The Hippo–YAP pathway regulates the proliferation of alveolar epithelial progenitors after acute lung injury

Regeneration of pulmonary epithelial cells plays an important role in the recovery of acute lung injury (ALI), which is defined by pulmonary epithelial cell death. However, the mechanism of the regenerative capacity of alveolar epithelial cells is unknown. Using a lung injury mouse model induced by hemorrhagic shock and lipopolysaccharide, a protein mass spectrometry‐based high‐throughput screening and linage tracing technology to mark alveolar epithelial type 2 cells (AEC2s), we analyzed the mechanism of alveolar epithelial cells proliferation. We demonstrated that the expression of Hippo‐yes‐associated protein 1 (YAP1) key proteins were highly consistent with the regularity of the proliferation of alveolar epithelial type 2 cells after ALI. Furthermore, the results showed that YAP1+ cells in lung tissue after ALI were mainly Sftpc lineage‐labeled AEC2s. An in vitro proliferation assay of AEC2s demonstrated that AEC2 proliferation was significantly inhibited by both YAP1 small interfering RNA and Hippo inhibitor. These findings revealed that YAP functioned as a key regulator to promote AEC2s proliferation, with the Hippo signaling pathway playing a pivotal role in this process.     

CMTM6, the newly identified PD-L1 regulator,correlates with PD-L1 expression in lung cancers

Modulation of Immune check point regulators, especially the PD-1/PD-L1 axis, plays a critical role in successful management of a small proportion of lung cancer patients, but not so effective in the rest of lung cancer patients. A better understanding of immunotherapy non-responsive or resistant patients therefore warranted for future development of novel therapeutics. The newly identified regulator CMTM6 (CKLF-like MARVEL transmembrane domain containing 6) has been reported to serves as the stabilizer of PD-L1 and enhances the inhibitory effect of PD-L1 on immune system in both cell line and animal models, but its clinical relevance associated with PD-L1 is unknown and the current study is designed to address this question. The study using immunohistochemistry demonstrated that CMTM6 positivity from 15 out of 19 types of cancers with our in-house tissue microarray, and PD-L1 expression is always found only in CMTM6 positive cancers. CMTM6 and PD-L1 expression were analyzed in 81 lung cancer patient sample, and we observed that CMTM6 expression correlated with cancer histotypes and inversely correlated with cancer metastases, but not with patients’ age and gender. No PD-L1 expression was observed in negative CMTM6 samples. Higher expression PD-L1 is also associated with higher CMTM6 expression. In summary, CMTM6 expression is associated with PD-L1 expression, as well as lung cancer histotypes and metastasis. The results thus for the first time confirmed earlier reports on CMTM6/PD-L1 connection, from a clinical aspect of analysis.     

脓毒症患者中可溶性髓样细胞触发受体1、降钙素原、核因子κB水平与肠道菌群失调的相关性

本研究旨在探讨脓毒症患者中血清可溶性髓样细胞触发受体1(soluble myeloid cell trigger receptor 1,sTREM1)、降钙素原(procalcitonin,PCT)、核因子κB(nuclear factor­κB,NF­κB)水平与肠道菌群失衡的相关性。将2016年5月-2019年6月重庆市綦江区人民医院消化内科收治的86例脓毒症患者纳入脓毒症组,同期收治的未并发脓毒症患者50例纳入非脓毒症组,同期到本院进行体检的健康人50例纳入对照组,比较3组之间肠道菌群α多样性、肠道菌群含量,以及血清sTREM1、PCT、NF­κB水平的差异,并进行Spearman相关性分析。结果显示,脓毒症组Ace指数、Chao指数、Shannon指数显著低于非脓毒症组和对照组,Simpson指数显著高于非脓毒症组和对照组(P<0.05);脓毒症组类杆菌、双歧杆菌含量显著低于非脓毒症组和对照组,肠球菌、大肠埃希菌、葡萄球菌含量显著高于非脓毒症组和对照组(P<0.05);脓毒症组血清sTREM1、PCT、NF­κB水平显著高于非脓毒症组和对照组(P<0.05)。Spearman相关性分析显示,脓毒症患者中sTREM1、PCT、NF­κB水平与Ace指数、Chao指数、Shannon指数均呈负相关(P<0.05),与Simpson指数呈正相关(P<0.05)。结果提示,脓毒症患者中血清sTREM1、PCT、NF­κB水平与肠道菌群失衡有关联。     

拟南芥AtDAD1 超量表达植株对H2O2抗性的研究

构建拟南芥AtDAD1超量表达载体,以农杆菌介导的方法转化拟南芥哥伦比亚生态型,比较AtDAD1超量表达植株和野生型植株表现型的差异,以及两者对H2O2抗性的不同。实验显示,AtDAD1转基因拟南芥生长较野生型拟南芥更为强壮,对高浓度H2O2有较强的耐受力。测定两者糖含量,发现AtDAD1转基因拟南芥叶片糖的含量明显高于野生型拟南芥叶片。以上结果表明,AtDAD1基因可能参与植物生长发育,并可能在拟南芥抵抗凋亡的过程中发挥重要的作用。     

一种经microRNA敲低PD-1的新型慢病毒载体在CAR-T细胞中的应用

本研究将microRNA插入EF1α启动子的内含子中,构建携带沉默PD-1基因的miRNA的新型慢病毒载体,并将其应用于CAR-T细胞。通过流式细胞术检测慢病毒载体转导效率和PD-1沉默效率;Westernblotting检测PD-1蛋白表达差异;荧光定量PCR检测microRNA相对表达情况;荧光素酶生物发光法和流式细胞术检测CAR-T细胞的能力。结果显示与U6转录microRNA的载体相比较,将microRNA插入到EF1-α内含子中的病毒载体转导效率更显著,对PD-1的敲低效率均达90%以上,且Westernblotting结果验证了PD-1的敲低效果。另外通过荧光定量PCR,可显示出转导该新型慢病毒载体的Jurkat细胞内microRNA的相对表达量。荧光素酶生物发光法证实了CAR-T细胞针对靶细胞的特异杀伤性,流式细胞术结果表明沉默PD-1的CAR-T细胞相较于正常CAR-T细胞显示出更强的特异性杀伤能力。本研究成功构建了经microRNA敲低PD-1的新型慢病毒载体并验证了其转导效率的优越性,以及基于此载体表达的microRNA可高效地沉默PD-1;且应用此载体的CAR-T细胞能发挥更强的杀伤活性,从而为后续该CAR-T细胞治疗表达PD-L1的肿瘤奠定基础。     

利用基于CRISPR-Cas9 的基因编辑技术对miR-29a在GT1-7 细胞中进 行基因敲除

目的:本文利用CRISPR-Cas9技术在GT1-7细胞中对miR-29a基因进行基因编辑,用于构建miR-29a基因敲除GT1-7细胞模型。方法:通过构建Cas9稳转的GT1-7细胞株并转染sgRNA质粒用于在靶向miR-29a基因区域引发突变。然后构建EGFP与sgRNA共表达质粒并转染Cas9稳转GT1-7细胞,利用流式细胞仪富集表达绿色荧光蛋白的阳性细胞和分选阳性单克隆细胞。最后利用实时荧光定量PCR(realtimefluorescencequantitativePCR)对富集细胞和单克隆细胞进行miR-29a表达量检测。结果:T7E1检测结果显示CRISPR-Cas9系统有效地在miR-29a基因区域引发了突变。荧光定量PCR结果显示,与对照组相比,富集后阳性细胞miR-29a的表达量整体下降了50%左右(P0.05)。此外,通过流式筛选获得了一个纯合miR-29a基因敲除细胞克隆,与对照组相比,其miR-29a的表达量下降了75%左右(P0.05)。结论:本文建立了一种有效编辑GT1-7细胞基因的方法,并采用该方法构建了miR-29a稳定敲除细胞模型。     

TNF家族成员TALL-1及其研究进展

TALL-1是TNF家族成员中最近发现的一个新型细胞因子,由单核细胞和巨噬细胞产生. 人TALL-1由285个氨基酸残基组成,而鼠TALL-1由309个氨基酸残基构成,两者均为Ⅱ型穿膜蛋白质. 人sTALL-1是长为152个氨基酸残基的胞外区片段,对应于C端134～285位氨基酸残基. 重组人sTALL-1具有刺激B细胞增殖、激活NF-κB和JNK以及抑制肿瘤细胞生长等生物学活性. 另外,TALL-1在转基因鼠中的过量表达可引起严重的B细胞增生以及与狼疮有关的自身免疫性疾病. 因此,TALL-1是一个具有多种生物学活性的细胞调控因子.     

沉默FNBP1表达诱导7703细胞形态重塑

FNBP1在真核细胞中广泛表达,能通过诱导细胞膜管状化、内陷或变形及后续膜曲率依赖性肌动蛋白聚合过程,参与细胞内吞和运动;还可参与端粒维持及FasL与溶酶体结合过程的调控。研究发现,在肝癌细胞7703中,利用RNA干扰（RNA interference technology,RNAi）技术使内源FNBPI基因表达沉默以后,7703细胞形态发生重塑,由上皮样转变为树状分枝的纤维状,表明FNBP1在细胞形态维持过程中亦具有不可或缺的潜在作用;通过对已知FNBP1蛋白相互作用情况的分析推断,FNBP1可能是通过对肌动蛋白骨架动态组装过程的分子调控参与7703细胞形态的控制。     

血清4型禽腺病毒Fiber蛋白在杆状病毒中的表达及其在抗体检测中的应用

为建立检测血清4型禽腺病毒(FAdV-4)抗体快捷特异方法,本研究首先分别构建含FAdV-4纤突蛋白Fiber-1和Fiber-2的两个重组杆状病毒rBv-Fiber-1和rBv-Fiber-2。在利用IFA以及Western blot鉴定重组杆状病毒分别高效表达Fiber-1和Fiber-2蛋白的基础上,以Ni柱纯化蛋白,并作为特异性识别FAdV-4抗体的包被抗原构建间接ELISA方法。特异性试验表明,间接ELISA仅特异地检出FAdV-4阳性血清,而不与Ⅰ群其他血清型禽腺病毒及其他禽源病毒阳性血清反应。间接ELISA检测灵敏度高于常规IFA方法,批间和批内重复性变异系数均小于10%。临床血清检测结果表明,间接ELISA可有效检出感染FAdV-4或免疫FAdV-4灭活疫苗的鸡群中抗Fiber抗体,且检测结果与BioChek商品化ELISA试剂盒一致。结果表明,本研究利用杆状病毒系统表达的Fiber蛋白及基于表达产物所构建的间接ELISA方法在FAdV-4感染预警和免疫评估有良好的应用前景。