中药“固真方”对ROS17/2.8细胞株PTH受体结合率的影响

 <正> 中药“固真方”是补肾益精方药“还精煎”的小复方,有延缓衰老的作用,其机理迄今未明。鉴于甲状旁腺素(PTH)为调节钙磷代谢的主要激素,而老年人由于肾功能衰退,尿排钙增高,脱钙是衰老的重要症状之一。大鼠成骨肉瘤细胞株ROS17/2.8含有PTH受体,本文观察了在活细胞培养过程中,中药“固真方”提取液对PTH受体结合率的影响。     

核苷对ROS17/2.8细胞株腺苷酸环化酶活性的影响

 大鼠成骨肉瘤细胞株(ROS17/2.8)系甲状旁腺素(PTH)的靶细胞。当该细胞质膜上的PTH受体与PTH结合后,可激发腺苷酸环化酶(AC)的活性。腺苷及四种不同的核苷酸(AMP、GMP、UMP、CMP)单独对AC无明显效应,但却可抑制PTH对AC的刺激作用。而鸟苷或木糖腺苷则可显著增强PTH对AC的刺激作用。提示核苷的不同代谢物在代谢调节中的多样化作用。     

中药固真方对一些与细胞增殖有关基因表达的影响

 中药固真方对一些与细胞增殖有关基因表达的影响姚明忠,顾文聪,丁卫,韩志芬,杜国光（上海中医药大学生物化学教研室,上海２０００３２）（北京医科大学生物化学与分子生物学系,北京１０００８３）中药固真方（ＶＲＦ）具有补肾益精、延缓衰老的作用［１］．能提高成...     

甲状旁腺素和胰岛素样生长因子促成骨样细胞ROS 17/2.8增殖分化中的协同作用

 间歇性小剂量地给予甲状旁腺素 (parathyroid hormone,PTH)可促进成骨 .胰岛素样生长因子 - I(insulin- like growth factor- I,IGF- I)由成骨细胞所产生并贮存于骨基质中 ,可促进成骨细胞的增殖分化 .为进一步了解向钙性激素和骨源性生长因子对骨生长的影响 ,利用成骨样细胞 ROS1 7/ 2 .8进行体外实验 ,观察了 PTH和 IGF- I这两种在骨生长和代谢中有重要作用的激素和因子相互作用的效果 ,并对其相互作用机制作出初步探讨 .结果显示 :联合使用 IGF- I及 PTH(间歇性给药 )时 ,(1 ) SRB(sodium rhodamine B,SRB)染色显示经 PTH(1 0 -9mol/ L,间歇给药 )和 IGF- I(1 0 -9mol/ L)联合处理的细胞 ,其数目明显增加 ,且明显高于单独处理组 ;(2 ) 3H- Td R参入增加 ,也明显高于单独处理组 ;(3)与增殖相关的原癌基因 (c- fos,c- jun,c- ki- ras)的表达增强 ,明显高于单独处理组 ;(4)骨钙素 (osteocalcin)基因 m RNA表达增强 ,明显高于单独处理组 ;(5) IGF- I(1 0 -8mol/L,1 0 -9mol/ L)可使 PTH受体基因 m RNA表达增强 .这些结果提示 PTH和 IGF- I在成骨样细胞ROS 1 7/ 2 .8增殖分化中具有协同作用 ,原癌基因的表达增强可能是其作用的一个环节 .此外 ,IGF- I可能通过增强 PTH受体表达 ,使细胞对 PTH的反应性增强     

肿瘤坏死因子α对ROS17/2.8细胞甲状旁腺素受体的下行调节

肿瘤坏死因子α(TNFα)是激活的单核巨噬细胞分泌的蛋白质,分子量17kD。其多功能性和选择性抑制肿瘤细胞生长的作用受到高度重视。我们的实验表明:TNFα(3×10~(-10)-1×10~(-7)mol/L)能显著降低大鼠成骨肉瘤细胞株ROS17/2.8的甲状旁腺素(PTH)受体总结合率,比对照降低7.47-37.45％,且与TNFα的浓度呈正相关。时间曲线显示,TNFα作用时间越长,受体总结合率降低越明显。Scatchard作图表明PTH受体数目降低而其亲和力无显著变化。细胞周期分析显示,TNFα(3.83×10~(-10) mol/L作用3天)能抑制S期DNA合成。可见TNFα通过减少PTH受体数目以调节骨代谢。同时通过抑制DNA的合成以调节骨细胞的增殖。     

染料木黄酮对成骨细胞增殖分化的影响

目的：研究染料木黄酮对体外培养乳鼠颅盖骨成骨细胞增殖分化的影响。方法：取乳鼠颅盖骨,采用胶原-胰蛋白酶消化法,进行颅骨成骨细胞培养,取第二代成骨细胞,添加10^-5～10^-7mol／L染料木黄酮,在CO2孵箱中培养48h和72h后MTT比色法测定细胞增殖,培养72h采用^3H-TdR和^H-Pro掺入实验测定DNA和胶原合成。用试剂盒检测细胞裂解液碱性磷酸酶(ALP)活性。结果：染料木黄酮明显增加成骨细胞MTT的吸光度值、^3H-TdR和^3H-Pro的掺入,增加成骨细胞碱性磷酸酶活性。结论：染料木黄酮促进体外培养的乳鼠颅盖骨成骨细胞DNA和胶原的合成,促进增殖和分化。     

中药固真方对老年鼠睾丸间质细胞(Leydig细胞)LH受体的下行调节

<正> 中药固真方是由首乌、苁蓉等六味中药组成的复方,具有补肾益精、延缓衰老及调整下丘脑-垂体-性腺-胸腺轴的功能,实验还证明它能显著增加老年大鼠大脑皮质和海马组织中的肾上腺素能受体(α_1和β)、多巴胺受体、胆碱能受体和γ-氨基丁酸受体。本文比较了     

甲状腺素及维甲酸对成骨样细胞甲状旁腺素受体的调节作用

研究了甲状腺素（T3／T4）及维甲酸（RA）对大鼠成骨样细胞ROS17／2．8细胞林甲状旁腺素（PTH）受体的调节作用．实验结果表明：细胞经T3／T4处理后,可显著增高PTH受体结合率及碱性磷酸酶活性,以及PTH受体mRNA的表达．细胞经RA处理后,则相反地降低PTH受体结合率及碱性磷酸酶活性．     

Colorimetric Evaluation of Cultured Osteoblast-like Cells (ROS 17/2.8)

We have developed a colorimetric method for evaluating the number of osteoblastic cells in culture without destroying the cells. This assay is based on the staining of basophilic cellular compounds with methylene blue. The dye bound by the cells is released at low pH and measured in a spectrophotometer at 662 nm. Linear correlations exist between the absorbance measured by the methylene blue assay and the number of cells seeded, the total cellular protein content, and thymidine labeling. This colorimetric method has the advantage of preserving cell integrity. After destaining, scanning electron microscopy can be performed on well preserved cell morphology.     

中药固真方对成骨样细胞UMR106增殖分化的影响

 中药固真方具有补肾益精、延缓衰老的作用 .为了进一步探讨其作用机理 ,研究观察了固真方对成骨样细胞 UMR1 0 6DNA合成、原癌基因 c- fos,c- ki- ras m RNA表达以及骨钙素 (osteocal-cin)基因 m RNA表达的影响 .结果表明 :固真方可明显促进 DNA合成 ,且在 1 0 -5稀释度时达高峰 ;增殖相关的原癌基因 (c- fos,c- ki- ras) m RNA表达增强 ;成熟成骨细胞特征性标志蛋白——骨钙素基因 m RNA表达增强 .结果提示 :中药固真方可能通过影响一些与增殖相关的原癌基因及骨钙素基因表达而促成骨样细胞 UMR1 0 6的增殖和分化成熟     

Effect of overexpressing fibroblast growth factor 2 protein isoforms in osteoblastic ROS 17/2.8 cells

Fibroblast growth factor-2 (FGF-2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF-2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF-2 protein that is exported from cells. Since FGF-2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF-2 as a novel means of assessing different FGF-2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb alpha1(I)-collagen 5' regulatory elements (Col 3.6). A set of expression vectors, called Col3.6-Fgf2 isoforms-IRES-GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF-2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6-IRES-GFPsaph) versus each of the Col3.6-Fgf2-IRES-GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF-2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF-2 isoforms. A neutralizing FGF-2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF-2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF-2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF-2 proteins isoforms is important in modulating OB function.     

Rotation in clinostat results in apoptosis of osteoblastic ROS 17/2.8 cells

Clinostat is an effective, ground-based tool which can be used to verify data from space flight, and to test hypotheses and experimental conditions for eventual space flights. Rotation in clinostat appears to mimic the microgravity environment by nulling the gravitational vector by continuous averaging. In the present study, we exposed osteoblast-like ROS 17/2.8 cells to a vector-averaged gravity environment in a clinostat and found that the cells undergo apoptotic death during the first 24 hr of clino-rotation. We suggest that apoptosis might be one of the mechanisms for reduced bone formation as observed in actual space flights.