Effect of Shortening Day Length on Immune Function in Siberian Hamsters

In order to understand the effect of gradual changes in photoperiod on immune function, adult female Siberian hamsters (Phodopus sungorus) were randomly divided into the control group (12L:12D, Con, n = 11) and the shortening day length group (SD, n = 11), in which day length was reduced from 12:12 h to 8:16 h light-dark cycle at the pace of half an hour every week. Meanwhile the winter immunoenhancement hypothesis, which holds that animals’ immune function would be enhanced in winter or winter-like conditions, was tested. Gradual shortening day length had no effect on body mass and body composition including wet carcass mass, the subcutaneous, retroperitoneal, mesenteric and total body fat masses in Siberian hamsters. The masses of liver and small intestine with contents were higher in the SD group than in the Con group, however other organ masses such as brain, heart, kidney and so on did not differ between the two groups. Phytohemagglutinin (PHA) response after 24 h of PHA injection was enhanced by the shortening photoperiod, which supported the winter immunoenhancement hypothesis. The masses of spleen and thymus, white blood cells, bacteria killing capacity indicative of innate immunity were not affected, which did not support this hypothesis. In summary, gradually decrease in day length increased cellular immunity, but had no effect on other immunological parameters in Siberian hamsters.

Comparative studies between humans and golden Syrian hamsters via thromboelastography

Background:Thromboelastography(TEG)is a widely utilized clinical testing method for real-time monitoring of platelet function and the thrombosis process.Lipid metab-olism disorders are crucial risk factors for thrombosis.The lipid metabolism charac-teristics of hamsters resemble those of humans more closely than mice and rats,and their relatively large blood volume makes them suitable for studying the mechanisms of thrombosis related to plasma lipid mechanisms.Whole blood samples from golden Syrian hamsters and healthy humans were obtained following standard clinical pro-cedures.TEG was employed to evaluate coagulation factor function,fibrinogen(Fib)function,platelet function,and the fibrinolytic system.Methods:The whole blood from hamster or healthy human was isolated following the clinical procedure,and TEG was employed to evaluate the coagulation factor func-tion,Fib function,platelet function,and fibrinolytic system.Coagulation analysis used ACLTOP750 automatic coagulation analysis pipeline.Blood routine testing used XN-2000 automatic blood analyzer.Results:TEG parameters revealed that hamsters exhibited stronger coagulation fac-tor function than humans(reaction time[R],p=0.0117),with stronger Fib function(alpha angle,p<0.0001;K-time[K],p<0.0001).Platelet function did not differ signifi-cantly(maximum amplitude[MA],p=0.077).Hamsters displayed higher coagulation status than humans(coagulation index[CI],p=0.0023),and the rate of blood clot dissolution in hamsters differed from that in humans(percentage lysis 30 min after MA,p=0.02).Coagulation analysis parameters indicated that prothrombin time(PT)and activated partial thromboplastin time(APTT)were faster in hamsters than in hu-mans(PT,p=0.0014;APTT,p=0.03),whereas the Fib content was significantly lower in hamsters than in humans(p<0.0001).No significant difference was observed in thrombin time(p=0.1949).Conclusions:In summary,TEG could be used to evaluate thrombosis and bleeding parameters in whole blood samples from hamsters.The platelet function of hamsters closely resembled that of humans,whereas their coagulation function was signifi-cantly stronger.

Gene-modified leucoconcentrate for personalized ex vivo gene therapy in a mini pig model of moderate spinal cord injury

We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases,neurotrauma,and stroke.In this study,using a mini pig model of spinal cord injury,we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy.Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector(Ad5/35 F)that carried an enhanced green fluorescent protein(EGFP)reporter gene in the plastic blood bag.The day after blood donation,the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate.A week after gene-modified leucoconcentrate therapy,fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury.In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed.In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes.Thus,the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions.This paper presents a proof-of-concept of simple,safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion.The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee(approval No.5)on May 27,2014.

Identification of optimal reference genes in golden Syrian hamster with ethanol-and palmitoleic acid-induced acute pancreatitis using quantitative real-time polymerase chain reaction

Background:Acute pancreatitis(AP)is a severe disorder that leads to high morbidity and mortality.Appropriate reference genes are important for gene analysis in AP.This study sought to study the expression stability of several reference genes in the golden Syrian hamster,a model of AP.Methods:AP was induced in golden Syrian hamster by intraperitoneal injection of ethanol(1.35 g/kg)and palmitoleic acid(2 mg/kg).The expression of candidate genes,including Actb,Gapdh,Eef2,Ywhaz,Rps18,Hprt1,Tubb,Rpl13a,Nono,and B2m,in hamster pancreas at different time points(1,3,6,9,and 24 h)posttreatment was analyzed using quantitative polymerase chain reaction.The expression stability of these genes was calculated using Best Keeper,Comprehensive Delta CT,Norm Finder,and ge Norm algorithms and Ref Finder software.Results:Our results show that the expression of these reference genes fluctuated during AP,of which Ywhaz and Gapdh were the most stable genes,whereas Tubb,Eef2,and Actb were the least stable genes.Furthermore,these genes were used to normalize the expression of TNF-αmessenger ribonucleic acid in inflamed pancreas.Conclusions:In conclusion,Ywhaz and Gapdh were suitable reference genes for gene expression analysis in AP induced in Syrian hamster.

Characterization of SHARPIN knockout Syrian hamsters developed using CRISPR/Cas9 system

Background : SHARPIN (SHANK- associated RH domain interactor) is a component ofthe linear ubiquitination complex that regulates the NF- κB signaling pathway. To betterunderstand the function of SHARPIN, we sought to establish a novel geneticallyengineered Syrian hamster with SHARPIN disruption using the CRISPR/Cas9 system.Methods : A single- guide ribonucleic acid targeting exon 1 of SHARPIN gene was designedand constructed. The zygotes generated by cytoplasmic injection of the Cas9/gRNA ribonucleoprotein were transferred into pseudopregnant hamsters. Neonatalmutants were identified by genotyping. SHARPIN protein expression was detectedusing Western blotting assay. Splenic, mesenteric lymph nodes (MLNs), and thymicweights were measured, and organ coefficients were calculated. Histopathologicalexamination of the spleen, liver, lung, small intestine, and esophagus was performedindependently by a pathologist. The expression of lymphocytic markers and cytokineswas evaluated using reverse transcriptase- quantitative polymerase chain reaction.Results : All the offspring harbored germline- transmitted SHARPIN mutations.Compared with wild- type hamsters, SHARPIN protein was undetectable in SHARPIN −/−hamsters. Spleen enlargement and splenic coefficient elevation were spotted inSHARPIN −/− hamsters, with the descent of MLNs and thymuses. Further, eosinophilinfiltration and structural alteration in spleens, livers, lungs, small intestines, and esophagiwere obvious after the deletion of SHARPIN. Notably, the expression of CD94 and CD22 was downregulated in the spleens of knockout (KO) animals. Nonetheless,the expression of CCR3, CCL11, Il4 , and Il13 was upregulated in the esophagi. Theexpression of NF- κB and phosphorylation of NF- κB and IκB protein significantly diminishedin SHARPIN −/− animals.Conclusions : A novel SHARPIN KO hamster was successfully established using theCRISPR/Cas9 system. Abnormal development of secondary lymphoid organs andeosinophil infiltration in multiple organs reveal its potential in delineating SHARPINfunction and chronic inflammation.

基于肠道菌群探讨粗壮女贞总苷对高脂血症金黄仓鼠的降脂作用机制

目的评价粗壮女贞总苷(total phenylpropanoid glycosides extracted from Ligustrum robustum(Roxb.)Blume,LRTPG)对高脂血症金黄仓鼠的降脂作用并探讨其对肠道菌群的调节作用。方法60只仓鼠随机分为对照组、模型组、阳性药组、LRTPG-L组、LRTPG-M组、LRTPG-H组。高脂饲料诱导造模成功后连续给药4周,进行血脂和肝脂含量检测,并采集对照组、模型组和LRTPG-H组仓鼠结直肠部位成形粪便进行16S rDNA测序。结果LRTPG对高脂血症仓鼠具有降低血清TG、TC、LDL-C水平和肝脏TG、TC含量的作用。肠道菌群测序结果显示,与模型组比较,LRTPG在门水平降低厚壁菌门相对丰度,增加拟杆菌门和疣微菌门相对丰度(P<0.01);科水平上明显升高克里斯滕森菌科、消化球菌科、疣微菌科相对丰度(P<0.05或P<0.01);属水平上明显升高颤螺菌属、颤杆菌克属、解黄酮菌属和阿克曼氏菌属的相对丰度(P<0.05或P<0.01)。这些菌群的变化均有益于LRTPG降脂作用的发挥。结论LRTPG可能通过改善高脂饮食导致的金黄仓鼠肠道菌群紊乱发挥降脂功效。

微小膜壳绦虫感染仓鼠小肠组织病理学观察及MUC2表达的分析

为了解微小膜壳绦虫感染仓鼠小肠组织的病理学特征及黏蛋白2(MUC2蛋白)的表达情况,试验从贵阳市宠物市场随机选取60只宠物仓鼠,采用饱和盐水浮聚法检测仓鼠粪便中的虫卵并进行鉴定,根据仓鼠感染微小膜壳绦虫的情况将仓鼠分为未感染组、无症状感染组、普通型感染组和重型感染组,解剖所有仓鼠获取肠道内寄生虫及小肠组织,制备小肠组织切片并进行苏木精-伊红(H.E.)染色、阿利新蓝-过碘酸-雪夫(AB-PAS)染色及免疫组织化学染色,通过实时荧光定量PCR(qPCR)扩增分析仓鼠小肠MUC2基因相对表达情况。结果表明:微小膜壳绦虫在宠物仓鼠中的感染率为74.5%,其感染可引起仓鼠小肠绒毛损伤、上皮细胞变性坏死、杯状细胞异常、白细胞浸润等病理改变;与未感染组相比,无症状组杯状细胞数量及MUC2基因相对表达量差异不显著(P>0.05),普通型感染组的杯状细胞数量极显著增加(P<0.01),重型感染组的杯状细胞极显著减少(P<0.01),同时重型感染组的MUC2基因及MUC2蛋白表达水平极显著增高(P<0.01)。说明微小膜壳绦虫感染会导致仓鼠小肠绒毛结构破坏,杯状细胞数量及形态异常,并引起杯状细胞分泌MUC2蛋白增多,且重型感染组杯状细胞数量减少,但其分泌的MUC2蛋白量增加。

无镁诱导仓鼠原代皮质神经元电生理学特性

目的无镁细胞外液建立癫痫放电模型,利用全细胞膜片钳技术,检测仓鼠原代皮质神经元的电生理学特性。方法采用生后1~2 d新生叙利亚仓鼠,分离大脑皮质培养原代神经元培养至第12天,分别予有镁细胞外液(有镁组)和无镁细胞外液(无镁组)孵育3 h,3 h后均更换为正常孵育液继续培养24 h。利用全细胞膜片钳技术,电压钳模式下记录神经元兴奋性突触后电流(excitatory postsynaptic currents,EPSC),电流钳模式下记录神经元兴奋性突触后电位(excitatory postsynaptic potentials,EPSP)。结果与有镁组相比,无镁组仓鼠原代皮质神经元EPSC[(124.38±75.15)Hz vs.(33.93±22.32)Hz,P<0.001]及EPSP[(37.05±38.37)Hz vs.(5.63±9.52)Hz,P<0.01]的频率升高,且差异有统计学意义;而两组之间EPSC及EPSP的振幅、曲线下面积和半宽度的差异无统计学意义(P>0.05)。结论无镁处理后仓鼠原代皮质神经元兴奋性升高,仓鼠原代皮质神经元可用于构建癫痫细胞模型。

复方石榴皮药膜对金黄地鼠皮脂腺斑形态学及雄激素受体表达的影响

目的:观察复方石榴皮药膜对金黄地鼠皮脂腺斑大小、组织形态及雄激素受体的影响,探讨其在痤疮治疗中的作用机制。方法:将15只天生具有雄激素依赖性的雄性金黄地鼠随机分为空白组、阳性对照组、药膜组,每组5只。分别于两侧皮脂腺斑处涂抹蒸馏水、0.025%维A酸乳膏、复方石榴皮药膜,连续给药28 d,于第0、14、28天测量金黄地鼠双侧皮脂腺斑面积大小并取部分皮脂腺斑行组织病理检测,免疫组织化学检查雄激素受体(AR)表达水平。结果:药膜组给药第14、28天分别与给药前比较,皮脂腺斑面积减小,差异均有统计学意义(P<0.05)。阳性对照组给药第28天与给药第14天比较,皮脂腺斑面积减小,差异有统计学意义(P<0.05)。给药第28天,药膜组与阳性对照组皮脂腺斑面积大小比较,差异有统计学意义(P<0.05)。给药第28天,与空白组比较,药膜组、阳性对照组皮脂腺斑厚度减小,差异均有统计学意义(P<0.05);药膜组、阳性对照组皮脂腺斑AR表达减少,差异均有统计学意义(P<0.05)。结论:复方石榴皮药膜可减小金黄地鼠痤疮模型皮脂腺斑面积、减轻皮脂腺斑厚度,其作用机理可能与抑制雄激素受体表达有关。

噪声对SPF级金黄地鼠繁殖性能的影响

为了检测噪声对SPF级金黄地鼠繁殖性能的影响,试验首先将48只饲养在≤60 dB(A)噪声环境中的10周龄金黄地鼠随机分为60 dB(A)以下组、60~70 dB(A)组、80~90 dB(A)组、100~110 dB(A)组,每组雄鼠和雌鼠各6只并按1∶1比例合笼直到分娩,每天08:30—11:30在相应的噪声环境中暴露3 h,连续25 d,计算各组妊娠率、单只孕鼠平均产崽数和幼崽5日龄存活率。然后将20只饲养在≤60 dB(A)噪声环境中距分娩还有1~3 d的金黄地鼠随机分为60 dB(A)以下组、60~70 dB(A)组、80~90 dB(A)组、100~110 dB(A)组,每组5只,幼崽从出生到10周龄每天08:30—11:30分别置于相应的噪声环境中暴露3 h,计算幼崽存活率;幼崽10周龄时,摘除眼球采血并采用ELISA法检测血清抗缪勒管激素、抑制素B含量,采用计算机辅助精子分析系统检测雄鼠精子活力和精子的曲线速度、直线速度和平均路径速度,计算睾丸指数和卵巢指数,制作睾丸和卵巢的石蜡切片并进行H.E.染色,观察睾丸和卵巢的组织病理学变化;在每组幼崽中选取非近亲关系的雄鼠和雌鼠各5只,按1∶1比例进行组内交配,计算各组妊娠率、单只孕鼠平均产崽数和幼崽5日龄存活率。结果表明:从雄雌合笼到分娩,各组妊娠率、单只孕鼠平均产崽数和幼崽存活率均差异不显著(P>0.05)。幼崽从出生到10周龄,80~90 dB(A)组、100~110 dB(A)组的幼崽存活率均显著低于60 dB(A)以下组(P<0.05)。幼崽10周龄时,各组抗缪勒管激素含量、抑制素B含量、睾丸指数、卵巢指数、精子的曲线速度、直线速度、平均路径速度、妊娠率、单只孕鼠平均产崽数和幼崽5日龄存活率均差异不显著(P>0.05);100~110 dB(A)组精子活力极显著低于其他组(P<0.01);除60 dB(A)以下组外,其他各组睾丸出现间质稀疏、生精细胞排列紊乱、空泡化等不同程度的病理变化,卵巢中的闭锁卵泡数量增多,80~90 dB(A)组可见次级卵泡和成熟卵泡。幼崽组内交配后,各组妊娠率、单只孕鼠平均产崽数和幼崽5日龄存活率均差异不显著(P>0.05)。说明金黄地鼠从出生开始每天08:30—11:30暴露于噪声环境中3 h,噪声超过80 dB(A)会影响睾丸和卵巢的组织形态结构,噪声超过100 dB(A)会显著降低精子活力。

真孕受体用于金黄仓鼠胚胎移植的方法建立与优化

目的 优化金黄仓鼠胚胎移植技术,探索金黄仓鼠真孕受体最佳移植胚胎数目及发育时期。方法 建立真孕白化金黄仓鼠种群作为胚胎移植受体,比较胚胎发育期、受体减胚及二次移植受体对产仔率、供胚得仔率及后代存活率的影响,确定真孕受体最佳胚胎移植枚数及移植方法。结果 相对于野生型金黄仓鼠作为移植受体,真孕白化受体可快速判定子代来源,并适用于多种以胚胎移植为基础的生殖类实验方案,采用受精卵或二细胞胚胎对移植效果无显著影响(P>0.05);减胚组能显著提高供胚得仔率(22%,P<0.05),二次移植受体的未孕率显著提高(42%,P<0.01);移植6~10枚胚胎的供胚得仔率最高(27%,P<0.01),存活率正常(89%)。结论 6~10枚供体胚胎有助于提高供胚得仔率和存活率,基于白化真孕受体的胚胎移植方法,为金黄仓鼠的应用与开发提供了技术支撑。

近交系自发性2型糖尿病中国地鼠山医群体血液生理生化指标的检测分析

目的对课题组前期建立的近交系自发性2型糖尿病(T2DM)中国地鼠山医群体的血液生理生化指标进行检测并分析。方法选取12月龄近交系自发性T2DM中国地鼠山医群体及正常地鼠各10只,进行空腹采血,应用Sysmex XT自动血液分析仪及Hitachi全自动生化仪对血液15项生理指标及16项生化指标进行测定并进行统计分析。结果与对照组相比,糖尿病组白细胞(WBC)、红细胞(RBC)、血小板(PLT)、血红蛋白(HGB)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、血糖(GLU)、总胆固醇(TC)、甘油三酯(TG)、尿酸(UA)等指标差异具有统计学意义(P<0.05)。结论对课题组前期建立的近交系中国地鼠自发性T2DM模型进行血液生理生化指标检测分析,其变化符合人类T2DM发病的趋势,为该模型的应用及发展提供了基础数据。

高纤维食物对黑线仓鼠哺乳期能量收支的影响

繁殖期是小型哺乳动物最重要的生活史阶段之一,哺乳期是母体能量需求最高的时期。为满足后代的能量需求,母体通常显著增加能量摄入,达到最大持续能量摄入(maximal sustained energy intake,mSusEI)。动物消化道形态和消化机能具有可塑性,然而消化系统是否限制了哺乳期mSusEI,尚不确定。本文以高纤维食物饲喂哺乳期黑线仓鼠(Cricetulus barabensis),通过测定体重、摄食量、摄入能和消化率、代谢率、泌乳能量输出,以及消化系统重量和消化酶活性等,分析哺育不同胎仔数的母体能量摄入与繁殖输出,比较在不同能量需求的条件下,消化酶活性的变化。结果发现,黑线仓鼠哺乳期的能量收支与其哺育后代的数量有关,哺乳期mSusEI未受高纤维食物的显著影响。饲喂高纤维食物未影响摄入能,但显著降低了消化能和消化率,母乳能量输出也显著减少,不能满足后代幼体的能量需求,导致幼体发育变缓。高纤维食物使胃、小肠、大肠和盲肠重量显著增加,小肠淀粉酶、麦芽糖酶和氨基肽酶活性显著增强,但未受胎仔数的显著影响。结果表明,哺乳期mSusEI的瓶颈可能来自消化系统,支持中心限制假说。由于“中心限制”的存在,食物中纤维素含量升高可能会降低动物繁殖价值。

基于位点特异性整合评价CHO细胞表达系统启动子活性的研究

工业生产上中国仓鼠卵巢细胞(CHO)药物蛋白质的表达水平受多种因素影响:调控元件对转录翻译的影响、基因组整合位点的影响以及表达系统的影响等。转录作为基因表达第一步,较大程度影响蛋白质的表达,其中启动子在转录起始发挥至关重要的作用。启动子筛选大部分通过瞬时转染或随机整合,但存在拷贝数不明确或整合位点随机等而无法准确评价启动子活性。位点特异性整合在一定程度上降低位置效应对外源基因造成的影响,并有可能提高外源基因的表达水平。本课题组前期在CHO细胞基因组中发现并验证了多个可稳定表达外源蛋白质的位点,本研究选择其中1个位点(2c6)用于启动子活性的评价。采用CRISPR/Cas9基因编辑技术分别将猿猴病毒早期启动子(SV40)、小鼠延伸因子-1α(mEF-1α)、鸡β-肌动蛋白(cACTB)启动子和人磷酸甘油酸激酶启动子(hPGK)调控的报告基因(EGFP)表达盒定点整合至2c6位点上。通过流式分选仪分析细胞平均荧光强度,qPCR检测EGFP的mRNA水平,综合评价启动子的活性。结果表明,mEF-1α和cACTB启动子的活性优于SV40和hPGK。2次流式分选结果表明,位点特异性整合可以更为精确评价CHO细胞表达系统启动子活性。

不同固定液对光镜下金黄地鼠海马体神经元的影响

目的比较不同固定液对光镜下金黄地鼠海马体神经元的影响,为金黄地鼠海马体神经元固定液的选择提供参考。方法将21只11周龄雄性SPF级金黄地鼠随机分为7组,分别使用4%多聚甲醛、10%中性甲醛、Bouin’s、Carnoy、Davidson’s、Zenker、Helly溶液进行心脏灌注,剥离脑壳取出完整大脑,在对应的固定液中固定48 h,并制作成石蜡切片,经HE染色后在光镜下观察海马体神经元的形态特征。通过匈牙利3DHISTECH公司数字切片扫描系统测量尺测量神经元细胞直径,比较固定液对细胞收缩的影响。结果4%多聚甲醛、10%中性甲醛、Bouin’s溶液展现了良好的固定效果,固定后的大脑硬度适中,HE染色后的切片着色适中、色泽鲜艳、神经元各种细胞形态对比清晰,4%多聚甲醛因固定后切片无龟裂而更为理想。不同固定液固定后的海马体神经元存在细胞收缩方面的极显著差异(P<0.01),使用Carnoy、Davidson’s固定后的神经元各种细胞收缩最严重。结论4%多聚甲醛、10%中性甲醛、Bouin’s溶液是金黄地鼠海马体神经元较为理想的固定液,4%多聚甲醛溶液建议作为首选。

啮齿类动物Playfighting的行为学研究进展

Playfighting是一种常见的社会性互动形式,具有互惠性和合作性等特点。其贯穿于动物的整个生命周期,并且会通过影响动物的社交、认知和运动等进一步影响大脑的执行功能。啮齿类动物的playfighting行为因具有易识别、易量化等特点,而被广泛研究。本文主要以大鼠和金仓鼠为例,整理概述了近年来啮齿类动物playfighting行为的研究现状,包括行为模式及其相关影响因素,希望为今后进一步研究playfighting行为提供参考。

无菌金黄地鼠模型建立

目的使用剖宫产净化(cesarean section purification)方法建立无菌金黄地鼠模型。方法供胎母鼠选择SPF级金黄地鼠,雌雄按1∶1进行交配,见栓后与雄鼠分开饲养。代乳母鼠的合笼时间应早于供胎母鼠一周。怀孕的金黄地鼠临产时在超净工作台内进行子宫摘除术,传入净化用隔离包内,剥离子宫获得待净化乳鼠,分别使用无菌级ICR小鼠和无菌级SD大鼠进行代乳,成功离乳后转入饲养用隔离包内。每月对饲养用隔离包进行无菌状态检测。结果共实施3次剖宫产手术,第1次和第2次代乳失败,第3次代乳成功,获得仔鼠18只,存活率88%,离乳存活率66%,依照GB/T 14926.41-2001进行无菌金黄地鼠状态检测均为合格。结论通过剖宫产净化技术,使用无菌级ICR小鼠和无菌级SD大鼠进行代乳,达到微生物净化目的,获得无菌化金黄地鼠模型。

哺乳动物细胞表达系统研究进展

哺乳动物细胞表达系统具有强大的翻译后修饰功能,表达的重组蛋白更接近人源构象,因此被广泛用于治疗性重组蛋白的生产,其中应用最广泛的是中华仓鼠卵巢细胞。基因组学、转录组学、蛋白质组学以及代谢组学等研究的不断深入促进了哺乳动物细胞表达系统的不断完善。本文立足于哺乳动物细胞表达系统的研究现状,对表达载体的优化、宿主细胞和位点特异性重组等方面的最新进展进行综述。

基于非标记分析方法应用四极杆-静电场轨道阱-线性离子阱三合一高分辨质谱仪鉴定中国仓鼠卵巢细胞膜蛋白

目的 探讨应用四极杆-静电场轨道阱-线性离子阱(Orbitrap fusion lumos)三合一高分辨质谱仪鉴定中国仓鼠卵巢(Chinese hamster overy,CHO)细胞的全蛋白质组和膜蛋白质组的非标记(label-free)分析方法,并评价其应用效果,为研究膜蛋白相关传染病的发生机制提供方法学基础。方法 贴壁培养中国仓鼠卵巢细胞,提取、富集和酶解处于对数生长期细胞的全蛋白和膜蛋白,用高效液相色谱仪联用四极杆-静电场轨道阱-线性离子阱三合一高分辨质谱仪对酶解后的多肽进行分析,利用Maxquant软件和Uniprot数据库进行搜库及鉴定,最后对鉴定膜蛋白进行基因本体论(gene ontology,GO)功能分类。结果 全蛋白组一共检测出14 300个肽段,搜库得到2708个蛋白,其中可信蛋白2002个(FDR<1%且特异性肽段>1);膜蛋白组一共检测出13 179个肽段,搜库得到2594个蛋白,其中可信蛋白1873个。在鉴定的全蛋白组和膜蛋白组中定量结果前20的蛋白中,全蛋白组中与膜相关的蛋白有4个,占比20%;膜蛋白组中与膜相关的蛋白有10个,占比50%。在膜蛋白组中,GO功能分类表明分子功能上主要涉及结合和催化活性,在生物学进程上主要涉及细胞进程、代谢过程等。结论 膜蛋白组中与膜相关的蛋白显著高于本实验的全蛋白组,基于非标记分析方法应用Orbitrap fusion lumos的分析技术是鉴定细胞膜蛋白的有效方法。

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.