背景与目的:肿瘤的发生、发展过程中会发生代谢重编程,1-酰基甘油-3-磷酸O-酰基转移酶(1-acylglycerol-3-phosphate O-acyltransferase,AGPAT)作为三酰甘油(triacylglycerol,TAG)从头合成的关键酶,与肿瘤的进展密切相关。但目前作为亚...背景与目的:肿瘤的发生、发展过程中会发生代谢重编程,1-酰基甘油-3-磷酸O-酰基转移酶(1-acylglycerol-3-phosphate O-acyltransferase,AGPAT)作为三酰甘油(triacylglycerol,TAG)从头合成的关键酶,与肿瘤的进展密切相关。但目前作为亚型之一的AGPAT5在癌症中的研究还十分有限,本研究深入剖析AGPAT5在肝癌发生、发展中发挥的作用及潜在的分子机制,旨在为肝癌诊断和治疗策略提供新思路。方法:利用慢病毒感染将多种肝癌细胞系中的AGPAT5敲减,并通过锥虫蓝计数、划痕、transwell及平板克隆等实验在体外检测AGPAT5对肝癌细胞增殖、迁移及抗失巢凋亡能力的影响。通过回复野生型或酶活性缺失型的AGPAT5,探究其作为代谢酶是否发挥经典代谢作用调控肝癌细胞迁移。构建BALB/c裸鼠尾静脉注射移植瘤模型,从体内层面验证体外的细胞表型。采用免疫沉淀质谱联用(immunoprecipitation mass spectrum,IP-MS)鉴定出与AGPAT5相互作用的蛋白,并进行免疫共沉淀(co-immunoprecipitation,coIP)验证。蛋白质翻译后通过修饰鉴定分析AGPAT5潜在的修饰位点,通过体外实验探究点突变前后对肝癌细胞迁移的影响。通过coIP探究该位点突变前后AGPAT5与相互作用蛋白结合的情况。通过敲低相互作用蛋白确定其在细胞表型中的作用。通过回复实验验证AGPAT5是否通过相互作用蛋白发挥作用。检测野生型肝癌细胞系中的AGPAT5和相互作用蛋白的表达水平,检验两者之间是否具有相关性。结果:肝癌细胞敲减AGPAT5后会更加耐受无血清饥饿,并促进细胞迁移,但不会影响细胞增殖和失巢凋亡。而酶活性缺失并不影响AGPAT5对肝癌细胞迁移的抑制。敲减AGPAT5可促进肝癌细胞在裸鼠体内的肺转移和肝转移。AGPAT5可以与原纤维蛋白(fibrillarin,FBL)相互作用,并在无血清饥饿刺激下加强两者的结合。遏制FBL的表达会抑制肝癌细胞迁移,且效果与过表达AGPAT5相似。抑制FBL的表达可削弱敲低AGPAT5对肝癌细胞迁移的促进作用。在已检测的肝癌细胞系中,AGPAT5和FBL在蛋白水平上并不存在相关性。K201位点突变使AGPAT5对肝癌细胞迁移的抑制作用减弱,并使AGPAT5与FBL的结合减弱。结论:敲低AGPAT5能够显著提高肝癌细胞迁移能力。AGPAT5可以与FBL相互作用,在无血清饥饿刺激下,AGPAT5或通过K201位点的乙酰化加强与FBL的结合,从而更有效地遏制FBL,进而抑制肝癌细胞迁移。但这种抑制作用并非来自AGPAT5的代谢酶活性,而是由非代谢作用所驱动。展开更多
目的:探讨原纤维蛋白-1(Fibrillin-1)、纤维蛋白-5(Fibulin-5)及类赖氨酰氧化酶-1(lysyl-oxidase-1,LOXL-1)在盆底器官脱垂(pelvic organ prolapse,POP)患者阴道前壁组织中的表达及意义。方法:选取2018年1月至2019年2月在海南省妇幼保...目的:探讨原纤维蛋白-1(Fibrillin-1)、纤维蛋白-5(Fibulin-5)及类赖氨酰氧化酶-1(lysyl-oxidase-1,LOXL-1)在盆底器官脱垂(pelvic organ prolapse,POP)患者阴道前壁组织中的表达及意义。方法:选取2018年1月至2019年2月在海南省妇幼保健院手术治疗的盆底器官脱垂患者108例作为观察组,其中POP-QⅡ度患者67例,POP-QⅢ度患者41例;同时选取因宫颈上皮内瘤变或妇科良性病变行子宫全切术患者92例作为对照组。术中均取阴道前壁组织,采用免疫组化法检测组织Fibrillin-1、Fibulin-5、LOXL-1蛋白表达情况,RT-PCR定量检测Fibrillin-1、Fibulin-5、LOXL-1 m RNA在组织标本中的表达水平,并进行对比分析。同时分析盆底器官脱垂患者阴道前壁组织中Fibrillin-1、Fibulin-5、LOXL-1表达的关系。结果:观察组阴道组织Fibrillin-1、Fibulin-5、LOXL-1蛋白阳性表达率及mRNA的相对表达量均明显低于对照组,经卡方检验及t检验,差异均有统计学意义(均P=0.000);POP-QⅡ度患者阴道前壁组织Fibrillin-1、Fibulin-5、LOXL-1蛋白阳性表达率及mRNA的相对表达量均高于POP-QⅢ度患者,经卡方检验及t检验,差异均有统计学意义(P=0.031,P=0.017,P=0.008,P=0.000,P=0.000,P=0.013)。POP患者阴道前壁组织Fibrillin-1与Fibulin-5、Fibrillin-1与LOXL-1、Fibulin-5与LOXL-1蛋白表达及mRNA的相对表达量经Spearman秩相关及Pearson相关分析均呈显著正相关(均P=0.000)。结论:盆底器官脱垂患者阴道组织Fibrillin-1、Fibulin-5及LOXL-1表达降低,且均与脱垂程度密切相关,三者间通过交联作用而呈显著相关性。展开更多
Background: Fibulin- 5 was recently found as a secreted extracellular matrix protein that functions as a scaffold for elastic fibres. However, the distribution of fibulin- 5 in human skin and its changes during the ag...Background: Fibulin- 5 was recently found as a secreted extracellular matrix protein that functions as a scaffold for elastic fibres. However, the distribution of fibulin- 5 in human skin and its changes during the ageing process are not known. Objectives: To explore the involvement of fibulin- 5 in skin ageing, the age- dependent changes in fibulin- 5 localization in human skin were examined compared with those of other elastic fibre components including elastin, fibrillin- 1 and fibulin- 2. Methods: The distribution of elastin, fibrillin- 1, fibrillin- 2, fibulin- 2 and fibulin- 5 was investigated by means of immunohistochemistry using their specific antibodies. Skin samples were recovered from 12 healthy subjects undergoing plastic surgery. Ultraviolet (UV) B- irradiated or control nonirradiated buttock skin samples were obtained from two healthy volunteers at 2 days after the irradiation at 2 minimal erythemal doses. Results: In the reticular dermis of young sun- protected skin from the upper arm, fibulin- 5 colocalized with the other elastic fibre components, while in the papillary dermis fibulin- 5 showed candelabra- like structures perpendicular to the epidermiswith an unstained area just beneath the epidermis, which was similar to that of elastin but not fibrillin- 1. Fibulin- 5 in the reticular dermis decreased and disappeared with age even in sun- protected skin from the thigh, abdomen and upper arm. In sun- exposed skin, fibulin- 5 was extremely reduced in the dermis of cheek skin even from a 20- year- old man. UVB irradiation reduced fibulin- 5, fibulin- 2 and elastin markedly, moderately and weakly, respectively, compared with levels in control nontreated skin. Interestingly, the deposition of fibulin- 5 was increased in solar elastosis, like that of other elastic fibre components. Conclusions: These results suggest that fibulin- 5 is a good marker of skin ageing and that the earlier loss of fibulin- 5 may involve age- dependent changes in other elastic fibre components.展开更多
文摘背景与目的:肿瘤的发生、发展过程中会发生代谢重编程,1-酰基甘油-3-磷酸O-酰基转移酶(1-acylglycerol-3-phosphate O-acyltransferase,AGPAT)作为三酰甘油(triacylglycerol,TAG)从头合成的关键酶,与肿瘤的进展密切相关。但目前作为亚型之一的AGPAT5在癌症中的研究还十分有限,本研究深入剖析AGPAT5在肝癌发生、发展中发挥的作用及潜在的分子机制,旨在为肝癌诊断和治疗策略提供新思路。方法:利用慢病毒感染将多种肝癌细胞系中的AGPAT5敲减,并通过锥虫蓝计数、划痕、transwell及平板克隆等实验在体外检测AGPAT5对肝癌细胞增殖、迁移及抗失巢凋亡能力的影响。通过回复野生型或酶活性缺失型的AGPAT5,探究其作为代谢酶是否发挥经典代谢作用调控肝癌细胞迁移。构建BALB/c裸鼠尾静脉注射移植瘤模型,从体内层面验证体外的细胞表型。采用免疫沉淀质谱联用(immunoprecipitation mass spectrum,IP-MS)鉴定出与AGPAT5相互作用的蛋白,并进行免疫共沉淀(co-immunoprecipitation,coIP)验证。蛋白质翻译后通过修饰鉴定分析AGPAT5潜在的修饰位点,通过体外实验探究点突变前后对肝癌细胞迁移的影响。通过coIP探究该位点突变前后AGPAT5与相互作用蛋白结合的情况。通过敲低相互作用蛋白确定其在细胞表型中的作用。通过回复实验验证AGPAT5是否通过相互作用蛋白发挥作用。检测野生型肝癌细胞系中的AGPAT5和相互作用蛋白的表达水平,检验两者之间是否具有相关性。结果:肝癌细胞敲减AGPAT5后会更加耐受无血清饥饿,并促进细胞迁移,但不会影响细胞增殖和失巢凋亡。而酶活性缺失并不影响AGPAT5对肝癌细胞迁移的抑制。敲减AGPAT5可促进肝癌细胞在裸鼠体内的肺转移和肝转移。AGPAT5可以与原纤维蛋白(fibrillarin,FBL)相互作用,并在无血清饥饿刺激下加强两者的结合。遏制FBL的表达会抑制肝癌细胞迁移,且效果与过表达AGPAT5相似。抑制FBL的表达可削弱敲低AGPAT5对肝癌细胞迁移的促进作用。在已检测的肝癌细胞系中,AGPAT5和FBL在蛋白水平上并不存在相关性。K201位点突变使AGPAT5对肝癌细胞迁移的抑制作用减弱,并使AGPAT5与FBL的结合减弱。结论:敲低AGPAT5能够显著提高肝癌细胞迁移能力。AGPAT5可以与FBL相互作用,在无血清饥饿刺激下,AGPAT5或通过K201位点的乙酰化加强与FBL的结合,从而更有效地遏制FBL,进而抑制肝癌细胞迁移。但这种抑制作用并非来自AGPAT5的代谢酶活性,而是由非代谢作用所驱动。
文摘目的:探讨原纤维蛋白-1(Fibrillin-1)、纤维蛋白-5(Fibulin-5)及类赖氨酰氧化酶-1(lysyl-oxidase-1,LOXL-1)在盆底器官脱垂(pelvic organ prolapse,POP)患者阴道前壁组织中的表达及意义。方法:选取2018年1月至2019年2月在海南省妇幼保健院手术治疗的盆底器官脱垂患者108例作为观察组,其中POP-QⅡ度患者67例,POP-QⅢ度患者41例;同时选取因宫颈上皮内瘤变或妇科良性病变行子宫全切术患者92例作为对照组。术中均取阴道前壁组织,采用免疫组化法检测组织Fibrillin-1、Fibulin-5、LOXL-1蛋白表达情况,RT-PCR定量检测Fibrillin-1、Fibulin-5、LOXL-1 m RNA在组织标本中的表达水平,并进行对比分析。同时分析盆底器官脱垂患者阴道前壁组织中Fibrillin-1、Fibulin-5、LOXL-1表达的关系。结果:观察组阴道组织Fibrillin-1、Fibulin-5、LOXL-1蛋白阳性表达率及mRNA的相对表达量均明显低于对照组,经卡方检验及t检验,差异均有统计学意义(均P=0.000);POP-QⅡ度患者阴道前壁组织Fibrillin-1、Fibulin-5、LOXL-1蛋白阳性表达率及mRNA的相对表达量均高于POP-QⅢ度患者,经卡方检验及t检验,差异均有统计学意义(P=0.031,P=0.017,P=0.008,P=0.000,P=0.000,P=0.013)。POP患者阴道前壁组织Fibrillin-1与Fibulin-5、Fibrillin-1与LOXL-1、Fibulin-5与LOXL-1蛋白表达及mRNA的相对表达量经Spearman秩相关及Pearson相关分析均呈显著正相关(均P=0.000)。结论:盆底器官脱垂患者阴道组织Fibrillin-1、Fibulin-5及LOXL-1表达降低,且均与脱垂程度密切相关,三者间通过交联作用而呈显著相关性。
文摘Background: Fibulin- 5 was recently found as a secreted extracellular matrix protein that functions as a scaffold for elastic fibres. However, the distribution of fibulin- 5 in human skin and its changes during the ageing process are not known. Objectives: To explore the involvement of fibulin- 5 in skin ageing, the age- dependent changes in fibulin- 5 localization in human skin were examined compared with those of other elastic fibre components including elastin, fibrillin- 1 and fibulin- 2. Methods: The distribution of elastin, fibrillin- 1, fibrillin- 2, fibulin- 2 and fibulin- 5 was investigated by means of immunohistochemistry using their specific antibodies. Skin samples were recovered from 12 healthy subjects undergoing plastic surgery. Ultraviolet (UV) B- irradiated or control nonirradiated buttock skin samples were obtained from two healthy volunteers at 2 days after the irradiation at 2 minimal erythemal doses. Results: In the reticular dermis of young sun- protected skin from the upper arm, fibulin- 5 colocalized with the other elastic fibre components, while in the papillary dermis fibulin- 5 showed candelabra- like structures perpendicular to the epidermiswith an unstained area just beneath the epidermis, which was similar to that of elastin but not fibrillin- 1. Fibulin- 5 in the reticular dermis decreased and disappeared with age even in sun- protected skin from the thigh, abdomen and upper arm. In sun- exposed skin, fibulin- 5 was extremely reduced in the dermis of cheek skin even from a 20- year- old man. UVB irradiation reduced fibulin- 5, fibulin- 2 and elastin markedly, moderately and weakly, respectively, compared with levels in control nontreated skin. Interestingly, the deposition of fibulin- 5 was increased in solar elastosis, like that of other elastic fibre components. Conclusions: These results suggest that fibulin- 5 is a good marker of skin ageing and that the earlier loss of fibulin- 5 may involve age- dependent changes in other elastic fibre components.