转化生长因子β mRNA在佛波酯刺激的小鼠表皮和化学性诱发的小鼠皮肤肿瘤中的高水平表达

转化生长因子β是一种细胞多功能调节肽,目前研究认为其和促癌过程有密切关系。本文利用Northern印迹分析技术研究了TGF-β mRNA在促癌物佛波酯刺激的小鼠表皮和化学性诱发的小鼠二阶段皮肤肿瘤中的表达情况。结果证明,巴豆油2—20 μL和TPA 5—40nmol一次局部涂用于小鼠皮肤,均可明显增加小鼠表皮TGF-β mRNA,呈较好的浓度依赖性。TGF-β mRNA表达在二甲基苯蒽(DMBA)和巴豆油诱发的二阶段小鼠皮肤乳头瘤和癌中均高于正常和瘤旁表皮。所检测到的TGF-βmRNA为2.5kb和1.9kb二种条带,但以2.5kb为主。     

促癌物TPA诱导小鼠表皮鸟氨酸脱羧酶mRNA表达和维甲类化合物对该…

强皮肤促癌物十四烷酰佛波醋酸酯局部应用时可触发一系列的生物化学改变，其中最明显的事件之一就是对ＯＤＣ活性的短暂而急剧的诱导，而这种诱导作用与其促癌作用密切相关。利用Ｎｏｒｔｈｅｒｎ印迹分析和条带印迹分析证明，１０ｎｍｏｌ／Ｌ ＴＰＡ一次局部处理小鼠背部皮肤可刺激ＯＤＣ ｍＲＮＡ（２．０ｋｂ大小）表达，在４ｈ左右最为明显，随后逐渐降低。１０ｎｍｏｌ／Ｌ ＴＰＡ多次处理小鼠皮肤（每２天一次，共４次）也     

转化生长因子βmRNA在佛波酯刺激的小鼠表皮和化学性诱发的小鼠…

转化生长因子β是一种细胞多功能调节肽,目前研究认为其和促癌过程有密切关系。本文利用Ｎｏｒｔｈｅｒｎ印迹分析技术研究了ＴＧＦ－βｍＲＮＡ在促癌笺佛波酯刺激的小鼠表皮和化学性诱发的小鼠二阶段皮肤肿瘤中的表达情况。结果证明,巴豆油２－２０μＬ和ＴＰＡ ５－４０ｍｍｏｌ一次局部涂用于小鼠皮肤,均可明显增加小鼠表皮ＴＧＦ－βｍＲＮＡ,呈较好的浓度依赖性。ＴＧＦ－βｍＲＮＡ表达在二甲基苯蒽和巴豆油诱发的二阶段     

褪黑激素体外对脂肪间充质干细胞增殖及向施万样细胞分化的影响

该文探究了褪黑激素(melatonin,MT)对脂肪间充质干细胞(adipose mesenchymal?stem cells,ADSCs)向施万细胞(Schwann cells,SCs)分化的影响。从小鼠腹股沟脂肪垫分离ADSCs,培养至第3代,进行成脂和成骨分化,流式细胞术鉴定细胞表面抗原;第3代ADSCs分别与不同浓度的MT孵育24 h,采用CCK-8法检测细胞增殖活性,确定MT合适浓度。实验分为对照组、50 nmol/L MT组、神经诱导液组和50 nmol/L MT+神经诱导液组,孵育11天后,采用qPCR和Western blot检测SCs表面标志GFAP和S-100的表达情况。结果显示,50和100 nmol/L MT对ADSCs的增殖活性显著高于其他组,且50 nmol/L MT促增殖效果更好。神经诱导液组和50 nmol/L MT+神经诱导液组GFAP、S-100的mRNA和蛋白表达水平均显著高于对照组,而与神经诱导液组相比,50 nmol/L MT+神经诱导液组GFAP、S-100的mRNA和蛋白表达水平显著升高。综上所述,50 nmol/L MT可以在体外显著促进...     

甘草黄酮体抗促癌,抗致突和抗氧化作用的研究

G9315是从胀果甘草[Glycyrrhizae inflata Bat(Ⅲ))中提取的含有6个黄酮体的复合物。2mg剂量明显抑制二甲基苯蒽(DMBA)合并巴豆油诱发的小鼠皮肤乳头瘤的生成,抑瘤作用主要在促癌阶段。1mg剂量显著抑制巴豆油诱发的小鼠耳部水肿。小鼠口服G_(9315)(0.5g/kg/d×7)明显对抗环磷酰胺诱发的骨髓微核细胞增加。20—40μg/ml显著抑制TPA促进的~(32)pi参入HeLa细胞的磷脂部分。20μg/ml显著抑制巴豆油诱发的Wistar大鼠中性粒细胞(PMN)和Balb/c新生小鼠皮肤表皮细胞的化学发光以及肝线粒体的脂质过氧化。G(9315)(10—20μg/ml)有效对抗CCl_4诱发的小鼠肝微粒体脂质过氧化。     

致癌剂DEN、促癌剂巴豆油对大鼠肝α_1抑制因子3基因表达的影响

本文对致癌剂二乙基亚硝胺(DEN)和促癌剂巴豆油对大鼠肝α_1-抑制因子3(α_1- I_s)基因表达的影响进行了观察。结果表明:在腹腔注射DEN(200mg/kg·b.w.)后2h,α_1- I_3 RNA水平即显著下降;4h左右有所回升,约为正常水平的四分之一;然后又下降至极低水平,并一直保持该水平至24h。同时,c-myc RNA在2h则被诱导,约为正常水平的2-3倍;之后,即在4至24h内虽有波动,但基本处于正常水平。在腹腔注射巴豆油(18mg/kg.b.w.)后1h,α_1-I_3RNA水平即下降,约为正常水平的1/8,此后有所回升,约为正常水平的1/3-1/4;在同样条件下,ODC和C-fos RNA能被巴豆油诱导而增加。我们以前的工作表明,在大多数(75％,12/16)由DEN所诱发的大鼠肝癌中,α_1- I_3RNA水平显著下降,且基因结构发生了变化。综合这些结果,说明α_1- I_3基因或与该基因表达有关的蛋白调控因子可能是致癌剂DEN或促癌剂TPA的作用靶分子。该基因表达异常的意义及机理正在深入研究中。     

小鼠受精卵早期发育过程中PKC对cdc2和cdc25C活性的影响

为研究小鼠受精卵细胞早期发育过程中PKC对cdc2和cdc2 5C活性的影响 ,采用免疫印迹和电泳迁移率差异分析的方法 ,观察PKC的激活剂TPA及其抑制剂星形孢子素对小鼠受精卵一细胞期cdc2和cdc2 5C活性的影响 .10nmol L的TPA作用 10min后 ,小鼠受精卵一细胞期卵裂率明显大于对照组 (P <0 0 5 ) ,而星形孢子素作用后卵裂率显著下降 (P <0 0 1) .TPA处理后 ,受精卵中呈去磷酸化状态的活性cdc2明显增加 ,没有活性呈磷酸化状态的cdc2 5C明显减少 ;而星形孢子素处理的受精卵中没有活性的cdc2明显增加 ,有活性的cdc2 5C明显减少 .结果表明 ,TPA短时间作用可以促进小鼠一细胞期受精卵分裂 ,星形孢子素抑制受精卵的分裂 ;TPA可以促进cdc2的去磷酸化以及cdc2 5C的磷酸化 ,从而促进G2 M转换 ,星形孢子素则抑制cdc2和cdc2 5C的活性 ,阻止受精卵由G2 期进入M期     

无血清培养上调N2a细胞钙反应性反式激活因子的表达

目的探讨钙反应性反式激活因子(calcium-responsive transactivator,CREST)是否与神经细胞的分化有关。方法选用在促分化因素作用下具有分化为神经元能力的小鼠神经母细胞瘤(N2a)细胞,用无血清培养(血清撤除)诱导N2a细胞分化。接种和培养N2a细胞12～24h后,将其分为对照组和无血清诱导组,对照组继续在含5%胎牛血清(FBS)的培养基中培养,无血清诱导组在不含FBS的培养基中培养。采用免疫印迹技术、免疫荧光技术和RT-PCR检测无血清诱导分化对N2a细胞CREST蛋白和mRNA表达的影响。结果在含5%胎牛血清(FBS)培养基培养的对照N2a细胞中,CREST蛋白水平在各时间点均无明显变化,CREST mRNA水平仅在培养48h后略有升高。而在无血清诱导分化的N2a细胞中,CREST蛋白表达水平在无血清诱导12h时无明显变化,但在诱导24h后明显升高,诱导48h后进一步升高;RT-PCR检测显示,CREST mRNA在无血清诱导12h后即开始升高,诱导24h和48h则升高更为明显。结论无血清诱导分化的N2a细胞中CREST的表达上调,提示CREST可能参与神经细胞的分化。     

皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶活性及其基因表达的影响

采用高效液相色谱和原位杂交技术研究了皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶 (ODC)活性及ODCmRNA表达的影响。结果显示 ,大鼠完整肝脏中ODC水平较低 ,2 / 3肝切除 (PH)后 3h ,不同处理组ODC活性开始升高 ,6h达到最高值 ,其中 ,去肾上腺 NaCl组和糖皮质激素受体拮抗剂RU4 86处理组的酶活性高于对照组 (去肾上腺假手术组 ) ,而去肾上腺 皮质酮处理组的酶活性低于对照组 ,36h恢复到肝切除前水平 ;完整肝脏的ODCmRNA水平极低 ,PH后表达量迅速增加 ,5h达到最大值 ,不同处理组mRNA水平的高低顺序与酶活性一致 ,12h降至肝切除前水平 ;在PH前 12h给大鼠注射RU4 86 (10mg/kg体重 ) ,取得了与去肾上腺 NaCl处理鼠相似的结果。以上结果表明 ,在PH诱导的再生肝细胞中 ,ODCmRNA表达量的增加和 /或减少是造成ODC活性改变的原因之一 ,皮质酮对ODC活性及其mRNA的表达具有抑制作用 ,主要表现在肝再生的早期 ,该作用可能是通过受体实现的     

胃肠道肿瘤TGF－α基因表达的研究

用分子杂交技术及流式细胞方法研究了转化生长因子α(TGFα)在人胃肠道肿瘤组织中的表达.结果表明,胃癌、结肠癌和直肠癌组织较相应癌旁组织TGFαmRNA水平增高.佛波脂(TPA 100μg/L)作用胃癌细胞MGc80-3后4h,可使细胞中TGFαmRNA水平增高,作用6h可使S期细胞百分比明显增加.这些结果提示:TGFα在胃肠道肿瘤中起重要作用,其表达可受TPA的调节.     

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin ornithine decarboxylase and protein kinase C by polyphenolics from grapes

Ornithine decarboxylase is the rate-limiting enzyme in the biosynthesis of polyamines, which are believed to play an essential role in diverse biological processes including cell proliferation and differentiation. We have previously reported [J. Bomser, K. Singletary, M. Wallig, M. Smith, Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds, Cancer Letters 135 (1999) 151-157] that pre-application of a grape polyphenolic fraction (GPF) to mouse skin epidermis inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, as well as 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated, TPA-promoted mouse skin tumorigenesis. The present studies were designed to further characterize the effect of time and dose of application of GPF on TPA-induced ODC activity and protein expression, and on protein kinase C activity in mouse skin epidermis. In addition, the effect of GPF on ODC kinetics in vitro was examined. Application of 5, 10, and 20 mg of GPF 20 min prior to treatment with TPA resulted in a significant decrease in epidermal ODC activity of 54, 53, 90%, respectively, compared with controls. Yet, ODC protein levels (Western blot) in the 10 and 20 mg GPF groups were significantly increased by 1.8 and 1.9-fold, respectively, compared with controls. A similar response was observed with the ODC inhibitor 2-difluoromethylornithine (DFMO), which served as a positive control. Application of grape polyphenolics (20 mg) at 60 and 30 min prior to treatment with TPA inhibited ODC activity by 62 and 68%, respectively, compared with controls (P<0.05). In contrast, application of grape polyphenolics (20 mg) at 60, 120 and 240 min after treatment with TPA resulted in no significant changes in ODC activity. A similar increase in epidermal ODC protein was observed in these GPF-treated animals, similar to that observed when GPF application preceded TPA. When applied to mouse skin prior to TPA, GPF was associated with a decrease in subsequent PKC activity compared with controls at 10 and 30 min following TPA treatment. The GPF-associated decrease in PKC activity preceded the decrease in ODC activity. In a separate in vitro study, kinetic analyses indicated that GPF is a competitive inhibitor of ODC activity. Collectively these data suggest that the grape polyphenolic fraction is effective as an inhibitor of ODC activity when applied before TPA, and that the magnitude of inhibition is independent of epidermal ODC protein content. In addition, GPF is a competitive inhibitor of ODC activity in vitro. The decrease in TPA-induced ODC activity due to GPF treatment is preceded by an inhibition of TPA-induced PKC activity. Thus, the polyphenolic fraction from grapes warrants further examination as a skin cancer chemopreventive agent that interferes with cellular events associated with TPA promotion.     

Haemosiderin-like properties of free-radical-modified ferritin.

Evidence was sought that the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mouse epidermal ornithine decarboxylase (ODC, EC 4.1.1.17) activity involves both increased ODC mRNA and ODC protein. Application of 10 nmol of TPA to mouse skin led to a dramatic increase in soluble epidermal ODC activity which paralleled an increase in amount of enzymically active ODC protein as determined by gel electrophoresis of immunoprecipitated difluoromethyl[3H]ornithine-bound ODC. Application of TPA to mouse skin also resulted in an increase in ODC mRNA measured by dot-blot analysis using a radiolabelled cDNA probe. ODC mRNA induction preceded the increase in ODC activity by TPA. TPA-increased ODC mRNA displayed a single major band of 2.1 kilobases in size identified by the Northern blotting procedure.     

Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.