三碘甲腺原氨酸对大鼠成骨细胞增殖的影响

目的:探讨三碘甲腺原氨酸(T3)对大鼠成骨细胞增殖的影响。方法:采用骨组织块法原代分离培养新生SD大鼠颅骨成骨细胞,然后配置含不同浓度(10-7mol/L、10-8mol/L、10-9mol/L)T3的培养基,与大鼠成骨细胞共培养4d,采用噻唑蓝(MTT)法检测细胞增殖能力,采用细胞化学染色法测定成骨细胞ALP活性。结果:T3可促进大鼠成骨细胞增殖,并呈剂量依赖性;随T3浓度的增高,成骨细胞表达ALP活性增强(P<0.05)。结论:10-7mol/L～10-9mol/L浓度的T3可通过刺激成骨细胞的增殖,对预防和治疗骨质疏松发挥一定作用。     

三碘甲腺原氨酸对大鼠成骨细胞增殖的影响

目的:探讨三碘甲腺原氨酸(T3)对大鼠成骨细胞增殖的影响.方法:采用骨组织块法原代分离培养新生SD大鼠颅骨成骨细胞,然后配置含不同浓度(10-7mol/L、10-8mol/L、10-9mol/L )T3的培养基,与大鼠成骨细胞共培养4d,采用噻唑蓝(MTT)法检测细胞增殖能力,采用细胞化学染色法测定成骨细胞ALP活性.结果:T3可促进大鼠成骨细胞增殖,并呈剂量依赖性;随T3浓度的增高,成骨细胞表达ALP活性增强(P＜0.05).结论:10-7mol/L～10-9mol/L浓度的T3可通过刺激成骨细胞的增殖,对预防和治疗骨质疏松发挥一定作用.     

不同温度下成骨细胞增殖及其OPG/RANKL mRNA表达的影响

目的:探讨不同温度下对小鼠成骨细胞MC3T3-E1的增殖以及OPG/RANKL表达水平的影响。方法:1.以小鼠成骨细胞MC3T3-E1为体外实验模型,MTT法检检测细胞的增殖情况。2.RT-PCR方法检测MC3T3-E1OPG/RANKL mRNA的表达水平。结果:设定对照组为37℃,高于对照组(38℃-39℃-40℃-41℃-42℃)分别作用于MC3T3-E1细胞1小时/天,连续1周,可刺激细胞增殖,OD值显著增加(P<0.05)。同时可增加OPG mRNA表达,降低RANKL mRNA表达,呈温度梯度依赖性。结论:热刺激促进MC3T3-E1细胞增殖,同时通过调节OPG/RANKL mRNA的表达,直接促进骨形成,抑制骨吸收。     

锌对铅染毒新生大鼠成骨细胞增殖分化的影响

目的:探讨铅锌联合染毒对乳鼠颅骨成骨细胞增殖分化的影响。方法:分离并培养原代成骨细胞,加入不同浓度铅、锌培养48h,检测其对成骨细胞增殖的作用;用碱性磷酸酶试剂盒检测ALP活力。结果:在染铅48h后,当铅浓度≥10μmol/L时,细胞增殖功能下降(P<0.05);加锌干预48h后,铅+锌组细胞增殖功能均高于各自单独染铅组,其中铅(1μmol/L、10μmol/L)+锌(50μmol/L)组、铅(10)+锌(100)组与对照组间的差异具有统计学意义(P<0.05)。铅干预48h后,100μmol/L铅组的ALP活力显著下(P<0.05),给予锌干预的铅锌联合染毒组,各组ALP活力均有增加,其中铅(1μmol/L、10μmol/L)+锌(50μmol/L)组ALP活力均高于对照组,而铅(100μmol/L)+锌(50μmol/L)组ALP活力低于对照组,差异均有统计学意义(P<0.05)。结论:铅对成骨细胞有毒性作用,影响其增殖和分化功能;50μmol/L锌在一定程度上可以拮抗铅对成骨细胞增殖和分化功能的损伤,且对ALP活力的作用更显著,为铅中毒骨病的防治提供一定的科学依据。     

甲状旁腺激素对成骨细胞中ClC-3 氯通道表达及成骨分化影响的研究

目的:观察甲状旁腺激素(PTH)对成骨细胞中Cl C-3氯通道表达及成骨分化影响,初步探索Cl C-3介导PTH在细胞成骨分化中的作用。方法:采用10-8M、10-9M、10-10M PTH持续刺激和间断刺激MC3T3-E1细胞72 h后,通过CCK-8试剂盒法检测MC3T3-E1细胞的增殖情况,Real-Time PCR法检测MC3T3-E1细胞中Clcn3及成骨相关基因Alp、Runx2的表达情况,免疫荧光法检测10-9M PTH不同给药方式下对Cl C-3蛋白表达的影响。结果 :经不同浓度PTH连续和间断处理72 h后,结果显示10-9 M PTH间断刺激的MC3T3-E1细胞的增殖能力最强,且其Alp、Runx2 m RNA表达均高于10-8 M组和10-10 M组(P<0.05),而相同浓度间断刺激的MC3T3-E1细胞成骨相关基因的表达均高于持续刺激组,以10-9M间断刺激组差异最显著(P<0.05),而10-8 M和10-10M均无统计学差异(P>0.05),10-9 M PTH刺激的MC3T3-E1细胞中Cl C-3蛋白表达也显著增加(P<0.05)。结论 :成骨细胞的Cl C-3氯通道能够响应PTH的刺激发生变化,并伴随着成骨相关基因Alp、Runx2表达的增强。     

模拟微重力通过调节AKT磷酸化抑制前成骨细胞的增殖

目的:研究模拟微重力对前成骨细胞的增殖作用及其分子机制。方法:利用2D回转器模拟失重条件培养前成骨细胞MC3T3-E1 24小时;将p-AKT激活剂SC79加入细胞培养基后模拟失重条件下培养前成骨细胞MC3T3-E1 24小时;利用Western blot技术分别检测细胞增殖相关蛋白PCNA以及AKT、p-AKT的表达变化情况。结果:(1)与对照组相比,模拟失重组前成骨细胞增殖受到抑制(P0.01),p-AKT/AKT比值减小(P0.01);(2)加入SC79组与对照组相比,p-AKT/AKT比值显著增加;(3)加入SC79的模拟失重组(MG+SC79)与模拟失重组相比(MG),p-AKT/AKT比值增加,PCNA蛋白表达增加(P0.01),成骨细胞增殖有所恢复。结论:模拟微重力可能通过抑制AKT的磷酸化形式抑制前成骨细胞的增殖,加入p-AKT激活剂可部分恢复前成骨细胞的增殖。     

雷奈酸锶在高糖诱导的成骨细胞损伤中的保护作用

目的:探究雷奈酸锶(SR)在高糖诱导的成骨细胞损伤中的保护作用。方法:体外培养MC3T3-E1成骨细胞,用MTT法筛选出能够诱导成骨细胞活性显著降低的高糖浓度,以及能够促进成骨细胞活性的最优SR浓度,随后观察SR对高糖诱导的成骨细胞的作用效果。采用MTT法检测细胞增殖情况,流式细胞仪检测凋亡水平,PCR和ELISA分别检测骨形成相关基因表达和蛋白分泌水平。结果:0.50 mmol/L的SR能够显著增强高糖诱导的MC3T3-E1成骨细胞的增殖能力和细胞活性,降低细胞凋亡水平,改善细胞形态,并显著促进Runx2、OCN、COL-1、BMP-2、IGF-1等的表达。结论:SR能够通过增加成骨细胞活性、抑制成骨细胞凋亡、促进骨形成相关因子的表达和分泌,发挥对高糖诱导的成骨细胞损伤的保护作用,为SR发挥糖尿病骨质疏松的治疗作用提供了实验依据。     

生物活性肽-蛋氨酸脑啡肽对成骨细胞增殖的影响

蛋氨酸脑啡肽（Methionine Enkephalin,MENK）作为内源性物质,通过与阿片受体结合,发挥阿片样物质的生物学作用。研究表明,MENK在成熟的骨组织和成骨细胞中均有分布,并且在成骨细胞表面发现了阿片受体。实验旨在进一步研究MENK对成骨细胞增殖的影响。体外培养MC3T3-E1成骨细胞,MTT方法检测MENK对成骨细胞增殖的影响。结果表明,10-7、10-8、10-9mol/L的MENK对成骨细胞的增殖具有促进作用（P〈0.05）。提示MENK可在骨缺损疾病中发挥促进骨组织重建的作用。     

雌激素对成骨细胞增殖及IGF-2表达的调控作用

目的 探讨雌激素对大鼠成骨细胞功能的影响及胰岛素样生长因子2（insulin-like growth factor 2,IGF-2）表达的调控机理。方法应用1×10^-10mol／L、1×10^-8mol／L、1×10^-6mol／L浓度的雌二醇（estradiol,172）分别作用原代培养的新生大鼠颅盖骨成骨细胞24h;采用MTT法和对硝基酚磷酸盐法检测成骨细胞增殖能力和细胞中碱性磷酸酶（ALP）活性;应用实时定量PCR和Western印迹杂交分析成骨细胞中IGF-2的mRNA和蛋白质的表达规律。结果1×10^-6mol／L浓度的磁使大鼠成骨细胞增殖能力和ALP活性分别增加了67％和55％,使IGF-2的mRNA与蛋白质的表达水平分别增加了90％和140％,差异有统计学意义（P〈0．05）。结论雌激素对成骨细胞中IGF-2基因的表达具有正性调控作用。成骨细胞中IGF-2的高表达可能与雌激素调节成骨细胞增殖和分泌功能相关。     

三维回转骨细胞条件培养基对成骨细胞功能的调节作用

骨细胞是骨组织中主要的力学感受器.研究失重条件下骨细胞对效应细胞的调控作用对于揭示失重引起的骨丢失机制具有重要意义.本研究拟采用三维回转器模拟失重,探讨模拟失重骨细胞条件培养基(RCM)对成骨功能的调节作用.小鼠骨细胞系MLO-Y4三维回转培养72h后,收集回转条件培养基(RCM)和未回转对照组的条件培养基(CCM),用四甲基偶氮唑盐比色(MTT)法、对硝基苯磷酸(pNPP)法和流式细胞术(FCM)分别检测RCM对小鼠成骨样细胞系MC3T3-E1增殖、周期及细胞分泌碱性磷酸酶(ALP)活性的影响.采用RT-PCR方法检测RCM对MC3T3-E1成骨相关基因表达的影响.结果显示,三维随机回转72h后的MLO-Y4RCM可促进MC3T3-E1增殖:条件培养基培养MC3T3-E124h和48h后,50%RCM组比CCM组分别增加了1.62和1.60倍,差异显著(*P0.05),培养72h后,100%RCM组比CCM组增加了1.69倍,差异显著(*P0.05);细胞周期检测结果表明,条件培养基培养24、48和72h后,RCM组部分恢复CCM引起的MC3T3-E1细胞周期阻滞;MC3T3-E1的ALP活性在RCM组和CCM组之间无差异;RT-PCR检测结果表明,100%MLO-Y4条件培养基培养MC3T3-E148h后,降低了成骨相关基因ALP、Runx2、OPN、OC的表达.差异显著(*P0.05,**P0.01,***P0.001).实验结果表明,三维随机回转模拟失重培养骨细胞72h后的条件培养基促进了成骨细胞增殖,抑制了成骨相关基因表达.     

Hyaluronate degradation in 3T3 and simian virus-transformed 3T3 cells

The cellular control of hyaluronate levels was examined in cultures of simian virus 40-transformed 3T3 (SV3T3) and 3T3 cells which are known to differ in their metabolism of hyaluronate. When [3H]hyaluronate was added to cultures of the two cell lines, four times more ligand was bound per mg of protein by the SV3T3 cells than by the 3T3 cells. Of the bound [3H] hyaluronate, 40% was degraded by the SV3T3 cells to oligosaccharides characteristic of the breakdown of hyaluronate, but only 2% was degraded by 3T3 cells. Hyaluronidase activity was found in the cell layer and medium of the SV3T3 cultures, but was not detectable in 3T3 cells. The SV3T3 enzyme was active only at acidic pH, but at neutral pH the secreted SV3T3 hyaluronidase was thermally more stable then the cell-associated enzyme. In contrast, both cell lines were found to contain similar amounts of beta-glucuronidase and beta-N-acetylglucosaminidase activity. We conclude that the elevated capacity of SV3T3 cells to degrade hyaluronate may be partially responsible for their lack of the hyaluronate-containing pericellular coat which is prominent around 3T3 cells.     

Ether-linked lipids of Balb/c3T3, SV3T3 and concanavalin A-selected SV3T3 revertant cells

Ether-linked lipids were analyzed in Balb/c3T3, SV3T3 and Concanavalin A-selected SV3T3 revertant cells. The three cell lines were found to contain significant quantities of alk-1-enyl- and alkyl-linked phosphatidylethanolamine (PE) and phosphatidylcholine (PC) and small amounts of alkyldiacylglycerols. Compared to 3T3 cells, SV3T3 cells contain a higher amount of alk-1-enyl-linked PC, while in SV3T3 revertant cells the concentrations of the various ether lipids are similar to those of 3T3 cells. The major difference in the composition of ether groups of SV3T3 cells, compared to 3T3 cells, is an increase of 18:0 accompanied by a decrease of 18:1 in the alk-1-enyl-linked PE and PC. Alk-1-enyl-linked PC of SV3T3 revertant cells also shows an increase of 18:0, while the decrease of 18:1 was not statistically significant.     

Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short- and long-lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA-3T3) or Rous sarcoma virus (RS-3T3), degraded short- and long-lived proteins at similar rates. Simian virus 40 (SV-3T3)- and benzo(a)pyrene (BP-3T3)-transformed cells had slightly lower rates of degradation of both short- and long-lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long-lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long-lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.     

Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly⁺ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transportspecific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly⁺ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na⁺-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na⁺-independent systems L and Ly⁺remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (V_max) rather than substrate concentration for half-maximal velocity (K_m). Transport activity of systems A and ASC was severalfold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.     

3-Bromo-3-deazaneplanocin and 3-bromo-3-deazaaristeromycin: Synthesis and antiviral activity

As an outgrowth of our program to explore 3-deazaadenine carbocyclic nucleosides, 3-bromo-3-deazaneplanocin (5) and 3-bromo-3-deazaaristeromycin (6) have been synthesized from a readily available cyclopentenol and cyclopentanone and either 4-amino- or 4-chloro-1H-imidazo[4,5-c]pyridine (6-amino- or 6-chloro-3-deazaadenine) in 5 steps and 7 steps, respectively. Antiviral analysis found 5 to display significant activity towards a number of (-)-ssRNA and a few dsDNA viruses. Compound 6 was less active than 5 against selected examples of those viruses affected by 5.     

Synthesis and biological activity of 3 beta-fluorovitamin D3: comparison of the biological activity of 3 beta-fluorovitamin D3 and 3-deoxyvitamin D3

The alteration in the biologic activity of the vitamin D3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3 beta-fluorovitamin D3 6 and its hydrogen analog, 3-deoxyvitamin D3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3 beta-fluorovitamin D3 was as active as vitamin D3 in vivo, we found that the fluoro-analog was less active than vitamin D3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3 beta-fluorovitamin D3 showed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D3. In the organ-cultured, embryonic chick duodenum, 3 beta-fluorovitamin D3 was approx 1/1000th as active as the native hormone, 1,25-dihydroxyvitamin D3, while 3-deoxyvitamin D3 was inactive even at microM concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D3 from a chick intestinal cytosol receptor, 3 beta-fluorovitamin D3 and 3 beta-deoxyvitamin D3 both showed very poor binding efficiencies when compared with vitamin D3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D3 7.     

Endogenous hyaluronate-cell surface interactions in 3T3 and simian virus-transformed 3T3 cells

3T3 cells have a large, pericellular coat which contains 30 times more hyaluronate than the amount of cell surface hyaluronate associated with simian virus 40-transformed 3T3 (SV-3T3) cells. On the other hand, SV-3T3 cells have high affinity binding sites for exogenously added hyaluronate, whereas 3T3 cells have much lower affinity sites. Removal of cell surface hyaluronate from SV-3T3 cells by treatment with hyaluronidase caused a reproducible increase in their maximum binding capacity for exogenous hyaluronate but no significant change in binding affinity or specificity. For 3T3 cells, however, the maximum amount of binding decreased and the affinity of binding increased after hyaluronidase treatment. When endogenous cell surface hyaluronate was labeled metabolically and then the cells incubated in the presence of exogenous unlabeled hyaluronate, the labeled cell surface hyaluronate was quantitatively displaced from the SV-3T3 cells but was not displaced from the 3T3 cells. Chondroitin sulfate and heparin did not displace cell surface hyaluronate from either cell type. Membranes isolated from SV-3T3 cells bound hyaluronate specifically and with high affinity, whereas membranes from 3T3 cells did not consistently bind a significant amount of hyaluronate. We conclude from these studies that the retention of endogenous hyaluronate on the surface of SV-3T3 cells is mediated by binding sites similar to those detected by the addition of exogenous hyaluronate, and the mechanism of retention of endogenous hyaluronate on the surface of 3T3 cells differs from SV-3T3 cells.