反常性痤疮一家系NCSTN基因新致病突变报道

目的:检测反常性痤疮(AI)一家系致病基因及突变位点。方法:该家系中三代4人发病,提取4例患者、9名健康亲属以及100名健康志愿者的外周血DNA;对先证者进行全基因组外显子测序,经生物信息学分析,获得致病变异;而后通过Sanger测序在全部患者、健康亲属及健康对照中进行验证。结果:家族中先证者及其他患者均存在位于NCSTN基因的一个剪接位点突变c.85+2T>C,9名健康亲属和100名健康志愿者未发现该突变,突变与AI疾病符合共分离。结论:本家系中NCSTN突变位点(c.85+2T>C)与反常性痤疮发病相关。

Nicastrin、N1ICD及hes1蛋白在小鼠肝脏及肝癌组织中的表达

目的 检测C57BL/6小鼠正常肝脏和肝癌组织中nicastrin、N1ICD和hes1蛋白的表达。方法 将12只6周龄雄性C57BL/6小鼠随机分为对照组和模型组,每组6只。模型组小鼠注射Hepa1-6肝癌细胞构建小鼠原位肝癌模型,对照组注射等量的生理盐水。HE染色观察肝脏癌变情况。IHC与Western blot检测nicastrin、N1ICD和hes1蛋白在正常肝脏组织及肝癌组织中的表达情况。结果 IHC显示,nicastrin、N1ICD及hes1蛋白在正常肝细胞中均定位于肝窦内皮细胞,肝细胞基本不表达;相比对照组肝脏组织,在模型组小鼠肝癌组织中,nicastrin蛋白在肝癌细胞中表达增多(P <0.01),N1ICD及hes1蛋白表达均减少(P <0.05,P <0.01)。结论 NCSTN基因在小鼠肝细胞癌中发挥促癌作用;notch1与hes1在小鼠肝细胞癌中可能发挥抑癌作用。

NCSTN基因沉默对HaCaT细胞增殖分化的影响

目的检测NCSTN基因稳定沉默的人永生化角质形成细胞(HaCaT)增殖活性及相关分化蛋白的改变,初步探索反常性痤疮发病的可能机制。方法构建慢病毒介导shRNA沉默NCSTN基因的HaCaT细胞模型(shRNA组),转染空载慢病毒的HaCaT细胞作为阴性对照组,实时定量PCR和Western印迹法检测NCSTN基因的沉默效率。CCK8法检测HaCaT细胞增殖活性,实时定量PCR和Western印迹法检测HaCaT细胞角蛋白(CK1、CK5、CK7、CK10、CK14、CK16、CK17、CK18、CK19、CK20)及其他分化分子(内披蛋白、兜甲蛋白)mRNA和蛋白的表达。两组计量资料的比较采用两独立样本t检验。结果shRNA组NCSTN mRNA及蛋白表达(0.42±0.19、0.30±0.07)均显著低于阴性对照组(1.00±0.34、1.00±0.26;t=5.196、2.637,P<0.001、<0.05),基因沉默效率达70%。与阴性对照组相比,shRNA组HaCaT细胞明显增殖活跃,CK16、CK19蛋白表达显著下调(t=3.787、3.817,P<0.01、<0.05),终末分化分子内披蛋白表达明显降低(t=2.904,P<0.05)。结论稳定沉默NCSTN基因表达会引起HaCaT细胞异常增殖分化,为后续探究NCSTN基因突变引起反常性痤疮提供新思路。

NCSTN Gene Silencing Inhibits the Retinoic Acid Signaling Pathway in Human Immortalized Keratinocytes

Objective:Acne inversa is a multifactorial chronic debilitating disease.Genetic factors are involved in 40%of patients,especially the nicastrin(NCSTN)gene.However,the role of the mutated NCSTN gene in the pathogenesis of acne inversa remains unclear.Retinoic acid is recommends to treat moderate to severe acne inversa,therefor we conduct this in vitro research to study the association between NCSTN gene mutation and the retinoic acid signaling pathway in human immortalized skin keratinocyte(HaCaT)cells.Methods:HaCaT cells were infected with a lentivirus-mediated short hairpin RNA(shRNA)expression plasmid specifically targeting the NCSTN gene.Real-time polymerase chain reaction(PCR)and Western blotting were used to detect the interference efficiency of NCSTN.RNA sequencing was used to detect differential genes in the NSCTN-deficient HaCaT cells.Based on bioinformatics analysis and clinical treatment data,the retinoic acid signal pathway was selected for screening.Quantitative PCR was used to verify the changes in the expressions of retinoic acid signaling pathway-related receptors and molecules in the HaCaT cell line after NCSTN silencing.The Student t test and one-way analysis of variance were used to evaluate intergroup differences.Results:Sequencing showed that the NCSTN-shRNA lentiviral recombinant expression plasmid was successfully constructed.After lentivirus infection of HaCaT cells,real-time PCR results showed significantly reduced NCSTN mRNA expression in the interference group compared with the negative control group,and the interference efficiency was 75.0%.Western blotting showed that the inhibition rate of NCSTN protein expression in the shRNA group was 71.7%.RNA sequencing revealed significant differential expression of some genes,and changes in signaling pathways.Compared with the control group,the group with the silenced NCSTN showed significantly decreased expression of retinoic acid receptors(RARα:F=23.482,RARβ:F=603.241,RXRα:F=69.689,and RARRES1:F=167.482,and all P<0.001),and peroxisome proliferator-activated receptorγ(F=8.138,P<0.01).Conclusion:Defective function of the NCSTN gene leads to an impaired retinoic acid signaling pathway in HaCaT cells,which suggests that the retinoic acid signaling pathway may play a role on the onset of acne inversa caused by NCSTN gene mutation.

Notch信号通路在肝癌相关疾病中的研究进展

Notch信号通路是细胞间一条进化保守的通路,由notch受体(notch1-4)、配体(Jag1、Jag2、Deltalike 1(Dll1)、Deltalike 3(Dll3)和Deltalike 4(Dll4))、细胞内效应分子、DNA结合蛋白及notch的调节分子等成分组成,决定细胞命运、参与胚胎和组织发育、组织功能及修复等生物学过程[1]。Notch信号通路的激活是一个级联反应,notch蛋白首先在S1位点被furin-like样蛋白切割,以异源二聚体的形式在细胞表面表达,与配体结合后在近细胞膜处S2位点被TACE或ADAM蛋白酶切割,然后在跨膜区的S3位点被γ分泌酶切割,此时产生活化分子NICD进入细胞膜内,继而入核与MAML、CSL形成复合物以调节靶基因的转录。

Nicastrin启动子区多态性与散发阿尔茨海默病相关性研究

目的探讨Nicastrin(NCSTN)基因启动子区多态性与散发阿尔茨海默病(sporadic Alzheimer s dis-ease,SAD)发病的关系及其机制。方法用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和直接测序的方法对中国北方汉族359例SAD患者和331名正常对照者NCSTN基因启动子区单核苷酸多态性位点(SNP)进行筛查,基因分型后,进行病例对照-相关分析。构建三种不同基因型启动子质粒,通过双荧光报告基因系统检测不同启动子质粒的转录活性。结果①中国北方人群中的NCSTN基因启动子区ATG上游2245 bp范围内存在3个SNP:-1216C/A(rs2147471),-796T/G(rs10752637)和-436C/T(rs1324738);②-1216C/A和-796T/G的基因型频率在各自的SAD组和正常对照组中的分布差异有统计学意义;③三种启动子质粒的转录活性差异无统计。结论NCSTN启动子区SNP:-796T/G和-1216C/A,与SAD的发病具有相关性,但未发现这两个位点具有病理学功能。