Hr突变体转基因小鼠的构建和表型分析

无毛基因（Hr）定位于8p12,在染色体上跨越14 kb,包含19个外显子。无毛基因的自发突变能引起人和动物毛发脱落及相关毛发疾病的产生。为深入研究Hr基因的功能,本文利用Gateway技术构建Hr表达载体,在该基因的3 427位引入点突变(G→A),通过显微注射建立转基因小鼠。采用PCR方法鉴定出阳性的转基因小鼠,确定首建鼠,通过与C57BL/6小鼠回交后互交数代建系。观察转基因小鼠毛发生长发育规律。结果表明,成功构建了pRP(Exp)-EF1A>mHairless mutant>IRES/EGFP真核表达载体,通过与野生型小鼠杂交获得阳性子代,进行同窝交配,第2代小鼠出生后14 d开始脱发,30 d左右脱落的毛发重新长出。取部分皮肤组织做石蜡切片,皮肤组织学观察发现,脱毛期无毛小鼠毛囊瓦解,真皮内形成大小不等的包囊,毛发重新生长时,真皮内见大量新生的毛囊。蛋白印迹实验表明,转基因小鼠脱发时HR蛋白表达量明显高于同龄阴性小鼠。本文成功建立稳定遗传的Hr突变的转基因小鼠品系,推测无毛基因突变引发转基因小鼠的脱发,为研究Hr基因的功能提供了良好的动物模型。     

Zygosity Determination in Hairless Mice by PCR Based on Hrhr Gene Analysis

We analyzed the Hr gene of a hairless mouse strain of unknown origin (HR strain, http://animal.nibio.go.jp/e_hr.html) to determine whether the strain shares a mutation with other hairless strains, such as HRS/J and Skh:HR-1, both of which have an Hr^hr allele. Using PCR with multiple pairs of primers designed to amplify multiple overlapping regions covering the entire Hr gene, we found an insertion mutation in intron 6 of mutant Hr genes in HR mice. The DNA sequence flanking the mutation indicated that the mutation in HR mice was the same as that of Hr^hr in the HRS/J strain. Based on the sequence, we developed a genotyping method using PCR to determine zygosities. Three primers were designed: S776 (GGTCTCGCTGGTCCTTGA), S607 (TCTGGAACCAGAGTGACAGACAGCTA), and R850 (TGGGCCACCATGGCCAGATTTAACACA). The S776 and R850 primers detected the Hr^hr allele (275-bp amplicon), and S607 and R850 identified the wild-type Hr allele (244-bp amplicon). Applying PCR using these three primers, we confirmed that it is possible to differentiate among homozygous Hr^hr (longer amplicons only), homozygous wild-type Hr(shorter amplicons only), and heterozygous (both amplicons) in HR and Hos:HR-1 mice. Our genomic analysis indicated that the HR, HRS/J, and Hos:HR-1 strains, and possibly Skh:HR-1 (an ancestor of Hos:HR-1) strain share the same Hr^hr gene mutation. Our genotyping method will facilitate further research using hairless mice, and especially immature mice, because pups can be genotyped before their phenotype (hair coat loss) appears at about 2 weeks of age.     

胚胎移植技术对特种突变小鼠的净化研究

目的通过净化特种突变小鼠来建立SPF级种子库。方法利用胚胎移植技术对四种突变无毛小鼠和一种白内障小鼠的桑葚胚进行子宫内移植。结果四种突变无毛小鼠和白内障小鼠均产出仔鼠,获得移植成功,其中白内障小鼠的产仔率最高为25.5%。结论建立了SPF级特种突变小鼠种群,为进一步研究与开发利用提供了可靠保证。     

豫医无毛小鼠分离近交系的建立及其遗传纯度测定

采用强迫杂合性史妹酱方式培育携带无毛突变基因的分离近交系,然后用生化标记法,皮肤移植实验和毛色基因测试法对其进行遗传监测。并对其基本生物学特性进行了研究。结果育成了具有独特生物学特性的豫医无毛小鼠分离近交系,现已达30代,生化标记法测定的9条染色体上13个生化标记位点全部纯合;同系异体间皮肤移植100天后,未见排斥现象,为同系组织遗传性;与DBA/2交配进行的毛色基因测试,杂交的F1代相同,全部为野生色,基因型为AABBccDD ,表明豫医无毛小鼠已成为一个达到国际标准的新品系。     

MRSA气溶胶诱导烫伤SKH-1无毛小鼠皮肤感染合并肺炎模型

目的探讨烫伤条件下,建立MRSA气溶胶致SKH-1无毛小鼠皮肤感染合并肺炎模型的方法及其评价指标。方法 48只SKH-1无毛小鼠,随机分成PBS对照组,单纯感染组和烫伤合并感染组。合并感染组小鼠皮肤烫伤后,MRSA（ST-239）气溶胶呼吸道感染,采用体重、血常规、菌血症率、皮肤和脏器荷菌量、病理组织学变化和分子生物学等指标对模型进行综合评价。结果与其他两组相比,合并感染组小鼠第3天体重下降明显;白细胞数显著增多;菌血症率达100%;皮肤和肺荷菌量较高,分别达108CFU/g和106CFU/g;镜下观察皮肤和肺炎症病变明显;免疫组化结果显示皮肤组织内CD138呈阳性表达;multi-PCR测定感染组织内nuc和mecA基因均为阳性。结论 SKH-1无毛小鼠烫伤再辅以MRSA气雾攻击的方式,可成功建立能够模拟临床上烧烫伤患者继发MRSA肺炎的动物模型,此模型对于抗MRSA药物药效评价研究具有重要临床应用价值。     

Protection by zinc against UVA— and UVB-induced cellular and genomic damage in vivo and in vitro

For many years, zinc salts have been used both topically and orally to treat minor burns and abrasions as well as to enhance wound repair in man and animals. In this study we describe the protective effects of zinc against UV-induced genotoxicity in vitro and against sunburn cell formation in mouse skin in vivo. Cultured skin cells from neonatal mice showed a dramatic increase in the number of micronuclei as a result of UVA and UVB irradiation. Inclusion of zinc at 5 μg/mL in the medium significantly reduced the frequency of micronuclei and of micronucleated cells. In hairless mice, topical application of zinc chloride for 5 consecutive days or a single application 2 h prior to UV exposure reduced the number of sunburn cells in the epidermis as did application of zinc 1 h after exposure. Application 2 h after irradiation also tended to have a protective effect, although there was a large variation between animals. It is proposed that an influx of zinc can protect epidermal cells against some of the more delayed effects of UV-induced damage.     

耐甲氧西林金黄色葡萄球菌诱导SKH-1无毛小鼠菌血症模型

目的建立SKH-1无毛小鼠感染MRSA致菌血症模型的方法及其评价体系。方法 62只SPF级SKH-1无毛小鼠随机分成PBS对照组、低剂量模型组、高剂量模型组及给药组,尾静脉注射无菌PBS至对照组小鼠体内,其余各组小鼠尾静脉注射相应剂量MRSA(ST-239)菌悬液,从体重、死亡率、血常规、血液细菌浓度、主要器官荷菌量及病理改变等方面对模型进行评价,并用抗生素替考拉宁对模型进行检验。结果各组感染小鼠体重均下降明显,血常规指标改变明显;高剂量组死亡率高,血液中细菌浓度较高,肝、肾等多组织器官播散严重,皮肤可见多发性脓肿灶;使用替考拉宁能显著降低小鼠死亡率,减轻主要器官的损伤程度。结论应用MRSA菌株可成功诱导建立SKH-1无毛小鼠菌血症模型,模型具有病理特征明显及观察简便的优点,为相关药物评价研究提供了一个理想的动物模型。     

The role of mannose-6-phosphate receptor and autophagy in influencing the outcome of combination therapy

Combining two different treatment modalities for targeting malignancies is gaining importance, with preclinical/clinical results indicating higher success rates in eradicating tumors or having longer remission periods. A better understanding of the synergy between the treatments helps in optimizing the dose and time of administration. We found that chemotherapy enhanced the levels of insulin-like growth factor 2 receptor/cation-independent mannose-6-phosphate receptor (IGF2R) on the surface of tumor cells, which leads to better tumor targeting by cytotoxic T cells (CTLs). Early evidence indicates that upregulation of IGF2R involves the autophagy pathway.     

Focus: A Multifaceted Battle Against Cancer: Evolution of Multidisciplinary Brain Metastasis Management: Case Study and Literature Review

Up to 40 percent of all cancer patients develop brain metastasis (BM) during the course of their disease. Despite advances in diagnosis and therapy, prognosis in patients with BM remains poor for many patients, but for some, survival can be of the order of several years in duration. Difficulty in predicting long-term survivors has created controversy in contemporary management of BM. Minimizing medical and neurocognitive side effects (disease borne or iatrogenic) to enhance functional independence and improving overall quality of life in these individuals requires a coordinated approach of first-line and salvage surgical, chemotherapeutic (cytotoxic, targeted, or immune based), and radiation (whole brain radiotherapy or stereotactic radiosurgery) modalities. This goal needs to be balanced against the more traditional targets of management such as symptom relief, reducing tumor burden, and local tumor control, thereby increasing progression-free survival. This case study and literature review demonstrates the role of various treatment modalities in the management of BM.     

Passive immunization targeting pathological phospho-tau protein in a mouse model reduces functional decline and clears tau aggregates from the brain

Targeting hyperphosphorylated tau by immunotherapy is emerging as a promising approach to treat tauopathies such as Alzheimer's disease and frontotemporal dementia. We have previously reported that active tau immunization clears tau aggregates from the brain and attenuates or prevents functional impairments in two different tangle mouse models. Here, we assessed the efficacy of passive immunization with the PHF1 antibody, which targets a phospho-epitope within one of our active immunogens. Homozygous female tangle mice (JNPL3, 2-3 months) were injected intraperitoneally once per week with PHF1 or pooled mouse IgG (250 μg/125 μL; n = 10 per group) for a total of 13 injections. Their behavior was assessed at 5-6 months of age and brain tissue was subsequently harvested for analyses of treatment efficacy. The treated mice performed better than controls on the traverse beam task (p < 0.03), and had 58% less tau pathology in the dentate gyrus of the hippocampus (p = 0.02). As assessed by western blots, the antibody therapy reduced the levels of insoluble pathological tau by 14-27% (PHF1, p < 0.05; PHF1/total tau, p < 0.0001) and 34-45% (CP13 or CP13/total tau, p < 0.05). Levels of soluble tau and sarkosyl soluble tau were unchanged, compared with controls, as well as total tau levels in all the fractions. Plasma levels of PHF1 correlated inversely with tau pathology in the brainstem (p < 0.01), with a strong trend in the motor cortex (p < 0.06) as well as with insoluble total tau levels (p < 0.02), indicating that higher dose of antibodies may have a greater therapeutic effect. Significant correlation was also observed between performance on the traverse beam task and PHF1 immunoreactivity in the dentate gyrus (p < 0.05) as well as with insoluble PHF1/total tau ratio on western blots (p < 0.04). These results show that passive immunization with tau antibodies can decrease tau pathology and functional impairments in the JNPL3 model. Future studies will determine the feasibility of this approach with other monoclonals and in different tangle models in which thorough cognitive assessment can be performed.     

Combined effects of avasimibe immunotherapy,doxorubicin chemotherapy,and metal–organic frameworks nanoparticles on breast cancer

CD8⁺ T cells play a vital role in cancer immunotherapy and can be shaped by metabolism. Avasimibe is an acyl coenzyme A-cholesterol acyltransferase (ACAT) inhibitor, which has been clinically verified safe in other phase Ⅲ clinical trials. It can potentiate the killing function of CD8⁺ T cells by modulating cholesterol metabolism. Doxorubicin (DOX) is an anticancer drug widely used in many cancers to induce tumor cell apoptosis. Unfortunately, DOX also can induce toxic and side effects in many organs, compromising its usage and efficacy. Herein, we report the combinational usage of avasimibe and a safe pH sensitive nano-drug delivery system composing of DOX and metal–organic frameworks nanoparticles (MNPs). Our findings demonstrated that DOX–MNPs treatment inhibited tumor growth with good safety profile and avasimibe treatment combined DOX–MNPs treatment exhibited a better efficacy than monotherapies in 4T1 breast cancer therapy.     

CD4+CD25+调节性T细胞与肿瘤免疫研究进展

调节性T细胞(Treg)是一类具有免疫调节功能的细胞群,在机体的免疫耐受中起着关键性作用。它们主要通过细胞-细胞直接接触的方式抑制CD4+和CD8+效应性T细胞的活化和增殖,来调节获得性免疫系统,阻止自身免疫疾病的发生。Treg中以自然产生的CD4+CD25+调节性T细胞(固有Treg细胞)研究最多。在人类,调控效能主要限于CD4+CD25high亚型。由于Treg独特的生物学功能,它在自身免疫性疾病的发生、移植耐受和肿瘤的发生和转归上越来越受到重视。该文就该类细胞的特点及其与肿瘤关系的研究进展作一综述。