Background::Pulmonary microvascular endothelial cells (PMVECs) were not complex, and the endothelial barrier was destroyed in the pathogenesis progress of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Previous studies have demonstrated that hepatocyte growth factor (HGF), which was secreted by bone marrow mesenchymal stem cells, could decrease endothelial apoptosis. We investigated whether mTOR/STAT3 signaling acted in HGF protective effects against oxidative stress and mitochondria-dependent apoptosis in lipopolysaccharide (LPS)-induced endothelial barrier dysfunction and ALI mice.Methods::In our current study, we introduced LPS-induced PMEVCs with HGF treatment. To investigate the effects of mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) pathway in endothelial oxidative stress and mitochondria-dependent apoptosis, mTOR inhibitor rapamycin and STAT3 inhibitor S3I-201 were, respectively, used to inhibit mTOR/STAT3 signaling. Moreover, lentivirus vector-mediated mTORC1 (Raptor) and mTORC2 (Rictor) gene knockdown modifications were introduced to evaluate mTORC1 and mTORC1 pathways. Calcium measurement, reactive oxygen species (ROS) production, mitochondrial membrane potential and protein, cell proliferation, apoptosis, and endothelial junction protein were detected to evaluate HGF effects. Moreover, we used the ALI mouse model to observe the mitochondria pathological changes with an electron microscope in vivo.Results::Our study demonstrated that HGF protected the endothelium via the suppression of ROS production and intracellular calcium uptake, which lead to increased mitochondrial membrane potential (JC-1 and mitochondria tracker green detection) and specific proteins (complex I), raised anti-apoptosis Messenger Ribonucleic Acid level (B-cell lymphoma 2 and Bcl-xL), and increased endothelial junction proteins (VE-cadherin and occludin). Reversely, mTOR inhibitor rapamycin and STAT3 inhibitor S3I-201 could raise oxidative stress and mitochondria-dependent apoptosis even with HGF treatment in LPS-induced endothelial cells. Similarly, mTORC1 as well as mTORC2 have the same protective effects in mitochondria damage and apoptosis. In in vivo experiments of ALI mouse, HGF also increased mitochondria structural integrity via the mTOR/STAT3 pathway. Conclusion::In all, these reveal that mTOR/STAT3 signaling mediates the HGF suppression effects to oxidative level, mitochondria-dependent apoptosis, and endothelial junction protein in ARDS, contributing to the pulmonary endothelial survival and barrier integrity.

目的:观察研究齐墩果酸（oleanolic acid，OA）对糖尿病心肌病（diabetic cardiomyopathy, DCM）大鼠心脏的保护作用及其机制。方法:50只健康4周龄雄性SD大鼠随机（随机数字法）分为正常对照组（CON）、糖尿病组（DM）、糖尿病+齐墩果酸组（DM+OA）、糖尿病+氯喹（chloroquine，CQ）组（DM+CQ）和糖尿病+齐墩果酸+氯喹组（DM+OA+CQ），每组10只。通过喂食高脂高糖食物（质量百分比35.5%）和腹腔注射链脲霉素建立糖尿病动物模型。OA 80 mg/（kg·d）灌胃喂药4周，CQ 10 mg/（kg·d）腹腔注射4周。于20周后进行超声心功能检测后麻醉取大鼠心脏组织。采用超声心动图检测SD大鼠心脏功能，并通过Western blot技术对自噬相关蛋白LAMP2、p-ULK1、p-p70S6K、p-raptor、LC3、p62进行蛋白半定量分析，观察大鼠心功能及心肌细胞中自噬相关蛋白表达的变化。正态分布计量资料多组间比较采用单因素方差分析，组间两两比较采用SNK- q检验。 结果:与CON组相比，DM组大鼠心脏舒张末期心肌前壁厚度、左室舒张末期容积、左室收缩末期容积[（7.58±0.25）mm、（376.6±13.6）μL、（132.6±10.8）μL]低于CON组[（8.37±0.11）mm、（458.3±16.4）μL、（166.6±12.8）μL]（均 P<0.05）。与DM组比较，DM+OA组大鼠心脏舒张末期心肌前壁厚度、左室舒张末期容积、左室收缩末期容积[（8.63±0.14）mm、（473.6±18.6）μL、（174.6±12.7）μL]高于DM组（均 P<0.05）。DM+OA+CQ组大鼠心脏舒张末期心肌前壁厚度、左室舒张末期容积、左室收缩末期容积[（7.97±0.17）mm、（393.6±15.6）μL、（147.6±10.2）μL]低于DM+OA组（均 P<0.05）。DM组心肌细胞自噬相关蛋白p-ULK1表达升高、p-p70S6K及p-raptor表达降低（均 P<0.05）。与DM组比较，OA治疗组心脏功能障碍减轻，伴随p62表达降低、LC3 Ⅱ/Ⅰ表达升高（均 P<0.05）。与DM+CQ组比较，DM+OA+CQ组p62表达降低，LC3 Ⅱ/Ⅰ表达升高（均 P<0.05）。 结论:OA对糖尿病大鼠的心肌具有保护作用，其机制可能与增强自噬有关。

目的 探究蛋白4.1R对肝细胞增殖、凋亡以及糖酵解的影响,并初步阐明其分子机制.方法 以肝细胞株HL-7702为材料,利用CRISPR/Cas9技术构建4.1R-/-HL-7702细胞株.设置对照组为正常培养的4.1R+/+HL-7702细胞,实验组为4.1R-/-HL-7702细胞.细胞在培养24、48以及72 h时,分别用CCK-8及EdU-488染色,然后利用酶标仪以及流式细胞术检测细胞的增殖能力.细胞经Annexin V-FITC/PI染色后,流式细胞术检测HL-7702细胞在培养24、48以及72 h时的凋亡水平.利用生化试剂盒分别检测HL-7702细胞葡萄糖摄取量、乳酸分泌量以及ATP生成的变化.pH计检测培养48 h HL-7702细胞上清培养基的pH值.qRT-PCR检测HL-7702细胞糖酵解过程关键调节酶HK2、PFKL、PKM2以及LDHA的mRNA表达量.Western blotting检测AMPK、p-AMPK、Raptor以及p-Raptor的蛋白表达量.结果 Western blotting以及基因测序结果表明4.1R-/-HL-7702细胞株构建成功.与对照组相比,4.1R-/-组的CCK-8和EdU-488实验结果均显示HL-7702细胞的增殖能力降低;细胞凋亡实验结果表明,4.1R-/-HL-7702细胞凋亡水平升高.蛋白4.1R的缺失导致HL-7702细胞葡萄糖摄取量(P<0.05)、乳酸分泌量(P<0.001)、ATP生成(P<0.001)下降,细胞上清培养基pH值(P<0.01)升高.qRT-PCR实验结果表明糖酵解过程关键调节酶的mRNA表达量均下降(P<0.001).相较于HL-7702细胞,4.1R-/-HL-7702细胞的AMPK和Raptor蛋白表达量下降,p-AMPK和p-Raptor蛋白表达量升高.结论 蛋白4.1R的缺失导致HL-7702细胞增殖能力降低、凋亡水平增加,糖酵解过程被抑制,其调控机制与下游AMPK-mTORC1信号通路的激活密切相关.

The highly conserved target of rapamycin(TOR)pathway plays an important role in aging across species.Previous studies have established that inhibition of the TOR complex 1(TORC1)significantly extends lifespan in Caenorhabditis elegans.However,it has not been clear whether TORC1 perturbation affects aging in a spatiotemporal manner.Here,we apply the auxin-inducible degradation tool to knock down endogenous DAF-15,the C.elegans ortholog of regulatory associated protein of TOR(Raptor),to char-acterize its roles in aging.Global or tissue-specific inhibition of DAF-15 during development results in various growth defects,whereas neuron-specific knockdown of DAF-15 during adulthood significantly extends lifespan and healthspan.The neuronal DAF-15 deficiency-induced longevity requires the intestinal activities of DAF-16/FOXO and PHA-4/FOXA transcription factors,as well as the AAK-2/AMP-activated protein kinase a catalytic subunit.Transcriptome profiling reveals that the neuronal DAF-15 knockdown promotes the expression of genes involved in protection.These findings define the tissue-specific roles of TORC1 in healthy aging and highlight the importance of neuronal modulation of aging.

目的:探讨4例46,XY性发育障碍发生的分子机制.方法:收集患者临床病历资料,采集其肝素抗凝外周血进行淋巴细胞培养,染色体G显带技术制备并分析染色体核型,提取外周血DNA行SRY基因检测和测序,SRY基因检测结果阴性者外送标本行性发育相关基因靶向测序,生物信息学方法分析测序结果.结果:查体显示4例患者社会性别均为女性,而外周血淋巴细胞核型分析结果提示核型均为男性(46,XY),社会性别与生物学性别不一致;性别决定基因SRY均为阳性,测序结果提示病例1为SRY基因编码区第5位碱基缺失(c.del5A),病例2为SRY基因编码区第5位碱基发生错义突变(c.5A>T),病例3为SRY基因编码区第6位碱基缺失(c.del6A),病例4的SRY基因检测范围内未见突变位点,靶向测序结果提示雄激素受体基因(AR)编码区发生错义突变(c.2117 A>G);生物信息学软件Mutation taster提示c.del5A、c.del6A和c.2117 A>G等3种突变可能导致疾病发生,c.5A>T突变可能为人群多态现象;Raptor X蛋白质三维结构预测软件分析结果显示SRY基因c.5A>T突变对SRY蛋白的三维构象没有影响,而AR基因c.2117 A>G突变可导致AR蛋白的三维结构明显变化,并增加两个氢键;PolyPhen-2显示SRY基因c.5A>T和AR基因c.2117A>G突变为可能致病;ClinvAR软件分析结果提示4种突变均未见临床病例报道.结论:基因突变可能是导致患者性器官发育异常的原因,基因检测有助于明确诊断以及家庭再生育咨询.

目的:探讨mTOR调节相关蛋白(regulatory-associated protein of mTOR,RAPTOR)对口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)迁移、侵袭和增殖能力的影响.方法:利用TCGA生物信息数据库查询在头颈部鳞状细胞癌(head and neck squamous cell carcinoma,HNSCC)组织与癌旁组织中差异表达的mRNA.West-ern blot检测RAPTOR在人口腔上皮细胞HOEC和OSCC细胞系中的表达.利用伤口愈合实验、Transwell实验、EdU实验检测各组细胞迁移、侵袭及增殖能力.生物信息学网站预测与RAPTOR靶向结合的微小RNA(mi-croRNA,miR).功能学实验验证miR-485-5p是否可以靶向RAPTOR影响OSCC细胞的迁移、侵袭和增殖.结果:RAPTOR在HNSCC组织中较癌旁组织表达增高.伤口愈合、Transwell和EdU实验结果示,RAPTOR有促进OSCC细胞的迁移、侵袭和增殖能力.miR-485-5p能与RAPTOR靶向结合,且miR-485-5p上调能逆转RAPTOR促进CAL27细胞迁移、侵袭和增殖能力.结论:miR-485-5p通过靶向RAPTOR抑制OSCC细胞迁移、侵袭和增殖能力.

目的:探讨没药甾酮增强化疗药物替莫唑胺抗脑胶质瘤的作用及其分子机制.方法:用没药甾酮、替莫唑胺单药及其联合处理U251 细胞后,采用平板克隆形成实验、5-乙炔基-2'-脱氧尿苷(EdU)实验检测各组细胞增殖能力;采用Transwell小室实验、划痕愈合实验检测各组细胞侵袭、迁移能力;采用Western Blot检测细胞中mTOR/自噬通路关键蛋白的表达水平.结果:低氧条件下(200 μmol/L的CoCl2 诱导 48 h),没药甾酮单药(10、30、100 μmol/L)和替莫唑胺单药(6.25、12.5、25 μmol/L)能显著抑制U251 细胞克隆形成数(P<0.001);与替莫唑胺单药组(6.25、12.5 μmol/L)比较,没药甾酮(10 μmol/L)联合替莫唑胺(6.25、12.5 μmol/L)抑制U251 细胞克隆形成的作用增强,但差异无统计学意义(P>0.05).没药甾酮单药(10、30、100 μmol/L)和替莫唑胺单药(100、200、400 μmol/L)显著下调了U251 细胞EdU阳性细胞率;与替莫唑胺单药组(200、400 μmol/L)比较,没药甾酮(30、100 μmol/L)联合替莫唑胺(200、400 μmol/L)显著下调了U251 细胞EdU阳性细胞率(P<0.05 或P<0.01).与替莫唑胺单药(400 μmol/L)比较,联用没药甾酮(30 μmol/L)显著抑制U251 细胞迁移、侵袭能力(P<0.001).与替莫唑胺单药(400 μmol/L)比较,联用没药甾酮(30 μmol/L)显著下调U251 细胞中Phospho-mTOR(Ser2448)、Phospho-4E-BP1(Ser65)蛋白表达,显著上调 MAPKAP1、p-P70(S6K)蛋白表达(P<0.05 或 P<0.01),对 mTOR、Phospho-mTOR(Ser2481)、Rictor、Raptor、GβL表达的下调也有一定的增强作用,但差异无统计学意义.与替莫唑胺单药(400 μmol/L)比较,联用没药甾酮(30 μmol/L)显著上调U251 细胞中自噬相关蛋白Beclin-1、LC3-Ⅱ/LC3-Ⅰ、P62的表达(P<0.05～P<0.001),对Atg12-5/free-Atg12 也有上调作用,但差异无统计学意义.结论:没药甾酮可通过调控mTOR/自噬信号通路增强替莫唑胺抗脑胶质瘤作用.

[目的]探讨mTOR蛋白对老龄大鼠肌腱干细胞肌腱损伤修复能力的影响.[方法]体外培养青年组和老年组大鼠肌腱干细胞,使用腺病毒转染调控mTOR表达,Western blot检测mTOR和相关蛋白.制作老龄大鼠髌腱损伤模型,分为mTOR表达、mTOR干扰、GFP(green fluorescent protein绿色荧光蛋白)和空白组,在2周和4周取大鼠修复肌腱组织,行组织学、组织免疫化学和生物力学检测.[结果]体外试验方面,Western blot结果显示,老年组mTOR[(1.8±0.1)vs(0.9±0.1),P＜0.05]、Raptor[(4.1±0.3)vs(2.2±0.3),P＜0.05]及 P-Raptor[(1.8±0.1)vs(0.9±0.1),P＜0.05]均显著高于青年组;而两组间 AKT、P-Akt、S6K、P-S6k的差异无统计学意义(P＞0.05).老年大鼠TSCs体外转染后mTOR、Raptor及P-Raptor表达从高到低的顺序:mTOR的表达组＞GFP组＞mTOR干扰组(P＜0.05).体内试验方面,与术后2周相比,术后4周四组HE组织评分均显著增加(P＜0.05),相应时间点分数从高到低的顺序依次为mTOR干扰组＞GFP组＞mTOR表达组＞空白组,差异均有统计学意义(P＜0.05).与术后2周相比,术后4周,Col1及SCX的表达均无显著变化(P＞0.05),相应时间点,四组间Col1、SCX的表达定量从高到低依次为mTOR干扰组＞GFP组＞空白组＞mTOR表达组,差异均有统计学意义(P＜0.05).与术后2周相比,术后4周四组最大损毁强度均显著增加(P＜0.05),相应时间点,最大损毁强度值从大到小依次为mTOR干扰组＞GFP组＞空白组＞mTOR表达组,差异均有统计学意义(P＜0.05).[结论]抑制mTOR蛋白可加强老龄大鼠肌腱干细胞的修复能力.

Large scavengers are strongly dependent on environmental conditions and carrion distribution and abundance,so season and breeding-related factors may influence the spatial ecology of species such as the Cinereous Vulture(Aegypius monachus),the largest European raptor.Iberia holds one of the biggest populations worldwide,but some aspects of the spatial ecology of the species in this region remain unknown.In this study,17 adult Cinereous Vultures were GPS-tracked in order to study their spatial ecology during the adult phase.The average monthly home ranges(95％Kernel Density Estimation,KDE)and core areas(50％KDE)were 6543±19,935 km2 and 1174±4004 km2,respectively.The average monthly home range fidelity ranged between 50 and 73％.Differ-ences in movement-related variables between the seasonal periods(incubation,chick-rearing and non-breeding)were found.During the chick-rearing period,the monthly accumulated distance was higher than during the other periods:3316±1108(chick-rearing)vs.1621±622(incubation)vs.1726±1159 km per month(non-breeding).Additionally,large home range sizes were more frequent during the chick-rearing period.There are two likely causes for these seasonal differences.Firstly,chick-rearing entails a higher energetic expenditure by the parental individuals in foraging activities,so larger movements and foraging areas are expected during this period.Secondly,the flight is favoured during spring and summer due to environmental conditions.Matching chick-rearing and warm months is a great evolutionary advantage for soaring-gliding raptors,as it allows them to cover larger areas with low energy expenditure.Furthermore,six individuals tagged as nestlings highlight the philopatric behaviour of the species:vultures settle their breeding areas 54±51 km from their natal nest(range=9-138 km).

目的 研究抑制了慢性排斥反应的大鼠模型中,T淋巴细胞中哺乳动物雷帕霉素靶蛋白-丝/苏氨酸蛋白激酶(mTOR)通路的各成分是否发生改变,探讨mTOR信号通路在抑制慢性排斥反应中的作用.方法 受体August-Copenhagen-Irish(ACI)大鼠与供体Wistar-Furth (WF)大鼠接受腹腔内异位心脏移植术.实验组大鼠给予慢性排斥反应消除处理,即术前给予经改造的Ⅰ类主要组织相容性复合物(MHC Ⅰ),并给予亚治疗量环孢素(CsA)(10 mg/kg,3d),治疗对照组于术后给予亚治疗量CsA(10 mg/kg,3 d),空白对照组不进行处理.将每组大鼠分为在术后1、3、7d处死的3个亚组,每个亚组大鼠数量为5只.分别在亚组对应天数处死大鼠,获取脾脏样本进行T细胞提取与蛋白免疫印迹(Western blot)分析.结果 蛋白免疫印迹结果表明,在消除了慢性排斥反应的移植心脏中(实验组),对雷帕霉素敏感的复合体1 (mTOR C1)的mTOR与mTOR调节相关蛋白(Raptor)下调,对雷帕霉素不敏感的复合体2(mTOR C2)的伴侣分子(Rictor)与哺乳动物应激活化蛋白激酶相互作用蛋白1 (Sin1)下调,mTOR调节因子(Deptor)与mTOR通路下游目标分子(Rac1)也受到抑制.结论 在消除了慢性排斥反应的大鼠心脏移植模型中,mTORC1和C2通路均受到影响,同时影响了细胞增殖调节(mTOR C1)和细胞运动调节(mTOR C2).因此,选择性地对T细胞肌动蛋白细胞骨架通路进行抑制,可成为新的免疫抑制剂发展方向.