<0.05)。Western blotting结果显示,敲低FBXO38之后,与PI3K-Akt通路相关的PTEN、P21和P53蛋白水平下降,而MDM2蛋白水平上升。qRT-PCR结果显示在FBXO38敲低细胞中P53转录水平显著下降(P<0.05),而MDM2转录水平显著上升(P<0.05)。结论·FBXO38通过PI3K-Akt信号通路参与调控眼部黑色素瘤细胞的增殖。展开更多
基金Supported by the Development Program for Guangxi Science andTechnology(0719004-3G)~~
文摘[ Objective] The study was to clone HN and F genes from GX1 strain of goose paramyxovirus and analyze their sequences. [ Method] According to the full nucleotide sequence of GPV-SF02 strain of goose paramyxovirus, two pairs of pdmers were designed to amplify the HN and F genes from GX1 strain of goose paramyxovirus isolated from diseased goose in Guangxi Zhuang Autonomous Region; the amplified products were ligated into pMD18-T vector and sequenced. [ Result ] HN and F genes of this strain tested were 1 716 and 1 662 bp in full nucleotide length, respectively; both showed the homologues of about 97.3% with GPV- SF02 strain, of 80.3% -97.5% with strains LaSota, F48E9 and JS, of just 84.8% with Miyadera strain. [ Conclusion] The results show that isolated strain BX1 matches to virulent APMV-1 strain, belonging to genotype Ⅶ of APMV-1 strain.
基金Supported by the National Natural Science Foundation of China, Nos. C03011402 and C30070690 and the Research and Technique Foundation of PLA during the 9~(th)-Five year plan period, No. 98D063 and the Launching Foundation for Student Studying Abroad of PLA, No. 98H038 and the Youth Research and Technique Foundation of PLA during the 10~(th)-Five Year Plan Period, No. 01Q138 and the Research and Technique Foundation of PLA during the 10~(th)-Five Year Plan Period, No. 01MB135
文摘AIM： To investigate the biological function of F protein by yeast two-hybrid system. METHODS： We constructed F protein bait plasmid by cloning the gene of F protein into pGBKT7, then recombinant plasmid DNA was transformed into yeast AH109 （a type）. The transformed yeast AH109 was mated with yeast Y187 （α type） containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-HisAde） containing X-α-gal for selection and screening. After extracting and sequencing plasmids from positive （blue） colonies, we underwent sequence analysis by bioinformatics. RESULTS： Thirty-six colonies were selected and sequenced. Among them, 11 colonies were zymogen granule protein, 5 colonies were zinc finger protein, 4 colonies were zinc-α-2-glycoprotein, 1 colony was sialyltransferase, 1 colony was complement control protein factor I, 1 colony was vitronectin, and 2 colonies were new genes with unknown function. CONCLUSION： The yeast two-hybrid system is an effective method for identifying hepatocyte proteins interacting with F protein of hepatitis C virus. F protein may bind to different proteins. 2005 The WJG Press and Elsevier Inc. All rights reserved
基金Supported by a grant from the Tianjin Science and Technology Committee, No. 023802911
文摘AIM： To clone human liver special F protein and to express it in a prokaryotic system. METHODS： Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this, cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein＇s cDNA was subdoned into the expression vector pET-15b and transformed into E. coli BL21 （DE3） pLyss. Isopropy-β-D-thiogalactoside （IPTG） was then used to induce expression of the target protein. RESULTS： The cDNA clone of human liver special F protein （1134bp） was successfully produced, with the cDNA sequence being published in Gene-bank： DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted. CONCLUSION： F protein expresses cDNA clone in a prokaryotic system, which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein.
基金Supported by the Natural Science Foundation of Jilin Province(201115194)Education Department of Jilin Province(2009.No.66)
文摘[Objective] This study aimed to construct the recombinant expression plasmids containing H and F protein genes of Canine distemper virus isolated from a mink and to express these two genes in prekaryotic cells as well as to study the reactogenieity of the expressed products. [ Method ] RT-PCR amplification was used to obtain H and F protein genes; TA cloning and subclonlng techniques were used to construct the cloning plasmids（pMD-18T-H and pMD-18T-F） and recombinant expression plasmids（pET28a-H and pET28a-F） ; SDS-PAGE and Western-blotting were adopted to verify whether the target proteins were successfully expressed. [ Result] The recombinant expression plasmids pET28a-H and pET28a-F containing H and F protein genes of Canine distemper virus isolated from a mink were successfully constructed, and both the expressed H and F proteins with respectively relative molecular mass of 31 400 and 38 200 produced positive reac- tion with the CDV standard positive serum. [ Conclusion] The H and F proteins expressed in prokaryotic cells were the same with the natural ones in terms of reac- togenicity, which can be utilized for diagnosis of a CDV＇s infection or for an epidemiological investigation. Meanwhile, they also provide a basis for developing ge- netically engineered subunit vaccines.
文摘目的:探讨血清抗着丝粒蛋白F抗体(anti-centromere protein F antibody,anti-CENPF)在乳腺癌中的临床价值。方法:收集100例初诊乳腺癌(breast cancer,BC)患者、40例乳腺良性疾病(non breast cancer,non-BC)患者和40名健康体检者(healthy control,HC)血清,采用酶联免疫吸附试验检测血清中的anti-CENPF水平,同时化学发光法测定血清糖类抗原153(carbohydrate antigen 153,CA153)的水平。结果:BC组血清anti-CENPF水平高于non-BC组和HC组,差异有统计学意义(P<0.05)。anti-CENPF和CA153诊断乳腺癌中的曲线下面积(area under the curve,AUC)分别为0.714和0.672,两者联合检测的AUC为0.739。有淋巴结转移、远处转移或者人表皮生长因子受体2(human epidermal growth factor receptor-2,HER-2)阴性的乳腺癌患者血清anti-CENPF浓度明显升高(P<0.05)。不同肿瘤大小、临床分期、分子分型乳腺癌患者血清anti-CENPF水平比较,差异有统计学意义(P<0.05)。组间比较显示Ⅳ期和Ⅲ期血清anti-CENPF浓度高于Ⅰ期和Ⅱ期,HER-2过表达型、Luminal B型血清anti-CENPF水平低于三阴性乳腺癌(triple negative breast cancer,TNBC)患者。雌激素受体(receptors estrogen,ER)阳性的乳腺癌患者中,HER-2阴性组的anti-CENPF浓度高于HER-2阳性组,差异有统计学意义(P=0.026)。结论:血清antiCENPF在乳腺癌的诊断、临床分期及分子分型中发挥了重要作用,其水平可能与乳腺癌预后呈负相关,有望成为乳腺癌潜在的疾病标志物。
文摘目的:探讨着丝粒蛋白F(centromere protein F,CENPF)在腺样囊性癌(adenoid cystic carcinoma,ACC)发生发展中的作用和分子机制。方法:通过小干扰RNA转染敲低ACC细胞中的CENPF。通过细胞计数试剂盒(cell counting kit-8,CCK-8)、划痕实验和Transwell测定评估敲低CENPF对ACC细胞增殖、迁移、侵袭的影响;通过蛋白免疫印迹实验分析改变CENPF表达后ACC细胞中磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/丝氨酸/苏氨酸蛋白激酶(serine/threonine protein kinase,AKT)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路相关蛋白表达变化,通过CCK-8、划痕实验和Transwell测定评估敲低CENPF联合PI3K抑制剂BKM-120对ACC细胞增殖、迁移、侵袭的影响。结果:敲低ACC-M中CENPF表达后,CCK-8实验证明其增殖能力减弱;划痕实验表明其迁移能力减弱,Transwell实验表明其侵袭能力减弱,PI3K/AKT通路关键蛋白p-PI3K、p-AKT和p-mTOR蛋白表达水平下降。在下调CENPF的ACC-M中加入PI3K抑制剂,PI3K/AKT通路关键蛋白表达水平进一步下降。此外,加入PI3K抑制剂后,CENPF下调的ACC-M增殖、迁移、侵袭能力较单纯下调CENPF的ACC-M进一步减弱。结论:CENPF通过调控PI3K/AKT信号通路参与ACC进展。
基金supported by the National Natural Science Foundation of China (No. 30770660,J0730860)
文摘Objective To determine if DNA excision repair enzymes oxoguanine glycosylase 1 （OGG1） and xeroderma pigmentosum group F protein （XPF） are involved in the pathogenesis of Parkinson＇s disease （PD） in a cell model. Methods PC12 cells were treated with 1-Methyl-4-phenylpyridine ion （MPP＋） for various periods of time to induce oxidative DNA damage. MTT assay was used to determine cell viability. Immunocytochemistry with antibody against 8-hydroxy-2＇- deoxyguanosine （8-oxodG） was used to evaluate oxidative DNA damage. Immunoblotting was used to detect the protein levels of OGG1 and XPF. Results MPP＋ treatment （1 mmol/L） for 18 h and 24 h reduced cell viability to 78.6% and 70.3% of the control, respectively, in a time-dependent way. MPP＋ increased the immunoreactivity of 8-oxodG in the cytoplasm at 3 h and in the nucleus at 24 h of treatment. With the treatment of MPP＋, the expression of OGG1 was significantly increased at 1 h, reaching a peak at 3 h, and then it was decreased at 24 h, as compared to that with vehicle treatment. The same effect was exerted on XPF level, except that the XPF level reached a peak at 18 h of MPP＋ treatment. Moreover, the maximally-increased protein level of OGG1 by MPP＋ was approximately 2-fold higher than that of XPF. Conclusion MPP＋ treatment could time- dependently induce increases in OGG1 and XPF expressions in PC12 cells. Also, this study indicates that the base and nucleotide excision repair pathways may be compensatorily activated in the early stage of pathogenesis in the cells after MPP＋ treatment.
文摘Objective Fusion protein is a subunit of the human respiratory syncytial virus(HRSV)and a potential vaccine candidate.Thus,a study on the genetic characteristics of F protein was considered important for further investigations in this field.The aim of this study was to determine the prevalence and genetic diversity of the F gene of HRSV infections in hospitalized pediatric patients in Beijing with acute lower respiratory tract infections and to compare the circulating genotypes that are currently found worldwide.Methods HRSV particles were amplified by RT-PCR and the PCR products were purified for sequencing.Further analysis was carried out by Bioedit and MEGA 3.0 biological software programs.Results Seventy-six samples(23.1%)were positive for HRSV.The percentage of cases in patients younger than 1year was 84.21%.Among the six Beijing isolates,four belonged to subgroup A,whose respective F genes shared97.0%-97.4%nucleotide sequence identity and 92.1%-93.0%amino acid sequence identity.The other two isolates belonged to subgroup B.Here,97.3%and 98.2%sequence identity were found at nucleotide and amino acid levels,respectively.Conclusions Phylogenetic analysis of nucleotide sequences revealed that those four isolates within subgroup A were monophyletic and closely related to each other,but those two within subgroup B distributed in two distinct clusters.Subgroup A and B strains co-circulated,indicating that two different transmission chains occurred in Beijing from 2003-2004.
文摘目的·研究F-box蛋白38(F-box only protein 38,FBXO38)对眼部黑色素瘤增殖的作用以及潜在的调控通路。方法·使用FBXO38短发夹RNA(short hairpin RNA,shRNA)和FBXO38过表达质粒构建FBXO38敲低以及过表达的人皮肤黑色素瘤A375和葡萄膜黑色素瘤OMM2.3细胞系,并通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和Western blotting在转录和蛋白水平验证FBXO38的敲低和过表达效率。通过克隆形成实验、BrdU免疫荧光染色和CCK8细胞增殖实验,探究FBXO38对黑色素瘤细胞增殖的影响。使用肿瘤基因组图谱计划数据库(The Cancer Genome Atlas,TCGA),分析FBXO38高表达和低表达组中的差异表达基因,并进行京都基因与基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集,揭示与FBXO38相关的信号通路。进一步通过CCK8细胞增殖实验检测信号通路抑制剂对不同FBXO38表达量细胞的抑制率。同时通过qRT-PCR和Western blotting,验证在敲低FBXO38之后该通路是否激活。结果·qRT-PCR和Western blotting验证A375和OMM2.3细胞系中的FBXO38的mRNA及蛋白质表达水平,发现与对照组相比敲低组的FBXO38表达水平下降,与野生型相比过表达组的FBXO38的表达水平提高(P<0.05)。克隆形成实验、BrdU免疫荧光染色和CCK8细胞增殖实验显示,敲低FBXO38显著增强A375和OMM2.3细胞的增殖能力(P<0.05),反之过表达FBXO38抑制A375和OMM2.3细胞增殖(P<0.05)。KEGG通路富集分析显示,在皮肤黑色素瘤和葡萄膜黑色素瘤中,FBXO38的表达影响磷脂酰肌醇3激酶/蛋白激酶B(phosphoinositide 3-kinase/protein kinase B,PI3K-Akt)通路激活。与对照组相比,PI3K抑制剂LY294002和mTOR1抑制剂Everolimus对FBXO38敲低组的抑制率显著提升(P<0.05),对FBXO38过表达组的抑制率则显著下降(P<0.05)。Western blotting结果显示,敲低FBXO38之后,与PI3K-Akt通路相关的PTEN、P21和P53蛋白水平下降,而MDM2蛋白水平上升。qRT-PCR结果显示在FBXO38敲低细胞中P53转录水平显著下降(P<0.05),而MDM2转录水平显著上升(P<0.05)。结论·FBXO38通过PI3K-Akt信号通路参与调控眼部黑色素瘤细胞的增殖。
