[目的]构建在中华按蚊Anopheles sinensis中的电转化显微注射技术平台,并利用该技术平台实现活体基因瞬时过表达对其进行验证,为开展系统的基因功能研究奠定基础.[方法]以CUY21 EDIT Ⅱ电转仪为主体构建中华按蚊中的电转化显微注射技术平台;使用同源重组法构建EGFP和Bm-iAANAT基因的瞬时过表达质粒,在中华按蚊化蛹3h时注射该质粒进入蛹体,随即使用电转仪对注射后的蛹进行电击,待注射个体发育至化蛹39 h时,利用体视荧光显微镜观察蛹体表皮着色和发光情况,并通过荧光定量PCR检测EGFP和Bm-iAANAT的表达.[结果]构建了用于显微注射的PIZ-modi-Aepub-EGFP-SV40和PIZ-modi-Aepub-AANAT-T2A-EGFP-SV40过表达载体.PIZ-modi-Aepub-EGFP-S V40注射组中华按蚊在化蛹39 h时约87.5％的个体成活并正常黑化,这其中约92.7％的个体的表皮检测到明显的绿色荧光,注射无启动子载体PIZ-modi-EGFP-SV40并进行电击的阴性对照组和注射过表达载体PIZ-modi-Aepub-EGFP-SV40但未进行电击的阳性对照组个体则没有明显的绿色荧光;并且荧光定量PCR结果显示,注射组中发光个体的EGFP基因明显高表达.PIZ-modi-Aepub-AANAT-T2A-EGFP-SV40注射组的蛹在化蛹39 h时有80.4％个体存活,这其中92.2％的个体相对于注射无启动子载体PIZ-modi-AANAT-T2A-EGFP-SV40的阴性对照组和注射过表达载体PIZ-modi-Aepub-AANAT-T2A-EGFP-SV40但未进行电击的阳性对照组个体黑化明显受阻,且有明显的绿色荧光;并且荧光定量PCR结果显示,报告基因Bm-iAAⅣAT和EGFP的表达明显提高.[结论]成功构建了在中华按蚊中的电转化显微注射技术平台;通过此技术平台能够便捷、快速和高效地实现报告基因在活蛹中的瞬时过表达,并产生了目的表型.这为中华按蚊功能基因组研究奠定了基础.

目的 用Streptomyces lividans GX28异源合成N-乙酰基色胺,并研究N-乙酰基色胺的抗赤潮藻活性.方法 前期研究发现Bacillus atrophaeus C89芳香族氨基酸脱羧酶(aromatic 1-amino acid decarboxylase,AADC)能将L-色氨酸脱羧成L-色胺.氨基酸多序列比对发现S lividans GX28基因组中存在使色胺乙酰化的酶.通过克隆、重组将aadc基因转入S.lividans GX28中构建重组菌株,HPLC检测目标产物,NMR鉴定化合物的结构.检测化合物对3种赤潮微藻的毒性.结果 S.lividans GX28中的蛋白WP_015609847.1含有芳烷基胺N-乙酰转移酶(alkyl amine N-acetyltransferase,AANAT)的底物结合位点(P64和F188)和催化位点(Y168),推测其为AANAT.宿主的发酵产物中分离得到N-乙酰基色胺.N-乙酰基色胺对墨西哥原甲藻的生长具有较强的抑制作用(IC50=9.56mg/L).结论 推测aadc基因在链霉菌中异源表达.重组链霉菌中检测到N-乙酰基色胺,并对N-乙酰基色胺的生物合成途径进行推测.N-乙酰基色胺对P.mexicanum的生长具有显著的抑制作用.本研究为抑藻化合物的生物合成研究奠定了基础.

Circadian rhythm disorder is a common, but often neglected, consequence of neonatal hypoxic-ischemic brain damage (HIBD). However, the underlying molecular mechanisms remain largely unknown. We previously showed that, in a rat model of HIBD, up-regulation of microRNA-325 (miR-325) in the pineal gland is responsible for the suppression of Aanat, a key enzyme involved in melatonin synthesis and circadian rhythm regulation. To better understand the mechanism by which miR-325 affects circadian rhythms in neonates with HIBD, we compared clinical samples from neonates with HIBD and samples from healthy neonates recruited from the First Affiliated Hospital of Soochow University (Dushuhu Branch) in 2019. We found that circulating miR-325 levels correlated positively with the severity of sleep and circadian rhythm disorders in neonates with HIBD. Furthermore, a luciferase reporter gene assay revealed that LIM homeobox 3 (LHX3) is a novel downstream target of miR-325. In addition, in miR-325 knock-down mice, the transcription factor LHX3 exhibited an miR-325-dependent circadian pattern of expression in the pineal gland. We established a neonatal mouse model of HIBD by performing double-layer ligation of the left common carotid artery and exposing the pups to a low-oxygen environment for 2 hours. Lhx3 mRNA expression was significantly down-regulated in these mice and partially rescued in miR-325 knockout mice subjected to the same conditions. Finally, we showed that improvement in circadian rhythm-related behaviors in animals with HIBD was dependent on both miR-325 and LHX3. Taken together, our findings suggest that the miR-325-LHX3 axis is responsible for regulating circadian rhythms and provide novel insights into the identification of potential therapeutic targets for circadian rhythm disorders in patients with neonatal HIBD. The clinical trial was approved by Institutional Review Board of Children's Hospital of Soochow University (approval No. 2015028) on July 20, 2015. Animal experiments were approved by Animal Care and Use Committee, School of Medicine, Soochow University, China (approval No. XD-2016-1) on January 15, 2016.

目的 探讨慢性肾脏疾病中microRNA-483-3p及其相关靶基因表达情况,明确过表达miR-483-3p的治疗效果.方法 大鼠分为对照组(假手术组,12只),模型组(5/6肾切除组,12只),miR-483-3p过表达组(5/6肾切除+miR-483-3p Agomir,12只)3组.miR-483-3p Agomir给予60mg/kg尾静脉注射,每两周一次.末次术后8周处死大鼠.测定血生化指标,使用ELISA方法测血清中hs-CRP及24小时尿蛋白含量;采用RT-PCR方法进一步筛查出miR-483-3p的下游靶基因;用RT-PCR和Western Blot方法测定三组大鼠肾组织中miR-483-3p及其靶基因表达变化.同时RT-PCR方法测定miR-483-3p宿主基因TGF-2的表达变化.结果 与对照组相比,模型组BUN、Cr明显升高,TC及LDL-C亦显著升高.干预组较模型组BUN、Cr明显降低,血脂水平治疗前后无明显变化.hs-CRP及24小时尿蛋白定量在模型组明显升高,干预组显著降低.RT-PCR结果显示与对照组相比,模型组肾脏组织中CTGF和EGFR/PTEN表达显著升高,而TGF-β、DPC4/Smad4及AANAT无明显变化.干预组较模型组表达显著降低.模型组较对照组肾脏组织中IGF-2的表达明显升高,干预组肾脏组织IGF-2的表达较模型组表达显著降低.结论 慢性肾脏疾病,miR-483-3p表达增加,通过抑制其靶基因CTGF、PTEN的表达,可延缓肾脏纤维化过程.

目的:观察蛇床子催眠活性组分(CHC)对对氯苯丙氨酸(PCPA)失眠大鼠行为学、褪黑素(MT)合成限速酶芳基烷基胺N-乙酰基转移酶(AANAT)的影响,探讨其对松果体的保护机制.方法:60只SPF级雄性SD大鼠随机分为正常、模型,MT阳性药,CHC高、中、低剂量6组,每组10只.除正常组其余各组腹腔注射4.5％PCPA混悬液,10 mL· kg-1,连续2d,建立大鼠PCPA失眠模型.造模后正常及模型组灌胃等体积2％聚山梨酯-80,MT组(10 mg· kg-1),CHC高、中、低(60,30,15 mg·kg-1)给予10 mL·kg-1给药体积的药物,每日1次,连续7d.给药4d后分别进行旷场活动、高架十字迷宫、戊巴比妥钠协同睡眠试验,采用酶联免疫吸附法检测血清MT;实时荧光定量聚合酶链式反应(Real-time PCR)测定松果体MT合成关键酶AANAT mRNA表达水平;蛋白免疫印迹法(Western blot)检测松果体AANAT蛋白表达变化.结果:与正常组比较,模型组大鼠旷场活动总距离、站立次数、中心区持续时间明显增加(P＜0.05,P＜0.01),高架开臂次数及时间比例下降(P＜0.05),入睡潜伏期显著延长(P＜0.01);与模型组比较,低剂量组无明显差异,其余各组大鼠活动总距离均减少(P＜0.05,P＜0.01),高架开臂次数百分比升高(P＜0.05),入睡潜伏期缩短、睡眠时间明显延长(P＜0.05,P＜0.01).与正常组比较,模型组MT含量显著下降(P＜0.01);与模型组比较,低剂量组差异无明显统计学意义,其余各组血清MT不同程度的升高(P＜0.05).与正常组比较,模型组AANAT mRNA表达量,AANAT蛋白表达量下降(P＜0.01);与模型组比较,MT组,CHC高剂量组表达量明显增加(P＜0.05).结论:CHC改善PCPA失眠行为学指标、增加松果体细胞合成分泌MT,提高血清MT水平,与上调松果体AANAT mRNA及其蛋白的表达有关.

Arylalkylamine N-acetyltransferase (aaNAT) catalyzes the acetylation of dopamine,5-hydroxy-tryptamine,tryptamine,octopamine,norepinephrine and other ary-lalkylamines to form respective N-acetyl-arylalkylamines.Depending on the products formed,aaNATs are involved in a variety of physiological functions.In the yellow fever mosquito,Aedes aegypti,a number of aaNATs and aaNAT-like proteins have been re-ported.However,the primary function of each individual aaNAT is yet to be identified.In this study we investigated the function of Ae.aegypti aaNAT1 (Ae-aaNAT1) in cuticle pigmentation and development of morphology.Ae-aaNAT1 transcripts were detected at all stages of development with highest expressions after pupation and right before adult eclosion.Ae-aaNAT1 mutant mosquitoes generated using clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 had no obvious effect on larval and pupal development.However,the mutant mosquitoes exhibited a roughened ex-oskeletal surface,darker cuticles,and color pattern changes suggesting that Ae-aaNAT1 plays a role in development of the morphology and pigmentation of Ae.aegypfi adult cu-ticles.The mutant also showed less blood feeding efficiency and lower fecundity when compared with the wild-type.The mutation of Ae-aaNAT1 influenced expression of genes involved in cuticle formation.In summary,Ae-aaNAT1 mainly functions on cuticular pig-mentation and also affects blood feeding efficiency and fecundity.

目的:观察电针对大鼠血清褪黑素含量及松果体褪黑素合成限速酶―芳烷胺-N-乙酰转移酶(AANAT)表达的影响,探讨电针"百会""神庭"减轻大鼠脑缺血再灌注损伤的机制.方法:SD大鼠随机分为假手术组、模型组、电针组和非经非穴组,每组12只.采用大脑中动脉闭塞法制备局灶性脑缺血再灌注损伤模型.电针组取"百会""神庭",非经非穴组取双胁下非经非穴点,每日电针20 min,1次/d,连续干预7 d.Longa法评估神经功能缺损情况,水迷宫实验检测各组大鼠认知功能,ELISA法检测血浆褪黑素含量,PCR及Western blot法检测大鼠松果体AANAT mRNA及蛋白的表达水平,免疫荧光法检测海马CA1区星形胶质细胞活化情况以及神经元损伤情况.结果:和假手术组比较,模型组Longa评分、水迷宫实验逃避潜伏期显著增高(P<0.01),穿越平台次数减少(P<0.01);夜间24:00褪黑素分泌显著降低(P<0.01);松果体内AANAT mRNA和蛋白表达水平显著降低(P<0.01);海马CA1区GFAP(星形胶质细胞标记物)阳性表达显著增加(P<0.01),NeuN(神经元标记物)阳性表达显著下降(P<0.01).和模型组比较,电针组Longa评分、水迷宫实验逃避潜伏期显著降低(P<0.01),穿越平台次数增加(P<0.01),夜间24:00褪黑素分泌显著升高(P<0.01),松果体内AANAT mRNA和蛋白的表达升高(P<0.01),海马CA1区GFAP阳性表达降低(P<0.01),NeuN阳性表达升高(P<0.01).与模型组比较,非经非穴组以上各指标无明显变化(P>0.01).结论:电针"百会""神庭"可以减轻脑缺血再灌注损伤模型大鼠神经功能和认知功能损伤,其机制可能和调控内源性褪黑素表达水平,抑制海马CA1区星形胶质细胞活化以及保护受损神经元有关.

目的 研究产前应激(PS)诱导的抑郁样子代大鼠大脑海马体的差异表达基因(DEG)及其表达规律和调节机制.方法 2020年3-10月选择SD大鼠建立慢性束缚应激模型进行研究.使用糖水偏好实验测试子代大鼠PS易感性(PS-S),分别采用旷场实验、强迫游泳实验、悬尾实验验证PS模型和糖水偏好实验筛选个体对应激反应的有效性.应用基于高通量测序的转录组学方法来表征PS-S子代大鼠海马体中的基因网络.在数据处理和质量控制之后,使用生物信息学分析筛选差异表达基因并进行分类.结果 鉴定出486个差异表达基因,其中230个上调,256个下调.GO功能富集分析显示,上调的基因中鉴别出20个GO注释,下调的基因中鉴别出17个GO注释.诸如"气体运输"、"血红蛋白复合物"、"氧气运输"、"氧载体活性"等GO注释在DEG中排名靠前.包括"谷氨酸与激酶活性"、"对含氧化合物的反应"和"视黄醇代谢过程"等7个GO注释可同时富集在上调和下调的DEG集.KEGG富集分析表明,这些DEG主要富集于色氨酸代谢、吞噬体、蛋白消化吸收、神经活性配体受体相互作用、果糖和甘露糖代谢等信号通路.分析发现TTR、CRABP2、FABP5、AANAT和TPH1等基因的表达差异可能在PS诱导的子代大鼠抑郁样行为中起关键作用.结论 视黄醇代谢过程、视黄酸代谢和色氨酸代谢通路相关基因的差异表达可能参与PS致子代抑郁行为的发病机制.

目的:观察针刺申脉、照海穴对模型大鼠睡眠-觉醒昼夜节律和松果体(PG)中芳基烷化胺-N-乙酰基转移酶(AANAT)的调节作用,初步探讨针刺调节睡眠-觉醒昼夜节律紊乱的效应机制.方法:成年雄性SD大鼠依据随机数字表随机分为对照组、模型组、针刺经穴组和针刺非经穴组,每组各35只,采用腹腔注射对氯苯丙氨酸(PCPA)的方法构建大鼠昼夜节律紊乱睡眠障碍模型.针刺经穴组针刺申脉、照海穴,每日1次,每次20 min,共5 d.利用自动化睡眠生物解析系统进行脑电及肌电数据采集,观察睡眠-觉醒昼夜节律变化,同时分别在ZT0、ZT4、ZT8、ZT12、ZT16、ZT20(ZT代表授时时间,将开灯时刻记为ZT0)6个时间点剥离PG,测定其中AANAT mRNA的相对表达量并进行节律分析.结果:睡眠-觉醒昼夜节律分析显示,造模后模型组大鼠觉醒(Wake)、非快速眼动睡眠(NREM sleep)昼夜节律消失(P>0.05),振幅减小(P<0.05),峰相位有后移趋势;干预后,与模型组和针刺非经穴组比较,针刺经穴组Wake中值减小(P<0.05),NREM sleep中值增高(P<0.05),振幅有升高趋势,峰相位有前移趋势.PG中AANAT表达量及节律分析显示,造模后模型组PG中AANAT mRNA相对表达量在ZT0、ZT12、ZT16、ZT204个时间点显著降低(P<0.05),AANAT mRNA昼夜节律消失(P>0.05),中值与节律振幅明显减小(P<0.05).干预后,与模型组比较,针刺经穴组在ZT12、ZT16、ZT20时间点AANAT mRNA显著升高(P<0.05);与针刺非经穴组比较,针刺经穴组在ZT16、ZT20时间点AANAT mRNA显著升高(P<0.05),AANAT mRNA表达昼夜节律恢复(P<0.05);中值与节律振幅显著升高(P<0.05).结论:针刺申脉、照海穴可调节模型大鼠睡眠-觉醒昼夜节律紊乱状态,其机制可能是通过调节PG中AANAT的相对表达量与表达节律来实现.

Arylalkylamine N-acetyltransferase(aaNAT),considered a potential new in-secticide target,catalyzes the acetylation of arylalkylamine substrates such as serotonin and dopamine and,hence,mediates diverse functions in insects.However,the origin of insect aaNATs(iaaNATs)and the evolutionary process that generates multiple aaNATs in mosquitoes remain largely unknown.Here,we have analyzed the genomes of 33 species to explore and expand our understanding of the molecular evolution of this gene family in detail.We show that aaNAT orthologs are present in Bacteria,Cephalochordata,Chon-drichthyes,Cnidaria,Crustacea,Mammalia,Placozoa,and Teleoste,as well as those from a number of insects,but are absent in some species of Annelida,Echinozoa,and Mollusca as well as Arachnida.Particularly,more than 10 aaNATs were detected in the Culicinae subfamily of mosquitoes.Molecular evolutionary analysis of aaNAT/aaNAT-like genes in mosquitoes reveals that tandem duplication events led to gene expansion in the Culici-nae subfamily of mosquitoes more than 190 million years ago.Further selection anal-ysis demonstrates that mosquito aaNATs evolved under strongly positive pressures that generated functional diversity following gene duplication events.Overall,this study may provide novel insights into the molecular evolution of the aaNAT family in mosquitoes.