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。     

健骨胶囊对成骨样细胞MC3T3-E1增殖及分化作用机制

目的 探讨国医大师刘柏龄“健骨胶囊”对MC3T3-E1成骨细胞分化及增殖的影响。方法 制备健骨胶囊水提物,采用CCK-8法和细胞迁移实验检测健骨胶囊提取物对MC3T3-E1细胞增殖和细胞迁移的影响;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、OCN、OPN、Col1a1、ALP、Bcl2、RASSF1A等mRNA表达水平;蛋白质印迹法Western blot检测Col1a1、Bcl2的蛋白表达水平。结果 通过实验结果比对得出,健骨胶囊提取物能促进MC3T3-E1细胞增殖、使细胞迁移率提高;同时健骨胶囊提取物组能明显提高MC3T3-E1细胞钙化能力(P＜0.01),促进Runx2、OCN、OPN、Col1a1、ALP、Bcl2的mRNA表达(P＜0.05),上调Col1a1、Bcl2蛋白量的表达。结论 健骨胶囊能促进成骨细胞MC3T3-E1的增殖及细胞迁移能力,并通过上调成骨基因的表达水平如Runx2、OCN、OPN、Col1a1、ALP、Bcl2等,提高MC3T3-E1细胞的成骨能力。     

THSD4基因在小鼠间充质干细胞及MC3T3-E1细胞成骨分化中的作用

目的 探讨THSD4基因对小鼠间充质干细胞和MC3T3-E1细胞成骨分化的影响。方法 提取绝经后骨质疏松症患者的骨髓间充质干细胞进行基因测序分析,与骨关节炎患者的骨髓间充质干细胞进行比较,分析基因表达差异。通过提取不同分化阶段的小鼠骨髓间充质干细胞（M-BMSC）及MC3T3-E1细胞的mRNA来检测THSD4 基因以及成骨分化的标志性基因（ALP、Runx2、Osx）的表达水平。通过构建慢病毒表达载体来实现对M-BMSC及MC3T3-E1细胞中THSD4的敲减及过表达,并观察其对M-BMSC及MC3T3-E1细胞成骨分化能力的影响。结果 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且通过KEGG以及GO富集分析发现THSD4基因可能与PI3K-AKT信号通路及Wnt信号通路相关。随着成骨诱导分化时间的延长,THSD4 mRNA和成骨分化标志性基因（ALP、Runx2、Osx）mRNA在MC3T3-E1以及M-BMSC中表达量均逐渐增加。过表达THSD4可以增强MC3T3-E1细胞和M-BMSC的成骨分化能力,而敲减THSD4则减弱了MC3T3-E1细胞和M-BMSC的成骨分化能力。结论 THSD4基因在绝经后骨质疏松症患者骨髓间充质干细胞中明显下调,且THSD4基因可以增强MC3T3-E1细胞以及M-BMSC的成骨分化能力。     

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。     

黄芩素通过BMP-2/Smad通路促进MC3T3-E1成骨作用的实验研究

目的 探讨黄芩素（BAI）对小鼠胚胎成骨细胞前体细胞（MC3T3-E1）成骨分化的作用及其分子机制。方法 将MC3T3-E1分为对照组（正常培养）和BAI组（以Baicalein处理）,在成骨分化条件培养下采用CCK-8检测BAI对MC3T3-E1细胞增殖的影响;分别以碱性磷酸酶染色（ALP）、茜素红染色（ARS）检测MC3T3-E1细胞成骨分化水平与矿化能力,实时荧光定量PCR检测成骨标志基因ALP、COL1A1、RUNX2、OSX的mRNA表达水平,通过免疫印迹法（Western-blot）检测MC3T3-E1细胞中BMP-2、Smad1、p-Smad1蛋白表达水平,通过免疫荧光技术（IF）检测RUNX2、COL1A1表达水平。结果 与对照组比较,BAI干预1 d后发现,BAI组COL1A1（P<0.001）、RUNX2（P <0.05）、OSX（P <0.05） mRNA表达水平在成骨分化中表达上升;干预3 d后发现,与对照组比较,BAI组ALP（P <0.05）、RUNX2（P <0.001）mRNA表达上升;干预7 d后发现,与对照组比较,BAI组COL1A1（P <0.05）mRNA表达水平较对照组上升,BMP-2、p-Smad1/Smad1蛋白表达水平上升（P <0.05）。免疫荧光中成骨标志蛋白RUNX2、COL1A1表达增多（P <0.05）。结论 BAI可通过激活BMP-2/Smad通路促进MC3T3-E1成骨分化。     

Cyclooxygenase inhibitors enhance cell growth in an osteoblastic cell line, MC3T3-E1

To elucidate the significance of endogenous prostaglandin E2 (PGE2) in osteoblastic cell function, we studied the effects of cyclooxygenase inhibitors on cell growth and alkaline phosphatase (ALP) activity in MC3T3-E1 cells. UMR-106 cells were also used as references in our experiments. MC3T3-E1 cells, cultured in alpha-minimal essential medium containing 10% fetal bovine serum, were shown to produce PGE2, which was markedly suppressed in the presence of indomethacin. Addition of indomethacin resulted in an increase in DNA content and [3H]thymidine incorporation. A similar growth stimulatory effect was observed when structurally different cyclooxygenase inhibitors, that is, acetyl salicylic acid (ASA), flurbiprofen, and piroxicam, were added. These cyclooxygenase inhibitors, however, differed in their effects on ALP activity. Indomethacin and ASA enhanced ALP activity, whereas flurbiprofen and piroxicam suppressed it. We then examined the effects of exogenous addition of PGE2. Although exogenous PGE2 at 6 x 10(-6) M slightly stimulated cell growth, it inhibited cell growth at 6 x 10(-8) M and 6 x 10(-7) M. ALP activity was reduced in a dose-dependent fashion by exogenous PGE2. These results suggest that PGE2 produced by MC3T3-E1 may be suppressing cell proliferation and that cyclooxygenase inhibitors, per se, may stimulate cell growth by inhibiting endogenous PGE2 production in MC3T3-E1 cells. UMR-106 cells also produced PGE2, although less than MC3T3-E1 cells. In UMR-106 cells, the cyclooxygenase inhibitors did not influence DNA content or ALP activity as distinctly as in MC3T3-E1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

MicroRNA-196a靶向调节HDAC9对MC3T3-E1细胞成骨分化的影响

目的 探究微小RNA（miR）-196a靶向调节组蛋白去乙酰化酶9（HDAC9）对MC3T3-E1细胞成骨分化的影响。方法 将MC3T3-E1细胞分为对照组（Cont）组、诱导组、miR-196a-mimics-NC组、miR-196a-mimics组、miR-196a-inhibitor-NC组、miR-196a-inhibitor组、miR-196a-mimics+pCMV-HDAC9-NC组、miR-196a-mimics+pCMV-HDAC9组,根据分组转染后进行成骨诱导。定量荧光PCR检测MC3T3-E1细胞中miR-196a、HDAC9表达量;试剂盒检测碱性磷酸酶（ALP）活性;茜素红染色观察矿化程度;Western blot检测HDAC9、ALP、Runt相关转录因子2（Runx2）、胶原蛋白I（COL1）、骨桥蛋白（OPN）、Histone H3、Histone H3（acetyl K9、K14和K23）表达量。结果 与Cont组相比,诱导组MC3T3-E1细胞中miR-196a表达、ALP、Runx2、COL1、OPN蛋白表达、ALP活性、矿化程度及Histone H3 K9、K14、K23位点乙酰化水平增高（P＜0.05）,HDAC9 mRNA和蛋白表达降低（P＜0.05）。转染miR-196a-mimics可明显增加miR-196a表达,降低HDAC9表达,并增加ALP、Runx2、COL1、OPN蛋白表达、ALP活性、矿化程度及Histone H3乙酰化,转染miR-196a-inhibitor则作用相反。miR-196a可靶向下调HDAC9表达,过表达HDAC9可部分逆转miR-196a mimics对MC3T3-E1细胞成骨分化的促进效应。结论 miR-196a可靶向下调HDAC9表达,增加组蛋白乙酰化水平,促进MC3T3-E1细胞成骨分化。     

PKCα suppresses osteoblastic differentiation

Protein kinase C (PKC) plays an essential role in cellular signal transduction for mediating a variety of biological functions. There are 11 PKC isoforms and these isoforms are believed to play distinct roles in cells. Although the role of individual isoforms of PKC has been investigated in many fields, little is known about the role of PKC in osteoblastic differentiation. Here, we investigated which isoforms of PKC are involved in osteoblastic differentiation of the mouse preosteoblastic cell line MC3T3-E1. Treatment with G?6976, an inhibitor of PKCα and PKCβI, increased alkaline phosphatase (ALP) activity as well as gene expression of ALP and Osteocalcin (OCN), and enhanced calcification of the extracellular matrix. Concurrently, osteoblastic cell proliferation decreased at a concentration of 1.0 μM. In contrast, a PKCβ inhibitor, which inhibits PKCβI and PKCβII, did not significantly affect osteoblastic differentiation or cell proliferation. Knockdown of PKCα using MC3T3-E1 cells transfected with siRNA also induced an increase in ALP activity and in gene expression of ALP and OCN. In contrast, overexpression of wild-type PKCα decreased ALP activity and attenuated osteoblastic differentiation markers including ALP and OCN, but promoted cell proliferation. Taken together, our results indicate that PKCα suppresses osteoblastic differentiation, but promotes osteoblastic cell proliferation. These results imply that PKCα may have a pivotal role in cell signaling that modulates the differentiation and proliferation of osteoblasts.     

Twisted gastrulation and chordin inhibit differentiation and mineralization in MC3T3-E1 osteoblast-like cells

Bone morphogenetic proteins (BMPs) are potent inducers of osteoblast differentiation. The accessibility of BMP ligands for binding to their receptors is regulated by secreted proteins Twisted gastrulation (Tsg) and Chordin (Chd). Tsg antagonizes BMP signaling by forming ternary complexes with Chd and BMPs, thereby preventing BMPs from binding to their receptors. In addition to the anti-BMP function, Tsg also has pro-BMP activity, partly mediated by cleavage and degradation of Chd, which releases BMPs from ternary complexes. The roles of Tsg and Chd in osteoblast differentiation are not known. Therefore, in the present study, we investigated the effect of exogenous Tsg and Chd on osteoblast differentiation and mineralization using a well-characterized subclone of MC3T3-E1 osteoblast-like cells. Our results show that Tsg and Chd are expressed in MC3T3-E1 osteoblast-like cells. While Tsg mRNA levels decrease during osteoblast differentiation, Chd levels are found to increase. Tsg and Chd proteins accumulate in the cell culture media as the osteoblasts differentiate. Exogenous Tsg and Chd inhibit osteoblast differentiation and mineralization. Osteocalcin (OCN) mRNA levels decrease following both Tsg and Chd treatment. Tsg and Chd also inhibit alkaline phosphatase (ALP) activity in a dose-dependent manner. To provide insight into the mechanism of Tsg and Chd action, we investigated the effect of Tsg and Chd on BMP activity by determining phosphorylated Smad1 (pSmad1) levels. We show that both Tsg and Chd can independently and in combination reduce pSmad1 levels in MC3T3-E1 cells treated with BMP4. Further, BMP2 partially reverses the inhibitory effect of Tsg and Chd on ALP activity. Taken together, these results suggest that Tsg and Chd are involved in osteoblast differentiation and mineralization by regulating BMP signaling.     

持续性甲状旁腺素抑制成骨细胞分化 的实验研究

目的探讨持续性给与甲状旁腺素(PTH)对成骨细胞分化过程的抑制作用。方法培养MC3T3-E1成骨前体细胞,持续性给与PTH处理,碱性磷酸酶(ALP)染色方法,检测细胞内ALP的分泌;免疫荧光方法检测细胞内Osterix(Osx)蛋白的表达;real-time RT-PCT法和Westernblot方法检测细胞内成骨因子基因和蛋白的表达。结果 MC3T3-E1成骨前体细胞具有自发向成骨细胞方向分化的特征,与对照组相比,经PTH持续性处理的细胞ALP染色强度明显降低,Osx免疫荧光强度较弱,细胞内成骨因子基因和蛋白的表达亦显著低于对照组。结论持续性给与PTH具有抑制成骨细胞分化的作用。     

唑来膦酸对小鼠胚胎成骨细胞增殖和分化的影响

目的研究唑来膦酸对小鼠胚胎成骨细胞体外增殖和成骨分化的影响。方法将小鼠胚胎成骨细胞体外传代培养,使用含不同浓度（1.0、0.1μmol/L）唑来膦酸的成骨诱导培养基干预细胞,不含唑来膦酸的培养基作对照,培养1、3、5 d采用CCK-8试剂盒检测唑来膦酸对成骨细胞增殖的影响;培养14 d行碱性磷酸酶（ALP）染色;培养21 d行茜素红染色;培养7 d,实时定量聚合酶链式反应（Real-time PCR）检测转录因子Runx2、成骨标志物Ⅰ型胶原（Collagen TypeⅠ）、ALP、骨钙素（OCN）基因的表达,免疫印迹法（Western Blotting）检测转录因子Runx2蛋白的表达。结果不同浓度的唑来膦酸干预细胞后,CCK-8实验检测吸光度值随天数增加而升高,各组之间差异无统计学意义（P〉0.05）。随着唑来膦酸浓度的升高,ALP染色和茜素红染色逐渐变浅,Runx2、Collagen TypeⅠ、ALP、OCN基因的表达量降低,Runx2蛋白的表达量降低,差异均有统计学意义（P〈0.05）。结论唑来膦酸在选定浓度（1.0、0.1μmol/L）下不影响成骨细胞的增殖,但对其分化功能的抑制作用随着浓度的增加而增强。     

Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation.

In the present study, we investigate the implication of the mitogen-activated protein kinases (MAPKs) Erk, p38, and JNK in mediating the effect of fetal calf serum (FCS) on the differentiation of MC3T3-E1 osteoblast-like cells. Erk is stimulated by FCS in proliferating, early-differentiating, as well as in mature cells. Activation of p38 by FCS is not detected in proliferating cells but is observed as the cells differentiate. JNK is activated in response to FCS throughout the entire differentiation process, but a maximal stimulation is observed in early differentiating cells. The roles of Erk and p38 pathways in mediating MC3T3-E1 cell differentiation was determined using specific inhibitors such as U0126 and SB203580, respectively. These experiments confirmed that the Erk pathway is essential for mediating cell proliferation in response to FCS, but indicated that this MAP kinase has little effect in regulating the differentiation of MC3T3-E1 cells. In contrast, p38 only marginally influenced proliferation, but appeared to be critical for the control of alkaline phosphatase (ALP) expression in differentiating cells. Finally, results obtained with high doses of SB203580, which also affected JNK activity, suggest that p38 and/or JNK are probably also involved in the control of type 1 collagen and osteocalcin expression in differentiating cells. The data indicate that MAPKs regulate different stages of MC3T3-E1 cell development in response to FCS. Distinct MAPK pathways seem to independently modulate osteoblastic cell proliferation and differentiation, with Erk playing an essential role in cell replication, whereas p38 is involved in the regulation of ALP expression during osteoblastic cell differentiation. JNK is also probably involved in the regulation of osteoblastic cell differentiation, but its precise role requires further investigation.     

通过p38MAPK信号通路探讨仙灵骨葆胶囊对MC3T3-E1成骨分化的影响

目的 探讨仙灵骨葆胶囊含药血清对小鼠胚胎成骨细胞前体细胞（MC3T3-E1）增殖分化的影响及与p38MAPK信号通路的关系。方法 用仙灵骨葆胶囊含药血清干预MC3T3-E1,CCK8法筛选最佳干预时间及浓度,试剂盒检测各组细胞ALP活性。将细胞分为A、B、C、D 4组,分别加入10%含药血清、10%空白血清、10%含药血清+SB203580、10%空白血清+SB203580,利用免疫印迹法（Western blot）检测p38丝裂原活化蛋白激酶（p38）、磷酸化p38（p-p38）、成骨相关转录因子2（Runx2）、骨形态发生蛋白2（BMP-2）的蛋白表达量,荧光定量PCR检测p38、Runx2和BMP-2 mRNA的表达水平。结果 与空白血清组相比,不同浓度的仙灵骨葆胶囊含药血清均能促进MC3T3-E1的增殖分化,提高ALP的活性,其中10%含药血清干预36 h的作用最为明显（P＜0.05）;与B组比较,A组p38、p-p38、Runx2、BMP-2蛋白及p38、Runx2、BMP-2 mRNA的表达明显增高（P＜0.05）;加入信号通路阻断剂SB203580后,C组与D组上述指标的表达明显降低（P＜0.05）;与C组相比,D组p38、p-p38、Runx2、BMP-2蛋白及p38、Runx2、BMP-2 mRNA表达更低（P＜0.05）。结论 仙灵骨葆胶囊含药血清能促进MC3T3-E1的分化生长,其作用机制可能与激活p38MAPK信号通路、上调成骨相关因子Runx2、BMP-2的表达有关。     

Restoration of Mineral Depositions by Dexamethasone in the Matrix of Nonmineralizing Osteoblastic Cells Subcloned from MC3T3-E1 Cells

The original osteoblastic cell line, MC3T3-E1, was derived from normal mouse bone tissue and mineralized without any specific factors in vitro. This cell line may be slightly unstable because of high differentiation, and some of these cells sometimes lost the ability for mineral deposition. In this study, a new cell line was cloned which lost the ability for mineral deposition from MC3T3-E1 cells. This cell line, termed MC3T3-NM4, was not observed to undergo mineral deposition for up to at least 36 days even in media containing beta-glycerophosphate. The alkaline phosphatase (ALP) activity was also not increased. The lack of calcifying ability was found to be restored by the addition of dexamethasone in the media. This restoration was accompanied by an increase in ALP activity and osteocalcin level. It was suggested that this restoration was not due to artificial mineralization resulting from cell death. Received: 3 March 1999 / Accepted: 25 May 2000 / Online publication: 22 September 2000