Identification of Suspected Stem/progenitor Cells in Bovine Mammary Epithelial Cells Culture System

To develop the potential function of dairy cow mammary stem cells (DCMECs) in regulation of lactation,we identify putative DCMECs which were BrdU label retaining epithelial cells,at the same time,analysis the location of two new mammary stem cells molecular marks FNDC3B and PROCR to verify the feasibility of them to indicate DCMECs.The mRNA levels of prolactin,growth hormone,insulin-like growth factor-1 and their receptors were detected along with cell passage by Real-time quantitative PCR.The results showed that the proportion of BrdU label-retaining epithelial cells was nearly 0.4% after 25 d continuous culture (passaged 4 times) and few cells were positive for FNDC3B or PROCR.Moreover,we observed the BrdU labelled epithelial cells by asymmetric division.The mRNA levels of prolactin,growth hormone,insulin-like growth factor-Ⅰ and their receptors in primary and passage cells were extremely significant difference(P<0.01).DCMECs would rapidly lose some physiological characteristics and the ability of milk synthesis when not under the condition of induction of lactation differentiation,but a certain percentage of mammary stem/progenitor cells will be retained,whose potential effects on the regulation of lactation and mammary acinar remodeling were worthy of attention.     

Effect Comparison of Adenovirus Vector Transfer into Different Buffalo Cells

Buffalo mammary epithelial cell,cumulus cell and fibroblasts were transfected by adenovirus vectors and compared their transfection efficiency.293 cells were transfected with pBHGloxdelE13cre and pDC316-eGFP by liposome,the virus was collected and titer was detected.Buffalo mammary epithelial cell,cumulus cell and fibroblasts were exposed to different multiplicity of infection (MOI) of adenovirus vectors.After 72 h,the cells were observed with inverted fluorescence microscope,and transfection efficiency was calculated.When the MOI was 25,50,100,200 and 400,the transfection efficiency of fibroblasts were 0.7%,7.0%,9.0%,12.5% and 34.0%,the transfection efficiency of cumulus cells were 42.5%,55.3%,57.4%,76.0% and 80.0%,the transfection efficiency of mammary epithelial cells were 88.7%,100%,100%,100% and 100%.The results showed that the transfection efficiency of mammary epithelial cell was the best,followed by cumulus cell,and fibroblast was poor.     

Interaction of NR2D subunit of NMDA receptor with MOCA

AIM: To identify the potential proteins interacting with NR2D subunit of NMDA receptor by yeast two-hybrid screening and to investigate the role of NR2D in excitotoxicity of the retina.METHODS: The Clontech GAL4 yeast two-hybrid system was used to screen the mouse brain cDNA library, and the bait plasmid containing C-terminus of NR2D was constructed. Physical interaction between 2 proteins was verified by co-immunoprecipitation assay. The subcellular localization of 2 proteins in the mouse retina was observed under microscope with immunofluorescence.RESULTS: Modifier of cell adhesion (MOCA) was identified as a new protein interacting with NR2D. MOCA and NR2D were co-expressed in the mouse retina. CONCLUSION: MOCA specifically interacts with NR2D, which provides the experimental basis for identifying the role of glutamate excitotoxicity in the retina neurodegeneration.     

Functional analysis of the venom allergen-like protein gene from pine wood nematode Bursaphelenchus xylophilus using a baculovirus expression system

The pine wood nematode, Bursaphelenchus xylophilus, infects pine trees, leading to fatal pine wilt disease. Here, recombinant venom allergen-like protein (VAP) was obtained by expressing Bx-vap-1 in insect cells. Three-year-old Pinus massoniana were inoculated with recombinant VAP, simulating B. xylophilus esophageal gland secretions. Recombinant VAP up-regulated α-pinene synthase gene expression, the trees showed disease symptoms 15 d after inoculation and the xylem pith revealed brown tissue discoloration, indicating that recombinant VAP could damage P. massoniana cells. Recombinant VAP did not, however, lead to cavitation, indicating that the VAP secreted from B. xylophilus acts as a defense response elicitor.     

不同激素对碧云茶树丛生芽增殖培养的影响实验

为了获得碧云茶树的组培技术,加快其优良品种繁殖速度,2010～2011年进行了不同浓度的细胞分裂素6-BA和生长素NAA对碧云茶树丛生芽增殖培养的影响。结果表明,以MS为基本培养基,当6-BA为2.0 mg/L,NAA为0.2 mg/L时,对碧云茶树芽的诱导和增殖最有利,增殖率可达2.84;其中NAA差异不显著,6-BA差异显著。     

In vitro effect of levamisole on the cell viability,phagocytosis and respiratory burst of Barbel chub (Squaliobarbus curriculus) macrophages

The present study was to determine in vitro effect of levamisole on the immune functions of Barbel chub (Squaliobarbus curriculus), head–kidney‐derived (HKM), spleen‐derived (SM) and peripheral blood monocyte‐derived macrophage (PBM). Macrophages were incubated with levamisole 10³, 10¹, 10⁻¹ and 10⁻³ ng mL⁻¹ to assay the cell viability, respiratory burst and phagocytosis. The results showed that macrophages treated with levamisole 10⁻³ ng mL⁻¹ gave a maximum respiratory burst response, whereas levamisole 10³ ng mL⁻¹ had no effect. Phagocytosis activity of the macrophages treated with levamisole enhanced significantly when compared to control, maximum response being at 10⁻³ ng mL⁻¹. While using the methylthiazoletetrazolium method to measure the cell activity for 12, 24, 36, 48, 60 h, there was no significant role on proliferation and the cell viability began to decline after 24 h. In conclusion, levamisole is a potent enhancer of Barbel chub macrophage activity with low concentration.     

岱衢族大黄鱼放流增殖试验

为了恢复舟山渔场本地种岱衢族大黄鱼（Pseudosciaenac Focea）资源，2000～2009年进行了岱衢族大黄鱼增殖放流试验。10年间共放流了经过人工繁育的岱衢族大黄鱼苗种1288．0×10^4尾，其中包括标志鱼62680尾。回捕标志鱼1432尾，平均回捕率2．73％。回捕试验结果表明，在岱衢洋进行本地大黄鱼的放流可行，体外挂牌法适合于大黄鱼的标志放流，放流鱼苗不仅可以成活、生长，还能够产卵、增殖并进行洄游；放流使舟山渔场本地岱衢族大黄鱼资源得到显著恢复，并产生了一定的经济效益。     

Vitamin C enhanced myocardial differentiation of dedifferentiated fat cells

AIM: In order to observe the myocardial differentiation capacity of the dedifferentiated fat (DFAT) cells treated with vitamin C in vitro. METHODS: DFAT cells were dedifferentiated from the mature rat adipocytes with ceiling adherent culture. The DFAT cells of passage 3 were used in the study. Vitamin C and/or neonatal rat heart tissue lysate were added into the culture medium to induce myocardial differentiation for 3 weeks. The cell morphology was observed under microscope. The myocardial-specific markers, such as cTnT, GATA-4 and NKx2.5, were examined by the methods of immunofluorescence, PCR and Western blot. RESULTS: Mature rat adipocytes dedifferentiated into fibroblast-like DFAT cells after ceiling adherent culture. The DFAT cells spontaneously differentiated into cardiomyocyte-like cells under normal culture condition with a low incidence. After treated with neonatal rat heart cell lysate, the DFAT cells became cardiomyocyte-like cells that had bigger size, longer shape and myotubule-structure. The expression of cTnT, GATA-4 and NKx2.5 was remarkably increased at both mRNA and protein levels as compared with the normal cultured DFAT cells. The expression of cTnT, GATA-4 and NKx2.5 was further increased in DFAT cells after treating with vitamin C. No spontaneous beating cell was observed. CONCLUSION: Vitamin C enhances the differentiation of DFAT cells into cardiomyocyte-like cells.     

藏绵羊KLF7基因表达特征分析及其过表达对前脂肪细胞增殖分化的影响

【目的】分析藏绵羊Krüppel样因子7(Krüppel-like factor 7,KLF7)基因表达特征,研究过表达该基因对前脂肪细胞增殖及分化的影响。【方法】从藏绵羊脂肪组织中分离前脂肪细胞进行培养及成脂诱导,应用实时荧光定量PCR技术检测KLF7基因在藏绵羊7个组织(大脑、皮下脂肪、肾脏、背最长肌、瘤胃、睾丸和回肠)和前脂肪细胞不同分化阶段(第0、2、4和8天)的mRNA相对表达水平;应用RT-PCR方法从藏绵羊脂肪组织中扩增KLF7基因CDS区序列,并将其连接到pcDNA3.1(+)真核表达载体获得pcDNA3.1-KLF7过表达质粒,转染前脂肪细胞;应用实时荧光定量PCR方法检测脂肪细胞增殖及分化标志基因mRNA表达水平;采用EdU和CCK-8方法分别检测过表达KLF7基因对EdU阳性细胞数和细胞活力的影响;采用油红O染色检测过表达KLF7基因后脂肪细胞脂滴生成量。【结果】KLF7基因在藏绵羊7个组织中均有表达,其中在大脑中的表达量最高,其次为皮下脂肪和肾脏,均显著高于其他组织(P<0.05);诱导分化第2、4和8天脂肪细胞mRNA表达量均显著高于分化前(P＜0.05),且分化第2天表达量最高;pcDNA3.1-KLF7过表达质粒转染前脂肪细胞2 d后显著或极显著抑制增殖标志基因CDK4、CyclinB1和CyclinD1的表达水平(P＜0.05;P＜0.01),极显著降低细胞活力及EdU阳性细胞数量(P＜0.01);pcDNA3.1-KLF7过表达质粒转染前脂肪细胞,诱导分化8 d后,脂肪细胞分化标志基因PPARγ、Glut4和ELOVL6的mRNA相对表达水平显著或极显著下调(P＜0.05;P＜0.01),且脂质沉积极显著减少(P＜0.01),表明过表达KLF7基因可抑制藏绵羊前脂肪细胞增殖及分化。【结论】KLF7基因在藏绵羊多个组织中广泛表达,且大脑、皮下脂肪、肾脏中表达量较高;诱导分化后脂肪细胞表达量显著高于分化前,且分化第2天表达量最高;过表达KLF7基因可抑制藏绵羊前脂肪细胞的增殖及分化。试验结果为阐明藏绵羊脂肪沉积的分子调控机制提供了基础数据。