低温冷冻对皮肤组织桥粒芯糖蛋白2表达的影响

目的:观察桥粒芯糖蛋白2(desmoglein2,Dsg2)在不同温度保存皮肤组织中的分布,以探讨其与皮肤冷冻损伤的关系。方法:用Dsg2抗体对5种保存条件下的皮肤组织作免疫组化染色,采用计算机图像定量分析方法测定表皮组织中的含量,并用H.E染色观察皮肤的组织结构变化。结果:与新鲜皮片组相比,-196℃温度条件下保存皮肤组织结构保存较好,Dsg2含量变化不明显,而4℃、-20℃、-80℃保存皮肤组织结构破坏明显,含量亦明显下降。结论:皮肤低温保存冷冻损伤可能与Dsg2破坏有关。

海藻糖对低温储存皮肤组织桥粒芯糖蛋白2表达及皮肤活力影响的实验研究

目的:比较海藻糖与传统低温保护剂丙二醇对皮肤低温保存后桥粒芯糖蛋白2(Dsg2)表达以及皮肤活力的影响,为寻求皮肤组织低温保护剂的优良配方提供实验依据。方法:经患者同意在烧伤后整形术中获取患者新鲜皮肤组织,分为对照组(新鲜皮肤,不作任何保护剂处理)、海藻糖/二甲基亚砜(T/D)组(皮肤以0.5mol/L T/D液浸泡)、二甲基亚砜/丙二醇(D/P)组(皮肤以D/P液浸泡),液氮冻存21 d,复温,Dsg2单克隆抗体免疫组化染色并用Image Pr05.O图像分析处理系统测定皮肤组织Dsg2表达的光密度值;用皮肤内琥珀酸脱氢酶(SDH)比色法检测皮肤组织SDH活性,以此表示皮肤活力。结果:低温保存21 d时,D/P组Dsg2表达(0.247±0.006)显著低于T/D组(0.2504±0.009,P<0.05)与对照组(0.254±0.007,P<0.05);对照组SDH水平为18.1±0.6,T/D组为17.4±0.9,D/P组为15.7±0.9,D/P组SDH明显低于T/D组(P<0.01)。结论:海藻糖对皮肤组织及细胞间桥粒连接的保护作用优于传统低温保护剂。

沉默A549细胞中的CD46和DSG2可抑制人3型和7型腺病毒的侵入及IL-8的释放

目的探讨沉默受体CD46、桥粒芯蛋白(DSG2)对人3型腺病毒(HAdV-3)和7型腺病毒(HAdV-7)的侵入及炎症因子释放的影响。方法通过RNA干扰技术沉默A549细胞中CD46、DSG2的表达。实验分为HAdV-3组、HAdV-3+siRNA-NC组(siRNA无关序列对照组)、HAdV-3+siRNA-CD46组(转染siRNA-CD46)、HAdV-3+siRNA-DSG2组(转染siRNA-DSG2);HAdV-7组、HAdV-7+siRNA-NC组(siRNA无关序列对照组)、HAdV-7+siRNA-CD46组(转染siRNA-CD46)、HAdV-7+siRNA-DSG2组(转染siRNA-DSG2)。HAdV-3、7感染A549细胞后0.5、2 h,通过激光共聚焦显微镜观察两种腺病毒与受体CD46、DSG2结合水平;体外转染siRNA-CD46、siRNA-DSG2后不同时间点,qRT-PCR技术检测HAdV-3、7拷贝数,ELISA检测转染后IL-8表达情况。结果腺病毒感染A549细胞后0.5、2 h,HAdV-3、7与其受体CD46、DSG2结合共定位;腺病毒入胞后,随时间延长,病毒拷贝数增加,转染siRNA-CD46后2、6、12、24 h,HAdV+siRNA-CD46组的病毒拷贝数较HAdV组降低(HAdV-3+siRNA-CD46 vs HAdV-3:6 h,P<0.05;12、24 h,P<0.0001;2 h,降低趋势无统计学差异;HAdV-7+siRNA-CD46 vs HAdV-7:2 h,P<0.01;6 h,P<0.001;12、24 h,P<0.0001)。转染siRNA-DSG2后2、6、12、24 h,同样明显降低了病毒载量(HAdV-3+siRNA-DSG2 vs HAdV-3,HAdV-7+siRNA-DSG2 vs HAdV-7,P<0.0001)。HAdV-3、7感染A549细胞后,炎症因子IL-8释放增多(P<0.0001),沉默CD46和DSG2的表达后2、6 h,炎症因子IL-8水平降低(P<0.0001)。结论HAdV-3、7与其受体CD46、DSG2结合后入胞,病毒拷贝数随时间延长而增加,沉默CD46、DSG2后,抑制HAdV-3、7型腺病毒的侵入及IL-8释放。

Reactivation of PPARαalleviates myocardial lipid accumulation and cardiac dysfunction by improving fatty acidβ-oxidation in Dsg2-deficient arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy(ACM),a fatal heart disease characterized by fibroadipocytic replacement of cardiac myocytes,accounts for 20%of sudden cardiac death and lacks effective treatment.It is often caused by mutations in desmosome proteins,with Desmoglein-2(DSG2)mutations as a common etiology.However,the mechanism underlying the accumulation of fibrofatty in ACM remains unknown,which impedes the development of curative treatment.Here we investigated the fat accumulation and the underlying mechanism in a mouse model of ACM induced by cardiac-specific knockout of Dsg2(CS-Dsg2^(-/-)).Heart failure and cardiac lipid accumulation were observed in CSDsg2^(-/-)mice.We demonstrated that these phenotypes were caused by decline of fatty acid(FA)β-oxidation resulted from impaired mammalian target of rapamycin(m TOR)signaling.Rapamycin worsened while overexpression of m TOR and 4EBP1 rescued the FAβ-oxidation pathway in CS-Dsg2^(-/-)mice.Reactivation of PPARa by fenofibrate or AAV9-Ppara significantly alleviated the lipid accumulation and restored cardiac function.Our results suggest that impaired m TOR-4EBP1-PPARa-dependent FAβ-oxidation contributes to myocardial lipid accumulation in ACM and PPARa may be a potential target for curative treatment of ACM.

Desmoglein 2(DSG2) Is A Receptor of Human Adenovirus Type 55 Causing Adult Severe Community-Acquired Pneumonia

Human adenovirus type 55(HAdV-B55) is a re-emergent acute respiratory disease pathogen that causes adult communityacquired pneumonia(CAP). Previous studies have shown that the receptor of HAdV-B14, which genome is highly similar with HAdV-B55, is human Desmoglein 2(DSG2). However, whether the receptor of HAdV-B55 is DSG2 is undetermined because there are three amino acid mutations in the fiber gene between HAdV-B14 and HAdV-B55. Here, firstly we found the 3T3 cells, a mouse embryo fibroblast rodent cell line which does not express human DSG2, were able to be infected by HAdV-B55 after transfected with pcDNA3.1-DSG2, while normal 3T3 cells were still unsusceptible to HAdV-B55 infection. Next, A549 cells with h DSG2 knock-down by siRNA were hard to be infected by HAdV-B3/-B14/-B55, while the control siRNA group was still able to be infected by all these types of HAdVs. Finally, immunofluorescence confocal microscopy indicated visually that Cy3-conjugated HAdV-B55 viruses entered A549 cells by binding to DSG2 protein.Therefore, DSG2 is a major receptor of HAdV-B55 causing adult CAP. Our finding is important for better understanding of interactions between adenoviruses and host cells and may shed light on the development of new drugs that can interfere with these processes as well as for the development of potent prophylactic vaccines.

Mutations of Desmoglein‑2 in Sudden Unexplained Death in the Chinese Han Population

Sudden unexplained death(SUD)remains a puzzle in forensic medicine.Desmoglein‑2(DSG2)has been linked to arrhythmogenic right ventricular cardiomyopathy which may cause life‑threatening ventricular arrhythmias and sudden death.Fatal arrhythmias resulting in sudden death also occur in the absence of morphologic cardiac abnormalities at autopsy.We hypothesized that DSG2 mutations may be responsible for certain Chinese SUD cases.We sequenced all 15 exons of DSG2 in DNA extracted from postmortem heart tissues of 25 Chinese patients dying from SUD.The primers were designed using the Primer Express 3.0 software.Direct sequencing for both sense and antisense strands was performed with a BigDye Terminator DNA sequencing kit on a 3130 Xl Genetic Analyzer.Mutation damage prediction was made using Mutation Taster,PolyPhen,and SIFT software.In 2 of 25 cases of Chinese SUD samples,two DSG2 heterozygous mutations(p.P927 L and p.T1070M)were identified,and one is probably damaging.We concluded that DSG2 mutations may be related to the occurrence of part of SUD cases in the Chinese Han population.