全面性癫痫伴热性惊厥附加症SCN1A基因G302D突变的膜片钳电生理研究

目的:探讨全面性癫痫伴热性惊厥附加症(GEFS+)SCN1A基因G302D突变的电生理机制。方法:采用体外定点诱变法构建携带有基因突变G302D的pCMV-SCN1A的表达载体,lipo2000脂质体转染法共转染pCMV-SCN1A-G302D质粒和pCD8-IRES-SCN1B质粒到HEK-293细胞系,进行全细胞膜片钳电生理实验记录Navl.1通道电流及动力学参数,由pClamp10.0以及OriginPro8.0软件分析。结果:SCN1A-G302D突变体与野生型相比,电流密度降低,激活速度减慢,失活后恢复时间延长。失活参数两者相比没有显著性统计学差异。结论:SCN1A基因G302D突变导致Navl.1通道功能部分丧失,可能是导致GEFS+的病因。     

海马与新皮质组织特异性GABRG2基因敲除小鼠模型的构建及其在遗传性癫痫伴热性惊厥附加症中的初步研究

建立基于Cre/loxp重组酶系统调控的海马和新皮质特异性GABA_A受体γ2亚基(GABRG2)基因条件基因敲除小鼠模型,为深入研究海马区和新皮质GABRG2在癫痫发生中的功能作用提供动物模型。将引进的GABRG2~(fl/wt)转基因小鼠与海马和新皮质特异性表达Cre~+/+重组酶工具鼠分别进行繁配和鉴定,然后再将2种小鼠进行杂交并对其子代小鼠的基因型进行鉴定,其子代基因型为GABRG2~(fl/wt)Cre~+的小鼠为构建的海马区和新皮质特异性GABRG2基因条件性敲除小鼠。利用PCR技术鉴定小鼠基因型,Real-Time PCR和Western blot技术检测GABRG2基因在小鼠海马和新皮质中的mRNA水平和蛋白质水平的表达情况。PCR结果显示子代小鼠基因型符合GABRG2~(fl/wt)Cre~+;海马与新皮质特异性GABRG2基因敲除小鼠海马和新皮质中GABRG2的mRNA水平和蛋白质水平显著低于对照组;热造模过程中,实验组小鼠癫痫发作更明显。利用Cre/Loxp技术成功构建了海马与新皮质GABRG2基因敲除小鼠,可为进一步研究GABRG2在癫痫发生中的作用机制奠定基础。     

海马皮质特异性敲除AEG-1基因小鼠的构建及其行为学初步研究*

星形胶质细胞上调基因-1(astrocyte upregulating gene-1,AEG-1)是HIV伴随老年痴呆患者脑组织中发现的星形胶质细胞上调基因之一,近年来研究表明其调控多种中枢神经系统疾病,但其在学习认知上的研究尚未见报道。海马和皮质在学习认知中起重要作用,利用CRISPR/Cas9技术结合Cre/loxp系统构建海马皮质特异性AEG-1敲除小鼠,在此模型鼠的基础上对AEG-1和学习认知的相关性进行初步研究。首先构建插入loxp位点的flox纯合型AEG-1^fl/fl小鼠,与海马、新皮层特异性表达Cre^+/+重组酶的工具鼠进行繁育,利用PCR技术筛选出子代基因型为AEG-1^fl/fl Cre⁺的海马皮质特异性AEG-1敲除小鼠;然后利用Western blot技术和免疫荧光技术检测AEG-1基因在小鼠海马皮质中的敲除效率;最后应用新物体识别箱和三腔社会互动箱并结合SMART 3.0分析系统,对海马皮质特异性AEG-1敲除小鼠的学习记忆和社会交互行为学进行初步评价。结果显示:成功获得子代基因型为AEG-1^fl/fl Cre⁺的基因敲除小鼠;AEG-1条件性敲除小鼠海马和皮质中AEG-1蛋白质表达水平较对照组显著降低;新物体识别结果表明AEG-1条件性敲除小鼠的区分系数明显低于对照组,表明AEG-1条件性敲除小鼠的学习记忆能力较弱,但是三腔交互结果表明AEG-1条件性敲除小鼠在社会交互上与对照组相比无明显差异。以上结果为AEG-1在学习认知方面的进一步研究奠定了基础。     

海马皮质特异性敲除AEG-1基因小鼠的构建及其行为学初步研究*

星形胶质细胞上调基因-1(astrocyte upregulating gene-1,AEG-1)是HIV伴随老年痴呆患者脑组织中发现的星形胶质细胞上调基因之一,近年来研究表明其调控多种中枢神经系统疾病,但其在学习认知上的研究尚未见报道。海马和皮质在学习认知中起重要作用,利用CRISPR/Cas9技术结合Cre/loxp系统构建海马皮质特异性AEG-1敲除小鼠,在此模型鼠的基础上对AEG-1和学习认知的相关性进行初步研究。首先构建插入loxp位点的flox纯合型AEG-1^fl/fl小鼠,与海马、新皮层特异性表达Cre^+/+重组酶的工具鼠进行繁育,利用PCR技术筛选出子代基因型为AEG-1^fl/fl Cre⁺的海马皮质特异性AEG-1敲除小鼠;然后利用Western blot技术和免疫荧光技术检测AEG-1基因在小鼠海马皮质中的敲除效率;最后应用新物体识别箱和三腔社会互动箱并结合SMART 3.0分析系统,对海马皮质特异性AEG-1敲除小鼠的学习记忆和社会交互行为学进行初步评价。结果显示:成功获得子代基因型为AEG-1^fl/fl Cre⁺的基因敲除小鼠;AEG-1条件性敲除小鼠海马和皮质中AEG-1蛋白质表达水平较对照组显著降低;新物体识别结果表明AEG-1条件性敲除小鼠的区分系数明显低于对照组,表明AEG-1条件性敲除小鼠的学习记忆能力较弱,但是三腔交互结果表明AEG-1条件性敲除小鼠在社会交互上与对照组相比无明显差异。以上结果为AEG-1在学习认知方面的进一步研究奠定了基础。     

肝脏特异性CD36基因敲除小鼠的制备及鉴定

目的:运用Cre/Loxp重组酶系统构建肝脏特异性CD36基因敲除小鼠并进行鉴定和验证,为研究CD36的生物学功能奠定基础。方法:构建CD36打靶载体,电转转染胚胎干细胞,通过长链PCR筛选出正确同源重组的阳性克隆,阳性胚胎干细胞克隆经扩增后,注射入C57BL/6J小鼠的囊胚中,获得嵌合小鼠,再与Flp小鼠交配筛选获得Flox杂合子小鼠,该小鼠与引进的Alb-Cre小鼠交配,在F3代获得CD36fl/fl:Alb-Cre+基因型小鼠,即为肝脏特异性CD36敲除小鼠。采用PCR鉴定小鼠基因型,PCR、实时荧光定量PCR和Western blot验证小鼠肝脏CD36敲除效果,Western blot检测小鼠肾脏、脂肪和心肌组织CD36表达情况,HE染色观察小鼠肝脏形态学改变。结果:建立了CD36基因的Flox杂合子小鼠,与Alb-Cre小鼠交配后,在F3代筛选出CD36fl/fl:AlbCre-和CD36fl/fl:Alb-Cre+基因型小鼠,DNA水平证实CD36fl/fl:Alb-Cre+基因型小鼠肝脏CD36基因通过Cre/Loxp重组酶系统被敲除。与CD36fl/fl:Alb-Cre-基因型小鼠相比,CD36fl/fl:Alb-Cre+基因型小鼠肝脏CD36mRNA和蛋白表达水平显著降低,肾脏、脂肪和心肌组织CD36蛋白表达无差别,肝脏形态学特征无明显差异。结论:通过Cre/Loxp重组酶系统成功构建了肝脏特异性CD36基因敲除小鼠,为研究CD36在肝脏代谢和肝脏疾病中的功能提供了动物模型。     

去泛素化酶OTUB1肝脏特异性基因敲除小鼠模型的构建与表型分析

目的:建立OTUB1肝脏特异性基因敲除小鼠模型,初步分析其表型并研究OTUB1基因与肝脏代谢的关系。方法:利用Cre/Loxp系统构建条件性基因敲除小鼠模型,即将OTUB1~(fl/fl)转基因小鼠与Alb-Cre小鼠杂交,子代自交,得到OTUB1肝特异性基因敲除小鼠并进行鉴定。取同窝对照小鼠(control,NC)和肝特异型基因敲除(hepatic-specific OTUB1 knockout,HCKO)小鼠,通过PCR和免疫印迹(Western blot),确证OTUB1肝脏特异性基因敲除小鼠模型是否成功构建。通过组织病理学方法,分析主要组织器官的形态以及是否存在自发的病变;通过血清生化指标检测肝脏脂代谢水平;通过血糖耐受实验(GTT)分析HCKO小鼠对血糖的控制。结果:基因组测序和Western blot检测结果显示HCKO小鼠肝脏中OTUB1被敲除,其他组织中OTUB1表达水平无变化,证明OTUB1肝脏特异性基因敲除小鼠模型构建成功。HCKO小鼠出生正常,各组织器官无异常,生化指标中总胆固醇水平明显降低,表明OTUB1影响肝脏脂代谢水平。糖耐受实验中HCKO小鼠血糖回落迅速,表明敲除OTUB1影响肝脏血糖调节稳态。结论:应用Cre/Loxp技术成功建立OTUB1肝脏特异性基因敲除小鼠模型,为研究OTUB1在肝脏的生理功能和调控机制提供了重要的动物模型。     

NPAS2基因敲除的HepG2细胞系构建及其对肝癌细胞凋亡的影响

目的:利用CRISPR/Cas9基因编辑技术构建生物节律基因NPAS2敲除的HepG2肝癌细胞系,并初步探讨NPAS2基因敲除对肝癌细胞凋亡的影响。方法:利用sgRNA在线设计工具,针对NPAS2设计两条sgRNA;利用PX459质粒构建分别含有两条sgRNA的敲除载体PX459-sgRNA1;PX459-sgRNA2;利用T7核酸内切酶I检测两条sgRNA活性;将活性较高的打靶载体瞬时转染HepG2细胞,经过药物筛选,克隆化培养及基因测序后得到NPAS2敲除的HepG2肝癌细胞系;利用Western blot检测NPAS2蛋白的表达和凋亡相关蛋白Caspase3的活化;利用流式细胞仪检测敲除细胞系的凋亡水平。结果:成功构建了针对NPAS2的打靶载体;并筛选得到了活性较高的打靶载体;经过药物筛选和克隆化培养得到的NPAS2敲除肝癌细胞系未检测到NPAS2蛋白的表达;进一步发现NPAS2敲除的肝癌细胞Caspase3明显活化,凋亡水平显著升高。结论:利用CRISPR/Cas9基因编辑技术成功构建了NPAS2基因敲除的HepG2肝癌细胞系,并发现NPAS2敲除可以促进肝癌细胞凋亡,为进一步研究生物节律基因NPAS2及其它相关基因在肝癌发生发展中的作用机制提供了有力的工具。     

Analysis of serotonin receptor 2A gene (HTR2A): Association study with autism spectrum disorder in the Indian population and investigation of the gene expression in peripheral blood leukocytes

Several studies suggest involvement of serotoninergic system in the pathophysiology of Autism Spectrum Disorder (ASD). The 5-HT receptor binding studies using ³H-lysergic acid diethylamide (³H-LSD) and linkage analysis provided evidences to consider HTR2A as a potential candidate gene for ASD. The three SNPs, −1438A/G (rs6311), 102T/C (rs6313) and 1354C/T (rs6314) of HTR2A have been well studied in the etiology of various neuropsychiatric disorders. But studies on association of this gene with ASD are limited to two reports from American and Korean populations. Additionally there are reports, which demonstrated paternal imprinting of HTR2A with expression from only one allele. So far no reports are available on HTR2A and its association with any neuropsychiatric disorders from Indian population. Therefore, the present study investigates association of the above mentioned three markers of HTR2A with ASD in Indian population using population and family-based approaches. The study also deals with allelic expression pattern of HTR2A in Peripheral Blood Leukocytes (PBLs) to understand the parental imprinting status. The genotyping analyses were carried out for probands, parents and controls. The subsequent association analyses did not show association of these markers with ASD. So, HTR2A is unlikely to be a genetic marker for ASD in Indian population. The expression analyses showed absence of monoallelic expression, suggesting lack of parental imprinting of HTR2A gene. However, we noticed methylation of the CpG sites at −1438A/G and 102T/C loci of HTR2A gene. Further bioinformatics analysis revealed absence of CpG islands in the promoter of the gene supporting biallelic expression pattern of HTR2A in PBLs.     

干旱高温复合胁迫下sHSPs基因在不同耐旱性玉米中的表达差异及其对ABA和H2O2的响应

以4个玉米(Zea mays L.)品种——‘郑单958’(耐干旱)、‘浚单20’(耐高温)、‘隆玉602’(耐干旱高温复合胁迫)和‘驻玉309’(对3种胁迫均敏感)为实验材料,分别采用脱落酸(ABA)及其抑制剂氟定酮(F)、H2O2及其清除剂碘化钾(I)和丙酮酸钠(P)预处理,探讨干旱高温复合胁迫下小热休克蛋白(sHSPs)在不同耐旱性玉米品种中的表达以及ABA和H2O2对其表达的影响。结果显示:(1)高温、干旱高温复合胁迫诱导的sHSP16.9、sHSP17.2、sHSP17.4、sHSP17.5、sHSP22和sHSP26等6个sHSPs基因表达增加量明显高于干旱和对照。(2)在6个sHSPs中,sHSP17.2基因仅在‘郑单958’中表达;sHSP16.9、sHSP17.4和sHSP26基因在‘隆玉602’中表达增加量最高,在‘驻玉309’中表达增量最低;sHSP17.5和sHSP22基因在‘郑单958’中表达增加量最高,在‘驻玉309’中最低。(3)ABA、F、I和P预处理后,对干旱高温复合胁迫诱导的6个sHSPs基因表达增加量仅有略微影响,但显著影响了sHSP26的蛋白表达。研究表明,sHSPs在不同耐旱性玉米品种中的表达存在显著差异,ABA和H2O2仅稍微提高了sHSPs基因表达,但显著提高了sHSP26蛋白表达,这些结果为进一步研究植物在多胁迫条件下耐逆机理奠定了基础。     

Quantification and Proteomic Characterization of β-Hydroxybutyrylation Modification in the Hearts of AMPKα2 Knockout Mice

AMP-activated protein kinase alpha 2 (AMPKα2) regulates energy metabolism, protein synthesis, and glucolipid metabolism myocardial cells. Ketone bodies produced by fatty acid β-oxidation, especially β-hydroxybutyrate, are fatty energy–supplying substances for the heart, brain, and other organs during fasting and long-term exercise. They also regulate metabolic signaling for multiple cellular functions. Lysine β-hydroxybutyrylation (Kbhb) is a β-hydroxybutyrate–mediated protein posttranslational modification. Histone Kbhb has been identified in yeast, mouse, and human cells. However, whether AMPK regulates protein Kbhb is yet unclear. Hence, the present study explored the changes in proteomics and Kbhb modification omics in the hearts of AMPKα2 knockout mice using a comprehensive quantitative proteomic analysis. Based on mass spectrometry (LC-MS/MS) analysis, the number of 1181 Kbhb modified sites in 455 proteins were quantified between AMPKα2 knockout mice and wildtype mice; 244 Kbhb sites in 142 proteins decreased or increased after AMPKα2 knockout (fold change >1.5 or <1/1.5, p < 0.05). The regulation of Kbhb sites in 26 key enzymes of fatty acid degradation and tricarboxylic acid cycle was noted in AMPKα2 knockout mouse cardiomyocytes. These findings, for the first time, identified proteomic features and Kbhb modification of cardiomyocytes after AMPKα2 knockout, suggesting that AMPKα2 regulates energy metabolism by modifying protein Kbhb.