红车轴草总异黄酮含药血清对MC3T3-E1细胞增殖、分化和矿化的影响

目的研究红车轴草总异黄酮含药血清对MC3T3-E1成骨样细胞增殖、分化和矿化的影响。方法大鼠灌胃红车轴草总异黄酮一定时间后,制备含药血清,并分别采用MTT法、碱性磷酸酶活性测定法、ELISA法和茜素红染色测定矿化结节的方法考察不同浓度红车轴草总异黄酮含药血清对MC3T3-E1成骨样细胞的增殖率、碱性磷酸酶活性、骨钙素生成以及矿化结节形成的影响。结果不同浓度的红车轴草总异黄酮含药血清都能显著促进MC3T3-E1成骨样细胞的增殖并显著提高MC3T3-E1成骨样细胞碱性磷酸酶的活性,且最佳的含药血清浓度为5%;5%的红车轴草总异黄酮含药血清处理MC3T3-E1成骨样细胞6 d和9 d可显著促进骨钙素的分泌;5%的红车轴草总异黄酮含药血清处理MC3T3-E1成骨样细胞6 d、12 d和18 d,形成的矿化结节均显著高于对照组。结论红车轴草总异黄酮含药血清可以显著促进MC3T3-E1成骨样细胞的增殖、碱性磷酸酶活性、骨钙素分泌和矿化,说明红车轴草总异黄酮含药血清确实可以促进MC3T3-E1成骨样细胞的分化成熟,该药物具有开发为抗骨质疏松症新药的潜力。     

牛骨胶原蛋白肽促进HOB增殖

目的 研究牛骨胶原蛋白肽(Bovine Collagen peptides,BCP)对人成骨细胞(Human Oseoblast,HOB)增殖的影响.方法 分离培养HOB,茜素红染色鉴定HOB的成骨特性,利用MTT实验检测胶原蛋白肽对HOB的增殖,流式细胞仪测定胶原蛋白肽对HOB细胞周期.结果 茜素红矿化染色结果表明,HOB具有成骨能力.MTT实验表明0.3 mg/ml胶原蛋白肽能显著促进细胞的增殖,并能增加G2 +S期的百分含量.结论 牛骨胶原蛋白肽能够促进HOB增殖,为骨质疏松的预防和治疗提供理论依据.     

续苓健骨汤含药血清对MC3T3-E1细胞分化及增殖的影响

目的探讨续苓健骨汤含药血清对MC3T3-E1成骨细胞分化及增殖的影响。方法制备续苓健骨汤含药血清,实验分为空白对照组、含药血清低剂量组、中剂量组和高剂量组。采用CCK-8法和流式细胞术检测续苓健骨汤含药血清对MC3T3-E1细胞增殖和细胞周期的影响;碱性磷酸酶(ALP)活性测定MC3T3-E1细胞的成骨分化能力;茜素红染色检测MC3T3-E1细胞的矿化能力;实时荧光定量PCR检测成骨分化基因Runx2、OC、Bmp2、Col1a1mRNA水平。结果与空白对照组比较,中、高剂量续苓健骨汤含药血清能促进MC3T3-E1细胞增殖、S期细胞比率和细胞增殖指数,并且呈现一定的剂量依赖性;同时中高剂量续苓健骨汤含药血清组能明显提高MC3T3-E1细胞ALP活性(P0.01)和钙化能力(P0.01),促进Runx2、OC、Bmp2、Col1a1 mRNA的表达(P0.05)。结论续苓健骨汤含药血清能促进成骨细胞MC3T3-E1的增殖,并通过上调骨形成相关基因Runx2、OC、BMP2、Col1a1的表达水平,提高MC3T3-E1细胞的成骨能力。     

黄瓜籽总皂苷对MC3T3-E1细胞分化及SPARC、OPG/RANKL表达的影响

目的观察黄瓜籽总皂苷提取物(cucumber seed saponins,CSS)对小鼠成骨细胞MC3T3-E1增殖、分化和矿化的影响,以及与骨质疏松相关的SPARC、OPG/RANKL/RANK信号通路的作用。方法通过MTT实验、碱性磷酸酶(alkaline phosphatase,ALP)活性检测、茜素红染色,考察不同浓度CSS对MC3T3-E1细胞增殖、分化及矿化的影响;采用RT-PCR方法检测SPARC、OPG/RANKL mRNA表达水平; Western blot检测SPARC、OPG/RANKL的蛋白表达量。结果与阳性对照组相比,CSS能明显促进MC3T3-E1细胞增殖(P0.05),CSS高、中剂量组能明显提高MC3T3-E1细胞ALP活性及钙化结节数量(P0.05);与空白组相比,CSS不同剂量组均明显上调SPARC、OPG/RANKL mRNA及蛋白表达水平(P0.05)。结论黄瓜籽总皂苷能够促进成骨细胞MC3T3-E1的增殖、分化及矿化能力,并通过上调SPARC、OPG/RANKL的表达水平提高MC3T3-E1细胞的成骨能力。     

STIM1调控Ras信号通路促进成骨细胞增殖的研究

目的成骨细胞增殖的抑制导致成骨细胞数量减少是骨质疏松症发生发展的重要原因。之前的研究发现介导受体依赖性钙离子内流的钙通道蛋白Orai1在调控成骨细胞增殖、分化、凋亡等细胞功能有非常重要的作用。STIM1是一类可激活Orai1的重要蛋白。然而,至今还未见STIM1是否参与调控成骨细胞增殖相关报道。本研究旨在明确STIM1是否参与调控成骨细胞增殖。方法应用靶向STIM1的siRNA在MC3T3-E1成骨细胞中沉默STIM1的表达,然后用MTT法检测细胞增殖变化、流式细胞仪检测细胞周期变化、Real-Time PCR检测CyclinD1、CyclinE、CDK4、CDK6的转录水平,并用Western Blot检测Ras信号通路活性。结果我们转染靶向STIM1的siRNA后,MC3T3-E1细胞中STIM1的表达和转录均明显降低(P0.05);接着,我们发现在MC3T3-E1细胞中沉默STIM1的表达后,MC3T3-E1细胞增殖抑制并且细胞周期阻滞在G0/G1期,并且调控细胞增殖的细胞周期蛋白CyclinD1的转录水平也显著下降(P0.05);进一步研究发现,在MC3T3-E1细胞沉默STIM1的表达后,明显抑制了钙离子依赖的Ras信号通路的活性(P0.05)。应用ARS-853抑制Ras信号通路的活性后,明显抑制了STIM1促进成骨细胞增殖的作用(P0.05)。结论 STIM1激活Ras信号通路发挥促进成骨细胞增殖的作用。     

GCM1通过激活Wnt3a/β-catenin信号通路改善绝经后骨质疏松症

目的 探究GCM1对小鼠胚胎成骨细胞前体细胞（MC3T3-E1）成骨分化及矿化的影响。方法 实验设置control组、BMP2诱导组、BMP2+ov-NC组、MP2+ov-GCM1组、BMP2+sh-NC组、BMP2+sh-GCM1组,BMP2用于诱导细胞成骨分化。通过碱性磷酸酶(ALP)染色及茜素红染色检测GCM1对MC3T3-E1成骨分化及矿化的影响;通过Western blot检测成骨分化相关蛋白及Wnt3a/β-catenin蛋白的表达变化。结果 GCM1在绝经后骨质疏松症患者血清中低表达。过表达GCM1能够促进BMP2诱导MC3T3-E1的成骨分化和矿化,而抑制GCM1表达则抑制了BMP2诱导MC3T3-E1的成骨分化和矿化。此外,过表达GCM1增加了Wnt3a/β-catenin蛋白表达,激活Wnt3a/β-catenin信号通路,抑制GCM1表达则与上述结果相反。结论 GCM1在绝经后骨质疏松症患者血清中低表达,过表达GCM1则可能通过激活Wnt3a/β-catenin信号通路、促进MC3T3-E1成骨分化及矿化,进而改善绝经后骨质疏松症。     

雷奈酸锶在模拟微重力环境对成骨细胞增殖功能的影响

目的 观察新型的抗骨质疏松药雷奈酸锶在模拟微重力环境下对成骨细胞增殖功能的影响.方法 利用沿水平轴连续回转(30 r/min)细胞培养系统模拟微重力环境,使用MTT比色法或台盼兰染色、细胞计数法观察小鼠成骨样细胞MC3T3-E1的增殖情况.结果 模拟微重力环境可以降低MC3T3-E1增殖功能,雷奈酸锶在模拟微重力环境可以增强MC3T3-E1增殖功能.结论 在模拟微重力环境下雷奈酸锶对成骨细胞增殖功能具有保护作用,为雷奈酸锶治疗微重力环境骨量丢失提供了理论和实验证据.     

芍药苷促进小鼠成骨分化抗骨质疏松作用的实验研究

目的 探讨芍药苷对MC3T3-E1成骨细胞分化以及小鼠骨质疏松模型的影响。 方法 体外细胞实验分为对照组、不同剂量芍药苷干预组。通过CCK-8法检测芍药苷对MC3T3-E1成骨细胞活力的影响;采用碱性磷酸酶（ALP）染色以及活性检测芍药苷促进MC3T3-E1成骨分化能力;通过茜素红染色检测芍药苷促矿化能力;运用荧光定量PCR、Western blot检测Runx2、OPG、RANKL、Col1α1的mRNA以及蛋白表达情况。选取8周龄C57BL/6小鼠24只,分为假手术组、骨质疏松模型组、药物干预组。选取各小鼠左侧股骨远端以及胫骨近端的区域进行苏木素-伊红（HE）染色、Runx2免疫组织化学以及micro-CT扫描。 结果 中、高剂量芍药苷干预组可以促进MC3T3-E1细胞ALP的活性（P<0.05）;高剂量芍药苷能够促进MC3T3-E1细胞的矿化能力（P<0.01）;同时中、高剂量能够促进OPG、Runx2蛋白的表达（P<0.05）,抑制RANKL的表达（P<0.05）。与假手术组比较,OVX组骨微结构破坏明显,Runx2蛋白表达显著减少（P<0.01）;芍药苷干预后骨质疏松模型小鼠骨小梁数量以及厚度显著增加（P<0.01）,骨小梁间隔减少明显（P<0.01）,Runx2蛋白提升明显（P<0.01）。 结论 芍药苷能够促进成骨细胞的分化,上调成骨分化基因,改善骨质疏松模型小鼠的骨微结构,具有抗骨质疏松治疗的潜在价值。     

小檗碱联合绿原酸抗骨质疏松的作用机制

目的 研究小檗碱联合绿原酸对MC3T3-E1细胞分化作用及对绝经后骨质疏松小鼠的干预作用,并探讨其可能的机制。方法 观察小檗碱、绿原酸及两药联用对MC3T3-E1细胞增殖和分化能力的影响,并检测细胞中Wnt3a、β-catenin及成骨分化相关基因mRNA的表达水平。在体构建绝经后骨质疏松症小鼠模型,随机分为模型组、小檗碱组、绿原酸组及两药联用组,同时设置假手术组为对照,尾静脉给药8周后处死小鼠,检测其血清BALP、BGP含量,Micro-CT扫描观察小鼠股骨微结构变化,免疫组织化学法检测小鼠股骨Wnt3a和β-catenin蛋白表达,HE染色观察小鼠重要器官的组织病理结构变化。结果 与正常组相比,小檗碱组、绿原酸组及两药联用组对MC3T3-E1细胞增殖率无明显影响,但均可促进MC3T3-E1细胞成骨分化能力和矿化水平,以两药联用组最明显;并且两药联用组能显著升高Wnt3a、β-catenin、ALP、Runx2、OPN和OCN mRNA的表达(P<0.01)。体内实验表明,与模型组相比,两药联用组小鼠血清BALP、BGP含量明显降低,股骨微观结构得到明显改善,股骨Wnt3a和β...     

Enamel matrix derivative inhibits TNF-alpha-induced apoptosis in osteoblastic MC3T3-E1 cells.

OBJECTIVE: The purpose of this study was to examine the effect of enamel matrix derivative (EMD) on TNF-alpha-induced apoptosis in osteoblastic MC3T3-E1 cells. STUDY DESIGN: MC3T3-E1 cells were cultured at an initial density of 5000/cm 2 in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and allowed to adhere for 24 hours. Medium was then changed into DMEM with 0.5% FBS. After 16 hours, cells were treated with EMD (100 microg/mL) alone, tumor necrosis factor alpha (TNF-alpha) (20 ng/mL) alone, transforming growth factor beta 1 (TGF-beta1) (10 ng/mL) alone, TNF-alpha plus TGF-beta1, or TNF-alpha plus EMD. Cells cultured with DMEM and 0.5% FBS served as control. Following 24-hour incubation, apoptosis was assessed by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay, and quantified by cell death enzyme-linked immunosorbent assay (ELISA). RESULTS: Both TUNEL assay and cell death ELISA show that TNF-alpha induces apoptosis in MC3T3-E1 cells. TNF-alpha increases cell death by approximately 2-fold, which is attenuated by both EMD and TGF-beta1. CONCLUSION: Like TGF-beta1, EMD protects osteoblasts from inflammation-induced apoptosis.     

Enhancement of human osteoblast proliferation and phenotypic expression when cultured in human serum

Traditionally, culture medium is supplemented with foetal bovine serum (FBS). However, in cultures of osteoblasts intended for human re-implantation, such serum presents potential risks of foreign protein contamination and transmission of viral or prionrelated material, if used. We cultured human osteoblasts from 16 patients in 10% autologous human serum, 10% pooled human serum, 10% FBS or 2% Ultroser G. Non-synthetic sera were tested in both heat-treated and non-heat-treated forms. We determined cell growth and osteoblast phenotype. Cell proliferation in all types of human serum was significantly greater than in FBS. This was most marked in heat-treated autologous human serum. Cells cultured in Ultroser G had less proliferation than all other groups. The phenotypic tests showed that cells cultured in human and foetal bovine serum displayed an osteoblast phenotype, with greater protein expression in cells cultured in human serum. We conclude that culture of human osteoblasts in autologous human serum enhances cell proliferation, while maintaining an osteoblast phenotype. These findings have implications for the use of cultured osteoblasts in self-cell therapy. Human osteoblast growth is supported by autologous human serum, which allows re-implantation of cultured cells, while avoiding the risk of foreign protein carry-over with enhancement of cell proliferation.     

Enhancement of human osteoblast proliferation and phenotypic expression when cultured in human serum

Traditionally, culture medium is supplemented with foetal bovine serum (FBS). However, in cultures of osteoblasts intended for human re-implantation, such serum presents potential risks of foreign protein contamination and transmission of viral or prionrelated material, if used. We cultured human osteoblasts from 16 patients in 10% autologous human serum, 10% pooled human serum, 10% FBS or 2% Ultroser G. Non-synthetic sera were tested in both heat-treated and non-heat-treated forms. We determined cell growth and osteoblast phenotype. Cell proliferation in all types of human serum was significantly greater than in FBS. This was most marked in heat-treated autologous human serum. Cells cultured in Ultroser G had less proliferation than all other groups. The phenotypic tests showed that cells cultured in human and foetal bovine serum displayed an osteoblast phenotype, with greater protein expression in cells cultured in human serum. We conclude that culture of human osteoblasts in autologous human serum enhances cell proliferation, while maintaining an osteoblast phenotype. These findings have implications for the use of cultured osteoblasts in self-cell therapy. Human osteoblast growth is supported by autologous human serum, which allows re-implantation of cultured cells, while avoiding the risk of foreign protein carry-over with enhancement of cell proliferation.     

Enhancement of human osteoblast proliferation and phenotypic expression when cultured in human serum

Traditionally, culture medium is supplemented with foetal bovine serum (FBS). However, in cultures of osteoblasts intended for human re-implantation, such serum presents potential risks of foreign protein contamination and transmission of viral or prion-related material, if used. We cultured human osteoblasts from 16 patients in 10% autologous human serum, 10% pooled human serum, 10% FBS or 2% Ultroser G. Non-synthetic sera were tested in both heat-treated and non-heat-treated forms. We determined cell growth and osteoblast phenotype. Cell proliferation in all types of human serum was significantly greater than in FBS. This was most marked in heat-treated autologous human serum. Cells cultured in Ultroser G had less proliferation than all other groups. The phenotypic tests showed that cells cultured in human and foetal bovine serum displayed an osteoblast phenotype, with greater protein expression in cells cultured in human serum. We conclude that culture of human osteoblasts in autologous human serum enhances cell proliferation, while maintaining an osteoblast phenotype. These findings have implications for the use of cultured osteoblasts in self-cell therapy. Human osteoblast growth is supported by autologous human serum, which allows re-implantation of cultured cells, while avoiding the risk of foreign protein carry-over with enhancement of cell proliferation.     

Fidgetin-like 1 gene inhibited by basic fibroblast growth factor regulates the proliferation and differentiation of osteoblasts.

The FIGNL1 gene was proven to be a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). In this in vitro study, the AAA proteins inhibited osteoblast proliferation and stimulated osteoblast differentiation. We showed that FIGNL1 may play some regulatory role in osteoblastogenesis. INTRODUCTION: The fidgetin-like 1 (FIGNL1) gene encodes a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). Although the FIGNL1 protein localizes to both the nucleus and cytoplasm, the function of FIGNL1 remains unknown. In a previous study, we identified several genes that mediate the anabolic effects of basic fibroblast growth factor (bFGF) on bone by using microarray data. FIGNL1 was one of the genes that downregulated >2-fold in MC3T3-E1 cells after treatment with bFGF. Therefore, this study was aimed to identify and confirm the function of FIGNL1 on osteoblastogenesis. MATERIALS AND METHODS: We examined the effect of the FIGNL1 gene on proliferation, differentiation, and apoptosis in mouse osteoblast cells (MC3T3-E1 and mouse primary calvarial cells) using flow cytometry, RT-PCR, cell proliferation assay, and cell death assay. MC3T3-E1 cells and mouse calvarial cells were transfected with small interfering RNA (siRNA) directed against the FIGNL1 or nontargeting control siRNA and examined by cell proliferation and cell death assays. Also, FIGNL1 was fused to enhance green fluorescent protein (EGFP), and the EGFP-fused protein was transiently expressed in MC3T3-E1 cells. RESULTS: Reduced expression of FIGNL1 by bFGF and TGF-beta1 treatment was verified by RT-PCR analysis. Overexpression of FIGNL1 reduced the proliferation of MC3T3-E1 and calvarial cells, more than the mock transfected control cells did. In contrast, siFIGNL1 transfection significantly increased the proliferation of osteoblasts, whereas overexpression of FIGNL1 did not seem to alter apoptosis in osteoblasts. Meanwhile, overexpression of FIGNL1 enhanced the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in osteoblasts. In contrast, siFIGNL1 decreased the expression of ALP and OCN. A pEGFP-FIGNL1 transfected into MCT3-E1 cells had an initially ubiquitous distribution and rapidly translocated to the nucleus 1 h after bFGF treatment. CONCLUSIONS: From these results, we proposed that FIGNL1, a subfamily member of the AAA family of proteins, might play some regulatory role in osteoblast proliferation and differentiation. Further analyses of FIGNL1 will be needed to better delineate the mechanisms contributing to the inhibition of proliferation and stimulation of osteoblast differentiation.     

Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development.

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.     

Purification and characterization of a growth factor from human bone matrix

A growth factor was purified from human bone matrix by extraction with EDTA, acetone treatment, gel filtration column chromatography, ion exchange column chromatography, and reversed-phase HPLC. Purified protein migrated as a single band to an area with a molecular weight of about 6,000 on SDS-polyacrylamide gel. The purified protein stimulated dose dependent osteoblast proliferation. The sequence of the first 30 N-terminal amino acids of the protein was identical to that of the human insulin-like growth factor-II (IGF-II). MC3T3-E1 cells reacted immunocytochemically to the monoclonal antibody against IGF-II. The culture medium of MC3T3-E1 cells contained immunoreactive IGF-II, which was measured by an enzyme immunoassay. These results indicate that IGF-II is contained in bone matrix, is effective in osteoblast proliferation, produced by the osteoblasts, and secreted from the osteoblasts. Taken together, these results show that IGF-II is present as a local growth factor in the bone system, playing an important role in bone formation following bone resorption.     

Down-Regulation of Osteoblastic Cell Differentiation by Epidermal Growth Factor Receptor

The role of epidermal growth factor receptors (EGF-R) in osteogenic cell differentiation was investigated using preosteoblastic MC3T3-E1 (MC3T3) cells and osteoblast-like ROS 17/2.8 (ROS) cells. When cultured in the presence of β-glycerophosphate (GP) and ascorbic acid (AA), MC3T3 cells underwent spontaneous differentiation into osteoblasts which was confirmed as they expressed osteoblast markers such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). Interestingly, the number of EGF-binding sites decreased during their differentiation into osteoblasts, and the osteogenic protein-1 (OP-1) treatment, which accelerated their differentiation, lowered the number of EGF-binding sites even further. On the other hand, ROS cells with high expression levels of osteoblast markers and no EGF-R, after being transfected with human EGF-R cDNA (EROS cells), expressed numerous EGF-binding sites as well as EGF-R mRNA and protein; in the process, they ceased to express osteoblast markers, indicating their dedifferentiation into osteoprogenitor cells. Both MC3T3 and EROS cells showed increased cell growth in response to EGF, whereas ROS cells did not. These results imply that the EGF/EGF-R system in osteogenic cells has a crucial function in osteoblast phenotype suppression and osteogenic cell proliferation.