Objective:To study evolutionary relationship of the 5'untranslated regions(5'UTRs) in low passage dengue3 viruses(DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok dur...Objective:To study evolutionary relationship of the 5'untranslated regions(5'UTRs) in low passage dengue3 viruses(DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year-evolution(1977-2000) comparing to the DEN3prototype(H87).Methods:The 5'UTRs of these Thai DEN3 and the H87 prototype were amplified by RT-PCR and sequenced.Their multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software.Replication of five Thai DEN3 candidates comparing to the 1187 prototype were done in human(HepG2) and the mosquito(C6/36) cell lines.Results:Among these Thai DEN3,the completely identical sequences of their first 89 nucleotides,their high-order secondary structure of 5'UTRs and three SNPs including the predominant C90 T,and two minor SNPs including A109 G and A112 G were found.The C90 T of Thai DEN3.Bangkok isolates was shown predominantly before 1977.Five Thai DEN3 candidates with the predominant C90 T were shown to replicate in human(HepG2) and the mosquito(C6/36) cell lines better than the H87 prototype.However,their highly conserved sequences as well as SNPs of the 5'UTR did not appear to correlate with their disease severity in human.Conclusions:Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence,the high-order secondary structure and the predominant C90 T of the 5'UTR of these Thai DEN3 during 24 year-evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3.展开更多
目的:探讨缺氧对脑内胆固醇代谢稳态及神经元衰老的影响。方法:8~12周龄的C57BL/6野生型雄性小鼠随机分为常氧(Normoxia)组和缺氧(Hypoxia)组;以缺氧处理小鼠海马神经元HT22细胞并分为对照(Control)组和缺氧(Hypoxia)组;以10µmol/...目的:探讨缺氧对脑内胆固醇代谢稳态及神经元衰老的影响。方法:8~12周龄的C57BL/6野生型雄性小鼠随机分为常氧(Normoxia)组和缺氧(Hypoxia)组;以缺氧处理小鼠海马神经元HT22细胞并分为对照(Control)组和缺氧(Hypoxia)组;以10µmol/L剂量的24-羟基胆固醇(24S-Hydroxycholesterol,24S-OHC)处理HT22细胞并分为Control组和24S-OHC组。苏木精-伊红染色(hematoxylin-ensin,HE)和尼氏(nissl)染色观察脑组织结构;酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)检测血清、脑组织和细胞24S-OHC水平;微量法检测脑组织和细胞总胆固醇(total cholesterol,TC)与甘油三酯(triglyceride,TG)水平;蛋白质免疫印迹(western blot,WB)法检测脑组织和细胞胆固醇代谢相关酶3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl-coenzyme A reductase,HMGCR)、细胞色素P450家族46亚家族A成员1(cytochrome P450 family 46 subfamily A member 1,CYP46A1)、衰老相关蛋白细胞周期蛋白依赖性激酶抑制因子2A(cyclin-dependent kinase inhibitor 2A,CDKN2A/p16)、细胞周期蛋白依赖性激酶抑制因子1A(cyclin-dependent kinase inhibitor 1A,CDKN1A/p21)、磷酸化组蛋白(phospho-H2AX,γH2AX)和脂质合成相关分子抗硬脂酰辅酶A去饱和酶1(stearoyl-Coenzyme A desaturase 1,SCD1)、脂肪酸合酶(fatty acid synthase,FASN)、固醇调节元件结合蛋白1c(sterol regulatory element-binding protein 1c,SREBP1c)的表达水平;免疫组织化学染色检测脑内CYP46A1和γH2AX的表达;检测各组小鼠大脑组织中基因表达谱,筛选目标差异表达基因(differentially expressed genes,DEGs),对DEGs进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析;衰老相关β半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-Gal)染色评估细胞衰老情况;BODIPY染色观察细胞脂滴蓄积情况。结果:与常氧组比较,缺氧组脑湿重差异无统计学意义(P=0.573);Nissl染色显示存活神经元数目减少;血清24S-OHC水平升高,脑内24S-OHC和TG含量增多,TC含量减少(均P<0.05);胆固醇合成酶HMGCR表达降低,胆固醇分解酶CYP46A1表达升高,p16、p21和γH2AX表达增加(均P<0.05);转录组学结果显示,在GO分析中炎症通路与脂质相关通路富集,在KEGG分析中磷脂酰肌醇3-激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein kinase B,PI3K-Akt)信号通路和肿瘤蛋白p53(tumor protein 53,p53)信号通路等与衰老相关通路富集。缺氧处理HT22细胞实验中,与Control组相比,Hypoxia组细胞内TC含量减少,24S-OHC含量增多(均P<0.05);HMGCR表达降低,CYP46A1表达升高,p16、p21、γH2AX表达增加(均P<0.05);SA-β-gal阳性细胞明显增多。24S-OHC处理HT22细胞实验中,与Control组相比,24S-OHC组细胞内TC和TG含量均增多(均P<0.05);细胞内脂滴含量明显增多(P<0.05);HMGCR和CYP46A1表达降低,SCD1、FASN和SREBP1c表达升高,p16、p21和γH2AX表达升高(均P<0.05)。结论:慢性缺氧通过下调HMGCR和上调CYP46A1表达,诱导24S-OHC异常蓄积并触发SREBP1c/SCD1信号通路介导的脂毒性衰老。展开更多
恶性肿瘤严重威胁人类健康,其发病机制复杂多样。分化簇抗原(cluster of differentiation,CD)24是一种近年来备受关注的糖基化膜蛋白,其在人体正常组织中参与细胞生理功能调控、免疫稳态维持、组织发育与分化等生理过程,而在乳腺癌、卵...恶性肿瘤严重威胁人类健康,其发病机制复杂多样。分化簇抗原(cluster of differentiation,CD)24是一种近年来备受关注的糖基化膜蛋白,其在人体正常组织中参与细胞生理功能调控、免疫稳态维持、组织发育与分化等生理过程,而在乳腺癌、卵巢癌、肺癌等多种恶性肿瘤组织中呈现异常高表达。CD24为肿瘤干细胞(cancer stem cell,CSC)核心标志物,与肿瘤细胞的恶性生物学行为、肿瘤恶性进展、治疗耐受及不良预后密切相关。CD24可通过CD24/唾液酸结合免疫球蛋白样凝集素-10(sialic acid-binding immunoglobulin-like lectin-10,Siglec-10)通路抑制巨噬细胞吞噬、重塑肿瘤免疫抑制微环境、激活促癌信号通路、调控肿瘤细胞黏附转移等多重机制参与肿瘤发生和发展,且在血液肿瘤中表现出独特的功能双重性。系统探究CD24在不同恶性肿瘤中的表达特征、作用机制及预后价值,发现靶向CD24的单克隆抗体等治疗策略有良好的临床应用前景,且CD24相较经典免疫检查点具有低血液毒性的潜在优势。研究证实CD24可作为恶性肿瘤精准诊断、预后评估的潜在标志物及个体化治疗的新靶点,具有重要的基础研究意义与临床转化价值。展开更多
基金supported by two research grants of Associate Professor Dr.W.Attatippaholkun:Grant No.493-5600-G-00-3461,Program in Science and Technology Cooperation,Human Capacity Development,Bureau for Global Programs,Field Support and Research,US Agency for International Development,Washington,DCThe Royal Golden Jubilee-Ph.D Program,Thailand Research Fund,Thailand
文摘Objective:To study evolutionary relationship of the 5'untranslated regions(5'UTRs) in low passage dengue3 viruses(DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year-evolution(1977-2000) comparing to the DEN3prototype(H87).Methods:The 5'UTRs of these Thai DEN3 and the H87 prototype were amplified by RT-PCR and sequenced.Their multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software.Replication of five Thai DEN3 candidates comparing to the 1187 prototype were done in human(HepG2) and the mosquito(C6/36) cell lines.Results:Among these Thai DEN3,the completely identical sequences of their first 89 nucleotides,their high-order secondary structure of 5'UTRs and three SNPs including the predominant C90 T,and two minor SNPs including A109 G and A112 G were found.The C90 T of Thai DEN3.Bangkok isolates was shown predominantly before 1977.Five Thai DEN3 candidates with the predominant C90 T were shown to replicate in human(HepG2) and the mosquito(C6/36) cell lines better than the H87 prototype.However,their highly conserved sequences as well as SNPs of the 5'UTR did not appear to correlate with their disease severity in human.Conclusions:Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence,the high-order secondary structure and the predominant C90 T of the 5'UTR of these Thai DEN3 during 24 year-evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3.
文摘目的:探讨缺氧对脑内胆固醇代谢稳态及神经元衰老的影响。方法:8~12周龄的C57BL/6野生型雄性小鼠随机分为常氧(Normoxia)组和缺氧(Hypoxia)组;以缺氧处理小鼠海马神经元HT22细胞并分为对照(Control)组和缺氧(Hypoxia)组;以10µmol/L剂量的24-羟基胆固醇(24S-Hydroxycholesterol,24S-OHC)处理HT22细胞并分为Control组和24S-OHC组。苏木精-伊红染色(hematoxylin-ensin,HE)和尼氏(nissl)染色观察脑组织结构;酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)检测血清、脑组织和细胞24S-OHC水平;微量法检测脑组织和细胞总胆固醇(total cholesterol,TC)与甘油三酯(triglyceride,TG)水平;蛋白质免疫印迹(western blot,WB)法检测脑组织和细胞胆固醇代谢相关酶3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl-coenzyme A reductase,HMGCR)、细胞色素P450家族46亚家族A成员1(cytochrome P450 family 46 subfamily A member 1,CYP46A1)、衰老相关蛋白细胞周期蛋白依赖性激酶抑制因子2A(cyclin-dependent kinase inhibitor 2A,CDKN2A/p16)、细胞周期蛋白依赖性激酶抑制因子1A(cyclin-dependent kinase inhibitor 1A,CDKN1A/p21)、磷酸化组蛋白(phospho-H2AX,γH2AX)和脂质合成相关分子抗硬脂酰辅酶A去饱和酶1(stearoyl-Coenzyme A desaturase 1,SCD1)、脂肪酸合酶(fatty acid synthase,FASN)、固醇调节元件结合蛋白1c(sterol regulatory element-binding protein 1c,SREBP1c)的表达水平;免疫组织化学染色检测脑内CYP46A1和γH2AX的表达;检测各组小鼠大脑组织中基因表达谱,筛选目标差异表达基因(differentially expressed genes,DEGs),对DEGs进行基因本体(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析;衰老相关β半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-Gal)染色评估细胞衰老情况;BODIPY染色观察细胞脂滴蓄积情况。结果:与常氧组比较,缺氧组脑湿重差异无统计学意义(P=0.573);Nissl染色显示存活神经元数目减少;血清24S-OHC水平升高,脑内24S-OHC和TG含量增多,TC含量减少(均P<0.05);胆固醇合成酶HMGCR表达降低,胆固醇分解酶CYP46A1表达升高,p16、p21和γH2AX表达增加(均P<0.05);转录组学结果显示,在GO分析中炎症通路与脂质相关通路富集,在KEGG分析中磷脂酰肌醇3-激酶/蛋白激酶B(phosphatidylinositol 3-kinase/protein kinase B,PI3K-Akt)信号通路和肿瘤蛋白p53(tumor protein 53,p53)信号通路等与衰老相关通路富集。缺氧处理HT22细胞实验中,与Control组相比,Hypoxia组细胞内TC含量减少,24S-OHC含量增多(均P<0.05);HMGCR表达降低,CYP46A1表达升高,p16、p21、γH2AX表达增加(均P<0.05);SA-β-gal阳性细胞明显增多。24S-OHC处理HT22细胞实验中,与Control组相比,24S-OHC组细胞内TC和TG含量均增多(均P<0.05);细胞内脂滴含量明显增多(P<0.05);HMGCR和CYP46A1表达降低,SCD1、FASN和SREBP1c表达升高,p16、p21和γH2AX表达升高(均P<0.05)。结论:慢性缺氧通过下调HMGCR和上调CYP46A1表达,诱导24S-OHC异常蓄积并触发SREBP1c/SCD1信号通路介导的脂毒性衰老。
