Physiological and pathological roles of mast cells in adipose tissue

Mast cells are important cells for the innate immunity that reside in tissues including adipose tissue and are involved in various physiological and pathological processes by producing a range of biological mediators. Adipose tissue not only acts as an energy depot and regulator of energy homeostasis that can deposit excess energy and dissipate energy through heat, but also is an active endocrine organ capable of producing hormones and adipokines. The dysfunction of adipose tissue is highly correlated with metabolic disorders, such as obesity, type 2 diabetes mellitus and so on. This review focuses on the physiological and pathological roles of mast cells in adipose tissue.     

Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

AIM To construct the mouse embryonic stem cell （ESC） line with stable pancreatic and duodenal homeobox 1 （Pdx1） expression by Tet-On system, which may lay a foundation for further research on the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells. METHODS The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC. The cells were divided into 3 groups： blank control group （ESC group）, empty lentivirus control group （PDX1^- ESC group） and Pdx1 lentivirus transfection group （PDX1⁺ ESC group）. Flow cytometry was used to detect the transfected cells after screening by doxycycline （DOX）. The function of Tet-On system and the expression of Pdx1 gene were detected. The transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were sorted by flow cytometry, and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified. RESULTS （1） The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were 90.72% and 94.01% after screening by DOX, respectively. The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group was 97.84% and 98.13% after sorting by flow cytometry, respectively. （2） With DOX, green fluorescence was observed in PDX1^- ESC group and PDX1⁺ ESC group. The mRNA and protein expression of Pdx1 was significantly increased in PDX1⁺ ESC group （P<0.05）. Without DOX, no green fluorescence was observed in the cells of the 3 groups, and no significant difference in the mRNA and protein expression of Pdx1 was observed （P>0.05）. （3） After 3 months of cryopreservation, the cell lines still survived in resuscitation culture and were regulated by DOX. CONCLUSION Using Tet-On system, the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells.     

Estrogen treatment enhances mesenteric lymphatic contractility in rats after hemorrhagic shock

AIM To investigate the effects of 17β-estradiol （E2） treatment on the mesenteric lymphatic microcirculation and isolated lymphatic contractility in rats after hemorrhagic shock, and to explore the relationship between contractility and the difference between intra- and extracellular calcium ion concentrations （［Ca²⁺］） of lymphatic smooth muscle cells （LSMCs）. METHODS Male Wistar rats were divided into sham group, shock group and shock+E2 group. The rats were subjected to hemorrhage ［（40±2） mmHg for 90 min］ and resuscitation with or without subcutaneous injection of E2 （2 mg/kg）. After resuscitation for 3 h, the mesenteric lymphatic microcirculation in vivo was observed. Moreover, the isolated mesenteric microlymphatic rings were prepared for the observations of lymphatic contractility evaluated by the indexes including end-systolic diameter, end-diastolic diameter, contraction frequency （CF） and passive diameter. Meanwhile, the difference between intra- and extracellular ［Ca²⁺］ of LSMCs was recorded during lymphatic contraction. RESULTS Treatment with E2 significantly enhanced the CF, total contractile fraction and lymphatic dynamics index in vivo in the rats after hemorrhagic shock, and increased the CF, the fractional pump flow and the difference between intra- and extracellular ［Ca²⁺］ of LSMCs in isolated lymphatics from the shocked rats （P<0.05）. CONCLUSION Estrogen treatment enhances lymphatic contractility in rats after hemorrhagic shock, which is related to enhancement of difference between intra- and extracellular ［Ca²⁺］ of LSMCs.     

鸡ES细胞体外定向诱导分化特性的研究

旨在探索鸡胚胎干细胞(ES细胞)体外定向诱导的多向分化潜能.分离鸡X期胚盘细胞,体外培养传代,经鉴定后,采用地塞米松(DEX)、β-甘油磷酸钠(β-GP)和维生素C(VitC)诱导ES细胞向成骨细胞分化;采用维甲酸(RA)、3-异丁基-1-甲基黄嘌呤(IBMX)诱导ES细胞向神经元样细胞分化;采用DEX、胰岛素、IBMX诱导ES细胞向脂肪细胞分化,比较不同浓度组合诱导剂的诱导效果,分别用Von Kossa's染色法、甲苯胺蓝染色法、免疫组化法、油红O染色法和RT-PCR鉴定诱导产生的细胞.结果显示,ES细胞被诱导3～21 d后,分化为成骨细胞,诱导率为40％～72％;被诱导4～7 d后,分化为神经元样细胞,诱导率为71％～84％;被诱导2～21 d后,分化为脂肪细胞,油红O染色阳性,并表达PPARγ基因.结果表明,在体外条件下,ES细胞可被特异的化学物质定向诱导分化为成骨细胞、神经元样细胞和脂肪细胞,具有多向分化潜能.     

藏山羊和藏绵羊小肠黏膜免疫相关细胞的比较研究

为了探讨藏山羊和藏绵羊在低氧环境中小肠黏膜免疫屏障结构的适应特征,本试验采用组织化学方法和图像分析法对4只成年藏山羊和4只成年藏绵羊小肠不同肠段的上皮内淋巴细胞、杯状细胞和肥大细胞的数量进行比较研究。结果显示:藏山羊小肠各段杯状细胞和肥大细胞的数量均高于藏绵羊(P<0.05),其中藏山羊十二指肠、空肠和回肠的上皮内杯状细胞数量分别比藏绵羊多37.87%(P<0.05)、21.43%(P>0.05)和31.68%(P<0.05),藏山羊十二指肠、空肠和回肠的肥大细胞数量分别比藏绵羊多85.20%(P<0.05)、50.73%(P<0.05)和22.52%(P>0.05);而藏山羊小肠各段上皮内淋巴细胞的数量(36.35±0.98)低于藏绵羊(48.84±2.12)(P<0.05)。结果提示,成年藏山羊的小肠黏膜免疫屏障功能强于藏绵羊;藏绵羊小肠中上皮内淋巴细胞起重要的黏膜防御功能,而在藏山羊小肠中杯状细胞和肥大细胞起重要的黏膜防御功能。     

徐淮山羊PPAR基因克隆及其表达产物亚细胞定位的研究

研究旨在克隆徐淮山羊过氧化物酶体激活增殖受体(PPAR)基因的cDNA,并通过EGFP融合蛋白对该基因产物亚细胞水平定位。采用RT-PCR方法克隆徐淮山羊脂肪组织PPAR基因cDNA,并构建含有增强型绿色荧光蛋白(EGFP)报告基因的融合表达载体pEGFP-C1-PPAR,聚乙烯亚胺(PEI)介导基因转染NIH-3T3细胞,48h后荧光倒置显微镜下观察基因表达产物的分布,RT-PCR检测mRNA在体外水平的表达。结果表明:首次成功克隆出徐淮山羊PPAR基因的全序列cDNA,大小为1428bp,GenBank登录号为GU082382;构建了融合表达载体pEGFP-C1-PPAR;RT-PCR检测mRNA表达明显;EGFP-PPAR融合蛋白定位在NIT-3T3细胞质中。体外克隆的徐淮山羊PPAR基因cDNA,在单细胞水平上可表达于细胞质中,结果为进一步研究PPAR的生物学功能奠定基础。     

猪卵巢颗粒细胞分离培养及鉴定

为构建猪卵巢颗粒细胞的体外培养体系,研究毒素的生殖毒性奠定基础,采用机械分离法从1～5 mm的卵泡中提取猪卵巢颗粒细胞,苔盼蓝染色计算细胞存活率,Giemsa染色和结晶紫染色观察细胞形态,免疫细胞化学染色方法检测促卵泡刺激素受体(FSHR)表达来鉴定颗粒细胞,MTT方法测定细胞生长曲线.结果显示分离提取的猪卵巢颗粒细胞存活率为65%左右,细胞纯度＞97%,细胞结构完整,边缘分明,胞核呈卵圆形位于靠近胞质的中央.另外细胞生长曲线表明,细胞从24 h开始进入对数生长期,并于96 h达到最高密度,之后进入平台期.分离培养的颗粒细胞生长状态良好,且细胞纯度＞97%,是理想的适合进行毒素生殖毒理学的细胞培养体系.     

Clinical,histological and immunophenotypic findings in a mare with a mammary lymphoma associated with anaemia and pruritus

An 18‐year‐old Quarter Horse mare presented with an 18 week history of dermatological lesions characterised by recurrent urticaria and recent signs of enlarged mammary glands, pyrexia, alopecia and intense pruritus. Haematological and serum biochemical analyses revealed anaemia, leucocytosis, thrombocytopenia and hyperfibrinogenaemia. Mammary discharge was evaluated and revealed neutrophilia. Cytological examinations of fine needle aspirates from the parotid lymph nodes were normal. Common causes of anaemia were ruled out by specific tests. The initial diagnosis was mastitis and 2 weeks later the animal returned to the hospital with worsening of clinical signs, intermittent fever, intense pruritus and generalised alopecia. Blood samples were collected for haematological and serum biochemical analyses, and the mammary gland and parotid lymph nodes were biopsied. Rectal palpation revealed a large irregular mass near the left kidney with adhesions of surrounding structures. The animal died before the laboratory results were ready. This report details the clinical, histological and immunophenotypic findings of a case of large lymphoma in a mare.