G蛋白耦联受体激酶结合蛋白1通过调节细胞外调节激酶1/2的活性促进成骨细胞迁移

目的探索G蛋白耦联受体激酶结合蛋白1(GITI)在成骨细胞迁移中的作用,并分析其机理。方法通过Western blot方法检测GIT1蛋白在鼠的成骨细胞内的表达；用免疫荧光染色方法确定：在血小板衍生生长因子(PDGF)不刺激和刺激的条件下,GIT1和细胞外调节激酶1/2(ERK1/2)在成骨细胞内的位置；用共同免疫沉淀的方法测定GIT1和ERK1/2相互结合,并且用免疫荧光双染的方法确定这两种蛋白相互结合的位置；用包含GIT1-RNA发夹结构的腺病毒感染成骨细胞后,用免疫荧光染色方法确定磷酸化ERK1/2(pERK1/2)在成骨细胞内的位置,用划痕愈合法检测在PDGF刺激下的迁移能力。结果在成骨细胞内,PDGF刺激导致了GIT1和ERK1/2的相互结合,并且这种结合发生在成骨细胞的局部粘附内。包含GIT1-RNA发夹结构的腺病毒明显抑制了pERK1/2招募至成骨细胞局部粘附内以及PDGF所刺激的成骨细胞的迁移。结论在PDGF刺激下,GIT1招募pERK1/2至成骨细胞的局部粘附内,从而促进成骨细胞的迁移。     

G蛋白偶联受体激酶结合蛋白1的SHD结构域对成骨细胞内局部黏附激酶活性的影响

目的 探讨G蛋白偶联受体激酶结合蛋白1(GITl)的SHD结构域对成骨细胞内局部黏附激酶(FAK)活性的影响,并分析其机制.方法 取1～2dSD大鼠颅盖骨进行成骨细胞原代培养,传至第3代.体外构建野生型GIT1和删除了SHD结构域的GIT1的质粒并组成慢病毒.血小板衍生生长因子(PDGF)刺激成骨细胞后,采用免疫共沉淀法检测GIT1、删除了SHD结构域的GIT1与FAK的相互关系,采用免疫荧光法检测GIT1和FAK在成骨细胞内的位置、删除了SHD结构域的GIT1对成骨细胞内FAK位置和活性的影响.结果 PDGF刺激成骨细胞后,GIT1与FAK的相互结合明显增加,且随着时间的增加,结合量逐渐增加(F=293.105,P=0.000).免疫荧光染色结果表明:PDGF刺激后,成骨细胞局部黏附内活性形式的FAK(FAK酪氨酸397磷酸化)明显增加.PDGF刺激成骨细胞后,删除了SHD结构域的GIT1与FAK的结合明显被抑制,且随着时间的增加,二者的相互关系无明显变化(F=0.322,P=0.737);删除了SHD结构域的GIT1抑制成骨细胞局部黏附内FAK酪氨酸397的磷酸化,同时降低FAK在局部黏附内的表达.结论 删除了SHD结构域的GIT1通过降低与FAK的相互结合,从而抑制成骨细胞局部黏附内FAK酪氨酸397的磷酸化,影响成骨细胞的功能.     

血小板衍生性生长因子(PDGF)与胰岛素样生长因子Ⅰ(IGF-Ⅰ)对成骨细胞增殖及分化的影响

目的探讨PDGF与IGF-Ⅰ对体外培养成骨细胞增殖与分化的影响。方法将兔成骨细胞分别与PDGF(10ng/ml),IGF-Ⅰ(100ng/ml),PDGF(10ng/ml)+IGF-Ⅰ(100ng/ml)共同培养,于1、3、5d分别进行3H-TdR、3H-Proline的掺入量以及碱性磷酸酶合成量的检测。结果PDGF与IGF-1联合应用对3H-TdR、3H-Proline的掺入量与单独使用因子组、对照组相比,在各个时段均存在显著性差异(P<0.01);PDGF与IGF-1联合应用对碱性磷酸酶合成量与对照组、PDGF组相比有显著性差异(P<0.01),与IGF-1组相比,未见明显差异(P>0.05)。结论PDGF与IGF-Ⅰ联合应用可显著促进成骨细胞DNA、胶原蛋白、碱性磷酸酶的合成,对促进成骨细胞的增殖与分化功能具有协同作用。     

丹参对血小板源生长因子刺激血管平滑肌细胞增生的影响

目的：研究丹参对血小板源生长因子（PDGF）刺激血管平滑肌细胞（SMC）增生的影响。方法：体外培养大鼠动脉SMC，设对照组、丹参组（3个浓度组：2.0mg/ml，0.4mg/ml，0.08mg/ml）、PDGF组（10ng/ml）及PDGF(10ng/ml)加丹参（3个浓度，同上）组，测定各组SMC数量^H-TdR掺入量。结果：PDGF可刺激SMC数量及^3H-TdR掺入明显增加，分别为基础状态的3倍和2.5倍；丹参呈剂量依赖性地抑制基础状态及PDGF刺激下SMC数量及^3H-TdR掺入的增加。结论：丹参可抑制基础状态及PDGF作用下血管SMC的增生。     

PDGF-BB对成年雌性大鼠成骨细胞雌激素受体α蛋白质表达影响的实验研究

目的通过研究PDGF—BB与ERα的关系，探讨PDGF—BB在骨代谢中的作用机理，并对其在骨质疏松症中的应用前景进行展望。方法①利用酶消化法分离培养成年雌性大鼠的成骨细胞；②对培养的成骨细胞及其雌激素受体α进行鉴定；③分别加入浓度为0ng／ml、0．1ng／ml、1ng／ml、10ng／ml的PDGF—BB，共同培养7d，并设阳性对照组和阴性对照组；④Western blotting化学发光法检测ER蛋白质，GIS-2010凝胶图象处理系统分析结果。结果①培养的细胞为成骨细胞，免疫组化显示ERα位于胞核；②SDS-PAGE电泳显示在66kD处有清晰的蛋白条带；③Western blot显示10ng／ml组、1ng／ml组、0．1ng／ml组ER。蛋白条带较0ng／ml组清晰，且10ng／ml组最明显。结论PDGF—BB具有促进成骨细胞雌激素受体α蛋白表达的作用，雌激素受体α蛋白含量随着PDGF—BB浓度的增加而呈增加的趋势。     

骨组织工程中促细胞粘附因子bFGF的研究

目的 针对骨组织工程中成骨细胞与基质材料间促粘附这一重要问题,研究碱性成纤维细胞生长因子(bFGF)对成骨细胞粘附特性的影响。方法 取兔骨髓基质干细胞来源的成骨细胞体外培养,分别用Ong/ml、10ng/ml、100ng/ml浓度的bFGF诱导培养,观察接种后各时间点成骨细胞粘附情况,体视学计量粘附细胞数量。结果 10ng/ml bFGF诱导成骨细胞24小时后,细胞粘附数量增加最明显,显著高于对照组及100ng/ml组(P<0.01)。实验还发现,成骨细胞粘附最快发生在接种后4小时内。结论 一定浓度碱性成纤维细胞生长因子具有促进成骨细胞粘附特性的作用。     

胰岛素样生长因子-1对成骨细胞生长影响的实验研究

目的:探讨胰岛素样生长因子-1对成骨细胞生长增殖的影响.方法:采用酶消化法获取兔骨膜成骨细胞,取第三代成骨细胞与0.1ng/ml、1ng/ml、10ng/ml的胰岛素样生长因子-1共同体外培养,在第1～7d观察细胞的形态、生长特点,MTT法测定细胞增殖情况,并通过细胞计数绘制细胞生长曲线.结果:不同浓度的胰岛素样生长因子-1对成骨细胞的生长增殖与对照组比较均有显著性差异(P＜0.01),各浓度之间对成骨细胞的生长增殖亦存在显著性差异(P＜0.05).结论:胰岛素样生长因子-1对成骨细胞生长增殖具有促进作用,在0.1～10ng/ml范围内此种作用与浓度呈正相关.     

胰岛素样生长因子-1对成骨细胞生长影响的实验研究

目的探讨胰岛素样生长因子-1对成骨细胞生长增殖的影响.方法采用酶消化法获取兔骨膜成骨细胞,取第三代成骨细胞与0.1ng/ml、1ng/ml、10ng/ml的胰岛素样生长因子-1共同体外培养,在第1～7d观察细胞的形态、生长特点,MTT法测定细胞增殖情况,并通过细胞计数绘制细胞生长曲线.结果不同浓度的胰岛素样生长因子-1对成骨细胞的生长增殖与对照组比较均有显著性差异(P＜0.01),各浓度之间对成骨细胞的生长增殖亦存在显著性差异(P＜0.05).结论胰岛素样生长因子-1对成骨细胞生长增殖具有促进作用,在0.1～10ng/ml范围内此种作用与浓度呈正相关.     

纤维粘连蛋白与碱性成纤维细胞生长因子对成骨细胞黏附的协同刺激作用

目的研究纤维粘连蛋白（fibronection,FN）与碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）单独或联合应用时对成骨细胞在生物衍生骨支架上黏附的影响,并探索其潜在机制。方法取第3代成骨细胞,以bFGF（0.1、1、10和100ng/ml）刺激,接种于FN（0.1、1、10和100μg/ml）或多聚赖氨酸（Polylysine）修饰的生物衍生骨支架上,MTT法检测组间细胞黏附效率差异,电镜观察细胞密度与形态。GRGDS肽封闭后再次重复上述步骤。结果单独应用FN与bFGF均可增加成骨细胞在生物衍生骨支架上的黏附效率（P〈0.05）,联合应用时,产生的效应显著增强,两者存在交互作用（P〈0.01）。而Polylysine与bFGF无此交互作用（P〉0.05）。电镜观察发现,两者联合应用时细胞密度、铺展效果均优于单独应用。应用GRGDS肽后,FN联合bFGF产生的黏附促进作用被显著阻断。结论bFGF与FN对成骨细胞在生物衍生骨三维支架上的黏附存在协同刺激作用,细胞黏附产生这一效应的机制很可能是由RGD-整合素α5β1途径来介导的,需深入研究探讨。     

淫羊藿甙对成骨细胞Smad4 mRNA作用的实验研究

目的检测淫羊藿甙对体外培养的成骨细胞MC3T3-E1中Smad4mRNA的影响。方法用0、10、20、40ng/ml的淫羊藿甙刺激MC3T3-E1细胞。用倒置相差显微镜观察细胞生长情况。通过四甲基噻唑蓝(MTT)法测绘细胞生长曲线和计算细胞群体倍增时间;描述刺激MC3T3-E1细胞的增殖能力。用速率分光光度法测定ALP的含量,免疫组织化学法观察Ⅰ型胶原的产生,用以判断细胞的分化情况。用RT-PCR测定Smad4mRNA的数量。结果10、20ng/ml浓度淫羊藿甙可使MC3T3-E1细胞的生长曲线明显上移(P<0.01);0、10、20和40ng/ml组群体倍增时间分别为(39.37±2.35)h、(26.76±1.78)h、(29.30±2.12)h和(36.35±2.34)h,各组间比较差异有统计学意义(P<0.01);0、10ng/ml可使MC3T3-E1细胞产生ALP和Ⅰ型胶原量增加,刺激ALP作用呈现时间和剂量的依赖性(P<0.01);RT-PCR显示10、20ng/ml浓度淫羊藿甙可以刺激MC3T3-E1细胞Smad4mRNA的数量增加。上述作用以10ng/ml浓度最强(P<0.01)。结论淫羊藿甙刺激成骨细胞MC3T3-E1增殖与分化,可能是通过提高MC3T3-E1细胞Smad4mRNA的量而产生作用。     

G蛋白偶联受体激酶结合蛋白1发夹结构通过影响Paxillin的功能抑制成骨细胞迁移

目的探索G蛋白偶联受体激酶结合蛋白1（Gprotein coupled receptor kinase interacting protein 1，GIT1）的RNA发夹结构（hairpin）（GIT1-RNAh）对成骨细胞迁移的影响，并分析其机制。方法取培养至第6代的鼠成骨细胞，随机分成两组，分别用包含GIT1-RNAh（实验组）和绿色荧光蛋白（green fluoresence protein，GFP）的RNA发夹结构（GFP—RNAh）的腺病毒（对照组）感染12h后，再将每组分成有、无血小板源性生长因子（platelet drived growth factor，PDGF）刺激的两组。免疫荧光染色方法检测成骨细胞内源性GIT1蛋白表达和Paxillin的位置。Western Blot检测Paxillin的磷酸化。构建包含蓝色荧光蛋白的GIT1-RNAh（CFP—GIT1-RNAh）实验组和GFP—RNAh（CFP—GFP—RNAh）（对照组），免疫荧光双染的方法检测CFP—GIT1-RNAh和CFP—GFP—RNAh对Paxillin位置的特异性作用。用划痕愈合法检测GIT1-RNAh（实验组）和GFP—RNAh（对照组）腺病毒对成骨细胞在PDGF刺激下的迁移能力的影响。结果免疫组织化学观察，实验组和对照组比较，明显抑制成骨细胞内源性的GIT1蛋白表达和扰乱Paxillin的分布。Western Blot观察，和对照组相比，实验组明显抑制了Paxiliin的磷酸化（P〈0．05）。划痕愈合法检测观察，和对照组相比，实验组明显抑制成骨细胞的迁移。结论GIT1-RNAh通过扰乱Paxillin的分布和其磷酸化从而抑制成骨细胞的迁移。     

Experimental study of Composix mesh for ventral hernia

We aimed to compare conventional single-layer mesh and composite mesh in terms of the degree of tissue repair on the abdominal wall side of the mesh and the degree of mechanical adhesion to the intestine and to confirm the stability of composite mesh. We used a single-layer polypropylene (PP) mesh and a two-layer Composix mesh (E/X type) consisting of a PP mesh and an expanded polytetrafluoroethylene mesh. Twenty rats were divided into two groups. Three months after mesh placement, histopathologically, ingrowth of granulation tissue into the mesh on the abdominal wall side was prominent without mesh shrinkage or shift in either group. In the PP mesh group, 50% of the rats had firm adhesions between the mesh and the intestine, whereas the Composix mesh group had no adhesions to the intestine. Unlike conventional PP mesh, Composix mesh prevented adhesions to the intestine on the peritoneal side without impairing tissue union with the visceral peritoneum, suggesting its usefulness in clinical onlay mesh repair for ventral defects.     

100Hz电刺激肩胛间区对家兔胆囊痛及其脊髓后角磷酸化细胞外信号调节蛋白激酶表达的影响

目的 探讨100 Hz电刺激肩胛间区对家兔胆囊痛及脊髓后角磷酸化细胞外信号调节蛋白激酶(phosphorylate extracellular signal-regulated kinases,pERK1/2)表达的影响.方法 家兔采用随机数字表法随机分为5组:对照组、生理盐水组、甲醛溶液组、肩胛间区电刺激组和非肩胛间...     

Role of protein kinase C alpha in primary human osteoblast proliferation.

Protein kinase C (PKC) isoforms have been shown to have specific expression profiles and individual isoforms are believed to play distinct roles in the cells in which they are found. The goal here was to determine which specific isoform(s) is involved in proliferation of primary human osteoblasts. In primary human osteoblasts, 10 microM of acute sphingosine-1-phosphate (S1P) treatment induced an increase in proliferation that correlated with an increase in PKCalpha and PKCiota expression. To further delineate which isoforms are involved in osteoblastic cell proliferation, the effect of low versus high serum culture conditions on PKC isoform expression was determined. Likewise, the effect of antisense oligodeoxynucleotides (ODNs) to specific PKC isoforms on proliferation and MAPK activation was studied. The effect of S1P on intracellular translocation of activated PKC isoforms was also evaluated. The results indicated that in primary human osteoblasts, PKCalpha was not expressed under conditions of low proliferative rate while PKCdelta and PKCiota expression was not affected. The specific inhibition of PKCalpha by antisense ODNs resulted in inhibition of MAPK activity leading to a significant decrease in proliferation. S1P up-regulated antisense ODN inhibited PKCalpha expression and MAPK activity and led to an increase in proliferation. Subsequent experiments using platelet-derived growth factor (PDGF) as an additional mitogen generated similar data. PDGF stimulation resulted in a significant increase in proliferation that correlated with an up-regulation of inhibited PKCalpha expression in antisense ODN-treated cells. Immunofluorescence methods showed that mitogenic stimulation of PKCa resulted in nuclear translocation. Our findings present original data that PKCalpha is the isoform specifically involved in the proliferation of primary human osteoblasts.     

Regulation by ultrasound treatment on the integrin expression and differentiation of osteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. However, the mechanism by which US achieves these outcomes is not clear. Here we investigated the effect of US stimulation on the differentiation of osteoblasts and osteoclastogenesis. The effect of different intensities of US stimulation (1 MHz, continuous wave) on the osteoblastic cell line MC3T3-E1 or primary cultured osteoblasts was examined. Flow cytometry showed that US stimulation at 125 mW/cm2 for 10 min transiently increased the surface expression of alpha2, alpha5, and beta1 integrins in both MC3T3-E1 and primary osteoblasts. Fluorocytochemistry showed that the actin cytoskeleton also reorganized in response to US stimulation. When the MC3T3-E1 cells were cultured in differentiation medium containing vitamin C and beta-glycerophosphate, long-term US stimulation (10 min/day for 11 days) increased mineralized nodule formation, collagen content, and alkaline phosphatase activity. The intensity at 125 mW/cm2 exerts the most prominent action. Effect of long-term US stimulation on the osteoclastogenesis was also examined. US stimulation at a power of 62.5 or 125 mW/cm2 markedly inhibited RANKL plus M-CSF-induced osteoclastic differentiation from bone marrow stromal cells. These findings suggest that US has a regulatory effect on the integrin expression and the differentiation of osteoblasts and osteoclastogenesis, which may contribute to the beneficial effects of US on the fracture repair.     

High-dose alendronate uncouples osteoclast and osteoblast function: a study in a rat spine pseudarthrosis model

The effect of alendronate on osteoclast and osteoblast function was studied in a novel spine pseudarthrosis model in rats. Sixty-three Sprague-Dawley rats were divided into three groups: control group (saline), therapeutic dose group (1 microg/kg/week), and one-log overdose group (10 microg/kg/week). Animals had L4-L5 posterior intertransverse process fusion with limited bone graft and were sacrificed at 2, 4, and 6 weeks. Manual palpation showed no notable differences among groups. Treatment group radiographic scores were equal to or better than control group scores and were higher than the overdose group at 2 and 6 weeks. Qualitatively, limited histologic remodeling and poor osteoclastic and osteoblastic function were noted in the alendronate treated groups. Quantitative histologic analysis showed fewer osteoclasts in the therapeutic and high-dose groups (p < 0.001). The percent osteoblasts per bone surface area was lower in the high-dose group (p < 0.05). The results suggest that the effect of alendronate was dose dependent and animal model dependent and that supranormal doses of alendronate had a deleterious effect on osteoclastic and osteoblastic function in this model.     

1，25（OH）2VD3诱导小鼠胚胎干细胞向成骨细胞分化的研究

目的 探讨1,25(OH)2VD3在诱导小鼠胚胎干细胞(embryonic stem cells,ESCs)向成骨细胞分化过程中的重要作用.方法 取2 d龄昆明小白鼠颅盖骨进行成骨细胞分离培养;参照并改进zur Nieden等的方法制备拟胚体(embryoid bodies,Ebs).将Ebs根据培养液不同分为4组:A组:不含白血病抑制因子Ebs培养液;B组:Ebs培养液中添加50μg/mL维生素C、50 mmol/L β-磷酸甘油;C组:培养液同B组,另每孔接种5×104个第3代成骨细胞;D组与C组相同,另添加4 × 10-9 mol/L 1,25(OH)2VD3.检测ALP活性,实时定量PCR检测骨钙素mRNA的表达,茜素红S染色进行矿化骨结节计数.结果 Ebs贴壁后前5 d,各组ALP表达未发生明显变化,5 d后ALP表达逐渐增加,其中D组在第20天表达水平最高.光镜下可见轮廓清晰大小不等的红色结节.茜素红S染色测得A、B、C、D组骨结节数分别为(20±8)、(18±5)、(31±1)、(50±1)个:实时定量PCR测得A、B、C、D组骨钙素mRNA分别为10.18±1.17、20.29±1.03、18.84±4.07、32.15±5.23:C、D组与A、B组比较差异均有统计学意义(P＜0.05),A、B组间比较差异无统计学意义(P＞0.05),C、D组间比较差异有统计学意义(P＜0.05).结论 在浓度为4 × 10-9 mol/L 1,25(OH)2VD3和维生素C、β-磷酸甘油共同作用下,有效促进了ESCs源性成骨细胞的产生.     

Inhibition of Src kinase can ameliorate renal interstitial fibrosis in unilateral ureteral obstruction mice

Objective To investigate the effect and mechanism of Src kinase in renal interstitial fibrosis of unilateral ureteral obstruction (UUO) mice. Methods Male C57BL/6J mice were randomly divided into 4 groups, including sham operation group (n=8), sham operation+PP2 group (n=8), UUO operation group (n=8) and UUO operation+PP2 group (n=8). The mice were injected 2 mg/kg PP2 by intraperitoneal everyday after surgery in sham+PP2 group and UUO+PP2 group. PP2 dissolved in 1% DMSO (formulated with normal saline). Sham and UUO group were given equal 1% DMSO. The mice were sacrificed at 7th day. Renal collagen was observed with Sirius red stain. The activities of Src, protein kinase B (PKB, AKT), p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and the protein expressions of α-smooth muscle actin (α-SMA) and fibronectin (FN) were detected by Western blotting. The expression of collagen I (COLⅠ) was detected by immunohistochemistry and the expressions of matrix metalloprotein 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6) were measured by ELISA. Results Compared with sham mice, UUO mice on 7th day displayed obvious renal fibrosis. Meanwhile, UUO mice had increased expressions of COLⅠ and FN, and activities of AKT, ERK and p38 MAPK (all P＜0.05). Their renal expressions of α-SMA, TGF-β1, MMP-9, TIMP-1, MCP-1 and IL-6 were also raised (all P＜0.05). Compared with those in UUO group, in UUO+PP2 group the activities of Src, AKT, p38 MAPK and ERK, and expressions of TGF-β1, MCP-1 and IL-6 decreased (all P＜0.05). Additionally, expressions of COLⅠ, FN and α-SMA, collagen deposition and renal fibrosis receded in UUO+PP2 group (all P＜0.05). However, the expressions of MMP-9 and TIMP-1 were not influenced by PP2 treatment. Conclusions Src kinase promotes myofibroblasts accumulation and inflammatory reaction through activating its downstream signaling pathway in the progressing of renal interstitial fibrosis.