DDPH对猪肺动脉平滑肌细胞PDGF·BB和bFGF表达的影响：免疫细胞化学研究

ＤＤＰＨ［１－（２．６－二甲基苯乙氧基）－２－（３．４二甲氧基苯乙胺基）丙烷盐酸盐］是南京药科大学合成的降压新化合物,也具有降低肺动脉高压和抑制肺动脉平滑肌细胞增殖作用。本实验用细胞培养、免疫细胞化学、图像分析、３Ｈ－ＴｄＲ、细胞周期测定等方法,进一步探讨ＤＤＰＨ对缺氧性肺动脉平滑肌细胞（ＰＡＳＭＣＳ）增殖的抑制机制。结果：缺氧促进肺动脉内皮细胞（ＰＡＥＣｓ）的ＰＤＧＦ·ＢＢ和ｂＦＧＦ两种生长因子的表达（积分光密度ＯＤ值）增高。缺氧内皮细胞条件培养液（ＨＥＣＣＭ）能促进ＰＡＳＭＣＳ的ＰＤＧＦ·ＢＢ的ＯＤ值增高,ｂＦＧＦ的ＯＤ值无明显改变。加药组（ＨＥＣ－ＣＭ＋ＤＤＰＨ）的ＰＤＧＦ·ＢＢ和ｂＦＧＦ的ＯＤ值均显著降低,尤以ＰＤＧＦ·ＢＢ的ＯＤ值减少最多．提示：ＤＤＰＨ能抑制ＨＥＣＣＭ引起ＰＡＳＭＣＳ的ＰＤＧＦ·ＢＢ和ｂＦＧＦ表达增多和细胞增殖。结果与大鼠实验观察相符。     

刺五加在大鼠实验性肝癌诱发过程中作用的探讨

目的探讨大鼠实验性肝癌发病中刺五加对肌体免疫功能和抗氧化酶活性的影响。方法４６只ＳＤ雄性大鼠被随机分成对照组（喂普通饲料）、３－甲基４－双甲氨基偶氮苯（３－Ｍｅ－ＤＡＢ）组（喂含０．０６％３Ｍｅ－ＤＡＢ饲料 １０周）和刺五加组（饲喂同 ３－Ｍｅ－ＤＡＢ外、另加入刺五加 ４．５ｇ／ｋｇ饲料,用常规方法检测全血谷光甘肽过氧化物酶（ＧＳＨ－ＰＸ）、血清超氧化物歧化酶（ＳＯＤ）活性和丙二醛（ＭＤＡ）含量,用微量化学发光造检测吞噬细胞活性（ＰＭＮ－ＣＬ）。结果１．ＰＭＮ－ＣＬ检测峰值、积分值和吞噬细胞指数,３－ＭｅＤＡＢ组较正常组和刺五加组均有显著升高（Ｐ＜０．０５和Ｐ＜０．０１）２．全血ＧＳＨ－ＰＸ活性、ＳＯＤ活性,刺五加组较３－ＭｅＤＡＢ组均有显著升高（Ｐ＜０．０５）。ＭＤＡ含量刺五加组和３－ＭｅＤＡＢ组均较正常组升高（均Ｐ＜０．０５）。结论刺五加在大鼠实验性肝癌诱发过程中有提高抗氧化酶活性和对抗致癌剂引起的机体中性粒细胞吞噬功能代偿性增高的作用。     

MnSOD基因转染对电离辐射诱发细胞凋亡的调控

电离辐射能诱发超氧阴离子自由基（Ｏ－２·）的产生,同时也是细胞凋亡发生的诱因之一。锰超氧化物歧化酶（ＭｎＳＯＤ）是一种存在于真核细胞线粒体、具有清除Ｏ－２·作用的抗氧化酶。为探讨ＭｎＳＯＤ在辐射诱发细胞凋亡中的作用,采用基因转染技术分别将有义和反义ＭｎＳＯＤｃＤＮＡ真核表达载体直接导入ＣＨＯ细胞,并用此细胞模型研究了ＭｎＳＯＤ对Ｘ射线诱发细胞凋亡的调控。结果发现,稳定表达有义ＭｎＳＯＤ的细胞克隆能明显抑制８ＧｙＸ射线诱发的细胞凋亡,而表达反义ＭｎＳＯＤ的细胞克隆对Ｘ射线诱发的细胞凋亡更加敏感,进一步的研究发现,线粒体膜电位的改变可能是ＭｎＳＯＤ调控细胞凋亡的机制之一。     

氧化修饰高密度脂蛋白对培养人动脉平滑肌细胞细胞周期蛋白D_1基因表达的影响

动脉平滑肌细胞（ｓｍ ｏｏｔｈ ｍ ｕｓｃｌｅ ｃｅｌｌ,ＳＭＣ）是动脉粥样硬化（ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ,ＡＳ）斑块中的主要细胞,它的增殖在ＡＳ形成过程中极其重要．利用体外培养的人主动脉ＳＭＣ,观察了天然高密度脂蛋白（ｎａｔｉｖｅ ｈｉｇｈ ｄｅｎｓｉｔｙ ｌｉｐｏｐｒｏｔｅｉｎ,Ｎ－ＨＤＬ）及氧化修饰ＨＤＬ（ｏｘｉｄｉｚｅｄ ＨＤＬ,ＯＸ－ＨＤＬ）对培养人主动脉ＳＭＣ ｃｙｃｌｉｎ Ｄ１（细胞周期蛋白Ｄ１）基因转录表达的影响．结果表明：（１）Ｎ－ＨＤＬ对ＳＭＣｃｙｃｌｉｎ Ｄ１基因表达无影响（Ｐ＞ ０．０５）;（２）ＯＸ－ＨＤＬ使ＳＭＣｃｙｃｌｉｎ Ｄ１基因表达显著增强（Ｐ＜０．０１）,其表达量随时间（２、１２、２４ ｈ）延长而增加．上述结果表明,ＯＸ－ＨＤＬ的致ＡＳ作用可能与其刺激ＳＭＣｃｙｃｌｉｎ Ｄ１基因表达增加有关．     

c—erbB2对大鼠黄体细胞hCG诱导的孕酮分泌的影响

采用离体细胞体外孵育法,研究反义ｃ－ｅｒｂＢ２寡脱氧核苷酸（ａｎｔｉｓｅｎｓｅ ｃ－ｅｒｂＢ２ ＯＤＮ）对大鼠黄体细胞ｈＣＧ诱导的孕酮分泌的影响,及其与外源性ｃＡＭＰ和Ｃａ＾２＋以及蛋白抑制剂放线菌酮（ＣＹＸ）之间的关系。结果表明,反义ｃ－ｅｒｂＢ２以剂量相关方式抑制黄体细胞ｈＣＧ诱导的孕酮的产生,同时使ｃ－ｅｒｂＢ２蛋白染色阳性的黄体细胞百分数下降,无义ｔａｔ ＯＤＮ没有相应的作用。１０＾－４     

血管活性肠肽对兔支气管上皮细胞抗臭氧损伤的保护作用

用支气管刷洗法收集新西兰兔支气管上皮细胞（ＢＥＣ）,以臭氧（Ｏ３）攻击培养的ＢＥＣ,建立细胞损伤模型。测定ＢＥＣ的３Ｈ释放率计算Ｏ３的细胞毒指数（ＣＩ）、测定细胞内丙二醛（ＭＤＡ）的含量反映细胞氧化性损伤的程度,测定细胞内过氧化氢酶（ＣＡＴ）活性及还原型和氧化型谷胱甘肽（ＧＳＨ和ＧＳＳＧ）的含量反映细胞抗氧化能力。观察血管活性肠肽（ＶＩＰ）预处理对ＢＥＣ的细胞保护作用并初步探讨其保护机制。观察到：ＢＥＣ的３Ｈ释放率与Ｏ３暴露时间成正比;Ｏ３暴露２ｈ使ＭＤＡ含量和ＧＳＳＧ含量明显增加,ＧＳＨ减少;ＶＩＰ预处理呈剂量依赖性降低Ｏ３暴露的ＣＩ值、降低ＭＤＡ和ＧＳＳＧ含量、增加ＧＳＨ及ＧＳＨ／ＧＳＳＧ比值、增加ＣＡＴ活性,显示出细胞保护效应;ＶＩＰ的保护效应可被放线菌素Ｄ（Ａ－Ｄ）或蛋白激酶Ｃ阻断剂Ｈ７部分取消。结果表明：Ｏ３暴露会导致ＢＥＣ损伤,ＶＩＰ可通过增强ＢＥＣ的抗氧化能力而保护ＢＥＣ,ＶＩＰ的信号在细胞内的转导途径与基因转录及依赖ＰＫＣ的酶蛋白磷酸化有关。     

植物胚胎发生过程的基因表达

植物胚胎发生过程的基因表达黄绍兴,阎隆飞（北京农业大学生物学院,北京１０００９４）ＧＥＮＥＥＸＰＲＥＳＳＩＯＮＤＵＲＩＮＧＰＬＡＮＴＥＭＢＲＹＯＧＥＮＥＳＩＳ￥ＨｕａｎｇＳｈａｏ－ｘｉｎｇ;ＹａｎＬｏｎｇ－ｆｅｉ（ＣｏｌｌｅｓｅｏｆＢｉｏｌｏｇｉｃａ...     

黑龙江铜木蚜蝇雌性的描述

黑龙江铜木蚜蝇雌性的描述ＤＥＳＣＲＩＰＴＩＯＮＯＦＴＨＥＦＥＭＡＬＥＣＨＡＬＣＯＳＹＲＰＨＵＳ（ＸＹＬＯＴＯＭＩＭＡ）ＡＭＵＲＥＮＳＩＳ（ＳＴＡＣＫＥＬＢＥＲＧ）（ＤＩＰＴＥＲＡ：ＳＹＲＰＨＩＤＡＥ）￥ＨＥＪｉｌｏｎｇ;ＣＨＵＸｉｐｉｎｇ（Ｄｅｐａｒ...     

Egr-1基因调控序列连接OSM cDNA表达质粒的构建及其在黑色素瘤细胞中的表达

ＯＳＭ是一种对黑色素瘤细胞显示抑制作用的细胞因子．为进行ＯＳＭ针对黑色素瘤的基因－放射治疗研究,构建了小鼠Ｅｇｒ－１基因调控序列引导入ＯＳＭｃＤＮＡ真核表达质粒（ｐＥＯ）,ｐＥＯ质粒转染小鼠Ｂ－１６黑色素瘤细胞,经Ｇ４１８和抗人ＯＳＭ抗体的筛选,获得了稳定表达ＯＳＭ的克隆细胞（ｐＥＯ－１细胞）,ＯＳＭ表达量可达５．９７ｎｇ每１０５细胞天,分子量为３２ｋＤ．ｐＥＯ－１细胞用一定浓度Ｈ２Ｏ２处理后ＯＳＭ表达量可提高６２％,表明ｐＥＯ重组质粒可在氧自由基的刺激作用下增强ＯＳＭ表达     

氧化修饰HDL刺激培养人主动脉平滑肌细胞增殖

平滑肌细胞（ｓｍｏｏｔｈ ｍｕｓｃｌｅ ｃｅｌｌ,ＳＭＣ）增殖在动脉粥样硬化（ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ,ＡＳ）形成中起着重要作用。氧化修饰ＨＤＬ（ｏｘｉｄｉｘｅｄ ＨＤＬ,ＯＸ－ＨＤＬ）可刺激＾３Ｈ－ＴｄＲ掺入培养人动脉ＳＭＣ的ＤＮＡ,促进ＳＭＣ增殖。以四甲工偶氮唑盐９ＭＴＴ）法直接观察ＯＸ－ＨＤＬ对培养人动脉ＳＭＣ增殖细胞数的影响。结果显示,天然ＨＤＬ（ｎａｔｉｖｅ ＨＤＬ Ｎ－ＨＤＬ）对     

Temporal Gene Expression Profile in Hippocampus of Mice Treated with <Emphasis Type="SmallCaps">d</Emphasis>-galactose

(1) Rodent chronically treated with D-galactose (D-gal) is increasingly used in pharmacological studies on aging; however, its mechanism remains unclear. The present study investigated the alterations of gene expression in the hippocampus of D-gal-treated mice. (2) C57 mice were subcutaneously injected with D-gal for 2, 4, and 8 weeks or vehicle, and then were subjected to behavioral tests. Gene expression profiles in hippocampus of each group were finally examined with cDNA microarray. (3) Both 4- and 8-week D-gal treatment led to a decrease of discrimination index in object recognition test, and 8-week D-gal-treated mice showed significant spatial learning & memory impairment in Morris water maze test. In comparison with the vehicle control group, the 2-, 4-, and 8-week D-gal treatment repressed the expression of 10, 13, and 30 genes by 2-fold or more, respectively. The early pattern was mainly characterized by down regulation of genes involved in ion transport. The delayed pattern included genes involved in protein biosynthesis, transport and signal transduction, which were highly related to synaptic functions. (4) These findings will contribute to the understanding of the mechanism of learning and memory impairment in mice treated with D-galactose.     

Resveratrol ameliorates thymus senescence changes in D-galactose induced mice

The thymic microenvironment plays an important role in the development of T cells. A decrease of thymic epithelial cells is the main cause of age-related thymic atrophy or degeneration. Resveratrol (RSV), a phytoalexin produced from plants, has been shown to inhibit the adverse effects of dietary obesity on the structure and function of the thymus. D-Galactose (D-gal) can induce accelerated aging in mice. In the present study, young mice (2 months old) were injected with D-gal (120 mg/kg/day) for 8 consecutive weeks to construct an accelerated aging model. Compared with normal control mice, the thymus epithelium of the D-gal treated mice had structural changes, the number of senescent cells increased, the number of CD4+ T cells decreased, and CD8+ T cells increased. After RSV administration by gavage for 6 weeks, it was found that RSV improved the surface phenotypes of D-gal treated mice, and recovered thymus function by maintaining the ratio of CD4+ to CD8+ cells. It also indicated that RSV enhanced the cell proliferation and inhibited cell senescence. Increased autoimmune regulator (Aire) expression was present in the RSV treated mice. The lymphotoxin-beta receptor (LTβR) expression also increased. These findings suggested that RSV intake could restore the alterations caused by D-gal treatment in the thymus via stimulation of Aire expression.     

Analysis of the herpes simplex virus genome during in vitro latency in human diploid fibroblasts and rat sensory neurons.

We have previously designed in vitro model systems to characterize the herpes simplex virus type 1 (HSV-1) genome during in vitro virus latency. Latency was established by treatment of infected human embryo lung fibroblast (HEL-F) cells or rat fetal neurons with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and was maintained by increasing the incubation temperature after inhibitor removal. Virus was reactivated by reducing the incubation temperature. We have now examined the HSV-1-specific DNA content of latently infected HEL-F cells and rat fetal neurons treated with (E)-5-(2-bromovinyl)-2'-deoxyuridine and human leukocyte interferon and increased temperature. The HEL-F cell population contained, on an average, between 0.25 and 0.5 copies of most, if not all, HSV-1 HindIII and XbaI DNA fragments per haploid cell genome equivalent. In contrast, the latently infected neurons contained, on an average, 8 to 10 copies per haploid cell genome equivalent of most HSV-1 BamHI DNA fragments. There was no detectable alteration in size or molarity of the HSV-1 terminal or junction DNA fragments obtained by HindIII, XbaI, or BamHI digestion of the latently infected neuron or HEL-F cell DNA, as compared with digestion of a reconstruction mixture of purified HSV-1 virion and HEL-F cell DNAs. These data suggest that the predominant form of the HSV-1 genome in either latently infected cell population is nonintegrated, linear, and nonconcatameric.     

Distinction between malignant L cells and normal mouse fibroblasts by rosette formation with sheep red blood cells.

Murine L cell fibroblasts, and derivatives were found to rosette with sheep red blood cells (SRBC). Primary fibroblast explants from the parent murine strain, C3H, did not possess this potential. No rosettes were observed with primary fibroblast explants from C57BL and B10Br mice, with a human fetal lung fibroblast, with baby hamster fibroblasts or their polyoma transormed derivative, or with a cell line, 1T-22, derived from BALB/c mice. Hybridization of 1T-22 and L cells, by Sendai virus-mediated cell fusion, suppressed the rosette potential of the L cell parent. The receptor for SRBC on L cells appears to result from the expression of a recessive characteristic.     

Glucocorticoids affect the synthesis of pulmonary fibroblast-pneumonocyte factor at a pretranslational level

Glucocorticoids accelerate fetal lung maturation by acting on the fetal lung fibroblast to induce the synthesis of fibroblast-pneumonocyte factor which in turn stimulates pulmonary surfactant synthesis by the alveolar type II cell. We have studied the site of glucocorticoid regulation of fibroblast-pneumonocyte factor synthesis in primary cultures of fetal rat lung fibroblasts. Conditioned media from fetal rat lung fibroblasts exposed to cortisol stimulate [Me-3H]choline incorporation into saturated phosphatidylcholine by primary cultures of fetal rat lung alveolar type II cells. This effect is blocked by the presence of actinomycin D during the first, but not the second, 24 h of incubation of the fibroblasts with cortisol. Cycloheximide blocks this effect if present during either the first or second 24 h of incubation. We fractionated mRNA from fetal rat lung fibroblasts incubated in the presence or absence of dexamethasone and observed that cell-free translation products from a fraction of approximately 500 bases possess biological activity in the bioassay. Such activity is only present in cell-free translation products of mRNA isolated from fibroblasts treated with dexamethasone. These results suggest that glucocorticoids act at a pretranslational level to induce production of fibroblast-pneumonocyte factor and that the primary translation products are biologically active.     

Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.

Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.     

Milk-derived growth factors as serum supplements for the growth of fibroblast and epithelial cells

Summary We have investigated the response of several epithelial and fibroblastic cells to a mitogenic extract of bovine milk. Cation exchange chromatography was used to produce a mitogen-rich fraction from an industrial whey source that, although comprising only 0.5% of total whey protein, contained the bulk of the growth factor activity. This fraction was a source of potent growth promoting activity for all mesodermal-derived cells tested, including human skin and embryonic lung fibroblasts, Balb/c 3T3 fibroblasts, and rat L6 myoblasts. Maximal growth of all these cell types exceeded that observed in 10% fetal bovine serum. Feline kidney and baby hamster fibroblasts and Chinese hamster ovary cells were less responsive, achieving a maximal growth response of 50–75% that observed in 10% fetal bovine serum. Maximal growth achieved in whey-extract-supplemented cultures of Balb/c 3T3 and human skin fibroblasts, and L6 myoblast cultures exceeded that seen in response to recombinant acidic or basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, or epidermal growth factor. Importantly, addition of low concentrations of fetal bovine serum to the whey-derived mitogenic fraction produced an additive response. However, concentrated milk-derived factors were found to be inhibitory to the growth of all epithelial lines tested, including rat intestinal epithelial cells, canine kidney epithelial cells, and mink lung cells. It is concluded that industrial whey extracted in this form constitutes an important source of potent growth-promoting agents for the supplementation of mesodermal-derived cell cultures.     

Lung-Derived Growth Factors: Ontogenic Shift in Parahormone Secretion in the Perinatal Rat Lung

Primary cultures of perinatal rat fibroblasts were found to produce at least three mitogenic activities which exhibited specificity for distinct cell types. One activity, lung growth factor(LGF), was a potent mitogen for chick embryonal fibroblasts, which also stimulated fetal rat lung fibroblasts to undergo DNA synthesis, provided that these cells were first exposed to a "competence" factor such as fibroblast growth factor or platelet-derived growth factor. Although LGF was active in the somatomedin-C (SmC) radioimmunoassay and resembled buffalo rat liver multiplication-stimulating activity (brlMSA) in molecular size, it appears to consist of a component that is neither SmC nor brlMSA. The second activity produced by perinatal rat lung cultures, pneumocyte-stimulating activity (PSA), stimulated mitosis in type II pneumocytes of postnatal rats, and was found to have physical attributes that are distinct from those of the other known pneumocyte-influencing factors. The third activity is a non-dialyzable substance which complements the mitogenic action of LGF on fetal lung fibroblasts, and appears to be a "competence" activity. An examination of the production of LGF and PSA by rat lung fibroblasts taken at various intervals of development revealed that fetal lung fibroblasts produce maximal levels of LGF but low levels of PSA, whereas, in neonatal lung fibroblasts, the situation is reversed. This ontogenic shift in the type of parahormone produced by the developing perinatal rat lung may be an important regulatory event in postnatal lung morphogenesis in this species.     

人胚肺二倍体成纤维细胞衰老过程中Wnt信号抑制因子DKK4的表达

Dickkopf家族蛋白DKK4是经典Wnt信号通路的抑制因子.为研究其在人胚肺二倍体成纤维细胞(2BS)复制性衰老过程中的作用机制及生理学意义,使用Wnt/β-联蛋白通路的激活剂氯化锂(LiCl)和抑制剂DKK1作用于2BS,同时利用免疫荧光技术分析衰老过程中细胞因子的定位.结果显示,在2BS复制性衰老的过程中,DKK4表达水平下降,而这种下降是β-联蛋白/TCF介导完成的.而在衰老过程或较高的过氧化物水平下,细胞核内转录因子FoxO4增多.由此得出结论:在衰老过程中,β-联蛋白/TCF下游靶基因DKK4表达下调,降低了对经典Wnt通路的抑制,使胞内β-联蛋白处于较高水平.较高水平的β-联蛋白在高过氧化物的微环境中,与FOXO家族转录因子相互作用,激活其下游靶基因,促进了衰老的发生发展.     

猪胎儿肾脏成纤维细胞体外培养体系的建立

本研究旨在建立猪胎儿肾脏成纤维细胞体外培养体系,并探讨其作为猪体细胞克隆供体的可能性。使用组织块培养法从体长为10cm以上的猪胎儿分离得到猪胎儿肾脏成纤维细胞,绘制了生长曲线,鉴定了细胞类型并且进行了细胞周期同期化效果的研究。结果表明:该培养体系可以支持猪胎儿肾脏成纤维细胞的体外生长,单个细胞均为梭形细胞,抗波形蛋白免疫荧光染色显示为阳性,而抗角形蛋白免疫荧光染色为阴性,分离到的细胞为胎儿肾脏成纤维细胞。使用血清饥饿法和接触抑制法诱导细胞进入G0/G1期,并且分别比较两者同期化效率,结果显示:血清饥饿2d和4d的同期化效率差异不显著,但都比8d组的高(88.97%和87.69%比82.45%,P<0.05);接触抑制4d、6d组间同期化效率差异不显著,但都比0d组的高(85.56%和85.89%比81.82%,P<0.05)。本研究在国内首次分离得到猪胎儿肾脏成纤维细胞,已经在体外传代培养到32代,其同期化效果好,可以作为体细胞克隆供体。