硼替佐米对多发性骨髓瘤患者破骨细胞生成的影响及机制研究

目的:观察多发性骨髓瘤(multiple myeloma,MM)患者破骨细胞在体外诱导分化成熟过程中,硼替佐米(Bortezomib,Btzmb)对其分化和功能的影响,以及破骨细胞分化过程中上游关键信号分子肿瘤坏死因子受体相关蛋白6(tumor necrosis factor receptor-associated factor 6,TRAF6)的改变.方法:患者外周血单个核细胞在经核因子KB受体活化因子配体(receptor activator of NF-kB ligand,RANKL)及巨噬细胞集落刺激因子(macmphage colony stimulating factor,M-CSF)诱导后向破骨细胞分化过程中,采用不同剂量的Btzmb进行处理,观察抗酒石酸酸性磷酸酶(tartrateresistant acid phosphatase,TRAP)染色阳性的破骨细胞数量,检测培养液中的TRAP活性,并观察骨片上骨陷窝数量的变化.采用Westem印迹和RT-PCR法,分析Btzmb对破骨前体细胞中TRAF6蛋白和mRNA的影响.结果:2.5和5 nmol/L Btzmb组破骨细胞数量分别为(157±21)和(98±15)个,较对照组(307±25)明显减少(P均＜0.05),骨陷窝形成数量分别为对照组的(53±24)%和(29±7)%(P均＜0.05),上清液中破骨细胞活性分别为对照组的(86±24)%和(60±25)%(P均＜0.05).破骨前体细胞经Btzmb处理后观察24 h,TRAF6蛋白活性逐渐降低,TRAF6 mRNA水平也有所下降.结论:Btzmb抑制MM患者破骨细胞的分化和功能,该作用可能与TRAF6有关.     

1,25(OH)2维生素D3诱导大鼠骨髓单核细胞形成破骨细胞

目的 观察1,25二羟维生素D3[1,25(OH)2 D3]诱导成年大鼠骨髓来源的单核细胞形成多核破骨细胞的作用.方法 实验分为单核细胞诱导组和全骨髓诱导组,每组各10只大鼠.密度梯度离心法分离大鼠骨髓中的单核细胞,α-MEM培养液中加入10-8 mol/L的1,25(OH)2 D3诱导单核细胞向破骨细胞分化.利用抗酒石酸酸性磷酸酶(TRAP)染色、倒置相差显微镜和扫描电镜,观察诱导不同时相TRAP阳性(TRAP )多核细胞的形态学特征、特异性酶表达、以及骨吸收陷窝.进而计数诱导的破骨细胞数量,鉴定其功能活性,并与全骨髓诱导方法比较.结果 骨髓单核细胞诱导组在培养第7 d出现TRAP 多核细胞,第14 d TRAP 多核细胞数量达峰值,第21 d TRAP 多核细胞数量开始减少.从培养第14 d到第23 d,单核细胞诱导组TRAP 多核细胞数量多于全骨髓诱导组(P＜0.05);培养第10 d到第23 d单核细胞诱导组骨吸收陷窝数量多于全骨髓诱导组 (P＜0.05).单核细胞诱导组破骨细胞数量高峰期可持续7 d,全骨髓诱导法仅持续3 d.结论 1,25(OH)2 D3能够诱导成年大鼠骨髓来源的单核细胞形成破骨细胞;所诱导的破骨细胞的数量和峰值持续时间、以及骨吸收活性均高于全骨髓诱导方法.     

Inhibitory effect of salmon calcitonin on bone resorption: Morphological study of the tibial growth plate in rats

Summary Salmon calcitonin (sCT) at doses of 100 and 50 UI given subcutaneously to growing rats produced in vivo evidence of osteoclastic activity inhibition. Histological assessment was carried out by measuring the perichondrial ring of Lacroix height, and a dose-correlated effect was found. These aspects were coupled with an increase in the osteoclast number and suggested that in studies with bone resorption inhibitors, morphological evaluation based on osteoclasts count is not reliable. The changes of the metaphysis suggested also that sCT affects the activity of hypertrophic chondrocytes of the growth plate. Plasma calcium levels did not differ significantly between treated rats and controls; an increased phosphatemia was observed in sCT-treated animals.     

Menatetrenone (Vitamin K<Subscript>2</Subscript>) Acts Directly on Circulating Human Osteoclast Precursors

It is still not certain what the direct effect of menatetrenone is on osteoclast precursors. In the present study, we investigated whether menatetrenone has a direct effect on circulating osteoclast precursors to influence osteoclast differentiation. Monocytes isolated from human peripheral blood were cultured with receptor-activated NF-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Menatetrenone or vitamin K₁ was then added to the cultures. Geranylgeraniol or phytol (the respective side chain) was also added to the cultures instead of menatetrenone or vitamin K₁, respectively. After 7 and 14 days incubation, cultures were evaluated for cytochemical and functional evidence of osteoclast formation. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) and the percentage area of lacunar resorption induced by RANKL and M-CSF were decreased when menatetrenone or geranylgeraniol was added to the cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when the cultures were treated with menatetrenone or geranylgeraniol. In contrast, vitamin K₁ or phytol did not affect the number of TRAP-positive MNCs nor the percentage area of lacunar resorption. These results indicate that menatetrenone not only influences osteoclast formation via bone stromal cells but also acts directly on circulating osteoclast precursors to influence osteoclast differentiation. These findings also suggest that geranylgeraniol, the side chain of menatetrenone, plays an important role in this inhibitory effect.     

Potent inhibitory effect of naturally occurring flavonoids quercetin and kaempferol on in vitro osteoclastic bone resorption

Several recent studies have suggested that flavonols, a class of phytochemicals with many biological activities, might exert a protective effect against post-menopausal bone loss. In the present study, we investigated the effects of quercetin and kaempferol, two of the major naturally occurring flavonols on the in vitro bone resorbing activity of osteoclasts. Our results indicate that both compounds, at concentrations ranging from 0.1 to 100 microM reduce bone resorption in a time and dose-dependent manner. Significant inhibitory effects were observed at concentrations as low as 0.1 microM especially with kaempferol. The IC(50)s, or concentration inhibitory of 50% of basal resorption, calculated for quercetin and kaempferol were 1.6 and 5.3 microM, respectively. Using highly purified rabbit osteoclasts, we showed that both flavonols directly induce apoptosis of mature osteoclasts in the same dose-range effective for inhibiting bone resorption. When osteoclasts were treated with 50 microM of quercetin and kaempferol, intracellular reactive oxygen species levels decreased significantly by 75 and 25%, respectively, indicating these molecules keep their antioxidant properties at this concentration. However, at concentrations below 50 microM, neither quercetin nor kaempferol exerted antiradical action, suggesting that antioxidant properties cannot fully explain the inhibitory effect on bone resorption. Finally, we report that kaempferol-, but not the quercetin-induced inhibition of bone resorption was partially abolished by the presence of the pure anti-estrogen ICI 182780 suggesting that kaempferol's estrogenic effect could be involved in the inhibition of bone resorption. The present study demonstrates that flavonols widely distributed in human diet such as quercetin and kaempferol, exert a potent inhibitory effect on in vitro bone resorption.     

针刺血清对体外培养破骨细胞数量的影响

目的:探讨针刺抑制骨吸收的细胞学机制。方法:40只12月龄雌性SD大鼠,随机分为假手术组、模型组、针刺预防组和针刺治疗组。针刺预防组和针刺治疗组分别在造模术后的3d、3个月开始针刺。取“肾俞”“脾俞”“足三里”“大椎”。自新生SD大鼠四肢长骨分离破骨细胞,接种于24孔培养板中,加入含10%上述各组大鼠血清的培养液培养48h后,抗酒石酸酸性磷酸酶(TRAP)染色并计数TRAP( )多核细胞数。结果:与假手术组比较,模型组TRAP( )多核细胞数明显增加(P<0.01);与模型组比较,针刺预防组和针刺治疗组TRAP( )多核细胞数明显减少(P<0.05)。结论:针刺血清能够减少破骨细胞的数量,预防组和治疗组之间的作用无统计学差异。     

Osteoclastic features of cells that resorb bone implants in rats

Summary The subcutaneous implantation of devitalized, mineral-containing bone particles in rats elicits the recruitment and differentiation of multinucleated cells and the rapid resorption of the bone. Cytochemical and ultrastructural characteristics were studied 12 days after implantation of bone. Tartrate-resistant acid phosphatase activity was demonstrated in these cells, especially at the cell/bone interface. Mast cells were seen vicinal to, but not in contact with the osteoclasts and the bone particles. Electron microscopic evaluation revealed that many multinucleated cells in lacunae on the surfaces of the bone particles displayed features characteristic ofin osso osteoclasts, including clear zones of attachment to the bone substrate, and ruffled borders overlying bone with frayed collagen fibers. Many cells contained multiple giant centrospheres; this finding suggests that fusion can occur between multinucleated cells. This model may be useful to characterize the differentiation and regulation of bone-resorbing cells.     

Elevated serum levels of creatine kinase BB in autosomal dominant osteopetrosis type II

Summary Serum levels of creatine kinase isoenzyme BB (CK-BB) were measured spectrophotometrically and by electrophoresis in 17 patients with autosomal dominant osteopetrosis (ADO) and compared with those of age- and sex-matched controls. Eight patients had ADO type I and nine patients had ADO type II. CK-BB was significantly increased (p<0.002) in type II but normal in type I compared with controls. This finding supports heterogeneity of ADO, and it may indicate a potential role for CK-BB as a marker of immature osteoclasts.