胆固醇缺乏对Jurkat细胞蛋白酪氨酸激酶活性的影响

目的：探讨胆固醇缺乏抑制Jurkat细胞增殖的分子机理,方法,用间接免疫荧光法,^3H-TdR掺入及流式细胞术等,观察其对蛋白酪氨酸激酶（PTKs)信号传递系统的影响,结果：经去脂血清培养基培养的Jurkat细胞和lovastatin处理3天后,^3H-TdR掺入率明显下降,细胞增残受抑,细胞受阻于G0／G1期,细胞内酪氨酸磷酸化蛋白含量明显下降,加入LDL可以部分逆转上述变化,结论：无论是内源性还是外源性胆固醇的缺乏都能使Jurkat细胞蛋白酪氨酸激酶活性下降,推测这一变化与细胞G0／G1期阻滞及细胞增残受抑有关。     

转化生长因子β在大豆异黄酮抑制乳腺癌细胞增殖中的作用

目的 :　探讨大豆异黄酮抑制 BCa P- 37乳腺癌细胞增殖的分子机制。方法 :　选择二羟异黄酮、三羟异黄酮处理 BCa P- 37细胞 ,采用生长曲线3H- Td R掺入试验及流式细胞分析等实验方法 ,观察大豆异黄酮对乳腺癌细胞 BCa P- 37增殖的抑制作用 ,同时用转化生长因子 (TGP) β拮抗试验、Western blot分析 ,检测 TGFβ1、TGFβ2 及受体的表达变化情况。结果 :　 (1～ 9)× 1 0 -5mol/L三羟异黄酮作用 3～ 4d可抑制 BCa P- 37细胞增殖 ,细胞受阻于 G1期 ,且 BCa P- 37细胞的TGFβ1、TGFβ2 及受体表达呈时间剂量依赖性增加。而二羟异黄酮处理组不明显。结论 :　三羟异黄酮抑制 BCa P- 37细胞增殖能力强于二羟异黄酮 ,三羟异黄酮可能通过诱导 TGF-β和 TGF-β受体表达的增加而抑制人乳腺癌细胞 BCa P- 37增殖     

姜黄素对TK6细胞的增殖抑制和凋亡诱导作用研究

目的 :　观察姜黄素 (curcumin)对人的类淋巴母细胞 TK6细胞的增殖抑制和凋亡诱导作用 ,为进一步探讨其抗诱变、抗肿瘤的机制和开发利用提供理论依据。方法 :　姜黄素处理TK6细胞 ,采用 MTT比色分析法、3 H- Td R掺入法和细胞周期测定以及细胞超微结构观察和流式细胞分析。结果 :　 2 0 μmol/L的姜黄素处理细胞 2 4 h,即可产生显著的细胞增殖抑制作用和凋亡诱导作用 ,且有剂量和时间依赖性。姜黄素引起细胞的凋亡主要在细胞周期的 DNA合成期 (S期 ) ,将细胞周期阻滞在静止期和 DNA合成前期 (G0 /G1期 )。结论 :　姜黄素对 TK6细胞具有显著的增殖抑制作用和凋亡诱导作用。     

活性维生素D_3对大鼠肾小球系膜细胞增殖影响

目的探讨活性维生素D3(1,25(OH)2D3)对大鼠系膜细胞增殖的影响。方法体外培养大鼠系膜细胞,随机分为正常对照组、表皮生长因子(EGF)(10 ng/m L)组、1,25(OH)2D3(10-8mol/L)组、EGF联合1,25(OH)2D3组,采用MTT法检测各组干预48 h后对大鼠系膜细胞增殖的影响,流式细胞术检测各组干预48 h后大鼠系膜细胞周期分布情况,免疫荧光法检测各组干预48 h后,大鼠系膜细胞中增生性细胞核抗原(PCNA)的表达情况。结果与正常对照组比较,EGF组能明显促进大鼠系膜细胞增殖,G0/G1期细胞减少,S期及G2/M期细胞增加,且PCNA表达增加,1,25(OH)2D3组大鼠系膜细胞增殖受抑制,G0/G1期细胞增加,S期及G2/M期细胞减少,且PCNA表达降低;与EGF组比较,EGF联合1,25(OH)2D3干预组系膜细胞增殖受抑制,G0/G1期细胞增多,S期及G2/M期细胞减少,且PCNA表达降低。结论 1,25(OH)2D3可阻滞大鼠系膜细胞的细胞周期并抑制大鼠系膜细胞的增殖和EGF对大鼠系膜细胞的促增殖作用。     

植物雌激素对乳腺癌细胞MCF-7增殖的影响

目的 :　探讨植物雌激素大豆异黄酮 (genistein,GS)和玉米赤霉烯酮 (zearalenone,ZEA)对乳腺癌细胞株 MCF- 7增殖的影响。方法 :　雌激素依赖性 MCF- 7细胞在 DMEM培养液(含小牛血清 1 0 % )中采用开放式单层贴壁培养 ,于加受试物前 5 d将细胞用 PBS洗涤后改为无酚红高糖 DMEM(含 5 %经活性碳 -葡聚糖苷处理的胎牛血清 ,CDT- FBS)培养 ,实验设溶剂对照、雌激素对照、抗雌激素对照及两种受试物各四个剂量组 ,采用噻唑蓝 (MTT)法、3H- Td R掺入法及流式细胞术对 MCF- 7细胞的增殖情况进行分析。结果 :　与溶剂对照组相比较 ,GS(96μmol/ L,2 4h)可明显抑制 MCF- 7细胞增殖和细胞 DNA合成 ,并将细胞周期阻滞在 G2 / M;8μmol/ L GS处理96 h也能产生类似的抑制效果。 96 nmol/ L ZEA处理 2 4 h可明显促进 MCF- 7细胞增殖和细胞DNA合成 ,并将细胞周期由 G0 / G1向 S期推进 ,提高细胞分裂增殖指数。结论 :　ZEA和 GS均属环境雌激素 ,但对乳腺癌细胞 MCF- 7增殖产生的影响不同 ,ZEA可促进 MCF- 7增殖 ,而 GS能够抑制 MCF- 7细胞的增殖 ,即 GS具有用于癌症预防及有关保健食品开发的价值     

番茄红素对前列腺癌细胞PC-3增殖和细胞周期的影响

目的研究番茄红素对体外培养的雄激素非依赖性前列腺癌细胞PC-3的抑制作用及其可能的作用机制。方法采用MTT法和H3-TdR掺入法观察番茄红素对癌细胞增殖的影响,流式细胞仪观察同步化的细胞经番茄红素作用后细胞周期及凋亡的变化,RT-PCR检测cyclin D1、bcl-2、bax的mRNA的表达的变化。结果番茄红素抑制PC-3细胞的增殖和DNA合成、诱导其凋亡、改变细胞周期分布(使G0/G1期细胞增多、而S期和G2/M期细胞减少)。RT-PCR结果显示,cyclin D1和bcl-2的mRNA表达水平下调,而bax的mRNA表达水平上调。上述作用呈剂量效应关系。结论番茄红素可诱导PC-3细胞凋亡、改变细胞周期分布、影响cyclin D1、bcl-2、bax的mRNA的表达,从而抑制肿瘤细胞增殖。     

外源性TGF-β_1抗体对SiO_2刺激的小鼠肺成纤维细胞增殖和细胞周期的影响

目的探讨外源性转化生长因子β1(TGF-β1)抗体对SiO2刺激的小鼠肺成纤维细胞生长的抑制作用及细胞周期的影响。方法实验分为3组:空白对照,SiO2(100 mg/L)组,SiO2(100 mg/L)+TGF-β1(1.0μg/ml)抗体组。应用MTT法检测小鼠肺成纤维细胞增殖的变化,流式细胞仪检测细胞周期。结果各处理组细胞增殖差异有统计学意义(P0.05),SiO2组细胞增殖最明显,其次为SiO2+TGF-β1抗体组;与空白对照组比较,SiO2组G0/G1期细胞百分率显著降低,S期及G2/M期细胞百分率增加,差异均有统计学意义(P0.05),经TGF-β1抗体处理后,G0/G1期细胞百分率与SiO2组比较明显增加,S、G2/M期细胞百分率显著降低(P0.05)。结论 TGF-β1抗体可能通过阻止SiO2刺激的小鼠肺成纤维细胞由G0/G1期进入S期,从而抑制肺成纤维细胞增殖。     

金雀异黄素抑制人胃癌细胞增殖与G_2/M期阻滞作用的体外研究

目的 :　研究金雀异黄素 (genistein,Gen)对人胃癌 SGC-790 1细胞增殖抑制作用及对细胞周期的影响。方法 :　采用3H-Td R掺入液闪计数法观察 Gen对人胃癌细胞增殖影响 ,流式细胞仪分析细胞周期分布 ,免疫组化和 Western Blotting分别检测 cyclin B、P2 1 waf1 / cip1 蛋白表达情况。结果 :　 Gen对胃癌细胞生长有显著抑制作用 ,使细胞生长停滞于 G2 / M期 ,并使细胞cyclin B、P2 1 waf1 / cip1蛋白表达增加 ,且呈剂量 -效应关系。结论 :　 Gen在此剂量下抑制胃癌细胞增殖、诱导 G2 / M期阻滞与其稳定 cyclin B蛋白和上调 P2 1 waf1 / cip1 蛋白表达有关     

苦参素对人食管癌Eca-109体外细胞增殖活性影响初探

目的:探讨苦参素对人食管癌Eca-109细胞增殖活性的影响.方法:运用MTT和流式细胞术分别检测细胞增殖率和细胞周期变化.结果:苦参素对Eca-109细胞持续作用48h后,细胞增殖率由100%降为9.64%(P<0.001);细胞周期结果显示,G0/G1期细胞明显增多,S期细胞明显减少,与阴性对照组和阳性对照组比较,差异具有显著性(P<0.05).结论:苦参素对人食管癌Eca-109细胞增殖活性具有抑制作用,其作用机制与细胞周期阻滞于G0/G1期有关.     

体外培养人卵巢癌细胞株SKOV-3、SKOV-3/DDP并探讨卵巢癌顺铂耐药机制

目的:通过对人卵巢癌细胞株SKOV-3、SKOV-3/DDP体外培养,探讨卵巢癌顺铂耐药机制。方法:用四甲基偶氮唑蓝(MTT)法、流式细胞仪测定SKOV-3、SKOV-3/DDP细胞顺铂耐药指数及细胞周期变化,并分析细胞内Bcl-2蛋白及细胞膜上Fas、Fas-L表达情况。结果:相对于SKOV-3细胞;SKOV-3/DDP细胞顺铂耐药指数约为4倍,其细胞周期主要分布于G0/G1期、S期,细胞内Bcl-2蛋白表达及细胞膜Fas、Fas-L水平明显升高。结论:细胞主要分布于潜伏期、细胞内凋亡抑制蛋白Bcl-2水平升高及细胞膜Fas、Fas-L表达增加,可能是卵巢癌顺铂耐药细胞株SKOV-3/DDP顺铂耐药的部分机理。     

降钙素对人成骨样细胞增殖与细胞膜流动性的影响

为了解降钙素（ＣＴ）对体外培养的人成骨样细胞ＯＳ－７３２增殖与细胞膜流动性的影响,并与１,２５（ＯＨ）２Ｄ３的作用进行比较,在培养液中添加１ＩＵ／ｍｌ ＣＴ前后观察ＤＮＡ合成速率、细胞增殖、细胞周期和细胞膜流动性的变化,结果表明ＣＴ有明显促进ＯＳ－７３２细胞增殖和ＤＮＡ合成的作用,使Ｇ２＋Ｍ细胞所占的百分比增加,而Ｓ期细胞的百分比下降。１,２５（ＯＨ２）Ｏ３可使细胞膜流动性增加,而１ＩＵ／ｍｌ Ｃ     

Mechanisms involved in Jurkat cell death induced by oleic and linoleic acids

BACKGROUND & AIMS: Previous study from our laboratory showed the toxicity of oleic (OA) and linoleic acids (LA) on Jurkat and Raji cells and human lymphocytes in vitro. The mechanisms involved in the toxicity induced by OA and LA on Jurkat cells were determined in vitro. METHODS: Jurkat cells were treated in the presence of OA and LA (25, 50, 100 and 200muM). The parameters investigated were: triglycerides and cholesterol ester concentrations determined by enzymatic assay, activation of peroxisome proliferator activated receptor (PPAR) by electrophoretic mobility shift assay, caspase 3, 6 and 8 activities by spectrofluorometric assay, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma production by enzyme linked absorbent assay (ELISA), expression of pro- (Bax) and anti- (Bcl-2) apoptotic genes by real time polymerase chain reaction and expression of pleiotropic genes by macroarray technique RESULTS: Evidence is presented herein that the increase in triglycerides concentrations induced by OA is more pronounced than that caused by LA in Jurkat cells. Importantly, triglycerides accumulation may be a mechanism to protect lymphocytes against the toxicity induced by fatty acids. Both fatty acids raised PPAR activation, caspase 3 and 6 activities and TNF-alpha production. LA in toxic concentrations modulated the expression of genes related to cell cycle, apoptosis, proliferation, oxidative stress, and cytokine receptors. CONCLUSION: The findings reported herein support the cell death induced by OA and LA involved triglycerides accumulation, PPAR activation, caspase 3 and 6 activities and TNF-alpha production.     

磷酸氯喹对心肌细胞膜损伤的保护作用

通过体外培养心肌细胞探讨磷脂酶 A2 (phospholipase A2 ,PL A2 )、脂多糖 (lipopolysaccharide,L PS)对心肌细胞膜的损伤及其磷酸氯喹 (chloroquine phosphate,CQP)对心机细胞膜的保护效应。实验结果表明 ,PL A2 、L PS在体外和培养心肌细胞一起孵育 ,能引起心肌细胞膜脂质过氧化损伤加重 ,降低细胞膜流动性 ,增加细胞膜通透性。L PS和体外培养心肌细胞一起孵育 ,可导致 PL A2 活性显著升高 ,细胞内脂质过氧损伤加重 ,细胞膜通透性增加 ,细胞膜流动性降低。而CQP可减轻心肌细胞膜的脂质过氧化损伤 ,抑制 PL A2 活性 ,改善细胞膜流动性 ,保护心肌细胞膜 ,减轻心肌细胞损伤     

Physiological Levels of Resveratrol Metabolites are Ineffective as Anti-Leukemia Agents Against Jurkat Leukemia Cells

Dietary resveratrol is metabolically transformed in vivo by the intestine and liver to produce resveratrol glucuronides and sulfates in humans. Little is known about the anticancer activities of these metabolic products. The majority of in vitro studies have investigated effects of resveratrol aglycone at supraphysiological levels. Physiological levels of resveratrol-3-O-glucuronide, resveratrol-4′-O-glucuronide, and resveratrol-3-O-sulfate, the major in vivo metabolites of dietary resveratrol, were evaluated as anticancer agents against Jurkat T leukemia cells. Propidium iodide was use to measure cell death and changes in cell cycle, and the mitochondrial membrane dye JC-1 was used to measure changes in mitochondrial membrane potential by flow cytometry. PKH67 was used to evaluate changes in proliferation of the cells by flow cytometry. Jurkat cells were exposed to 0, 2.5, 5, 10, 15, and 20 μM of each resveratrol metabolite, which are concentrations achievable in vivo. None of the resveratrol metabolites were able to kill Jurkat T leukemia cells or alter cell cycle or proliferation at these concentrations. Only resveratrol-3-O-sulfate induced depolarization of mitochondrial membranes but without induction of cell death. These results suggest that the in vivo transformation of resveratrol to these glucuronide and sulfate metabolites renders these agents ineffective against T leukemia cells.     

Acute in vivo elevation of intravascular triacylglycerol lipolysis impairs peripheral T cell activation in humans

BACKGROUND: Previous studies have shown suppressive effects of polyunsaturated fatty acids (PUFAs) on T cell proliferation, but the precise mechanism for this effect has not been fully investigated in vivo in humans. OBJECTIVE: The objective was to determine whether this effect is the result of altered T cell membrane properties and impaired CD3- and CD28-mediated signaling in vivo in humans. DESIGN: Peripheral T cells were isolated from healthy subjects before and 2 h after an intravenous infusion of heparin plus a PUFA-rich lipid emulsion during a euglycemic hyperinsulinemic clamp to induce a 2.5-fold elevation in plasma linoleic acid concentration without significant change in plasma total free fatty acid concentrations. RESULTS: Intravenous infusion of heparin plus the lipid emulsion reduced peripheral T cell membrane fluidity and altered lipid raft organization, both of which were associated with reduced T cell proliferation after stimulation with CD3 plus CD28. Tyrosine phosphorylation of linker of activated T cells and activation of protein kinase B in T cells were also impaired without a reduction in T cell receptor expression. In addition, acute PUFA elevation was associated with a reduction in T cell membrane cholesterol exchange with the cellular milieu ex vivo. CONCLUSIONS: A selective increase in plasma linoleic acid concentration and in intravascular lipolysis has a suppressive effect on peripheral T cell CD28-dependent activation, and this effect is associated with changes in plasma membrane properties. Our results have important implications for nutritional therapy in patients at high risk of septic complications and may also be of relevance to postprandial lipid metabolism disorders such as insulin resistance and type 2 diabetes.     

Assessment and Imaging of Intracellular Magnesium in SaOS-2 Osteosarcoma Cells and Its Role in Proliferation

Magnesium is an essential nutrient involved in many important processes in living organisms, including protein synthesis, cellular energy production and storage, cell growth and nucleic acid synthesis. In this study, we analysed the effect of magnesium deficiency on the proliferation of SaOS-2 osteosarcoma cells. When quiescent magnesium-starved cells were induced to proliferate by serum addition, the magnesium content was 2–3 times lower in cells maintained in a medium without magnesium compared with cells growing in the presence of the ion. Magnesium depletion inhibited cell cycle progression and caused the inhibition of cell proliferation, which was associated with mTOR hypophosphorylation at Serine 2448. In order to map the intracellular magnesium distribution, an analytical approach using synchrotron-based X-ray techniques was applied. When cell growth was stimulated, magnesium was mainly localized near the plasma membrane in cells maintained in a medium without magnesium. In non-proliferating cells growing in the presence of the ion, high concentration areas inside the cell were observed. These results support the role of magnesium in the control of cell proliferation, suggesting that mTOR may represent an important target for the antiproliferative effect of magnesium. Selective control of magnesium availability could be a useful strategy for inhibiting osteosarcoma cell growth.     

Iron deficiency alters the progression of mitogen-treated murine splenic lymphocytes through the cell cycle.

The influence of iron deficiency on the progression of mitogen-treated splenic lymphocytes through the cell cycle was studied in 16 control, 16 pair-fed, 15 iron-deficient (ID) and 16 ID mice that were repleted for up to 3 d (R3). The test and control diets differed only in iron concentrations (0.09 vs. 0.9 mmol/kg). When mice were killed (68 d of feeding), the hemoglobin concentration and liver iron stores of ID and R3 mice were <50% those of control mice (P < 0.05). Iron deficiency did not reduce the percentage of CD3(+) cells, but decreased CD3(+) cells/mg spleen (P < 0.05). In concanavalin A-treated and nonactivated cultures, there were no significant differences among groups in the percentages of cells in resting phase of the cell cycle (G0) to cell cycle initiation phase (G1), DNA synthesis phase (S) and exit from the S phase (G2) to mitosis phase (M) phases. In anti-CD3 and anti-CD3/anti-CD28-treated cultures, higher percentages of lymphocytes from ID and R3 mice than those from control and pair-fed mice were in the G0--G1 phase (P < 0.05). Conversely, lower percentages of activated cells from ID and R mice than those from control and pair-fed mice were in S and G2--M phases (P < 0.05). Incubation of lymphocytes with mitogens decreased the percentages of cells in G0--G1 phase from 90% to 80% in control and pair-fed but not in ID and R3 mice (P < 0.05). In activated cells, indices of iron status negatively correlated with the percentages of cells in G0--G1 (r = -0.306 to -0.597) but positively with those in S (r = 0.166--0.511) and G2--M phases (r = 0.265-0.59; P < 0.05). Data suggest that altered cell cycle progression likely contributes to impaired lymphocyte proliferation usually associated with iron deficiency.     

脂肪酸组成对化学物诱导的结肠癌变危险性机理的研究

大鼠喂以含１５％脂肪,但是脂肪酸组成不一致的５组半合成饲料,实验期给以甲基亚硝基脲诱导结肠肿瘤,观察脂肪组成对处于增生危险的大鼠结肠粘膜的增生细胞核抗原（ＰＣＮＡ）、碱性磷酸酶活性（ＡＬＰ）、膜脂流动性、前列腺素Ｅ２（ＰＧＥ２）及细胞动力学的影响。结果表明：含饱和脂肪酸和单不饱和脂肪酸成分最少而含ｎ－６多不饱和脂肪酸（ＰＵＦＡ）成分最高的第３组,ＰＣＮＡ和ＰＩ标志的Ｓ期细胞数最多,ＡＬＰ活性较高;含ｎ－３ＰＵＦＡ比例最高的第４组,ＰＣＮＡ和ＰＩ标志的Ｓ期细胞数、ＡＬＰ活性及ＰＧＥ２含量最少,而膜脂流动性最好。结果提示,含ｎ－３ＰＵＦＡ较高比例的脂肪酸组成降低由化学物诱导的结肠癌变危险性,其机制之一可能是,通过减少结肠粘膜ＰＣＮＡ、ＰＩ标志的Ｓ期细胞数、ＡＬＰ活性、保护膜脂流动性及减少ＰＧＥ２生成等进行。     

牛乳铁蛋白素对Jurkat细胞和HFL-I细胞生物学特性的影响

目的形态学观察牛乳铁蛋白素(LfcinB)对Jurkat细胞与人胚肺成纤维(HFL-I)细胞作用的程度,验证LfcinB诱导Jurkat细胞凋亡信号转导通路。方法用荧光显微镜观察Hoechst33258染色LfcinB处理过的Jurkat细胞和正常成纤维细胞,与阴、阳性对照组比较;用DNA断裂琼脂糖凝胶电泳分析LfcinB对Jurkat和HFL-I细胞作用差异;用流式细胞术分析Jurkat细胞早期凋亡和线粒体电势电位状况;Westernblotting研究Jurkat细胞接触LfcinB4、6和8h细胞内caspase-3、caspase-8、caspase-9和胞浆中细胞色素C含量的变化。结果在LfcinB作用Jurkat细胞DNA断裂琼脂糖凝胶电泳图中显示为阶梯状凋亡DNALadder,而LfcinB作用的HFL-I细胞无此现象;在荧光显微镜可以看到LfcinB作用过的Jurkat细胞经Hoechst33258染色细胞核呈致密浓染和HFL-I细胞的细胞核呈均匀染色;流式术分析结果为LfcinB作用Jurkat细胞2、4h的凋亡率分别为11.5%和17.8%,线粒体膜电位下降;caspase-3、caspase-9和细胞色素C的免疫印记分析显示,LfcinB能使Jurkat细胞胞浆中的细胞色素C含量增加,被激活caspase-3、caspase-9在胞浆中逐渐增多,对caspase-8没有影响。结论LfcinB诱导Jurkat细胞凋亡,对正常成纤维细胞没有作用;Jurkat细胞接触LfcinB2h以后细胞内caspase-3、caspase-9和胞浆中细胞色素C的含量累积增加,验证了LfcinB通过依赖caspase家族的细胞内信号通路诱导Jurkat细胞凋亡。