氟铝单独及联合染毒对MC3T3-E1细胞增殖能力及细胞周期的影响

目的 观察不同浓度的氟、铝对体外培养的小鼠颅顶前成骨细胞亚克隆14(MC3T3-E subclone 14)增殖及细胞周期的影响,以进一步阐明地方性氟中毒机制提供实验依据.方法 以10~(-9)～10~(-3)mol/LNaF染毒MC3T3-E1细胞,同时以50μmoI/L NaF及5μmol/L AICl3单独或者联合染毒MC3T3-E1细胞,培养72 h.应用CCK-8(cell counting kit-8)观察MC3T3-E1细胞增殖能力的影响;采用流式细胞术检测MC3T3-E1细胞周期变化情况.结果 氟无显著促进MC3T3-E1细胞增殖的作用,较高浓度的氟(1 mmol/L)抑制MC3T3-E1细胞增殖(P<0.01).氟铝联合染毒显著刺激MC3T3-E1细胞增殖(P<0.01):G_2/M期细胞明显增多,增殖指数(PI)升高(P<0.05),DNA相对含量增高(P<0.05).结论 氟对MC3T3-E1细胞无显著促增殖作用,氟铝联合能显著提高MC3T3-E1的增殖能力,使G_2/M期细胞明显增多,促进成骨细胞的增殖分裂,细胞处于活跃生长状态.

Abstract：

Objective To explore the effects of different concentrations of fluoride, aluminum alone and in combination exposure on mice parietal bone cell subclone 14 (MC3T3-E subclone 14), and to elucidate the pathogenesis of endemic fluorosis. Methods The proliferation of MC3T3-E1 cells exposed to 10~(-9)-10~(-3)mol/L NaF alone, 50 μmol/L NaF and 5 (μmol/L AlCl_3 aloneand in combination ,was measured by CCK-8, and the change of cell cycle was measured by flow cytometry after treatment with various concentrations of fluoride and aluminum. Results Fluoride alone did not promote osteoblast MC3T3-E1 cells proliferation, higher concentration fluoride inhibited MC3T3-E1 cells proliferation. Fluoride and aluminum combined exposure (50 μmol/L NaF +5 μmol/L AlCl_3) stimulated proliferation of MC3T3-E1 cells (P<0.01 ).and significantly induced increase of G_2/M phase, PI (proliferation index) and DNA relative content. Conclusion Fluoride does not promote the MC3T3-cells proliferation, aluminium plus fluoride may increase MC3T3-E1 cells proliferation and affect the cell cycle, which can significantly increase the number of G_2/M phase cells.     

氟对MC3T3-E1细胞矿化能力的影响

目的探讨氟对小鼠成骨细胞系MC3T3-E1细胞矿化的影响。方法在4×104个/孔的MC3T3-E1细胞培养液中加入0～10-4mol/L的氟化钠,于培养第14、28天,采用茜素红染色法检测矿化结节的形成情况。结果染毒14天,各氟化钠染毒MC3T3-E1细胞OD值间比较,差异均无统计学意义。与对照组相比,染氟28天时,10-7、10-6mol/L氟化钠染毒组MC3T3-E1细胞OD值较高,10-4mol/L氟化钠染毒组MC3T3-E1细胞OD值较低,差异均有统计学意义(P0.05)。且随着氟化钠染毒剂量的升高,MC3T3-E1细胞OD值呈先升高后降低的趋势。结论低浓度氟化钠(10-7～10-6mol/L)可促进MC3T3-E1细胞的矿化,而高浓度氟化钠则可抑制MC3T3-E1矿化结节的形成。氟对成骨细胞矿化过程起双向调节作用。     

慢性氟砷联合暴露对大鼠骨骼Runx2及其下游相关因子的影响

目的 研究慢性氟砷联合暴露对大鼠骨骼代谢Runx2及其下游相关因子的影响.方法 将54只8周龄清洁级SD大鼠按析因设计方法随机分成9组,每组6只,雌雄各半,分为对照组、低氟组、高氟组、低砷组、高砷组、低氟低砷组、低氟高砷组、高氟低砷组及高氟高砷组.使用氟化钠(NaF,低氟组5mg/kg、高氟组20 mg/kg)和亚砷酸钠(NaAsO2,低砷组2.5 mg/kg、高砷组10 mg/kg)灌胃染毒6个月;测定大鼠骨骼中Runx2、基质金属蛋白酶9(MMP-9)、成骨相关转录因子(Osterix)核因子-κB受体活化因子配基(RANKL)蛋白浓度.结果 对照组、低砷组、高砷组无氟斑牙出现,低氟组、高氟组氟斑牙比例(分别为5/6、6/6)与对照组(O)相比,差异均有统计学意义(x2=8.57、12.00,p＜0.05).骨氟含量随着氟染毒剂量的增加而升高,无氟染毒组[对照组、低砷组、高砷组的几何均数(最小值～最大值)分别为0.005(0.003 ～0.009)、0.006(0.003～0.021)、0.007(0.002～0.100)mg/g]、低氟组[低氟组、低氟低砷组、低氟高砷组分别为3.395(2.416 ～5.871)、3.809(1.471 ～7.799)、3.853(1.473～6.732)mg/g]、高氟组[高氟组、高氟低砷组、高氟高砷组分别为70.086(46.183～ 131.927)、69.925 (40.503 ～ 96.183)、67.950(52.622 ～ 89.487) mg/g]组间比较差异有统计学意义(P＜0.05).骨砷含量随着砷染毒剂量的增加而升高,其中低砷组[低砷组、低氟低砷组、高氟低砷组分别为7.195(5.060 ～9.860)、6.518(2.960 ～ 12.130)、6.970(3.400 ～9.730)μg/g]、高砷组[高砷组、低氟高砷组、高氟高砷组分别为8.823 (5.760 ～ 10.840)、9.470(7.230～12.860)、8.321(2.420 ～17.540)μg/g]浓度均高于无砷染毒组[对照组、低氟组、高氟组分别为1.785(0.300～3.750)、2.226(1.410～3.980)、2.030(1.040 ～3.850) μg/g],差异有统计学意义(P＜0.05),而低砷组与高砷组骨砷含量差异无统计学意义(P＞0.05).氟与Runx2、MMP-9、Osterix、RANKL蛋白含量间呈正相关(氟染毒量与蛋白含量间相关系数分别为0.647、0.354、0.582和0.613),骨氟含量与蛋白含量间相关系数分别为0.559、0.387、0.487、0.525,P值均＜0.01,砷染毒剂量与Runx2呈负相关(相关系数为-0.527,P＜0.05),与MMP-9、RANKL、Osterix无相关关系(P＞0.05).氟砷联合染毒与Runx2、MMP-9、RANKL、Osterix蛋白含量具有交互效应(F值分别为3.88、15.66、2.92、6.42,P值均＜0.05).结论 氟砷联合暴露对大鼠骨骼代谢Runx2及其下游相关因子的交互作用表现为拮抗作用.     

亚慢性氟中毒对大鼠骨组织Runx2 mRNA表达的影响

目的观察亚慢性氟中毒对大鼠骨组织中转录因子Runx2 mRNA表达的影响,探讨Runx2在氟骨症发生中的可能作用。方法 40只Wistar大鼠按体重随机分成4组,染氟组大鼠饮用含氟50、100、150 mg/L的自来水,对照组大鼠饮用自来水;3个月后测定大鼠血清及骨组织氟含量,并采用SYBR Green Real-time RT-PCR法检测骨组织中Runx2 mRNA表达情况,同时观察骨组织病理学改变。结果 HE染色显示染氟组大鼠呈现骨皮质增厚、骨小梁增多等骨硬化表现;染氟组大鼠骨组织中Runx2 mRNA表达显著高于对照组,且随染毒剂量升高,表达亦逐渐升高。结论过量的氟可能通过增加骨组织Runx2基因表达水平来促进间充质干细胞向成骨细胞系分化,使骨形成增加。     

氟和铝对小鼠胚胎成骨细胞骨保护蛋白和核因子κB受体活化因子配体mRNA表达的影响

目的观察氟和铝对小鼠胚胎成骨细胞株(MC3T3-E1)骨保护蛋白(OPG)和NF-κB受体活化因子配体(RANKL)mRNA表达的影响。方法 在细胞培养液中加入50μmol/L氟化钠或/和5μmol/L氯化铝;72 h后提取细胞总mRNA,用RT-PCR方法分析MC3T3-E1细胞OPG和RANKL的mRNA表达,进行半定量分析。结果与对照组比较,氟铝联合染毒可显著提高MC3T3-E1细胞OPG mRNA的表达(P<0.05),两者具有协同作用(P<0.05),但氟铝联合染毒对MC3T3-E1细胞RANKL mRNA的表达水平无显著改变;因此,与对照组相比,氟铝联合染毒组RANKL/OPG比值显著降低(P<0.05)。结论氟铝联合可能通过增加成骨细胞OPG基因表达水平来抑制破骨细胞的分化和成熟,从而抑制骨吸收,使骨形成大于骨吸收。     

游离脂肪酸对3T3-L1细胞分化及insig2 mRNA表达的影响

目的 观察游离脂肪酸对3T3-L1细胞分化及insig2 mRNA基因表达的影响,进一步了解影响3T3-L1细胞分化成熟及insig2表达的因素,探讨insig2基因在脂肪细胞分化与代谢中的作用.方法 将3T3-L1细胞分为高脂组与对照组,分别置于低糖高脂与低糖环境中分化培养12 d,油红O染色计算染色阳性细胞面积,RT-PCR法测定insig2、ap2 mRNA的表达.结果 分化培养12 d后,油红O染色显示两组细胞均得到明显分化,但高脂组油红O染色阳性细胞面积较对照组明显升高(P<0.05).分化后,两组细胞ap2、insig2表达水平均较分化前明显升高(P>0.05),且高脂组较对照组上调明显(P<0.05). 结论 游离脂肪酸可促进3T3-L1细胞分化成熟与insig2表达;insig2在细胞内的表达水平可能与脂肪细胞成熟及分化程度相关.     

MCHR1基因沉默对3T3-L1细胞诱导分化的影响

摘要:目的　运用RNA干扰技术沉默黑色素浓集激素受体1（Melanin-Concentrating Hormone Receptor 1,MCHR1）基因的表达,观察其对3T3-L1细胞诱导分化的影响,为肥胖症的基因治疗提供新思路。方法　用含绿色荧光蛋白的重组载体sh-MCHR1,通过脂质体法转染3T3-L1细胞。细胞诱导分化的同时用黑色素浓集激素（Melanin-Concentrating Hormone,MCH）干预,并在不同时点对脂滴进行油红O染色。采用RT-PCR以及Western blot分别检测过氧化物酶体增殖物激活受体γ（Peroxisome Proliferator-Activated Receptor γ,PPAR γ）、CCAAT/增强子结合蛋白-α（CCAAT/ Enhancer-Binding Protein α,C/EBPα）和脂肪酸结合蛋白2（adipocyte protein 2,ap2）mRNA和蛋白的表达。结果　重组载体sh-MCHR1成功转染;与阴性转染组相比,sh-MCHR1转染组d 10后,油红O 染液相对OD510值显著下降（P＜0.05）;PPARγ、C/EBPα和ap2 mRNA的表达量分别在d 6、d 8和d 8后显著下降（P＜0.05）;3者蛋白的表达量均在d 8后显著下降（P＜0.05）。结论　shRNA沉默MCHR1基因可减缓3T3-L1细胞诱导分化,其可能通过抑制PPARγ、C/EBPα和ap2的表达而实现。     

甲醛、苯单独和联合致NIH/3T3细胞转化的研究

目的探讨苯、甲醛单独和联合致NIH/3T3细胞转化活性。方法采用细胞灶法观察甲醛、苯单独和联合致NIH/3T3细胞的转化作用。结果苯、甲醛及苯和甲醛联合染毒组均有细胞转化灶形成,其形成率均呈现剂量依赖关系。联合作用分析显示,苯25μg/ml 甲醛25μg/ml、苯25μg/ml 甲醛50μg/ml、苯50μg/ml 甲醛25μg/ml、苯50μg/ml 甲醛50μg/ml,两化合物联合作用转化率实测值分别为3.6%、5.8%、5.7%和7.6%。而预期转化率分别为3.5%、5.6%、5.6%、7.7%。说明在等剂量原则下,苯、甲醛单独染毒的转化细胞灶形成率之和与两化合物同时染毒的转化细胞灶形成率非常接近;析因分析表明两者间无交互作用(F=0.07,P>0.05)。结论苯、甲醛具有细胞转化活性,二者联合作用类型呈相加作用。     

齿密螺旋体对小鼠T细胞IL-2mRNA表达水平及活性的影响

齿密螺旋体(Treponea denticola T．d)是一种能常在口腔内检出的微生物，T．d常在龈下菌斑定居，可产生一系列破坏性酶和细胞抑制因子破坏牙周组织．因此有些学者认为T．d可能是牙周炎的特定致病因子之一。本研究探讨T．d对小鼠T细胞中IL-2 mRNA表达水平及活性的影响作用．有助于进一步弄清T．d导致牙周炎发生的机理。     

Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells

Zinc is an essential trace element required for bone formation, however not much has been clarified yet for its role in osteoblast. We hypothesized that zinc would increase osteogenetic function in osteoblasts. To test this, we investigated whether zinc treatment enhances bone formation by stimulating osteoblast proliferation, bone marker protein alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 1, 3, 15, 25 uM) along with a normal osteogenic medium (OSM) as control for 1, 5, 10 days. As measured by MTT assay for mitochondrial metabolic activity, cell proliferation was stimulated even at low zinc treatment (1-3 µM) compared to OSM, and it was stimulated in a zinc concentration-dependent manner during 5 and 10 days, with the most pronounced effect at 15 and 25 uM Zn. Cellular (synthesized) alkaline phosphatase (ALP) activity was increased in a zinc concentration-dependent manner, so did medium (secreted) ALP activity. Cellular collagen concentration was increased by zinc as time went by, therefore with the maximum zinc stimulatory effect in 10 days, and medium collagen concentration showed the same pattern even on 1 and 5 day. This zinc stimulatory effect of collagen synthesis was observed in cell matrix collagen staining. The study results imply that zinc can increase osteogenic effect by stimulating cell proliferation, ALP activity and collagen synthesis in osteoblastic cells.     

Cellular Zn depletion by metal ion chelators (TPEN, DTPA and chelex resin) and its application to osteoblastic MC3T3-E1 cells

Trace mineral studies involving metal ion chelators have been conducted in investigating the response of gene and protein expressions of certain cell lines but a few had really focused on how these metal ion chelators could affect the availability of important trace minerals such as Zn, Mn, Fe and Cu. The aim of the present study was to investigate the availability of Zn for the treatment of MC3T3-E1 osteoblast-like cells and the availability of some trace minerals in the cell culture media components after using chelexing resin in the FBS and the addition of N,N,N'',N''-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN, membrane-permeable chelator) and diethylenetriaminepentaacetic acid (DTPA, membrane-impermeable chelator) in the treatment medium. Components for the preparation of cell culture medium and Zn-treated medium have been tested for Zn, Mn, Fe and Cu contents by atomic absorption spectrophotometer or inductively coupled plasma spectrophotometer. Also, the expression of bone-related genes (ALP, Runx2, PTH-R, ProCOL I, OPN and OC) was measured on the cellular Zn depletion such as chelexing or TPEN treatment. Results have shown that using the chelexing resin in FBS would significantly decrease the available Zn (p<0.05) (39.4 ± 1.5 µM vs 0.61 ± 10.15 µM) and Mn (p<0.05) (0.74 ± 0.01 µM vs 0.12 ± 0.04 µM). However, levels of Fe and Cu in FBS were not changed by chelexing FBS. The use of TPEN and DTPA as Zn-chelators did not show significant difference on the final concentration of Zn in the treatment medium (0, 3, 6, 9, 12 µM) except for in the addition of higher 15 µM ZnCl₂ which showed a significant increase of Zn level in DTPA-chelated treatment medium. Results have shown that both chelators gave the same pattern for the expression of the five bone-related genes between Zn- and Zn+, and TPEN-treated experiments, compared to chelex-treated experiment, showed lower bone-related gene expression, which may imply that TPEN would be a stronger chelator than chelex resin. This study showed that TPEN would be a stronger chelator compared to DTPA or chelex resin and TPEN and chelex resin exerted cellular zinc depletion to be enough for cell study for Zn depletion.     

姜黄素对脂肪细胞脂联素及抵抗素表达影响

目的 探讨姜黄素对3T3-L1脂肪细胞脂联素和抵抗素mRNA表达的影响。方法 在诱导3T3-L1前脂肪细胞分化过程中加入姜黄素,于第6 d收集细胞,或用姜黄素处理分化成熟3T3-L1脂肪细胞,24 h后收集细胞,采用逆转录聚合酶链反应(RT-PCR)方法检测脂肪细胞中脂联素和抵抗素mRNA水平。结果 在3T3-L1前脂肪细胞分化期间,姜黄素2.5,5,10,15和20μmol/L分别使脂联素mRNA表达增强48.7%,25.6%,89.7%,128.2%和207.7%,抵抗素mRNA表达下降了42.3%,35.4%,31.5%,58.3%,33.9%;20μmol/L姜黄素使成熟脂肪细胞脂联素mRNA表达增强203.3%;5,10,20μmol/L的姜黄素分别使抵抗素mRNA的表达降低15.2%,49.0%和55.6%。结论 姜黄素可增强脂肪细胞脂联素mRNA表达,降低抵抗素mRNA表达。     

沉默PLIN1基因与异丙肾上腺素对3T3-L1脂肪细胞脂解的机制探究

目的 研究沉默PLIN1基因与异丙肾上腺素(ISO)联合作用对3T3-L1脂肪细胞脂解的影响及机制探讨。方法 sh-PLIN1干扰载体成功转染后,以浓度为10μM的ISO处理3T3-L1脂肪细胞。采用油红O进行脂滴染色,观察脂滴形态;采用酶学方法测定甘油三酯(TG)和甘油含量,了解细胞脂解情况;采用Western-Blot法检测脂肪细胞中围脂滴蛋白A(PLIN1A)、脂肪甘油三酯脂肪酶(ATGL)、激素敏感性脂肪酶(HSL)和其磷酸化蛋白(p-HSL)的表达水平;双抗体夹心ABC-ELISA法检测细胞中环磷酸腺苷(cAMP)和蛋白激酶A(PKA)的浓度。结果 与空白组比较,ISO+sh-PLIN1组和ISO组脂滴变小,TG含量降低,甘油含量升高,有统计学意义(P<0.01)。同时,ISO+sh-PLIN1组和 sh-PLIN1组中ATGL 表达量均升高,有统计学意义(P<0.05)。ISO+sh-PLIN1转染组和ISO组中HSL、p-HSL、cAMP及 PKA 的表达量均上调(P<0.05)。结论 ISO促进脂解作用可能是cAMP/PKA通路介导,增加HSL和p-HSL的表达量来实现,而cAMP/PKA信号通路不能介导沉默PLIN1的促脂解作用。     

乳腺癌患者Runx3基因启动子甲基化状态及蛋白表达的关系

目的:探讨乳腺癌组织中Runx3基因启动子甲基化及蛋白的表达与乳腺癌生物学行为的关系,并分析Runx3基因启动子区甲基化与其蛋白表达的相关性.方法:采用甲基化特异性PCR(methylation-specific PCR,MSP)技术和免疫组织化学SP法检测48例患者乳腺癌组织、18例癌旁乳腺组织中Runx3基因启动子甲基化情况及蛋白的表达.结果:Runx3基因启动子在乳腺癌组织及癌旁乳腺组织中的甲基化阳性率分别为52.1％和3.7％ (P＜0.05); Runx3蛋白在乳腺癌及癌旁乳腺组织中的阳性率分别为33.3％和92.6％ (P＜0.05);Runx3基因启动子甲基化及蛋白的表达分别与乳腺癌的组织学分级、临床分期及淋巴结转移有关(P＜0.05);Runx3基因启动子甲基化与蛋白表达呈明显的负相关(P＜0.05).结论:Runx3基因启动子的异常甲基化是引起蛋白表达缺失的主要原因,Runx3基因启动子的异常甲基化与乳腺癌的发生、发展有关.