糖皮质激素对血液病患儿成骨细胞的影响及维生素D干预研究

目的探讨糖皮质激素对急性特发性血小板减少性紫癜和小儿急性淋巴细胞白血病惠儿成骨细胞功能的影响及应用维生素D后的变化。方法将急性特发性血小板减少性紫癜和急性淋巴细胞白血病患儿各分成两组,每组均为20例均应用糖皮质激素（每日给予泼尼松2mg／kg,治疗3周）,其中对照组单纯应用乳酸钙0．5g／d（n=20）,治疗组（n=20）应用乳酸钙0．5g／d,并每日口服维生素D800 IU,测定各组患儿治疗前后的骨代谢生化指标：血清Ⅰ型前胶原羧基端前肽（PICP）、血清骨钙蛋白（BGP）、总碱性磷酸酶（AKP）、钙（Ca）和磷（P）水平。结果治疗3周后,对照组患者的PICP、BGP、PICP、AKP、Ca较治疗前明显下降,差异有统计学意义（P〈0．01）,磷较治疗前明显上升,比较差异有统计学意义（P〈0．01）,而治疗组比较差异无统计学意义（P〉0．05）。结论血液病患儿应用糖皮质激素治疗后,成骨细胞合成明显受抑制,而联合比较应用维生素D后可改善糖皮质激素对成骨细胞的抑制。     

Effects of sodium fluoride treatment in vitro on cell proliferation,apoptosis and caspase-3 and caspase-9 mRNA expression by neonatal rat osteoblasts

Long-term excessive fluoride intake is linked to skeletal disease. Skeletal health is influenced by the balance between bone formation and resorption of which osteoblast function is critical. The objectives of this study were to determine the effect of fluoride treatment on osteoblast proliferation, apoptosis and caspase-3 and caspase-9 mRNA expression in vitro. Neonatal rat osteoblasts were cultured in the presence of varying concentrations (0.5–30 mg/l) of sodium fluoride and effects of treatments were determined. Treatment with sodium fluoride inhibited osteoblast proliferation in a dose-dependent fashion and effects were maximal after 120 h incubation. A significant increase in osteoblast apoptosis was observed (after 24 and 72-h treatment) in response to the lowest dose of sodium fluoride (0.5 mg/l) and osteoblast apoptosis was further increased in response to higher doses. Increased-osteoblast caspase-3 and caspase-9 mRNA was also observed in response to sodium fluoride treatment (5 mg/l) for 72 h. Results indicate that negative effects of excess fluoride on skeletal health may be mediated in part by inhibition of osteoblast survival.     

Formation of bone-like mineralized matrix by periodontal ligament cells in vivo: a morphological study in rats

Periodontal ligament (PDL) is a unique connective tissue that not only connects cementum and alveolar bone to support teeth, but also plays an important role in reconstructing periodontal tissues. Previous studies have suggested that PDL cells have osteogenic potential; however, they lack precise histological examinations. Here, we studied bone-like matrix formation by PDL cells in rats using morphological techniques. Rat and human PDL cells exhibited substantial alkaline phosphatase activity and induced mineralization in vitro. RT-PCR analyses showed that PDL cells expressed the osteoblast markers, Runx2, osterix, and osteocalcin. These results suggest that PDL cells share similar phenotypes with osteoblasts. To examine the bone-like matrix formation in vivo, PDL cells isolated from green fluorescent protein (GFP)-transgenic rats were inoculated with hydroxyapatite (HA) disks into wild-type rats. Five weeks after the implantation, the pores in HA disks were occupied by GFP-positive cells. Mineralized matrix formation was also found on the surface of HA pores. At 12 weeks, some of the pores were filled with bone-like mineralized matrices (BLMM), which were positive for the bone matrix proteins, osteopontin, bone sialoprotein, and osteocalcin. Immunohistochemical examination revealed that most of the osteoblast- and osteocyte-like cells on or in the BLMM were GFP-positive, suggesting that the BLMM were directly formed by the inoculated PDL cells. On the pore surfaces, Sharpey’s fiber-like structures embedded in cementum-like mineralized layers were also observed. These results collectively suggest that PDL cells have the ability to form periodontal tissues and could be a useful source for regenerative therapies of periodontal diseases.     

松梅乐注射液促进骨折愈合的机理研究

目的:通过观察松梅乐注射液治疗后骨折处细胞生长情况及生长因子的表达,探讨松梅乐注射液促进骨折愈合的机理。方法:80只新西兰大白兔随机分为高剂量组?低剂量组?对照组和空白组,双侧桡骨中段制造3 mm骨缺损模型,术后每日分别肌注0.2 ml/kg松梅乐注射液?0.1 ml/kg松梅乐注射液?0.2 ml/kg骨肽注射液和0.2 ml/kg生理盐水。术后1,2,4,6周分批处死动物取材行X线?组织学染色及生物力学测试。结果:高剂量组骨折处早期即有成骨细胞出现和大量生长因子染色阳性细胞;术后6周X线和组织学观察均显示骨折完全愈合,具有良好的抗压缩能力。结论:松梅乐注射液能促使骨折处早期分泌各种生长因子促进成骨细胞出现、成熟,进而缩短骨折愈合时间。     

脂多糖刺激单核巨噬细胞培养上清液对成骨细胞成骨特性的影响

目的 探讨建立牙周炎症的体外实验模型的可行性,为后续进行牙周炎症状态下应力对牙槽骨改建的研究奠定基础.方法 以牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)脂多糖(lipopolysaccharide,LPS)Pg-LPS刺激小鼠单核巨噬细胞(RAW264.7)的培养上清液作用于体外培养的小...     

不同浓度地塞米松体外对人成骨细胞和破骨细胞分化的影响

目的: 探讨地塞米松(DEX)体外对人成骨细胞(OBs)和破骨细胞(OCs)分化的影响。方法: 以DEX为主要成分的诱导剂体外刺激正常人骨髓间充质干细胞(MSCs),茜素红染色及碱性磷酸酶(ALP)定量法检测OBs间钙结节形成及OBs中ALP水平;改变DEX浓度后,再检测ALP水平以及用Western blotting检测OBs中磷酸化GSK-3β蛋白的表达;外源性核因子κB受体活化因子配基(RANKL)刺激人骨髓单个核细胞(BMMs)14 d后,用抗酒石酸酸性磷酸酶染色鉴定OCs形成情况、Western blotting检测BMMs中RANKL下游的信号途径;不同浓度DEX刺激MSCs 7 d后,用ELISA和Western blotting检测培养液中可溶性RANKL(sRANKL)水平及MSCs中RANKL蛋白表达。结果: 含0.1 μmol/L DEX的诱导剂刺激MSCs后可有钙结节形成,在DEX 0.001-0.1 μmol/L浓度范围内,OBs中ALP水平逐渐增高、磷酸化GSK-3β蛋白表达逐渐增强,当DEX浓度进一步增加后(0.1-10 μmol/L), ALP水平和磷酸化GSK-3β蛋白表达反而下降;外源性RANKL可以诱导BMMs分化为OCs,且BMMs中磷酸化IκBα、SPAK/JNK、p38 MAPK、p44/42 MAPK表达增强;在0.001-10 μmol/L浓度范围内,DEX刺激MSCs后,培养液中 sRANKL水平和MSCs中RANKL蛋白表达逐渐增高,且sRANKL水平和DEX浓度呈正相关(r=0.821,P<0.05)。结论: 在低浓度(0.001-0.1 μmol/L)范围内,DEX既促进OBs分化,又可促进OCs分化。在高浓度(0.1-10 μmol/L)范围内,仍能促进OCs分化,但失去了其对OBs的分化作用。     

淫羊藿素通过雌激素受体和骨形态发生蛋白信号诱导MC3T3-E1 subclone 14细胞分化

目的: 观察淫羊藿素(ICT)对MC3T3-E1 subclone 14前体成骨细胞株增殖、分化的影响,以及雌激素受体(ER)和骨形态发生蛋白(BMP)信号在分化中的作用。方法: WST-8、BrdU法检测ICT对MC3T3-E1 subclone 14细胞活力和增殖的影响;ICI182780阻断ER受体信号后,检测ICT和noggin对MC3T3-E1 subclone 14细胞碱性磷酸酶(ALP)活性、I型胶原(Col I)和骨钙素(BGP)的影响;实时荧光PCR检测ICT对BMP-2、4、7 mRNA表达的影响;Western blotting检测ER受体信号阻断后,ICT对Smad1/5/8蛋白磷酸化的影响。结果: ICT(0.1 μmol/L、1 μmol/L)可以提高MC3T3-E1 subclone 14细胞ALP、Col I、BGP和矿化结节数量(P<0.01或P<0.05),表明ICT有促分化的作用,但对细胞活力和增殖指数无明显影响;阻断ER受体信号后,ICT促分化作用明显下降(P<0.01);ICT可以提高BMP-2、4 mRNA的表达(P<0.01),但对BMP-7 mRNA无作用(P>0.01);阻断ER信号后,ICT 促Smad1/5/8磷酸化明显减弱(P<0.01)。进一步阻断BMP/Smad信号可以抑制ICT促分化的作用(P<0.01)。结论: ICT可以通过ER受体激活BMP/Smad信号通路,进而促进MC3T3-E1 subclone 14细胞的分化。     

Relevance of laser irradiance threshold in the induction of alkaline phosphatase in human osteoblast cultures

Induction of matrix synthesis by low-level laser has been demonstrated extensively. However, the question of dose- or power intensity-dependency is under-investigated. To address this issue we chose human osteoblast cell cultures and measured their alkaline phosphatase (ALP) activity after laser irradiation. The cell cultures were irradiated periodically by 690 nm radiation via optical transmission fiber-based laser needles, reaching into the culture dishes. The osteoblasts showed no induction of ALP activity when we used a single laser needle stimulation with a laser irradiance of 51 mW/cm², an increase of approximately 43% at 102 mW/cm² irradiance (two needles per well) and a ninefold increase at 204 mW/cm² irradiance (four needles per well), leaving the temperature of the culture medium unaffected. We concluded that the osteoblastic response in ALP activity to a laser stimulus shows a logarithmic relationship, with a distinct threshold, rather than a linear dose-dependency. Secondly, the laser irradiance, rather than the dose, is relevant for the impact of the laser.     

Histopathological and biochemical changes in the sutural region in craniosynostosis

Object: Histopathological observations and biochemical analysis of sutural bones in nine patients with craniosynostosis were compared with control subjects of the same age range. Methods: Microscopic examination in craniosynostosis showed formation of an active osseous front, with higher osteoblastic activity than in controls. Biochemical analysis revealed higher calcium, phosphorus, alkaline phosphatase, phospholipids and chon-droitin sulphate-A contents in sutural bones of the same patients. Conclusions: The present study systematically establishes a pre- mature increase in osteogenesis in the sutures of craniosynostosis patients. Received: 27 September 1999