50 Hz正弦交变磁场对体外培养大鼠成骨细胞分化及BMP-2和collagen-Ⅰ基因表达的影响

目的:研究频率为50 Hz不同强度正弦交变电磁场(electroagnetic fields,EMFs)对体外培养成骨细胞(Osteoblasts,OB)分化的影响.方法:体外分离培养大鼠颅骨成骨细胞,传代后随机分为9组,分别用频率50 Hz,磁场强度为0 mT(对照组)、0.9 mT、1.2 mT、1.5 mT、1.8 mT、2.1 mT、2.4 mT、2.7 mT和3.0 mT的正弦交变磁场处理,30 min/次/d.倒置相差显微镜下观察细胞形态;第3 d、6 d、9 d测定钙含量;第10 d茜素红钙化结节染色;RT-PCR法检测BMP-2和Collagen-Ⅰ基因的表达.结果:磁场处理了3d以后成骨细胞呈漩涡样分布;第9d磁场组钙盐沉淀量普遍高于对照组,尤以1.8mT显著高于对照(P<0.01)及其他各组(P<0.05);磁场处理组的BMP-2、Collagen-Ⅰ基因表达水平高于对照组.其中1.8 mT和2.1 mT组表达量明显较高.结论:本实验所用50 Hz,0.9 mT～3.0 mT的正弦交变磁场能促进体外培养成骨细胞分化成熟,并提高BMP-2和Collagen-I的基因表达水平,其中以1.8mT效果最为明显.     

单向交变拉伸应变及压应力对大鼠成骨细胞FOS蛋白表达及细胞定位的影响

分别对体外培养的大鼠成骨细胞施加单向交变拉伸应变和2atm的压力。利用免疫细胞化学技术，检测成骨细胞c—fos基因的表达。结果表明：施加两种不同的载荷后，成骨细胞c—fos基因表达FOS蛋白显著增加，FOS蛋白多集中在细胞核中，提示应力、应变在提高成骨细胞的增殖能力方面起着十分重要的作用。     

参脉注射液对缺血性大鼠下丘脑损伤的保护作用──c-fos免疫组化研究

缺血性脑损伤是一种常见病、多发病 ,研究其病因、发病机制、诊断和治疗手段具有重要意义 ;c- fos蛋白是 c- fos原癌基因在受刺激时快速、瞬间表达的一种 DNA结合蛋白 ,有研究提示 c- fos蛋白在缺血性脑损伤中具有一定作用。本文采用 c- fos蛋白免疫组织化学方法从分子水平探讨缺血性脑损伤的病理生理改变和参脉注射液的保护作用 ,为临床病理研究提供有关形态学依据。实验采用雄性 Wister大鼠 15只 (体重 2 0 0～ 2 5 0 g) ,随机分为对照组、缺血组和治疗组。缺血组和治疗组大鼠夹闭双侧颈总动脉 2 0分钟后再灌流 1小时 ,建立缺血性脑损伤…     

成骨细胞响应机械刺激的力化学转导机理

应力环境可调节骨组织的生长、吸收和重建。在细胞水平上,机械刺激可以影响成骨细胞的生理活性,如增殖、碱性磷酸酶活性、骨钙素合成等。力转导是将生物物理力转化为细胞的生化响应的过程,它是许多生理功能的基础,早期响应基因(c- fos,c- jun) ,第二信使系统(Ca2 ,NO,c AMP) ,力敏感阳离子通道等都参与了成骨细胞响应机械刺激的力化学转导过程     

人甲状旁腺素对新生大鼠成骨细胞护骨素基因表达的影响

目的研究人甲状旁腺激素(hPTH)对新生大鼠成骨细胞护骨素(OPG)基因表达的影响,探讨PTH对成骨细胞的作用机制.方法采用新生SD大鼠的颅骨行成骨细胞的原代培养,用hPTH于培养的不同阶段用不同浓度和不同方式进行刺激,以ELASA测定成骨细胞的分化指标;RT-PCR测定OPG基因的表达.结果与hPTH间歇刺激组相比,hPTH持续刺激组可明显抑制成骨细胞的分化成熟,且可明显抑制OPG mRNA的表达.这一作用在培养后6 d即出现,持续至第18 d.此外,hPTH抑制OPGmRNA的表达,以100ng/ml浓度最明显.结论持续PTH刺激可明显抑制成骨细胞的分化成熟和OPG基因的表达,这可能与慢性肾衰继发性甲状旁腺亢进骨病的发生密切相关.     

50Hz正弦交变磁场对体外培养大鼠成骨细胞分化及BMP-2和collagen-I基因表达的影响

目的:研究频率为50 Hz不同强度正弦交变电磁场(electromagnetic fields,EMFs)对体外培养成骨细胞(Osteoblasts,OB)分化的影响。方法:体外分离培养大鼠颅骨成骨细胞,传代后随机分为9组,分别用频率50 Hz,磁场强度为0 mT(对照组)、0.9 mT、1.2 mT、1.5 mT、1.8 mT、2.1 mT、2.4 mT、2.7 mT和3.0 mT的正弦交变磁场处理,30 min/次/d。倒置相差显微镜下观察细胞形态;第3 d、6 d、9 d测定钙含量;第10 d茜素红钙化结节染色;RT-PCR法检测BMP-2和Collagen-I基因的表达。结果:磁场处理了3 d以后成骨细胞呈漩涡样分布;第9 d磁场组钙盐沉淀量普遍高于对照组,尤以1.8 mT显著高于对照(P0.01)及其他各组(P0.05);磁场处理组的BMP-2、Collagen-I基因表达水平高于对照组,其中1.8 mT和2.1 mT组表达量明显较高。结论:本实验所用50 Hz,0.9 mT~3.0 mT的正弦交变磁场能促进体外培养成骨细胞分化成熟,并提高BMP-2和Collagen-I的基因表达水平,其中以1.8 mT效果最为明显。     

FGF10及其受体FGFR2 Ⅲb在小鼠肾脏发育中的表达

 摘要：目的 观察FGF10及其受体FGFR2 Ⅲb在小鼠肾脏发生发育中的表达和分布,探讨它们在肾脏发生发育中的作用。方法 选取胚龄12、14、16、18 d和生后1、7、14、21、42 d小鼠肾组织,用免疫组织化学和蛋白印迹技术（Western blot）,检测FGF10和FGFR2 Ⅲb的表达。结果 E12 d FGF10及FGFR2Ⅲb开始在输尿管芽微弱表达;随着肾脏发育成熟,FGF10及FGFR2Ⅲb主要表达于远端小管和集合管;FGF10在近曲小管表达,近直小管无表达;FGFR2Ⅲb在近端小管无阳性表达;生后肾组织及发育各阶段肾小体未见FGF10及FGFR2Ⅲb表达。Western blot显示随着胚（日）龄的增加,FGF10及FGFR2Ⅲb在肾脏的表达量先增后减。结论 FGF10和FGFR2Ⅲb在肾脏发生发育过程中发挥重要作用。     

Expression of FGF10 and FGFR2Ⅲb in development of mouse kindey

目的 观察FGF10及其受体FGFR2Ⅲb在小鼠肾脏发生发育中的表达和分布,探讨其在肾脏发生发育中的作用.方法 选取胚龄12、14、16及18 d和出生后1、7、14、21及42 d小鼠肾组织,用免疫组织化学和Western blot,检测FGF10和FGFR2Ⅲb的表达.结果 胚龄12 d组FGF10及FGFR2Ⅲb开始在输尿管芽微弱表达;随着肾脏发育成熟,FGF10及FGFR2Ⅲb主要表达于远端小管和集合管;FGF10在近曲小管表达,近直小管无表达;FGFR 2Ⅲb在近端小管无阳性表达;生后肾组织及发育各阶段肾小体未见FGF10及FGFR2Ⅲb表达.Western blot显示随着胚(日)龄的增加,FGF10及FGFR2Ⅲb在肾脏的表达量先增后减.结论 FGF10和FGFR2Ⅲb在肾脏发生发育过程中发挥重要作用.     

BMP-2和FGF-2对小鼠骨髓间充质干细胞向成骨细胞分化的影响

目的:观察骨形成蛋白(BMP-2)和成纤维细胞生长因子2(FGF-2)对小鼠骨髓间充质干细胞(MSCs)向成骨细胞分化的影响.方法:取3～18月雄性C57BL/6J小鼠(共50只)的骨髓细胞, 分离贴壁培养后, 用免疫磁珠法纯化, 并鉴定为MSCs后, 再进行贴壁培养24 h后, 分别在成骨细胞诱导培养液中加入100 μg/L BMP-2和0.5 nmol/L FGF-2持续诱导7、 14、 21 d后, 进行碱性磷酸酶染色、碱性磷酸酶活性检测, Vonkossa染色以及茜素红染色, 并用递转录荧光定量PCR法检测向成骨细胞分化的标志性基因(Runx2/cbfa1、 Alp、 collagen-1、 osteocalcin)的表达情况.结果:BMP-2刺激组的ALP活性以及钙化结节明显高于对照组, Runx2/cbfa1, ALP, collage-1, osteocalcin的mRNA呈高表达; FGF-2刺激组的ALP活性以及钙化结节也高于对照组, Runx2/cbfa1、 Alp、 collagen-1、 osteocalcin的mRNA表达量也高于对照组, 但不如BMP-2明显. 结论:BMP-2和 FGF-2在不同程度上促进体外培养的小鼠MSCs向成骨细胞方向分化.     

甘草酸对切口愈合模型大鼠促进切口愈合及防止瘢痕形成效果的研究

目的研究甘草酸促进胶原合成对切口愈合模型大鼠瘢痕形成及切口愈合的效果。方法选择50只SD健康大鼠,随机分为空白对照组、模型组、低剂量组、中剂量组和高剂量组,每组10只。模型组、低剂量组、中剂量组、高剂量组均建立切口愈合模型,30 min后对低剂量组、中剂量组、高剂量组大鼠分别静脉注射10 mg/kg、15 mg/kg、30 mg/kg甘草酸,术后3 d、7 d、14 d观察各组大鼠变化。进行组织切片、HE染色,观察大鼠病理组织学变化,检测创面组织中Ⅰ型胶原、Ⅲ型胶原、VEGF、b FGF、EGF mRNA表达量及Ⅰ型胶原、Ⅲ型胶原、PTEN/AKT/VEGF信号通路蛋白表达量,计算愈合瘢痕面积、创面愈合率及愈合时间,并进行组间比较。结果术后3 d、7 d、14 d低剂量组、中剂量组、高剂量组大鼠创面组织中Ⅰ型胶原、Ⅲ型胶原、VEGF、b FGF、EGF mRNA及Ⅰ型胶原、Ⅲ型胶原、VEGFR2、p-AKT蛋白表达量均高于模型组,PTEN蛋白表达量低于模型组,差异具有统计学意义(P 0. 05);术后3 d、7 d、14 d中剂量组、高剂量组大鼠创面组织中Ⅰ型胶原、Ⅲ型胶原、VEGF、b FGF、EGF mRNA及Ⅰ型胶原、Ⅲ型胶原、VEGFR2、p-AKT蛋白表达量均高于低剂量组,PTEN蛋白表达量低于低剂量组,差异具有统计学意义(P 0. 05);术后3 d、7 d、14 d高剂量组大鼠创面组织中Ⅰ型胶原、Ⅲ型胶原、VEGF、b FGF、EGF mRNA及Ⅰ型胶原、Ⅲ型胶原、VEGFR2、p-AKT蛋白表达量均高于中剂量组,PTEN蛋白表达量均低于中剂量组,差异具有统计学意义(P 0. 05)。术后3 d、7 d、14 d低剂量组、中剂量组、高剂量组大鼠愈合瘢痕面积、创面愈合率均高于模型组大鼠,创面愈合时间短于模型组,差异具有统计学意义(P 0. 05);术后3 d、7 d、14 d中剂量组、高剂量组大鼠愈合瘢痕面积、创面愈合率均高于低剂量组,创面愈合时间短于低剂量组,差异具有统计学意义(P 0. 05);术后3 d、7 d、14 d高剂量组大鼠愈合瘢痕面积、创面愈合率均高于中剂量组,创面愈合时间短于中剂量组,差异具有统计学意义(P 0. 05)。结论甘草酸通过作用于PTEN/AKT/VEGF信号通路,减轻切口创面炎性浸润,促进胶原合成、创面愈合,防止瘢痕形成,且呈剂量依赖。     

骨髓基质干细胞成骨分化过程中Wnt、骨形态发生蛋白2信号通路相关因子与辛伐他汀的作用

背景：辛伐他汀可促进体外培养的人或鼠骨髓基质干细胞向成骨细胞分化,但作用机制尚不清楚。 目的：观察辛伐他汀对大鼠骨髓基质干细胞向成骨细胞分化过程中Wnt与骨形态发生蛋白2信号途径中相关因子表达的影响。 方法：取6周龄雌性SD大鼠双侧股骨、胫骨全骨髓进行体外成骨细胞诱导培养。实验分为对照组及SIM组。SIM组加入浓度为10-7 mol/L辛伐他汀,对照组加入等量无水乙醇和PBS。培养14 d,行碱性磷酸酶染色,28 d时,行von Kossa染色观察细胞外基质矿化情况;培养14,21 d,免疫荧光细胞化学染色观察成骨细胞中β-catenin,Smad1/5,Cbfa1的表达及分布。  结果与结论：大鼠骨髓基质干细胞经体外诱导后可分化为具有碱性磷酸酶活性和矿化细胞外基质能力的成熟成骨细胞。辛伐他汀可显著上调骨髓基质干细胞成骨分化过程中碱性磷酸酶的表达。同时,与对照组比较,SIM组β-catenin,Smad1/5,Cbfa1表达明显增多(P < 0.05),且呈现明显的核内聚集趋势。说明辛伐他汀促进骨髓基质干细胞向成骨细胞分化的作用可能与调控Wnt与骨形态发生蛋白2信号通路中相关因子的表达及细胞内分布有关。     

辐射对体外培养的小鼠成骨细胞Ⅰ型胶原mRNA表达的影响

目的 肿瘤放射治疗诱发骨组织损伤,辐射后成骨细胞Ⅰ型胶原的基因表达分析揭示辐射对早期和晚期的成骨细胞功能的影响.方法 在体外诱导骨髓基质细胞生成成骨细胞,对其特性进行确定.用聚合酶链式反应(PCR)方法分析了1～4 Gy照射的早期和晚期成骨细胞的Ⅰ型胶原表达.结果与对照组相比,经1～3 Gy剂量照射后的早期成骨细胞Ⅰ型...     

Hyperbaric oxygen-stimulated proliferation and growth of osteoblasts may be mediated through the FGF-2/MEK/ERK 1/2/NF-κB and PKC/JNK pathways

We investigated whether the hyperbaric oxygen (O₂) could promote the proliferation of growth-arrested osteoblasts in vitro and the mechanisms involved in this process. Osteoblasts were exposed to different combinations of saturation and pressure of O₂ and evaluated at 3 and 7 days. Control cells were cultured under ambient O₂ and normal pressure [1 atmosphere (ATA)]; high-pressure group cells were treated with high pressure (2.5 ATA) twice daily; high-O₂ group cells were treated with a high concentration O₂ (50% O₂) twice daily; and high pressure plus high-O₂ group cells were treated with high pressure (2.5 ATA) and a high concentration O₂ (50% O₂) twice daily. Hyperbaric O₂ significantly promoted osteoblast proliferation and cell cycle progression after 3 days of treatment. Hyperbaric O₂ treatment stimulated significantly increased mRNA expression of fibroblast growth factor (FGF)-2 as well as protein expression levels of Akt, p70^S6K, phosphorylated ERK, nuclear factor (NF)-κB, protein kinase C (PKC)α, and phosphorylated c-Jun N-terminal kinase (JNK). Our findings indicate that high pressure and high O₂ saturation stimulates growth-arrested osteoblasts to proliferate. These findings suggest that the proliferative effects of hyperbaric O₂ on osteoblasts may contribute to the recruitment of osteoblasts at the fracture site. The FGF-2/MEK/ERK 1/2/Akt/p70^S6K/NF-κB and PKC/JNK pathways may be involved in mediating this process.     

Temporal pattern of stimulation of osteoblast-associated genes during mechanically-induced osteogenesis in vivo: early responses of osteocalcin and type I collagen.

Mechanical loading is an essential environmental factor in skeletal homeostasis, but the response of osteoblast-associated genes to mechanical osteogenic signal is largely unknown. This study uses our recently characterized in vivo osteoinductive model to analyze the sequence of stimulation and the time course of expression of osteoblast-associated genes in mechanically loaded mouse periodontium. Temporal pattern of regulation of osteocalcin (OC), alkaline phosphatase (ALP), and type I collagen (collagen I) was determined during mechanically-induced osteoblast differentiation in vivo, using a mouse tooth movement model earlier shown to induce bone formation and cell-specific regulation of genes in osteoblasts. The expression of target genes was determined after 1, 2, 3, 4, and 6 days of orthodontic movement of the mouse first molar. mRNA levels were measured in the layer of osteoblasts adjacent to the alveolar bone surface, using in situ hybridization and a relative quantitative video image analysis of cell-specific hybridization intensity, with non-osseous mesenchymal periodontal cells as an internal standard. After 24 hours of loading, the level of OC in osteoblasts slightly decreased, followed by a remarkable 4.6-fold cell-specific stimulation between 1 and 2 days of treatment. The high level expression of OC was maintained throughout the treatment with a peak 7-fold stimulation at day 4. The expression of collagen I gene was not significantly affected after 1 day, but it was stimulated 3-fold at day 2, and maintained at a similar level through day 6. The ALP gene, which we previously found to be mechanically stimulated during the first 24 hours, remained enhanced from 1.8- to 2.2-fold throughout the 6 days of treatment. Thus, in an intact alveolar bone compartment, mechanical loading resulted in a defined temporal sequence of induction of osteoblast-associated genes. Stimulation of OC 48 h after the onset of loading (and 24 h prior to deposition of osteoid) temporally coincided with that of collagen I, and was preceded for 24 h by an enhancement of ALP. Identification of OC as a mechanically responsive gene induced in functionally active osteoblasts in this study is consistent with its potential role in limiting the rate of mechanically-induced bone modeling. Furthermore, these results show that temporal progression of mechanically-induced osteoblast phenotype in this in vivo model occurs very rapidly. This suggests that physiologically relevant mechanical osteoinductive signal in vivo is targeting a population of committed osteoblast precursor cells that are capable of rapidly responding by entering a differentiation pathway and initiating an anabolic skeletal adaptation process.     

假体磨损颗粒钴铬离子影响成骨细胞增殖及RANKL、骨保护素的表达

背景：金属-金属假体置入体内后可以发生腐蚀或磨损,释放镍、钴、铬、钛等金属离子,诱导局部炎性因子的释放。 目的：观察Co²⁺、Cr³⁺对小鼠成骨细胞增殖的影响,以及成骨细胞暴露在Co²⁺ 、Cr³⁺条件下RANKL、骨保护素基因的表达。 方法：体外培养成骨细胞,实验分两组,对照组给予生理盐水,离子组给予钴、铬离子干预。 结果与结论：显微镜直接计数法显示干预后1~6 d对照组随时间推移细胞数目显著增加,离子组则增加不明显。共培养24,48 h后,RT-PCR结果显示离子组RANKL、骨保护素基因表达较对照组均增加,以RANKL增加更显著(P < 0.05),RANKL/OPG mRNA的比率也明显增加。提示金属离子对成骨细胞的增殖有显著抑制作用,且可刺激成骨细胞RANKL、骨保护素mRNA的表达。     

Effects of coumestrol on neonatal and adult mice osteoblasts activities

Estrogen replacement therapy has been shown to reduce postmenopausal osteoporosis. In the present study, we examined the effects of the phytoestrogen coumestrol on neonatal and adult osteoblasts metabolism. Two different sources of osteoblast cells (neonatal mice calvaria and adult mice long bone) cultures were used in this study. The effects of coumestrol on the cellular activities were analyzed by the mitochondrial tetrazolium (MTT) assay, secretion of alkaline phosphatase (ALP), intracellular calcium content (Ca), and the gene expression of bone matrix protein, estrogen receptors (ER-alpha, ER-beta), and osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL). The results showed that the proliferation of neonatal mice osteoblast cells was enhanced by treatment of coumestrol. In the presence of 10(-9)M coumestrol, the osteoblast proliferation attained 139.5% of the control and that the coumestrol can increase the intracellular calcium contents. Type I collagen gene expression was upregulated 167% at the 1st day's culture; ALP gene expression was upregulated 360% at the 7th day's culture; while the osteocalcin gene expression was upregulated 222% at the 14th day's culture. When adult mice osteoblasts were cultured in the presence of 10(-9)M coumestrol, the osteoblasts population increased significantly earlier and attained its maximal effect at the 21st day's culture with 207.4% of control group. The content of ER-beta and osteoprotegerin secretion by neonatal mice control cells gradually increased during osteoblasts differentiation, whereas the ER-alpha and OPGL content were decreased in this study. The cellular responses to the estradiol and counmestrol were quite different in the osteoblasts derived from different age.