中药医院制剂活血益气方对裸鼠抑瘤作用及小鼠急性毒性的实验研究

目的:测试首都医科大学宣武医院药剂科制剂活血益气方抗瘤丸的抑瘤作用及对小鼠的急性毒性。方法:将足够量的大鼠神经胶质瘤C6细胞注射至裸鼠右侧腋窝,建立裸鼠荷瘤模型,按瘤块体积随机分组,分别灌胃给予KLW的高、低剂量组,以替莫唑胺为阳性对照,以溶媒为阴性对照,期间测量裸鼠体重、瘤体积并观察给药前后动物症状;连续给药15d后,处死全部小鼠,称量裸鼠瘤重量;剥离瘤块,采用Western Blot法检测肿瘤组织Bcl-2,Bax蛋白。选用ICR(Institute of Cancer Research,ICR)小鼠1 d内2次灌胃给药KLW,肉眼观察给药前、每次给药后连续4 h及14 d内小鼠的一般情况,中毒反应及死亡情况,结束观察后解剖,肉眼观察各组织脏器的变化。结果:裸鼠给药期间全部存活,体重正常增长,一般行为学观察未见明显异常。给药15 d后,KLW低、高剂量组,瘤体积、瘤重量较模型对照组显著减小;Bcl-2/Bax的比例较模型对照组显著增加。急性毒性试验中所有给药组小鼠在观察期内全部存活,一般行为学观察未见明显异常。供试品组2个性别的小鼠体重与阴性对照组比较,差异无统计学意义(P0.05)。剖检各组小鼠,肉眼观察各组织脏器未见病理改变。结论:KLW低、高剂量组对C6细胞移植瘤的生长具有明显的抑制作用,该作用与诱导胶质瘤细胞凋亡相关,与阴性对照组比较,差异有统计学意义(P0.05)。KLW小鼠灌胃40 mL/kg BW,最大给药浓度(0.50 g/mL),相当于临床人用剂量的107倍,未见明显毒性反应,使用安全性高。     

B6AF1雌性小鼠免疫性卵巢早衰模型建立

目的：本文主要研究自身免疫性卵巢早衰小鼠模型的建立和鉴定方法,为治疗和预防免疫性卵巢早衰提供实验基础。方法：将A/J雄鼠和C57BL/6雌鼠进行杂交繁殖B6AF1小鼠,筛选出B6AF1雌性小鼠,运用透明带多肽片段ZP3免疫B6AF1雌性小鼠,对阴道脱落细胞进行巴士染色,第14天结束造模,第7天和第14天分别进行模型鉴定。将卵巢进行H&E染色和ZP3免疫荧光染色,采用ELISA法检测小鼠血清中E₂和FSH的含量,来观察小鼠动情周期、卵巢组织形态学、抗透明带抗体免疫荧光和相关性激素的变化,评判免疫性卵巢早衰模型小鼠建立情况。结果：利用透明带ZP3多肽免疫B6AF1雌性小鼠14天,小鼠动情周期紊乱,卵巢组织有炎细胞浸润,卵巢中有明显透明带出现,血清中性激素E2含量下降和FSH含量上升,第7天上述变化不明显。结论：利用透明带多肽ZP3免疫B6AF1雌性小鼠14天,能够成功建立自身免疫性卵巢早衰模型,该模型发病率高,方法简单,能为免疫性卵巢早衰的研究提供基础。     

脂肪干细胞来源外泌体促进糖尿病小鼠创面愈合的实验研究

目的探讨脂肪干细胞来源外泌体(adipose-derived stem cell released exosomes,ADSC-Exos)对糖尿病小鼠创面愈合的影响。方法取患者自愿捐赠脂肪组织,采用酶消化法分离培养ADSCs,并于第3代细胞上清液提取Exos(ADSC-Exos);透射电镜观察ADSC-Exos形态,Western blot检测膜表面标志性蛋白Alix、CD63,纳米颗粒跟踪分析仪检测粒径分布。取患者自愿捐赠皮肤组织,采用酶消化法分离培养成纤维细胞。将PKH26标记的ADSC-Exos与第5代成纤维细胞培养,共聚焦荧光显微镜观察其能否进入细胞,采用细胞计数试剂盒8(cell counting kit 8,CCK-8)及划痕法观察ADSC-Exos对成纤维细胞增殖及迁移的影响。取24只8周龄Balb/c雄性小鼠制备糖尿病模型后,背部制备直径8 mm全层皮肤缺损创面,实验组(n=12)及对照组(n=12)创面真皮层分别注射0.2 mL ADSC-Exos及PBS。第1、4、7、11、16、21天大体观察创面愈合情况,计算创面愈合率;第7、14、21天取材,行组织学(HE及Masson)及CD31免疫组织化学染色,观察创面结构、胶原纤维及新生血管情况。结果ADSC-Exos为边缘清晰、大小分布均匀的膜性囊泡;粒径为40~200 nm,平均102.1 nm;膜表面标志性蛋白Alix、CD63均呈阳性。ADSC-Exos与成纤维细胞复合培养观察示,ADSC-Exos可进入成纤维细胞胞内,并能促进成纤维细胞增殖及迁移。动物实验显示,实验组小鼠背部创面愈合明显快于对照组,各时间点创面愈合率差异均有统计学意义(P<0.05)。与对照组比较,实验组创面结构愈合更好,愈合前期新生微血管更多,愈合后期胶原纤维沉积更多;其中,第7、14、21天创面缺损长度,第7、14天微血管数量,第14、21天胶原纤维沉积百分比,组间差异均有统计学意义(P<0.05)。结论ADSC-Exos通过促进创面血管新生以及成纤维细胞增殖、迁移和胶原合成来促进糖尿病小鼠创面愈合。     

荧光腹腔镜下小鼠胃癌可视化成像的实验研究

目的探讨荧光腹腔镜下小鼠胃癌可视化成像在鉴别肿瘤和正常组织中的价值。方法共20只3~4周龄,体重20 g裸鼠。2只裸鼠右后肢皮下注射0.2 ml高表达HER-2基因的NCI-N87胃癌细胞悬液用于观察背部成瘤情况,1周后当瘤体直径达1 cm左右进行下一步实验观察。将0.2 ml高表达HER-2基因的NCI-N87胃癌细胞悬液注射于12只裸鼠胃黏膜下,建立裸鼠胃癌模型。将量子点与HER-2的单抗结合制作量子点纳米荧光探针。将12只胃癌模型裸鼠分为A、B组,每组6只,A组裸鼠尾静脉注射量子点纳米荧光探针100μl与裸鼠胃癌模型进行体内结合,B组裸鼠尾静脉先注射HER-2单克隆抗体再注射量子点纳米荧光探针100μl,C组6只正常生长的裸鼠尾静脉直接注射量子点纳米荧光探针100μl。最后利用改装后的荧光腹腔镜分别以15、30 min和1、2、4、8、12、24、30 h时间点观察不同时期裸鼠体内胃癌组织及其他部位的荧光显像,标本送检。结果裸鼠种瘤成功,胃癌组织在荧光腹腔镜下完成可视化成像,荧光探针结合后稳定性良好,荧光强度在1 h后达到峰值,30 h后荧光消失。发光组织病理切片证实为胃癌组织,免疫组化证实该组织高表达HER-2蛋白。结论量子点纳米荧光探针性质稳定,荧光强度1 h达到峰值,然后逐渐衰减,荧光腹腔镜下可清晰看到高表达HER-2的NCI-N87胃恶性肿瘤的边界和周围组织及淋巴结的转移情况。     

The effects of ageing on mouse muscle microstructure: a comparative study of time‐dependent diffusion MRI and histological assessment

The investigation of age‐related changes in muscle microstructure between developmental and healthy adult mice may help us to understand the clinical features of early‐onset muscle diseases, such as Duchenne muscular dystrophy. We investigated the evolution of mouse hind‐limb muscle microstructure using diffusion imaging of in vivo and in vitro samples from both actively growing and mature mice. Mean apparent diffusion coefficients (ADCs) of the gastrocnemius and tibialis anterior muscles were determined as a function of diffusion time (Δ), age (7.5, 22 and 44 weeks) and diffusion gradient direction, applied parallel or transverse to the principal axis of the muscle fibres. We investigated a wide range of diffusion times with the goal of probing a range of diffusion lengths characteristic of muscle microstructure. We compared the diffusion time‐dependent ADC of hind‐limb muscles with histology. ADC was found to vary as a function of diffusion time in muscles at all stages of maturation. Muscle water diffusivity was higher in younger (7.5 weeks) than in adult (22 and 44 weeks) mice, whereas no differences were observed between the older ages. In vitro data showed the same diffusivity pattern as in vivo data. The highlighted differences in diffusion properties between young and mature muscles suggested differences in underlying muscle microstructure, which were confirmed by histological assessment. In particular, although diffusion was more restricted in older muscle, muscle fibre size increased significantly from young to adult age. The extracellular space decreased with age by only ~1%. This suggests that the observed diffusivity differences between young and adult muscles may be caused by increased membrane permeability in younger muscle associated with properties of the sarcolemma.     

Exercising with blocked muscle glycogenolysis: Adaptation in the McArdle mouse

Background

McArdle disease (glycogen storage disease type V) is an inborn error of skeletal muscle metabolism, which affects glycogen phosphorylase (myophosphorylase) activity leading to an inability to break down glycogen. Patients with McArdle disease are exercise intolerant, as muscle glycogen-derived glucose is unavailable during exercise. Metabolic adaptation to blocked muscle glycogenolysis occurs at rest in the McArdle mouse model, but only in highly glycolytic muscle. However, it is unknown what compensatory metabolic adaptations occur during exercise in McArdle disease.

Novel non‐alcoholic steatohepatitis model with histopathological and insulin‐resistant features

Although several non‐alcoholic steatohepatitis (NASH) models have been reported to date, few of these models fully reflect the histopathology and pathophysiology of human NASH. The aim of this study was to establish a novel NASH model by feeding a high‐fat (HF) diet and administering both carbon tetrachloride (CCl₄) and the Liver X receptor agonist T0901317. Male C57BL/6J mice were divided into four groups (each n = 5): HF, HF + CCl₄, HF + T0901317, and the novel NASH model (HF + CCl₄ + T0901317). CCl₄ (0.1 mL/kg) and T0901317 (2.5 mg/kg) were intraperitoneally administered four times and five times, respectively. The livers of the novel NASH model group presented a whitish colour. The serum levels of TNF‐α and IL‐6 were significantly increased in the novel NASH model group, and mice in this group exhibited histopathological features and insulin resistance reflective of NASH, i.e., macrovesicular hepatic steatosis, ballooning hepatocytes, Mallory‐Denk bodies, lobular inflammation and fibrosis. The novel NASH model group presented significantly upregulated expression levels of mRNAs related to lipogenesis, oxidative stress, fibrosis and steatosis and significantly downregulated expression levels of mRNAs related to triglyceride export. We successfully established a novel experimental NASH model that exhibits similar histopathology and pathophysiology to human NASH.     

Modeling the complex etiology of holoprosencephaly in mice

Holoprosencephaly (HPE) is a common developmental defect caused by failure to define the midline of the forebrain and/or midface. HPE is associated with heterozygous mutations in Nodal and Sonic hedgehog (SHH) pathway components, but clinical presentation is highly variable, and many mutation carriers are unaffected. It is therefore thought that such mutations interact with more common modifiers, genetic and/or environmental, to produce severe patterning defects. Modifiers are difficult to identify, as their effects are context-dependent and occur within the complex genetic and environmental landscapes that characterize human populations. This has made a full understanding of HPE etiology challenging. We discuss here the use of mice, a genetically tractable model sensitive to teratogens, as a system to address this challenge. Mice carrying mutations in human HPE genes often display wide variations in phenotypic penetrance and expressivity when placed on different genetic backgrounds, demonstrating the existence of silent HPE modifier genes. Studies with mouse lines carrying SHH pathway mutations on appropriate genetic backgrounds have led to identification of both genetic and environmental modifiers that synergize with the mutations to produce a spectrum of HPE phenotypes. These models favor a scenario in which multiple modifying influences—both genetic and environmental, sensitizing and protective—interact with bona fide HPE mutations to grade phenotypic outcomes. Despite the complex interplay of HPE risk factors, mouse models have helped establish some clear concepts in HPE etiology. A combination of mouse and human cohort studies should improve our understanding of this fascinating and medically important issue.     

Regulatory T cells for tolerance

Regulatory T cells (Tregs) are critical mediators of immune homeostasis and hold significant promise in the quest for transplantation tolerance. Progress has now reached a critical threshold as techniques for production of clinical therapies are optimised and Phase I/II clinical trials are in full swing. Initial safety and efficacy data are being reported, with trials assessing a number of different strategies for the introduction of Treg therapy. It is now more crucial than ever to elucidate further the function and behaviour of Tregs in vivo and ensure safe delivery. This review will discuss the current state of the art and future directions in Treg therapy.