目的 分析2017～2021年陕西省部分实验动物使用单位无特定病原体(specific pathogen free,SPF)级大、小鼠微生物和寄生虫质量检测结果,了解各单位大、小鼠病原微生物学质量状况,为实验动物质量管理与监测提供参考依据.方法 按照现行国家标准GB 14922.2-2011、GB/T 14926-2001和GB/T 14926.21-2008,由陕西省实验动物质量监督检测中心对大、小鼠进行病原微生物和寄生虫抽样检测并分析质量状况.结果 2017年～2021年累计抽检109家单位的小鼠2549只,45家单位的大鼠503只.检测结果显示,小鼠体外寄生虫(螨虫)检出率由5.26％降为0.15％,肠道鞭毛虫由10.53％降为0.45％,肠道蠕虫由0.40％升至2.39％,沙门菌由2.78％降至无检出,泰泽病原体抗体阳性率由1.38％降至无检出;大鼠体外寄生虫(螨虫)检出率由7.07％降至无检出;肠道鞭毛虫由11.50％降为0.83％,蠕虫由3.79％升至7.50％,金黄色葡萄球菌由1.77％降至无检出.小鼠病毒血清抗体阳性率,2017年检出小鼠肝炎病毒(4.71％)、仙台病毒(1.39％)和小鼠细小病毒(2.22％),2021年出小鼠肝炎病毒(0.30％)和仙台病毒(0.15％),小鼠肺炎病毒阳性率则由1.11％升至2.39％,小鼠呼肠孤病毒Ⅲ型由0.17％升至0.45％.大鼠仙台病毒阳性率由7.07％降为0.83％,大鼠细小病毒和大鼠冠状病毒仅2017年和2018年在个别单位有检出,大鼠肺炎病毒则由0.88％增长为10.00％.结论 近5年的检测结果表明,陕西省实验动物使用单位的大、小鼠病原微生物质量逐年提升,但仍存在一定问题,应持续采取严格的生物安全防控措施,加强动物实验过程,确保各单位在实验周期内动物质量合格.

目的 研究七甲川菁(heptamethine carbocyanine)近红外(near-infrared fluorescence,NIRF)荧光染料与醋酸阿比特龙(abiraterone acetate,ABi)合成化合物(DZ1-ABi和783-ABi)对前列腺癌的治疗及其在活体成像中的应用.方法 化学合成两种化合物DZ1-ABi和783-ABi;将人前列腺癌细胞PC-3、LNcaP和C4-2培养至对数生长期后分别加入DZ1-ABi、783-ABi和ABi,观察三种化合物对肿瘤细胞增殖的抑制作用;将人源性前列腺癌PDX肿瘤(B45354)皮下移植裸鼠随机分组给予DZ1-ABi、783-ABi和ABi治疗并监测肿瘤生长状况.荷瘤鼠分别注射DZ1-ABi和783-ABi(0.1 nmol/只),活体成像监测两种化合物在小鼠体内的代谢情况和肿瘤靶向性.结果 相较于ABi和783-ABi,DZ1-ABi能更好地抑制人前列腺癌细胞PC-3、LNcaP和C4-2的增殖;在荷瘤鼠药敏实验发现,DZ1-ABi表现出较783-ABi和ABi更好的治疗效果;活体成像显示,无论是肿瘤的靶向性还是荧光信号强度,DZ1-ABi均表现出比783-ABi更为明显的优势.结论 DZ1-ABi相较于783-ABi和临床药物ABi在对肿瘤的抑制和活体成像中均表现出比较明显的优势,有望成为新型的肿瘤显像和治疗双重靶向药物.

目的 研究人胃癌转移裸鼠模型中人Alu基因的表达水平与组织脏器中胃癌转移程度的相关性,探索建立胃癌转移模型早期评估的分子生物学方法.方法 分别以人胃癌细胞SGC-7901、MKN45和正常胃黏膜细胞GES1基因组DNA为模板构建标准质粒,通过实时荧光定量PCR检测不同比例胃癌细胞SGC-7901中Alu基因的表达,获取相关性曲线;选取胃癌细胞SGC-7901和MKN45,通过裸鼠皮下接种的方式构建人胃癌细胞异种移植转移模型,实时荧光定量PCR方法检测模型鼠肝、脾、肺、肾和皮下肿瘤组织中的人Alu基因表达,获得Alu基因的表达与各器官组织中肿瘤转移程度的相关性曲线;将临床胃癌患者新鲜肿瘤标本皮下接种裸鼠构建异种移植模型,进一步验证模型鼠各组织中人Alu基因的表达与器官中肿瘤转移程度的相关性.结果 胃癌细胞SGC-7901的含量与其Alu基因的Ct值呈负相关(R2=0.9239);人胃癌细胞异种移植(CDX)裸鼠转移模型中,人Alu基因的表达在皮下肿瘤中处于较高水平,在肺转移瘤和肝转移瘤中的表达则介于正常裸鼠与皮下肿瘤之间,与组织病理学检查结果相符;胃癌病人肿瘤异种移植(PDX)模型中,已确定发生转移的脏器中虽未形成肉眼可见的转移灶,但其人Alu基因的表达(Ct值17.86)与正常裸鼠(Ct值22.18)差异显著(P<0.05);而对于肉眼可见的转移灶,其人Alu基因的表达(Ct值14.29)则差异极显著(P<0.01).结论 人胃癌裸鼠转移模型中,人Alu基因的表达与胃癌转移程度呈正相关,Alu基因表达越高,则该组织中转移的肿瘤细胞就越多,形成的转移灶越明显.

S-trans-trans-Farnesylthiosalicylic acid (FTS) is a Ras inhibitor that exhibits desirable anticancer property and currently undergoing clinical trials for pancreatic cancer (PC). However, its poor water solubility and low bioavailability have severely hampered clinical applications. A strategy to improve FTS bioavailability is to develop a suitable drug delivery method. Here, we use a near-infrared fluorescence (NIRF) heptamethine carbocyanine (HC) dye conjugated with FTS (to produce FTS-148) as a drug delivery system to enhance FTS bioavailability. We further investigate its tumor-targeting functions. FTS-148 displayed better bioavailability and photophysical property and selective recognition of cancer cells. FTS-148 significantly reduced PC cell proliferation, and more effective than FTS in restricting tumor growth both in a cell-derived xenograft (CDX) model and a patient-derived tumor xenograft (PDX) model. FTS-148 can specifically recognize PC cells in mice subcutaneous models or rabbit orthotopic models and allows real-time monitoring of the therapeutic effects by NIRF optical imaging. FTS-148 treatment significantly reduced Ras expression in PC cells and increased tumor tissue apoptosis. In short, FTS conjugated with HC dye had enhanced bioavailability and tumor-targeting property. It provides a potential agent for imaging and therapy of PC.

新冠疫情下开展实验动物学的线上教学是对传统教学模式的有效补充和完善,需要熟悉网络软件的操作,熟练运用网络实现授课意识的转变;教员应保持授课思路的连贯性,重点关注PPT的讲述,充分利用现有的慕课资源展示动物实验操作,完成实践课教学,教会学员在网络环境中如何有效掌握授课内容;同时在授课中应及时增加与新冠病毒相关的内容,激发学员的学习兴趣;完善课程学业考核方式,积极推进实验动物学教学改革,不断提升实验动物学网上教学效果.

目的 建立小鼠肝炎病毒(Mouse Hepatitis Virus,MHV)血清抗体ELISA检测方法,用于本地区MHV的日常监测,以便对SPF小鼠感染状况及时作出判断.方法 选取3种MHV毒株MHV1、MHV-JHM和MHV-A59,通过L929细胞扩增培养获得纯化浓缩抗原并筛选适合包被抗原类型;免疫BALB/C小鼠,获得高效价免疫阳性血清;优化ELISA反应体系建立规范化的ELISA试剂盒并用于临床小鼠血清样品的检测.结果 筛选出适合本地区的MHV包被抗原类型为MHV1,建立了病毒扩增及浓缩纯化方法,制备的MHV抗血清达到同批次大量高滴度的水平,可作为标准化质控血清.包被抗原、待检血清和酶结合物最佳工作浓度分别为4.0 μg/mL(10-7.73/0.1 mL TCID50)、1∶40和1∶4 000稀释;批次内和批次间平均变异系数分别为5.13％和5.57％;检测灵敏度为1∶4 000稀释;与小鼠仙台病毒(SV)、小鼠肺炎病毒(PVM)、呼肠孤病毒Ⅲ型(Re03)、小鼠细小病毒(MVM)和鼠痘病毒(Ect)阳性血清均无交叉反应.稳定性试验相对偏差小于10％.对165份血清样品进行检测,阳性血清相符率97.37％ (37/38),阴性血清相符率92.19％ (118/127).结论 本研究建立的ELISA方法检测小鼠血清MHV抗体具有较高的特异性、敏感性和结果可重复性,可以用于本地区MHV日常病原学监测,以便对小鼠感染状况作出准确判断.

目的 建立前列腺癌人源性异种移植(patient-derived xenograft,PDX)模型,评价不同治疗方案的抗肿瘤效果.方法 将人新鲜的前列腺癌手术标本与基质胶混合后移植入补充有外源性雄激素的裸鼠皮下,连续监测肿瘤生长,评估其保真度并连续传代;将荷瘤鼠分为四组:多西他赛组、去势组、多西他赛联合去势组以及对照组,治疗期间测量肿瘤体积及小鼠体重变化,治疗结束后,检测血清中总前列腺特异性抗原(total prostate specific antigen,tPSA)浓度及组织病理学变化,评估治疗效果.结果 成功建立了前列腺癌PDX模型,包括激素敏感型(D17225)和去势抵抗型(C40019)肿瘤,病理学分析发现移植瘤较好的保持了患者原发瘤的主要特征;病理组织学及血清tPSA检测发现多西他赛组及多西他赛联合去势组在D17225模型中显示出良好的治疗效果,且后者抑瘤效果更为明显.结论 成功建立了前列腺癌PDX模型并稳定传代,多西他赛单药或联合去势处理对激素敏感型(D17225)前列腺癌PDX模型具有显著治疗效果.

目的 利用双重sgRNAs构建miR-223全基因敲除小鼠.方法 针对miR-223基因设计双重sgRNAs,将体外转录的sgRNAs和Cas9 mRNA共同显微注射入C57BL/6小鼠受精卵细胞.小鼠出生后取其基因组DNA进行PCR扩增和测序以鉴定基因型,同时取小鼠肝脏研磨后提取总RNA,通过real-time PCR分析miR-223在肝脏中的表达.结果 设计了miR-223基因双重sgRNAs并对其进行了体外转录,纯化后显微注射小鼠受精卵细胞获得miR-223基因突变小鼠.测序结果表明突变小鼠有3种基因型,一种为6 bp的缺失突变,但未对miR-223序列产生影响;另外两种为162 bp和168 bp的缺失突变,完全删除miR-223前体和成熟区序列.与野生型相比,这两种小鼠肝组织中几乎不能检测到miR-223的表达.结论 设计双重sgRNAs并应用CRISPR/Cas9技术成功构建miR-223全基因敲除小鼠.

目的 初步评估七甲川菁染料衍生物(IR-783)和化疗药物吉西他滨(gemcitabine)的缀合物(NIRG)对肝癌模型荧光成像及靶向治疗效果.方法 首先使用肝癌细胞系Hep3B验证肝癌细胞对NIRG的特异性吸收,并制备该细胞的裸鼠皮下移植瘤模型,观察NIRG在CDX模型中的近红外荧光成像效果;制备三例肝癌(C64003、C34566和B66873)裸鼠皮下PDX模型,研究NIRG在此模型中的肿瘤靶向荧光成像效果以及治疗效果,同时通过免疫组织化学法观察肝癌特异性抗原AFP在三例肝癌PDX模型的肿瘤组织中的表达,以及观察与NIRF染料特异性吸收相关的抗原HIF1α和OATP的表达.结果 通过肝癌细胞系Hep3B观察到NIRG在肿瘤细胞内结合的部位分别为线粒体和溶酶体,在肝癌CDX皮下模型中发现NIRG特异性聚集肿瘤部位,随着肿瘤的生长NIRF信号增强,并与生物发光信号具有良好的相关性(R2=0.99716);免疫组织化学检测显示在三例肝癌PDX模型中的AFP、HIF1α和OATP均高表达;NIRG在三例肝癌PDX模型中荧光成像结果表明具有很好的肿瘤靶向性,同时能够抑制肿瘤的生长,显示了良好的治疗效果.结论 化合物NIRG兼具荧光成像和肿瘤靶向转运化疗药物的能力,可作为人类肝肿瘤的新型影像探针和靶向制剂.

Mammalian target of rapamycin (mTOR) kinase is vital to the regulation of cell growth and proliferation, and it has been taken as a promising target to develop cancer therapies. By reference to the crystal structure of mTOR-PP242, we explored to discover potential ATP-competitive inhibitors of mTOR. Through the integrated use of multiple in silico screenings, the tremendous amount of compounds from the SPECS database were finally reduced to 30. After several rounds of convincing biological tests in A549 cells, the newfound C-4 was identified as a potential ATP-competitive inhibitor of mTOR. Besides A549 cell proliferation suppression caused by C-4, autophagy was also determined through autophagosome observation and autophagy flux detection in C-4 treated A549 cells. We demonstrated that C-4 could inhibit cell growth and proliferation, and this inhibition may be associated with autophagy.

目的 建立基于临床肝癌手术标本的原位移植(patient-derived orthotopic xenograft PDOX)模型,研究近红外荧光(Near-infrared fluorescence,NIRF)活体成像和PET/CT在模型评估中的应用.方法 将临床新鲜肝癌手术标本接种于重度免疫缺陷NPG小鼠皮下建立肝癌PDX模型,通过组织形态观察、STR分型检测和免疫组化分析对PDX模型进行评估.进一步将PDX模型肿瘤组织进行裸鼠肝原位移植建立PDOX模型,注射近红外荧光染料IR-783,通过小动物活体成像检测肿瘤的发生;尾静脉注射18F-FDG,通过小动物PET/CT观察确认肝原位肿瘤的生长.结果 STR分型结果表明PDX肿瘤的人源性特征,组织形态观察和免疫组化检测表明PDX肿瘤保持了原发肿瘤病理学特征;近红外荧光活体成像检测到肝脏肿瘤的发生;PET/CT可清晰观察到小鼠肝脏部位18F-FDG分子探针富集.结论 成功建立了肝癌PDOX模型,通过小动物活体成像和PET/CT影像技术可对该模型进行评估,为肝癌的治疗和发病机制研究提供了良好的动物模型.

目的 观察Hedgehog通路抑制剂GANT61对人乳腺癌MCF-7细胞的抑制作用.方法 GANT61作用MCF-7细胞后,通过流式细胞术检测细胞死亡;提取处理细胞总RNA和蛋白.分别采用Real-time PCR和Westem blot检测Hedgehog通路SHH、Gli1、Gli2及自噬标志物LC3-Ⅱ表达;处理细胞固定后通过电子显微镜观察GANT61诱导的自噬体形态;自噬抑制剂3-MA处理细胞或自噬相关基因atg5 siRNA转染细胞后再用GANT61作用,流式细胞术检测细胞死亡数量.结果 GANT61能明显诱导MCF-7死亡,降低Hedgeho g通路SHH、Gli1、Gli2表达水平,提高LC3-Ⅱ蛋白表达,并可诱导MCF-7细胞产生自噬体.3-MA及atg5 siRNA可减弱GANT61诱导的细胞死亡.结论 GANT61通过诱导自噬性细胞死亡机制发挥抗乳腺癌细胞活性.

Near-infrared fluorescence (NIRF) dyes have recently emerged as promising tools for non-invasive imaging of different types of cancers. Here, we explored the potential utility of a NIRF DZ-1 dye, with dual imaging and tumour targeting functions, in hepatocellular carcinoma (HCC). We showed the preferential uptake of DZ-1 by HCC cells in vitro and in derived subcutaneous/orthotopic tumour xenografts, accompanied by a minimal effect on normal cells. DZ-1 simplified tumour growth profiling as well, since we were able to correlate NIRF signals with tumour volume and/or tumour-emitting luminescence in mice. Using both orthotopic tumour transplantation and cirrhosis models in parallel, we demonstrated the ability of DZ-1 to differentiate liver tumour from cirrhosis. DZ-1 showed superiority in HCC imaging over indocyanine green by demonstrating significantly enhanced tumour-targeting specificity. At the cellular level, DZ-1 was mainly retained in mitochondria and lysosomes. Additionally, DZ-1 fluorescence spectroscopy has been used for the intraoperative navigation of rabbit liver cancer, to determine surgical margins. We showed that tumor hypoxia and select organic anion-transporting polypeptide genes mediate NIRF dye uptake in HCC, which was supported by clinical evidence. All these findings represent the first evidence that DZ-1 is an effective molecular probe for tumour-specific imaging in HCC, and provide insights into the development of a new generation of imaging agents for intraoperative guidance of cancer surgery.

构建及鉴定卡介苗(BCG) Hsp16.3基因突变株,观察该突变株对小鼠巨噬细胞自噬功能的影响.通过聚合酶链反应(PCR)的方法扩增Hsp16.3基因两侧分子量分别为663 bp和684 bp的两个DNA片段,并将这两个目的片段分别插入pKO载体上相应的位点.通过硫酸卡那霉素筛选,利用双酶切以及测序的方法,获得阳性重组质粒pKO-Hsp16.3.将阳性重组质粒pKO-Hsp16.3电转入BCG感受态细胞中,经过硫酸卡那霉素和蔗糖两次筛选,得到BCG Hsp16.3基因突变株.将筛选培养的BCG Hsp16.3突变株用于小鼠巨噬细胞的感染实验,并通过Western Blot和细胞免疫荧光技术对自噬相关蛋白的表达水平的变化进行观察及分析.成功获得重组质粒pKO-Hsp16.3的阳性克隆;电转化该重组质粒,并经过硫酸卡那霉素筛选和蔗糖反筛选,成功得到BCG Hsp16.3基因突变株.通过Western Blot以及免疫荧光分析发现,BCG Hsp16.3基因的突变株促进了小鼠巨噬细胞自噬的发生.

目的 研究高能氙光传递窗对空气中和物品表面细菌的消毒效果.方法 分别用标准菌株金黄色葡萄球菌、枯草芽孢杆菌和大肠埃希菌制备细菌悬液并涂布血平板,模拟物品表面细菌;用普通营养琼脂平板培养空气落下菌.将两种平板经氙光传递窗和紫外线分别照射3 min后,与对照组均放置于37℃恒温培养箱24-48 h,计算杀菌率.结果 涂布于血平板的细菌悬液,经氙光照射3 min后,平均杀菌率达到93.2％;空气落下菌经氙光照射3 min后,平均杀菌率达到99.1％.两种平板经紫外线照射3 min后的杀菌率不到50％.结论 氙光传递窗能够高效、快速杀灭物品表面和空气中的细菌,平均杀菌率达到95％以上,氙光传递窗有可能成为常规传递窗的替代品.

According to relevant national laws and regulations, practitioner training was included into laboratory animal science teaching reform.By adjusting the training content and teaching method and use of animal models of typical human diseases, the transformation of training mode was realized and improved.By the assessment of basic theory in combination with practical operation, the thinking ability and hands-on skill of the practitioners are much improved. Through classroom instruction, experimental teaching, quality assessment and tracking survey, the evaluating process of the training quality of training teaching is performed.Therefore, the teaching reform of the laboratory animal science based on the training of practitioners is established.

Objective To knockout Rag2 and IL2rg genes and construct severe combined immunodeficiency mice based on CRISPR/Cas9 technology. Method Design and synthesis of 25 bp sgRNA were made according to the Rag2 and IL2rg sequences in Genbank. After annealing, sgRNA was cloned into pX330 vector. Recombination plasmid Rag2?sgRNA, IL2rg?sgRN and Cas9 were then transcribed into RNA, these RNA were microinjected into zygotes and the zygotes were transplanted into recipient ICR mice. F0 founders were born and mutated F0 founders mated with wild type mice to obtain F1 generation heterozygous mice. Mutated F1 mice were crossed and got F2 generation homozygous mice. Genotype and phenotype of the knockout mice were identified by sequencing, flow cytometry and xenograft model. Results Rag2?sgRNA and IL2rg?sgRNA recombination plasmids were constructed and transcribed into RNA. After microinjection and mat? ing, F0 founders were born and F2 homozygous mice were obtained. The results of sequencing showed that there were two types of genotype in IL2rg gene, 10 bp or 11 bp deletion;however, there was only one genotype in Rag2 gene, which was 8 bp deletion. Compared with wild?type BALB/c mice, the number of CD3 +, B220 + and NKp46 + cells in peripheral blood of the knockout mice was reduced significantly. After inoculation of human breast cancer cell line SKBR?2HL cells, tumor size in the xenograft mouse model was increased gradually along with time extension. Conclusion CRISPR/Cas9 is an efficient way to mutate Rag2 and IL2rg gene in mice in vivo, leading to aberrant T cells, B cells and NK cells.

目的 建立RAG2/IL2 RG双基因缺陷的CRG小鼠杂交群体.方法 将RAG2基因缺陷小鼠与IL2RG基因缺陷小鼠分别进行繁殖,选取RAG2基因缺陷雄鼠RAG2(-/-)与IL2RG基因缺陷雌鼠IL2RG(-/-)进行配对,培育杂交后代,通过PCR扩增基因组RAG2和IL2RG基因进行鉴定;应用流式细胞仪分析检测RAG2/IL2RG双基因缺陷小鼠外周血T细胞(CD3+)、B细胞(CD19+)和NK细胞(CD49b+)含量;并测定8-12周龄RAG2/IL2 RG双基因缺陷小鼠血液生理生化及主要脏器重量指标.结果 成功繁育RAG2基因缺陷小鼠和IL2 RG基因缺陷小鼠,筛选获得稳定的RAG2/IL2 RG双基因缺陷小鼠并成功保种和扩群;该双基因缺陷小鼠外周血淋巴细胞中T细胞、B细胞,NK细胞比例显著降低(P＜0.05);与相同周龄野生型C57B L/6相比9项血清生化指标无明显变化(P＞0.05);18项血液生理指标中红细胞(RBC)和血红蛋白(HGB)含量显著降低(P＜0.05);白细胞(WBC)、淋巴细胞数(LYMPH)、淋巴细胞比率(LYM)、单核细胞、中性细胞数(NEUT)降低极显著(P＜0.01);脾脏重量显著均低于野生型C57B L/6小鼠(P＜0.05);人肝肿瘤传代细胞移植于RAG2/IL2RG双基因缺陷小鼠后,肿瘤移植成功率和生长速率均明显高于亲本单基因缺陷小鼠(P＜0.05),成功获得了临床肝癌病人肿瘤组织的异种移植模型.结论 成功构建筛选出RAG2/IL2RG双基因缺陷小鼠杂交群体.

Near-infrared fluorescence (NIRF) imaging agents are promising tools for noninvasive cancer imaging. Here, we explored the tumor-specific targeting ability of NIRF heptamethine carbocyanine MHI-148 dye in cultured gastric cancer cells, gastric cancer cell-derived and patient-derived tumor xenograft (PDX) models. We show that the NIRF dye specifically accumulated in tumor regions of both xenograft models, suggesting the potential utility of the dye for tumor-specific imaging and targeting in gastric cancer. We also demonstrated significant correlations between NIRF signal intensity and tumor volume in PDX models. Mechanistically, the higher cellular uptake of MHI-148 in gastric cancer cells than in normal cells was stimulated by hypoxia and activation of a group of organic anion-transporting polypeptide (OATP) genes. Importantly, this NIRF dye was not retained in inflammatory stomach tissues induced by gastric ulcer in mice. In addition, fresh clinical gastric tumor specimens, when perfused with NIR dye, exhibited increased uptake of NIR dye in situ. Together, these results show the possibility of using NIRF dyes as novel candidate agents for clinical imaging and detection of gastric cancer.