5 Aza dC对奶牛乳腺上皮细胞PPARγ表达的影响

为阐明DNA甲基化对奶牛乳腺泌乳功能调控的机制,研究了甲基化抑制剂5氮杂2′脱氧胞苷（5 Aza dC）对奶牛乳腺上皮细胞中PPARγ基因启动子甲基化状态及其表达的影响,以体外培养的奶牛乳腺上皮细胞为模型,采用0.1、0.5、1.0、5.0μmol／L的5 Aza dC对奶牛乳腺上皮细胞进行处理,用EpiQuikTM DNA methyltransferase assay试剂盒进行甲基转移酶活性检测,采用CASY细胞分析仪检测细胞活力和增殖能力,利用亚硫酸氢盐测序（BSP ）技术检测了PPARγ启动子的甲基化特征,qRT PCR法检测PPARγmRNA表达的变化,Western blot检测PPARγ蛋白表达的变化。结果显示：与空白对照组相比,0.1μmol／L的5 Aza dC对奶牛乳腺细胞生长无毒性,处理96 h甲基转移酶活性极显著降低,奶牛乳腺细胞的PPARγ基因启动子甲基化程度降低,PPARγ表达升高。表明5 Aza dC可降低奶牛乳腺细胞中PPARγ启动子区域的甲基化程度,促进PPARγ表达。     

奶牛乳腺上皮细胞的不同培养方法比较及激素和细胞因子对β-酪蛋白mRNA表达的诱导

本试验旨在建立一种高效的奶牛乳腺上皮细胞培养方法,并研究不同激素和细胞因子对其表达β-酪蛋白mRNA的诱导作用.分别采用组织块培养法和机械破碎法培养奶牛乳腺上皮细胞,利用成纤维细胞和乳腺上皮细胞对胰酶的敏感性不同,对获得的细胞进行纯化.通过细胞计数法测定纯化细胞的生长曲线,通过免疫荧光组织化学染色法检测角蛋白18的表达,通过实时定量PCR法检测8种不同激素和细胞因子组合培养液诱导细胞表达β-酪蛋白mRNA的效果.结果表明:通过机械破碎法可以分离得到增殖旺盛的奶牛乳腺上皮细胞,与组织块培养法相比,具有细胞迁出速度快等优点.细胞生长曲线呈典型的“S”型.在纯化的乳腺上皮细胞及第20代乳腺上皮细胞中都检测到角蛋白18的表达.100 ng/mL类胰岛素生长因子Ⅰ(IGF-Ⅰ)显著提高了乳腺上皮细胞中β-酪蛋白mRNA的表达(P＜0.05).由结果可知,本试验采用的机械破碎法是一种高效的奶牛乳腺上皮细胞培养方法,100 ng/mL IGF-Ⅰ对细胞中β-酪蛋白mRNA表达的诱导效果最好.     

Expression of TMEM16A as a calcium-activated chloride channel in Fischer rat thyroid follicular epithelial cells and its electrophysiologic pro-perties

AIM：To investigate the expression of transmembrane protein 16A(TMEM16A) in Fischer rat thyroid follicular epithelial (FRT) cells and its electrophysiologic properties. METHODS：The eukaryotic expression vector of pUB6/V5-TMEM16A was constructed and transfected into FRT cells by liposome-mediated transfection. In order to obtain the high efficiency of gene transfection and expression, the quantity and ratio of lipid/DNA complexes were optimized. The FRT cells stably expressing TMEM16A were gained by the selection with blasticidin and confirmed by the techniques of RT-PCR and immunofluorescence. The expression and location of TMEM16A in the FRT cells were observed under an inverted fluorescence microscope. TMEM16A protein was associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent protein and patch-clamp technique. RESULTS：The results of double digestion and sequencing indicated that TMEM16A was cloned into pUB6/V5. The results of RT-PCR and immunofluorescence confirmed that TMEM16A was expressed in the FRT cells after transfection with TMEM16A. The classical calcium-activated chloride channel currents were recorded in the FRT cells stably expressing TMEM16A by the technique of patch-clamp and halide-sensitive fluorescent protein YFP-H148Q/I152L. CONCLUSION：The protein expression of TMEM16A in the FRT cells was observed. TMEM16A is the molecular identity of calcium-activated chloride channels.     

Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT‐MECs)

Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post‐parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT‐MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF‐7 cells. hT‐MECs on passage 42 grew vigorously and ‘S’ sigmoid curves of growth were observed. Moreover, hT‐MECs maintained a normal chromosome modal number of 2n = 60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta‐casein protein was synthesized and secreted. Beta‐casein expression was enhanced by prolactin (P < 0.05). Lipid droplets were found in hT‐MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT‐MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT‐MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time.     

High‐level exogenous trans10, cis12 conjugated linoleic acid plays an anti‐lipogenesis role in bovine mammary epithelial cells

Primary bovine mammary epithelial cells (BMECs) were treated by 0, 37.5, 75, 112.5, 150 μmol/L trans10, cis12 conjugated linoleic acid (CLA) to evaluate the effects of different level trans10, cis12 CLA on lipogenesis in BMEC. Addition of 75–150 μmol/L trans10, cis12 CLA reduced significantly the triacylglycerol (TAG) content (P < 0.05), but did not have inhibiting action on cell proliferation (P > 0.05). Treatment with 150 μmol/L trans10, cis12 CLA for 48 h resulted in a 17.1% reduction (P < 0.0001) of medium chain fatty acids (MCFA, C14 < C < C16), a 26.5% reduction (P < 0.0001) of unsaturated fatty acids (UFA) and a corresponding reduction of the mRNA abundance of acetyl coenzyme A (acetylCoA) carboxylase (ACC) (P = 0.046), fatty acid synthase (FAS) (P = 0.017) and stearoylCoA desaturase1 (SCD1) (P = 0.002). Another finding was that trans10, cis12 CLA elevated expression of diacylglycerol acyltransferase2 (DGAT2) (P = 0.020) and long chain acylCoA synthetases (ACSL) (P = 0.032). In conclusion, higher trans10, cis12 CLA, not low trans10, cis12 CLA, inhibited milk fat synthesis and changed fatty acid composition by regulating the expression of FAS, ACC, SCD1, DGAT2 and ACSL.     

Smad泛素化调节因子2siRNA的筛选及转染原代肾小管上皮细胞条件的优化简

试验旨在优化Smad泛素化调节因子2（Smurf2） siRNA最佳转染条件,筛选最佳siRNA干扰片段,进而实现对Smurf2基因的瞬时沉默,为研究Smurf2基因在马兜铃酸肾病（aristolochic acid nephrohathy,AAN）中的作用奠定基础。本研究通过培养小鼠原代肾小管上皮细胞（renal tubular epithelial cells,RTECs）,并以脂质体Lipofectamine^TM 2000为转染介质,将Smurf2 siRNA转染入RTECs;通过观察绿色荧光的表达量及Real-time PCR反应优化转染条件,转染后Real-time PCR检测mRNA表达抑制率。CCK-8法检测Smurf2 siRNA复合物对RTECs活性的影响;同时,用Real-time PCR和Western blotting检测不同位点Smurf2 siRNA对Smurf2 表达的影响。结果显示,当Lipofectamine ^TM 2000与siRNA比例为1.5 μL∶30 pmol时,转染效率最高,为70%～80%;Smurf2-619 siRNA干扰效果最明显;与正常组相比,转染siRNA组的Smurf2蛋白表达水平显著下降（P<0.05）。最终确定了Smurf2 siRNA最佳转染条件,其中Smurf2-619 siRNA对Smurf2 表达的抑制率最高。     

Culture of mantle epithelial cells expressing shell matrix proteins from scallop Patinopecten yessoensis

ABSTRACT:   In molluscs, mantle epithelial cells secrete organic matrix proteins to form shells. In this study, we established a culture of mantle epithelial cells by using the mantle pallial layer of scallops. We aimed to identify the mantle epithelial cells expressing scallop shell matrix proteins and establish a culture system of epithelial cells. After the mantle pallial layer was carefully isolated from the mantle tissue, explant culture was performed at 4°C. Most cells that migrated from the explant tissue were round cells. Most of the adhered cells retained round morphology, while some of the cells adhered to the dish and showed morphology similar to that of epithelial-like and fibroblast-like cells. When the cultured cells were immunostained with a polyclonal antibody against the shell matrix protein, the antibody recognized many of the adhered cells. An estimation of the number of epithelial cells revealed that approximately 70% of the adhered cells were epithelial cells. This is the first report to describe epithelial cells in cultured mantle cells, which express shell matrix proteins. This culture system may be a useful method for characterization of the mantle epithelial cells.     

成纤维上皮细胞炎症反应模型的建立

利用培养金黄色葡萄球菌培养基过滤液建立成纤维上皮细胞炎症反应模型，用于观察和研究炎症反应过程中细胞形态的变化。将培养金黄色葡萄球菌培养基除菌过滤冻干，用细胞培养基稀释，按照一定蛋白含量加到上皮细胞的培养基中，制作细胞炎症反应模型。结果显示，成纤维上皮细胞发生炎症反应，高浓度细菌滤过液的细胞形态发生变化，细胞边缘模糊不清，细胞出现空泡，随着培养时间的延长低浓度的大量大型细胞开始增加；在添加金黄色葡萄球菌培养基滤过液培养24h后利用^3H—TdR方法测定细胞增殖率，细胞增殖差异是显著的。结果表明，建立细胞炎症反应模型是可行的，并为以后炎症反应机理研究和抗炎性反应药物研究奠定了基础。     

研究不同氨基酸模式对奶牛乳腺上皮细胞生长的影响

本试验以泌乳奶牛乳腺组织为原料,采用组织块培养技术,研究培养基内不同氨基酸模式对奶牛乳腺上皮细胞增殖的影响。结果表明,随着培养天数的增加,细胞数量逐渐增加,第10天各组细胞数量分别达到最高,乳腺上皮细胞生长曲线呈"S"形。不同氨基酸模式对乳腺细胞生长有显著影响,与氨基酸不平衡组相比,氨基酸平衡组、理想组更能促进细胞生长,各组间差异显著(P0.05)。     

牛初乳酶解物对离体犊牛小肠上皮细胞增殖和吸收功能的影响

初乳经胃蛋白酶水解处理90分钟后所产生的水解物对离体犊牛小肠上皮细胞有明显的促增殖作用(P<0.01) ,而初乳经胰蛋白酶处理则不表现促增殖活性。初乳酶解物对促进细胞吸收葡萄糖的作用随着酶解处理时间的延长而逐渐增强 ,初乳经胰蛋白酶水解处理90分钟或经胃蛋白酶水解处理150分钟时的产物对细胞吸收葡萄糖的促进作用分别达到最强(P<0.01)。试验结果表明 ,初乳酶解物即小分子蛋白质(肽)具有刺激离体小肠上皮细胞增殖和功能发育的活性。