小鼠不同泌乳期乳腺脂肪合成相关基因的mRNA表达

为了研究小鼠不同泌乳期乳脂肪合成相关基因的表达规律,文章采用荧光定量PCR检测了小鼠乳腺中与脂肪合成和分泌相关20个基因的mRNA相对表达丰度和表达差异。结果表明,在乳腺中脂蛋白脂酶(LPL)、乙酰辅酶A羧化酶(ACACA)、硬脂酰辅酶A去饱和酶(SCD)、黄嘌呤脱氢酶(XDH)、嗜乳脂蛋白(BTN)、脂肪酸分化蛋白(ADFP)基因都具有高mRNA表达丰度(表达丰度>5%),脂肪酸转运体(CD36)、脂肪酸合成酶(FASN)、1-酰基甘油磷酸酰基转移酶(AGPAT6)和甘油酰基转移酶(DGAT)基因具有中等mRNA表达丰度(5%>表达丰度>1%),与妊娠期乳腺基因的mRNA表达相比,在泌乳期这些基因的mRNA表达均有显著上调(P<0.05),并且ACACA、SCD、FASN、AGPAT6和DGAT等脂肪合成酶基因的表达在泌乳中期(12 d)最高,而在泌乳初期(6 d)和泌乳末期(18 d)较低,呈现低-高-低的表达模式。转录因子固醇调节元件结合蛋白(SREBF)基因在泌乳开始时mRNA表达增加,在泌乳中期(12 d)表达有10倍上调,其变化规律与脂肪合成酶基因的表达模式相同,说明SREBF基因在小鼠乳腺脂肪合成酶基因的表达调控中发挥重要调节作用。     

小鼠不同泌乳期乳腺脂肪合成相关基因的mRNA表达

为了研究小鼠不同泌乳期乳脂肪合成相关基因的表达规律, 文章采用荧光定量PCR检测了小鼠乳腺中与脂肪合成和分泌相关20个基因的mRNA相对表达丰度和表达差异。结果表明, 在乳腺中脂蛋白脂酶(LPL)、乙酰辅酶A羧化酶(ACACA)、硬脂酰辅酶A去饱和酶(SCD)、黄嘌呤脱氢酶(XDH)、嗜乳脂蛋白(BTN)、脂肪酸分化蛋白(ADFP)基因都具有高mRNA表达丰度 (表达丰度>5%), 脂肪酸转运体(CD36)、脂肪酸合成酶(FASN)、1-酰基甘油磷酸酰基转移酶(AGPAT6)和甘油酰基转移酶(DGAT)基因具有中等mRNA表达丰度(5%>表达丰度>1%), 与妊娠期乳腺基因的mRNA表达相比, 在泌乳期这些基因的mRNA表达均有显著上调(P<0.05), 并且ACACA、SCD、FASN、AGPAT6和DGAT等脂肪合成酶基因的表达在泌乳中期(12 d)最高, 而在泌乳初期(6 d)和泌乳末期(18 d)较低, 呈现低-高-低的表达模式。转录因子固醇调节元件结合蛋白(SREBF)基因在泌乳开始时mRNA表达增加, 在泌乳中期(12 d)表达有10倍上调, 其变化规律与脂肪合成酶基因的表达模式相同, 说明SREBF基因在小鼠乳腺脂肪合成酶基因的表达调控中发挥重要调节作用。     

CRISPR/Cas9敲除PLIN1基因增强3T3-L1脂肪细胞的脂解作用

应用CRISPR/Cas9技术敲除3T3-L1前脂肪细胞plin1,观察PLIN1缺失对脂肪细胞中脂肪水解的影响并探究可能机制。常规培养3T3-L1前脂肪细胞,电穿孔法转染plin1敲除载体,嘌呤霉素培养基挑选plin1敲除细胞,观察转染及筛选后的细胞存活率。\"鸡尾酒\"法诱导3T3-L1前脂肪细胞分化,酶法测定甘油和TG含量,油红O染色观察脂滴形态及数目的变化。Western blotting检测PLIN1、PPARγ、Fsp27和脂肪酶的蛋白表达;RT-PCR检测PLIN1和脂肪酶的mRNA表达。对照组细胞诱导分化后,微小脂滴数目较少,单房脂滴数目较多并围绕细胞核呈环型排列。相较于对照组,敲除组细胞诱导分化后微小脂滴数目增加,单房脂滴体积缩小,数目减少;细胞中PLIN1mRNA及蛋白表达被显著抑制(P0.05);甘油水平显著上升(0.0984±0.0076),TG含量显著下降(0.031 0±0.005 3);HSL和ATGL两种脂肪酶的mRNA及蛋白表达均升高(P0.05);PPARγ和Fsp27的表达未有明显变化。上述结果表明plin1敲除后通过暴露脂滴中脂质以及上调脂肪酶等效应增强了3T3-L1脂肪细胞的脂解作用。     

人胰岛素原基因在酵母中的分泌表达

 我们研究了外源基因——合成的人胰岛素原基因在酵母α因子系统中的表达和分泌。用表达载体YTI-15转化酵母63号株可得到每升约2毫克的人胰岛素原分泌产物。     

Generation of an immortal differentiated lung type-II epithelial cell line from the adult H-2KbtsA58 transgenic mouse

Summary This paper describes a new fully differentiated Type-II alveolar epithelial cell line designated T₇, derived from transgenic H-2K^b-tsA58 mice, capable of being passaged as an immortalized cloned cell line in culture. H-2K^b-tsA58 mice harbor a temperature-sensitive (ts) mutant of the simian virus 40 (SV40) large tumor antigen (T antigen) under the control of the γ-interferon (INF)-inducible mouse major histocompatibility complex H-2K^b promoter. When cultured under permissive conditions (33°C and in the presence of γ-INF) cells isolated from H-2K^b-tsA58 mice express the large T antigen, which drives the cells to proliferate. However, upon withdrawal of the γ-INF and transfer of the cells to a higher temperature (39°C), T antigen expression is turned off, the cells stop proliferating and differentiate. The T₇ cell line is a clonal cell line originally derived from a Type-II cell-rich fraction isolated from lungs of H-2K^b-tsA58 mice. The T₇ cells form confluent monolayers, and have a polarized epithelial cell morphology with tight junctions and apical microvilli. In addition, the T₇ cells have distinct cytoplasmic lamellar bodies, which become more numerous and pronounced when the cells are grown under nonpermissive conditions. The T₇ cells synthesize and secrete phosphatidylcholine and the three surfactant proteins, SP-A, SP-B, and SP-C. The T₇ cell line is unique in that it is the first non-tumor-derived Type-II cell line capable of synthesizing and secreting the major components of surfactant. Based on the criteria studied, the T₇ cell line is phenotypically very similar to normal Type-II cells. The T₇ cell line, therefore, should prove a valuable experimental system to advance the study of the cell biology/physiology of surfactant metabolism and secretion as well as serve as a model for other studies of Type-II cell physiology.     

Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro

Haloperidol, a typical antipsychotic, has been shown to inhibit cholesterol biosynthesis by affecting Δ⁷-reductase, Δ^8,7-isomerase, and Δ¹⁴-reductase activities, which results in the accumulation of different sterol intermediates. In the present work, we investigated the effects of atypical or second-generation antipsychotics (SGA), such as clozapine, risperidone, and ziprasidone, on intracellular lipid metabolism in different cell lines. All the SGAs tested inhibited cholesterol biosynthesis. Ziprasidone and risperidone had the same targets as haloperidol at inhibiting cholesterol biosynthesis, although with different relative activities (ziprasidone > haloperidol > risperidone). In contrast, clozapine mainly affected Δ²⁴-reductase and Δ^8,7-isomerase activities. These amphiphilic drugs also interfered with the LDL-derived cholesterol egress from the endosome/lysosome compartment, thus further reducing the cholesterol content in the endoplasmic reticulum. This triggered a homeostatic response with the stimulation of sterol regulatory element-binding protein (SREBP)-regulated gene expression. Treatment with SGAs also increased the synthesis of complex lipids (phospholipids and triacylglycerides). Once the antipsychotics were removed from the medium, a rebound in the cholesterol biosynthesis rate was detected, and the complex-lipid synthesis further increased. In this condition, apolipoprotein B secretion was also stimulated as demonstrated in HepG2 cells. These effects of SGAs on lipid homeostasis may be relevant in the metabolic side effects of antipsychotics, especially hypertriglyceridemia.     

木聚糖酶基因克隆、表达与分泌及定点诱变研究进展

木聚糖酶专一性水解木聚糖分子中β14糖苷键,在制浆造纸、食品、纺织、饲料、能源工业中具有广阔的应用前景。随着各种新技术的发展与应用,木聚糖酶基因的研究取得了很大进展,不仅克隆了许多不同来源的木聚糖酶基因,研究了木聚糖酶基因在同源和异源寄主中的表达和分泌,而且还使用定点诱变技术探讨了木聚糖酶的结构与功能的关系并对酶的性质进行改造以满足工业生产的需要 。     

Biological activities of 7-dehydrocholesterol-derived oxysterols: implications for Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a metabolic and developmental disorder caused by mutations in the gene encoding the enzyme 7-dehydrocholesterol reductase (Dhcr7). This reductase catalyzes the last step in cholesterol biosynthesis, and levels of 7-dehydrocholesterol (7-DHC), the substrate for this enzyme, are elevated in SLOS patients as a result of this defect. Our group has previously shown that 7-DHC is extremely prone to free radical autoxidation, and we identified about a dozen different oxysterols formed from oxidation of 7-DHC. We report here that 7-DHC-derived oxysterols reduce cell viability in a dose- and time-dependent manner, some of the compounds showing activity at sub-micromolar concentrations. The reduction of cell survival is caused by a combination of reduced proliferation and induced differentiation of the Neuro2a cells. The complex 7-DHC oxysterol mixture added to control Neuro2a cells also triggers the gene expression changes that were previously identified in Dhcr7-deficient Neuro2a cells. Based on the identification of overlapping gene expression changes in Dhcr7-deficient and 7-DHC oxysterol-treated Neuro2a cells, we hypothesize that some of the pathophysiological findings in the mouse SLOS model and SLOS patients might be due to accumulated 7-DHC oxysterols.     

Evidence of Mucin Secretion in Human Lung Adenocarcinoma Cell Lines NCIH650 and NCIH2077 and Effect of Select Secretagogues on Mucin Secretion

Mucins comprise an important class of tumor-associated antigens. The objectives of the present study were (a) to establish an in vitro model system using human non-small cell lung adenocarcinoma cell lines NCIH650 and NCIH2077 (b) provide evidence that these cell lines secrete mucin in culture conditions and (c) investigate the effects of select secretagogues on mucin secretion. The cell lines were established in ACL-4 medium containing several growth factors and retinoic acid and 5% fetal calf serum. The high molecular weight glycoconjugates secreted in the culture medium were purified by ammonium sulfate precipitation and Superose 6 and Superose 12 FPLC chromatography. The purified high molecular weight glycoconjugate fraction and the carcinoma cells were shown to have mucin by dot blot, Western blot and immunohistochemical analysis, respectively, using specific antibodies to purified major mucin, HTM-1. Also, incorporation experiments with mucin precursor ³H-glucosamine demonstrated that the cells indeed synthesize high molecular weight mucins. The effects of secretagogues such as, 8-bromocyclic AMP, ionomycin, phorbol-12-myristate-13-acetate and neutrophil elastase on mucin secretion were also investigated. Only 8-bromocyclic AMP and neutrophil elastase influenced mucin secretion. These studies provided strong evidence that the lung adenocarcinoma cell lines secrete high molecular weight mucins in culture conditions and only two of the four tested secretagogues significantly increased mucin secretion. Thus, this in vitro model system may be useful in determining alterations in mucin structure, if any, in lung adenocarcinomas as well as in studying the regulation of mucin gene expression.