二(口恶)英对胰岛素样生长因子Ⅱ表达的调控

目的 探讨二(口恶)英(TCDD)对成骨细胞胰岛素样生长因子Ⅱ(insulin-like growth factor II,IGF-II)基闪表达的影响.方法 应用1×10-9、1×10-8、1×10-7mol/L的TCDD作用于原代培养的大鼠颅盖骨成骨细胞,采用实时定量聚合酶链反应(PCR)和Western印迹杂交分析成骨细胞中IGF-II mRNA及蛋白质的表达.结果 TCDD增加了成骨细胞中IGF-II的基因表达,1×10-9、1×10-8、1×10-7mol/L TCDD组IGF-II mRNA表达水平分别为10.85±0.18、11.82±0.16、11.92±0.18,与对照组(5.06±0.02)比较,差异有统计学意义(P＜0.05);1×10-7 mol/L剂量的TCDD使IGF-II的mRNA和蛋白质的表达分别上调130%和280%.结论 TCDD在基因转录和翻译水平上正性调控成骨细胞内IGF-II基因的表达.     

2,3,7,8-四氯二苯并二噁英对人芳香烃受体和转化生长因子α mRNA表达的影响

目的 研究2,3,7,8-四氯二苯并二嗯英(TCDD)染毒对表皮细胞芳香烃受体(AhR)和转化生长因子(TGF-α)表达的影响.方法 利用半定量逆转录PCR技术(RT-PCR)对24 h TCDD染毒组进行AhR和TGF-α基因表达的测定,并分析两者之间的相互关系.结果 以β-actin为内参照,测定细胞中AhR和TGF-α mRNA表达的相对含量.结果 显示随着TCDD浓度的增高,细胞中AhR mRNA的相对表达量呈现下降的趋势,r=-0.548,各染毒组与对照组相比差异有统计学意义(F=4.124,P=0.021),两两比较7×10-10 mol/L组(0.0620±0.0085)和7×10-9 mol/L组(0.0518±0.0194)与对照组(0.1138±0.9227)之间的差别有统计学意义(t=-3.48,P＜0.05;t=-4.17,P＜0.01),且其相对表达量分别为对照组的55%和45%.TGF-α mRNA的相对表达量随浓度的增加则呈现上升的趋势,相关系数r=0.695(P＜0.01),各组之间差异有统计学意义(F=15.789,P=0.000),在7×10-10 mol/L组(0.1474±0.0390)和7×10-9 mol/L组(0.2133±0.0364)相对表达量与对照组(0.0561±0.0100)的差别具有统计学意义(t=4.49,P＜0.01;t=7.74,P＜0.01),且其相对表达量是对照组的2.6倍和3.8倍.同时对AhR与TGF-α mRNA相对表达水平进行相关分析,两者呈负相关,r=-0.561(P＜0.05).结论 TCDD对细胞增殖过程中可影响基因表达,AhR表现为下调,TGF-α表现为上调,且两基因表达量之间存在负相关关系.     

镉通过类雌激素样作用促进人乳腺癌MCF-7细胞增殖

[目的]研究镉对人乳腺癌MCF-7细胞增殖及雌激素受体(estrogen receptor,ER)蛋白表达水平的影响及其可能机制。[方法]用1×10~(-6)、1×10~(-7)、1×10~(-8)、1×10~(-9)、1×10~(-10)、1×10~(-11) mol/L的17β-雌二醇(后称雌激素)培养不同来源的A、B两株MCF-7细胞4 d,应用CCK8法筛选敏感细胞株并确定雌激素促细胞增殖作用最强的浓度。用1×10~(-6)、1×10~(-7)、1×10~(-8)、1×10~(-9)、10~(-10)、1×10~(-11) mol/L的镉溶液染毒敏感细胞株,确定镉促进细胞增殖的最大浓度。设置阴性对照组(去雌激素培养液)、实验组(1×10~(-8) mol/L镉)、阳性对照组(1×10~(-9) mol/L雌激素),通过克隆形成实验检测镉对MCF-7细胞集落形成能力的影响。利用ER抑制剂(ICI182780)初步探讨镉致细胞增殖的可能机制,增设实验抑制剂组(1×10~(-8) mol/L镉+1×10~(-7) mol/L ER抑制剂)、阳性抑制剂组(1×10~(-9) mol/L雌激素+1×10~(-7) mol/L ER抑制剂),通过CCK8法、流式细胞术和Western blot分别检测各组的细胞增殖率、细胞周期S期比例和ER表达水平。[结果]实验所用B株MCF-7细胞为雌激素敏感细胞株,且当雌激素浓度为1×10~(-9) mol/L时,对MCF-7细胞的促进增殖作用[(203.55±36.65)%]最大(P0.001)。与阴性对照组(100%)相比,1×10~(-8)~1×10~(-10) mol/L镉溶液可促进MCF-7细胞增殖[(163.78±31.90)%~(176.88±10.06)%](P0.001)。1×10~(-8) mol/L镉可促进细胞集落形成(P0.05),增加细胞S期比例(P0.001),提高细胞ER蛋白表达水平(P0.001)。与实验组相比,加入ER抑制剂能够抑制镉对MCF-7细胞的促增殖作用[由(135.17±23.96)%降至(107.66±7.64)%](P0.01),抑制镉诱导的细胞S期比例增加[由(24.17±0.53)%降至(12.36±0.43)%](P0.001),拮抗镉诱导的ER表达增加[由(56.19±3.67)%降至(38.84±1.04)%](P0.05)。[结论]镉可以促进人乳腺癌细胞MCF-7增殖和ER表达,这种作用可被ER抑制剂所抑制,提示镉可能具有雌激素样作用。     

氟虫腈对PC12细胞凋亡及Bcl-2蛋白表达的影响

目的 研究氟虫腈对神经细胞凋亡的影响并初步探讨其神经毒作用机制.方法 设3.13×10~(-6)、1.25×10~(-5)、5.00×10~(-5)mol/L 3个氟虫腈染毒组和1个对照组,用荧光显微镜检测各组PC12细胞凋亡荧光强度并用流式细胞仪对凋亡率进行测定,并对3个染毒组Bcl-2蛋白表达进行荧光强度检测.结果 细胞凋亡荧光强度检测发现,3.13×10~(-6)mol/L染毒组细胞凋亡率为17.00%±0.57%,5.00×10~(-5)mol/L染毒组细胞坏死率为13.95%±1.95%,均明显高于对照组(分别为9.80%±0.30%、3.10%±0.40%),差异均有统计学意义(P<0.05).对Bcl-2蛋白表达含量进行定性检测,结果显示,3.13×10~(-6)mol/L染毒组Bcl-2蛋白表达明显低于对照组,其他染毒组Bcl-2蛋白表达与对照组比较无明显差异.结论 氟虫腈在较低剂量时可能是通过影响Bcl-2蛋白表达来诱导PCI2细胞凋亡,而在较高剂量时可能通过其他非细胞凋亡途径直接致细胞坏死发生.     

氯氰菊酯对人乳腺癌细胞MCF-7增殖的影响

目的通过观察氯氰菊酯(cypermethrin,CYP)对人乳腺癌细胞株MCF-7细胞增值的影响,探讨氯氰菊酯促进细胞增殖的生物学机制。方法 MCF-7细胞在加受试物前7 d改为无酚红完全培养基,以耗尽细胞内储存的雌激素。实验设为6组:无水乙醇(EtOH)溶剂对照组、雌二醇(E2)雌激素对照组、他莫西芬(TAM)抗雌激素对照组及氯氰菊酯3个浓度梯度(1.0×10-7mol/L、1.0×10-8mol/L、1.0×10-9mol/L)实验组。采用CCK8法检测各组细胞的增值情况,采用Hoechst 33342和Propidium Iodide双染荧光显微镜观察氯氰菊酯对雌激素耗尽诱导细胞凋亡作用影响。结果第4、5天细胞增殖实验,与EtOH溶剂对照组相比,除TAM抗雌激素组与EtOH溶剂对照组细胞增殖活力低外,其他4组细胞增殖活力低,依次为E2雌激素对照组、氯氰菊酯1.0×10-7mol/L组、氯氰菊酯1.0×10-8mol/L组、氯氰菊酯1.0×10-9mol/L组,P均<0.05;第3天与EtOH溶剂对照组比,E2雌激素对照组MCF-7细胞增殖活力高,P<0.05,第2天差异无统计学意义。荧光显微镜下见,EtOH溶剂对照组相比和TAM抗雌激素组细胞的凋亡作用明显,后者强于前者,而E2雌激素对照组、氯氰菊酯1.0×10-7mol/L组、氯氰菊酯1.0×10-8mol/L组、氯氰菊酯1.0×10-9mol/L组对细胞凋亡作用不明显。结论氯氰菊酯有可能是通过抑制乳腺癌细胞凋亡而发挥其促进细胞增殖作用的。     

氟化钠对成骨样细胞功能表达影响的研究

目的探讨氟对成骨样细胞功能表达的影响.方法用细胞生长曲线和四唑盐比色(MTT)法测定细胞增殖,对硝基苯基磷酸二钠盐比色(PNPP)法检测碱性磷酸酶(ALP)活性及放免法测定骨钙素分泌量,研究不同浓度(10-7mol/L～10-3mol/L)氟化钠(NaF)对成骨样细胞UMR106增殖和分化功能的影响.结果NaF对UMR106细胞增殖及早期分化呈双向调节作用,主要表现为低浓度促进,高浓度抑制.染氟一定时间后,与对照组相比10-7mol/L、10-6mol/L、10-5mol/L NaF组细胞增殖及ALP活性均增加(P＜0.05),而10-4mol/L、10-3mol/L NaF组则表现为抑制细胞增殖及ALP活性(P＜0.05).但是,NaF对UMR106细胞晚期分化呈单一作用,上述不同浓度的NaF均可促进骨钙素分泌,与对照组相比有统计学意义(P＜0.05).而且,随NaF浓度升高骨钙素分泌量增加.结论NaF对成骨样细胞的增殖呈双向调节,但对其分化因暴露时间和浓度不同而呈多样性.     

有机碘和无机碘对人甲状腺细胞凋亡的影响

目的 研究有机碘和无机碘对体外培养的人甲状腺细胞形态和凋亡相关蛋白Bcl-2和Bax表达的影响.方法 取正常人甲状腺细胞进行培养,分别设对照(培养液)组和KI组(碘浓度分别为10-7、10-5、10-3 mol/L)及3,5-二碘酪氨酸(DIT)组(碘浓度分别为10-7、10-5、10-3 mol/L),分别加入相应浓度用培养液稀释的DIT和KI溶液,继续培养48 h.采用免疫组织化学技术测定细胞凋亡相关蛋白表达的情况.结果 与对照组相比,10-5、10-3 mol/L KI组和10-3 mol/L DIT组甲状腺细胞Bcl-2表达量较低,10-5、10-3 mol/L KI及DIT暴露甲状腺细胞Bax表达量较高,差异均有统计学意义(P＜0.05,P＜0.01);且随着KI或DIT暴露浓度的升高,甲状腺细胞Bcl-2表达量呈下降趋势,Bax表达量呈上升趋势.与相同剂量KI组相比,10-5、10-3 mol/L DIT组甲状腺细胞Bcl-2表达量较高,Bax表达量较低,差异均有统计学意义(P＜0.05,P＜0.01).结论 高碘可引起甲状腺细胞形态的改变并增强其细胞凋亡的发生,且DIT对甲状腺细胞的影响弱于KI.     

氟对原代培养的大鼠成骨细胞分化相关指标的影响

目的研究氟对原代培养的大鼠成骨细胞的细胞活性及对成骨细胞分化相关指标的影响。方法取出生24 h内的大鼠胎鼠的颅盖骨,采用胰酶和Ⅱ型胶原酶交替消化的方法获得原代成骨细胞。分别加入终浓度为0(对照组)、10-7、10-6、10-5、10-4mol/L氟化钠进行染毒72 h,采用MTT法检测细胞活性;染氟72 h后,采用Real-time RT-PCR法检测成骨细胞分化标志物COL1A1、ALP和ON mRNA的表达情况;染氟5 d后,采用染色法检测成骨细胞ALP蛋白的表达情况。结果与对照组相比,10-6~10-4 mol/L染氟组成骨细胞存活率及10-7~10-5 mol/L染氟组成骨细胞ALP蛋白的表达水平均较高,差异有统计学意义(P0.05,P0.01);且随着NaF染毒浓度的升高,成骨细胞存活率和ALP蛋白的表达水平均呈先升高后下降的趋势。与对照组相比,各浓度NaF染毒组成骨细胞ALP、ON mRNA的表达水平及10-7、10-6 mol/L NaF染毒组成骨细胞COL1A1 mRNA的表达水平均升高,差异有统计学意义(P0.05,P0.01)。结论本实验条件下,较低剂量的氟可促进大鼠成骨细胞的分化。     

PCB153对INS-1细胞毒性作用及机制

目的 探讨2,2＇,4,4＇,5,5＇-六氯联苯(PCB153)对体外培养胰岛β细胞株(INS-1)细胞毒性作用及机制.方法 PCB153设3个剂量组(1、3、6μmol/L),二甲基亚砜(DMSO)为溶剂对照组,染毒INS-1细胞24 h后,检测细胞存活率、凋亡、活性氧(ROS)水平、Caspase 3和Caspase 12等凋亡相关基因表达水平.结果 3、6 μmol/L PCB153剂量组细胞存活率分别为(80.9±8.7)％和(42.2±4.3)％,与对照组比较均有下降(P＜0.05);3μmol/L PCB153剂量组ROS为(9.2±0.4)、凋亡率为(30.7±3.4)％,6μmol/L PCB153剂量组ROS为(13.7±1.6)、凋亡率为(40.4±1.3)％,与对照组比较,ROS水平和细胞凋亡率均增加(P＜0.05);与对照组比较,3 μmol/L PCB153剂量组Caspase 3及3、6μmol/L PCB153剂量组Caspase 12基因表达水平均有增加(P＜0.05).结论 PCB153可诱导INS-1细胞凋亡,氧化应激和内质网信号通路可能参与PCB153对INS-1细胞的毒性作用.     

氟化钠对大鼠成骨肉瘤细胞增殖活性及碱性磷酸酶活力和骨钙素mRNA表达的影响

目的探讨氟化钠(NaF)对成骨样细胞大鼠骨肉瘤(UMR-106)细胞增殖活性及主要成骨活性标志物碱性磷酸酶(ALP)活力和骨钙素(BGP)mRNA表达的影响。方法将处于对数生长期的UMR-106细胞分别暴露于含终浓度为0(对照)、5×10、10~2、5×10~2、1×10~3、5×10~3、1×10~4、2×10~4、4×10~4、8×10~4μmol/L NaF的DMEM培养液中孵育24、48 h,采用CCK-8检测细胞活性。将处于对数生长期的UMR-106细胞分别暴露于含终浓度为0(对照)、1×10~3、2×10~3、4×10~3μmol/L NaF的DMEM培养液(不含FBS)继续培养12、24、48 h,测定细胞培养液和细胞ALP的活力及细胞中BGP mRNA的表达水平。结果与对照组比较,1×10~3~8×10~4μmol/L NaF染毒24 h和5×10~8×10~4μmol/L NaF染毒48 h时大鼠UMR-106细胞的存活率均较低,差异有统计学意义(P0.05)。5×10、1×10~3、5×10~3、1×10~4、2×10~4μmol/L NaF染毒48 h时大鼠UMR-106细胞的存活率均低于染毒24 h时,差异有统计学意义(P0.05)。各浓度NaF染毒组UMR-106细胞培养液和细胞中ALP活力均较对照组高,除1×10~3μmol/L NaF染毒24 h的细胞培养液和1×103μmol/L NaF染毒48 h的细胞内ALP活力外,差异有统计学意义(P0.05)。随着NaF染毒浓度的升高,UMR-106细胞培养液和细胞中ALP活力均呈上升趋势;随着NaF染毒时间的延长,细胞培养液中ALP的活力呈上升趋势,而细胞中ALP的活力呈先升高后下降的趋势。2×103、4×103μmol/L NaF染毒组UMR-106细胞的BGP mRNA表达水平均较对照组高,差异有统计学意义(P0.05)。随着NaF染毒浓度的升高,UMR-106细胞BGP mRNA的表达水平呈上升趋势;随着NaF染毒时间的延长,UMR-106细胞BGP mRNA的表达水平呈下降趋势。结论在本实验浓度下,NaF对UMR-106细胞的增殖呈抑制作用,但可促进其成骨活性标志物ALP活力及BGP mRNA的表达。     

溴氰菊酯对原代培养大鼠星形胶质细胞存活率和胞内游离钙浓度的影响

目的　观察溴氰菊酯 (DM)对原代培养大鼠神经星形胶质细胞存活率及胞内游离钙离子浓度 ([Ca2 + ]i)的影响。方法　以锥虫蓝染料排斥试验检测细胞染DM后存活率的变化 ;以Fura 2 /AM为荧光指示剂 ,RF 5 30 1PC荧光分光光度计测定细胞内 [Ca2 + ]i的变化。结果　 1× 10 -5mol/L组染毒DM 72h后细胞存活率下降为 91.9% ,与对照组的差异有显著性 (P <0 .0 5 ) ,1× 10 -4mol/L组DM染毒4、12、4 8、72h后细胞存活率分别为 89.0 %、84 .8%、81.2 %和 79.2 % ,差异有显著性 (P <0 .0 1) ;1× 10 -7、1× 10 -6、1× 10 -5mol/L组DM染毒 5min后胞内 [Ca2 + ]i分别上升至 (45 1.4± 4 2 .3)、(5 36 .9± 4 7.5 )、(870 .9± 10 0 .5 )nmol/L ,与染毒前及对照组比 ,差异有显著性 (P <0 .0 1)。以后各染毒组胞内 [Ca2 + ]i逐渐下降 ,15min时 1× 10 -8、1× 10 -7、1× 10 -6、1× 10 -5mol/L染毒组胞内 [Ca2 + ]i分别降至 (12 4 .3± 6 .0 )、(131.3± 19.1)、(118.9± 1.4 )、(136 .6± 3.8)nmol/L ,与染毒前及对照组比差异均有显著性 (P <0 .0 5或P <0 .0 1)。结论　DM在体外可降低神经胶质细胞的存活率并短时升高胞内 [Ca2 + ]i。     

两种有机磷酸酯对人成神经细胞瘤细胞生长与钙摄取的影响

目的　探讨具有迟发性神经毒性的有机磷酸酯 (organophosphates,OPs)对人成神经细胞瘤细胞SH SY5Y的毒性作用。方法　采用四甲基偶氮唑蓝 (MTT)比色法测定SH SY5Y细胞活力 ;用培养的SH SY5Y细胞与45CaCl2 和磷酸三邻甲苯酯 (tri o cresylphosphate ,TOCP)或甲胺磷共育后 ,洗脱 ,裂解 ,经液闪仪计数测定细胞对钙的摄取情况。结果　甲胺磷在较低浓度 (7× 10 -7～ 7× 10 -6mol/L)时可刺激SH SY5Y细胞的生长 ,在 7× 10 -7mol/L浓度时细胞的增长比对照增加了 2 8% ,而在高浓度 (7× 10 -4～ 7× 10 -3mol/L)时抑制细胞生长 ,在 7× 10 -3 mol/L时的抑制率为 6 2 % ;但TOCP只在高浓度 (7× 10 -3 mol/L)时才对细胞的生长有抑制作用 (抑制率为 34% )。TOCP在几乎所有的测试浓度下都对SH SY5Y细胞的钙摄取量有影响 :在低浓度 (1× 10 -9～ 1× 10 -7mol/L)时刺激细胞的钙摄取 ,细胞的钙摄取量最高增加 2 4 1% ,但在高浓度 (1× 10 -6～ 1× 10 -4mol/L)时抑制细胞的钙摄取 ,最高的抑制率达 5 5 %。结论　OPs的神经毒性可能涉及阻遏神经细胞生长和干扰细胞钙平衡。     

The distribution of [14C]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and its effect on the vitamin A content in parenchymal and stellate cells of rat liver

Isolated liver cells from male Sprague-Dawley rats given a single dose of [14C]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 micrograms, 0.9 microCi/kg body wt in corn oil, p.o.) or vehicle only were separated into parenchymal and nonparenchymal cell fractions 4 h, and 1, 4, 7, 25, 50, and 147 d after treatment. Vitamin A content and TCDD-derived radioactivity were estimated in the parenchymal cells and in the stellate cells, which were identified and quantified in these fractions. Similar levels of vitamin A (0.3 +/- 0.4 nmol per million cells or 0.5 +/- 0.7 mumol per liver; values are mean +/- SD for 56 rats) were found in parenchymal cells from both control and TCDD-treated rats. However, while the vitamin A content of stellate cells increased from 14 to 46 nmol per million cells (i.e., from 1.7 to 7.7 mumol per liver) in control rats over the course of the study, stellate cells from TCDD-exposed rats showed no increase in vitamin A level until at least 25 d after exposure and remained at a level about 30% below the controls thereafter. TCDD-derived radioactivity resided mainly in the parenchymal cell compartment, although stellate cells contained more radioactivity per cell. Most of the radioactivity in parenchymal cells was eliminated with a half-life of 13 d, whereas the remainder persisted with an elimination half-life of 70 d. The elimination half-life in stellate cells was estimated to be 52 d. Thus, TCDD inhibited storage of vitamin A in stellate cells until 60-90% of the TCDD-derived radioactivity had been eliminated from the liver.     

Genistein modulates the effects of parathyroid hormone in human osteoblastic SaOS-2 cells

Genistein and parathyroid hormone (PTH) are anabolic agents that stimulate bone formation through their direct actions in osteoblastic cells. In the present study, we aimed to determine whether genistein modulates the actions of PTH in human osteoblastic SaOS-2 cells in an oestrogen-depleted condition. The present results showed that genistein (10(-8) to 10(-6) m) induced alkaline phosphatase (ALP) activity and osteoprotegrin (OPG) expression in SaOS-2 cells in a dose-dependent manner. These effects could be completely abolished by co-treatment with oestrogen antagonist ICI 182780 (7alpha-[9-[(4,4,5,5,5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3,17beta-diol). Genistein (at 1 microM) could stimulate the mRNA expression of receptor activator of NF-kappaB ligand (RANKL). As OPG and RANKL are known to modulate osteoclastogenesis, the ability of genistein to modulate OPG and RANKL expression in SaOS-2 cells suggested that it might modulate osteoclastogenesis through its direct actions on osteoblastic cells. PTH (at 10 nM) stimulated ALP activity, induced RANKL mRNA expression and suppressed OPG mRNA expression in SaOS-2 cells, confirming its bi-directional effects on osteoblastic cells. Pre-treatment of SaOS-2 cells with genistein and oestrogen not only enhanced PTH-induced ALP activity, but also attenuated PTH up regulation of RANKL mRNA expression and PTH down regulation of OPG mRNA expression. Taken together, the present study provides the first evidence that genistein could modulate the actions of PTH in human osteoblastic SaOS-2 cells in an oestrogen-depleted condition.     

三种环境雌激素对卵巢癌PEO4细胞增殖及凋亡相关基因表达的影响

目的　探讨几种常见的环境类雌激素壬基酚 (NP)、双酚A(BisA)和邻苯二甲酸二丁酯 (DBP)对人雌激素受体阳性卵巢癌细胞PEO4增殖和凋亡相关基因PCNA ,bcl 2和baxmRNA及相应蛋白质表达的影响。方法　PEO4细胞在DMEM培养液 (含 1 0 %小牛血清 )中进行常规传代培养 ,实验前将细胞转移至无酚红DMEM(含 5 %活性碳葡聚糖苷处理过的FBS)中继续培养 5d ,收集细胞 ,PBS洗涤后接种于内置血盖片的六孔板或 75ml培养瓶 ,32× 1 0 - 7mol LBisA和NP及 32× 1 0 - 6 mol LDBP对PEO4细胞分别处理 72h ,用半定量RT PCR技术观察其对核增殖抗原PCNA、bcl- 2及baxmRNA表达的影响 ,并用免疫组化方法对结果进行验证。结果　与溶剂对照组相比 ,32× 1 0 - 7mol LNP和BisA对PEO4处理 72h可促进核增殖抗原PCNA和抗凋亡基因bcl 2mRNA表达 ,但对baxmRNA的表达没有影响 ;DBP可促进PCNA的表达 ,但对bcl 2和bax的表达均没有明显的影响。随后进行的免疫组化实验结果显示 ,NP和BisA可促进Bcl 2蛋白的表达 ,并抑制Bax蛋白的表达。结论　对PCNA和bcl 2表达的影响可能是环境类雌激素调节卵巢癌细胞PEO4增殖和凋亡的重要机制之一。     

双酚A等化学物对耗尽内源性雌激素诱导T47D细胞凋亡的影响

目的　在乳腺癌细胞株T4 7D细胞内采用雌激素耗尽的方法诱导其发生凋亡 ,然后观察对 壬基酚 (n 4 noniphenol,NP)、双酚A (bisphenolA ,BisA )和邻苯二甲酸酯二丁酯(dibutylphthalate ,DBP)对这种凋亡的影响 ,探讨环境雌激素是否能够抑制雌激素耗尽所诱导的细胞凋亡作用。方法　T4 7D细胞在达克尔培养液 (DMEM ,含 10 %小牛血清 )中进行常规传代培养 ,于实验前 4d将细胞用磷酸盐缓冲盐水洗涤后改为在无酚红DMEM培养 ,目的是耗尽细胞内源性雌激素。实验设溶剂对照、雌激素对照 (E2 )、抗雌激素他莫昔芬 (TAM)对照及 3个试验组 ,采用流式细胞术 ,琼脂糖凝胶电泳和HE染色镜检观察环境雌激素对雌激素耗尽所诱导的细胞凋亡的影响。结果　流式细胞仪分析结果表明 ,雌激素耗尽能够诱导T4 7D细胞发生凋亡 ,凋亡率为 34 5 % ,此时往培养液中分别加入 32× 10 -7mol/LNP和 32× 10 -7mol/LBisA ,对细胞处理 72h ,可显著抑制细胞凋亡作用 (凋亡率分别变为 2 0 1%和 16 5 % ) ;琼脂糖凝胶电泳显示 ,细胞凋亡特有的DNA ladder消逝 ;HE染色镜检表明 ,细胞出现活跃核分裂相。加入 5× 10 -7mol/LTAM可加重细胞的凋亡现象 (细胞凋亡率为 5 5 6 % )。结论　NP和BisA均可抑制雌激素耗尽所诱导的T4 7D细胞的凋亡作用 ;     

Influence of short-term protein malnutrition of mice on the phenotype and costimulatory signals of lymphocytes from spleen and Peyer's patches

The objective of this study was to investigate the impact of short-term protein malnutrition (PM) on immunoglobulin A (IgA) production and on the number and phenotype of lymphocytes in Peyer's patches (PP) and in the spleen. Balb/c mice were fed for 4, 7, or 10 d with a protein-deficient diet (0.1% protein). We determined B lymphocytes (CD40(+)), T lymphocytes (CD3(+)), T-helper (CD4(+)), and T-suppressor (CD8(+)) cells and the expression of costimulatory signals B7.1 (CD80) and B7.2 (CD86) on B cells and their counter receptors CD28 and CTLA-4 on T cells by fluorescence-activated cell-sorting analysis. Luminal IgA concentration in the small intestine was determined by an enzyme-linked immunosorbent assay. Four days of PM caused a significant reduction in the number of mononuclear cells in the spleen (5.6 x 10(7) +/- 1 x 10(7) versus 2. 4 x 10(7) +/- 0.5 x 10(7), P < 0.001) and the PP (13 x 10(6) +/- 3 x 10(6) versus 8.6 x 10(6) +/- 2 x 10(6), P < 0.01). There was a relative increase of T cells in the spleen and a relative increase of B cells in the PP. Luminal IgA content of small intestine was significantly reduced after 4 d of PM (242 +/- 55 microg versus 173 +/- 39 microg, P < 0.05) and remained at about this level until day 10 of PM. Four days after PM, the costimulatory signals B7.1 and B7. 2 on B cells were upregulated in the PP but markedly downregulated in the spleen, which was inversely related to the expression of the counter receptor CD28 on T-helper cells. We conclude that short-term PM increases the activation of B cells in the PP but reduces the relative number and activation state of splenic B cells. Only 4 d of PM caused a systemic and intestinal immunodepression, as indicated by a markedly decreased content of mononuclear cells in the PP and the spleen.     

Flow cytometric analysis of lymphocyte subpopulations in the spleen and thymus of mice exposed to an acute immunosuppressive dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

The objective of the present studies was to determine if acute exposure to an immunotoxic dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces alterations in the expression of lymphocyte surface markers as measured by multiparameter flow cytometry. The immunotoxicity of a single oral dose of TCDD was assessed by the anti-SRBC PFC response; an ED50 of 0.74 micrograms/kg was determined. Subpopulations in the spleen and thymus of C57B1/6 mice were analyzed 2 days following exposure to 2 micrograms/kg TCDD. In addition, splenic lymphocyte subsets were examined on Days 1-4 following SRBC challenge of mice treated with 0, 2, or 5 micrograms/kg TCDD. T and B cells were identified by single parameter analysis of Thy 1.2 and Ig expression. T cell subsets were defined by dual parameter analysis of CD4 and CD8 expression. In TCDD-treated mice, the percentage and the total number of double-positive CD4+ CD8+ thymocytes were significantly decreased while the percentage but not the total number of double-negative CD4- CD8- thymocytes was significantly increased. No changes in the percentage or total number of single positive (CD4+ CD8- or CD4- CD8+) thymocyte subsets were observed. In contrast to the thymus, lymphocyte subsets in the spleen were not significantly altered in percentage or total number 2 days following acute TCDD exposure. When splenic lymphocytes were analyzed daily following SRBC challenge, Ig+, Thy 1.2+, and CD4+ CD8- subpopulations remained relatively unchanged in both control and TCDD-treated animals. A small but significant decrease in the percentage of CD4- CD8+ T cells was observed on Day 3 in mice treated with 2 or 5 micrograms/kg TCDD when compared to that of vehicle-treated mice. The total number of CD4- CD8+ splenocytes was also significantly lower in the 5-micrograms/kg group on Day 3. However, this effect appeared to result from an elevation of the CD4- CD8+ subset in the controls rather than from a reduction in the TCDD-treated groups. Double-positive (CD4+ CD8+) lymphocytes were not detected in either control or TCDD-treated spleens. These results indicate that an acute dose of TCDD which reduced the splenic anti-SRBC response by 65-80% did not cause detectable changes in major splenic lymphocyte subpopulations. This is an important finding from the standpoint of utilizing lymphocyte subset analysis to screen for potential immunotoxic effects of TCDD. Specifically, the absence of subset changes does not preclude the presence of functional immunosuppression.