牵张作用对成骨样细胞MG63的线粒体跨膜电位的影响

目的:探讨在低渗透压形成的静牵张应力环境下线粒体跨膜电位与细胞增殖分化及凋亡的关系。方法:用成骨样细胞MG63细胞株进行体外培养、扩增,在对数生长期采用不同的低渗透压对细胞进行刺激,检测不同作用务件下线粒体跨膜电位(ΔΨm)、细胞增殖比例(S期百分比)以及凋亡指数。结果:240mOsm组ΔΨm呈上升趋势,4h达到峰值,6h逐渐下降,但仍高于对照组;163 mOsm组ΔΨm在6 h时明显降低。277和240 mOsm组S期百分比在6 h和8 h达到峰值(26.54±0.71,28.10±0.39:26.96±0.33,28.55±0.26)。三个实验组的凋亡峰均提前,且大于对照组,尤以163 mOsm组为最(54.87±0.78)。结论:成骨样细胞MG63ΔΨm的变化与时间和力学刺激强度有一定的依赖性,预示线粒体跨膜电位的变化与细胞增殖活性之间存在一定的关系。     

表达小鼠白细胞介素21的Sp2/0细胞体外对鼠肿瘤特异性淋巴细胞增殖及功能的影响

目的:研究表达小鼠白细胞介素21(mIL-21)的Sp2/0细胞与用Sp2/0细胞预先免疫的小鼠淋巴细胞体外共培养,是否对预致敏淋巴细胞增殖及功能有影响.方法:获取灭活Sp2/0细胞免疫的小鼠淋巴细胞,在mIL-2存在的条件下,以miL-21转染的Sp2/0细胞为刺激细胞,用流式细胞术检测CFSE标记的淋巴细胞增殖和7-AAD标记的细胞毒活性;用ELISpot法确定分泌IFN-y/的淋巴细胞数量.结果:转染mIL-21的Sp2/0细胞对预致敏的淋巴细胞增殖有明显影响,活化的淋巴细胞对靶细胞的杀伤率(39.57%±4.72%)与对照组(23.18%±2.94%)相比有较大的提高(P＜0.05),且分泌IFN-y的细胞数量明显增加.活化增殖后的淋巴细胞回输至环磷酰胺预处理的小鼠,能延长小鼠的成瘤时间.结论:表达mIL-21的Sp2/0细胞可有效促进肿瘤抗原特异性淋巴细胞活化及增殖,并增强其对肿瘤细胞的杀伤功能.     

Cytohesins介导EGFR 通路在结直肠癌细胞增殖中的作用

目的:研究Cytohesins介导EGFR通路在结直肠癌细胞增殖中的作用。方法:选择人结直肠癌细胞系HT-29作为研究细胞,采用免疫荧光检测结直肠癌细胞系HT29中EGFR表达情况,Secin H3阻断结直肠癌细胞系HT29细胞内cytohensins观察细胞内EGFR通路激活情况,MTT法检测细胞增殖情况。结果:EGF+Secin H3组细胞内p-EGFR水平显著低于EGF组(P0.05)。第3、5天,各组细胞增殖情况存在显著差异(F=38.072,P0.05),其中EGF组显著高于Secin H3阻断组(P0.05)和对照组(P0.05),对照组吸光度值显著高于Secin H3阻断组(P0.05)。结论:EGF与结直肠癌细胞的增殖密切相关,通过阻断Cytohesins介导EGFR通路能够显著抑制结直肠癌细胞的增殖。     

组织工程化软骨与富血小板血浆复合可促进软骨缺损修复

为探讨组织工程化软骨与富血小板血浆复合修复软骨缺损的效果,本研究选取了8周龄健康新西兰兔24只,依据随机数表法分为观察组(组织工程化软骨与富血小板血浆复合)和对照组(单纯软骨缺损模型),发现术后4周、8周、12周,观察组实验动物的大体评分均明显高于对照组(p0.05)。观察组实验动物的Collagen TypeⅠ、Collagen TypeⅡ相对表达水平明显高于对照组(p0.05)。两组实验动物的Collagen TypeⅩ相对表达水平无显著差异(p0.05)。观察组实验动物的软骨缺损直径和缺损深度分别为(1.02±0.35)mm、(0.96±0.27)mm,对照组实验动物的软骨缺损直径和缺损深度分别为(4.27±1.09)mm、(5.43±1.85)mm(p0.05),表明组织工程化软骨与富血小板血浆复合修复软骨缺损效果明显,能够刺激软骨相关基因表达,缩小软骨缺损范围,促进缺损软骨愈合。     

氧化应激对腺病毒E1A蛋白活化NF-κB转录活性的影响及其机制研究

目的:探讨腺病毒E1A蛋白对细胞内抗氧化物质谷胱甘肽(GSH)水平的影响及氧化应激对腺病毒E1A蛋白介导的核因子-κB(NF-κB)转录活化的影响。方法:构建稳定表达E1A蛋白的大鼠肺泡上皮细胞(E1A组)及对照质粒转染细胞(对照组),每组5×105个细胞,试验重复3次。采用H2O2刺激细胞,检测细胞内GSH水平。脂多糖(LPS)和肿瘤坏死因子-α(TNF-α)进行刺激,丁胱亚磺酰亚胺(BSO)进行干预,Western blot法检测NF-κB和AP-1蛋白的表达。结果:E1A+细胞内GSH基础水平与E1A-比较无显著差异,但在H2O2作用后没有诱导出GSH含量的上升,表现为下降而低平的趋势,去除氧化剂后,仍低于正常水平。E1A-细胞在氧化剂的作用后呈明显的上升趋势,去除氧化剂后仍高于基础水平。细胞内NF-κB蛋白表达(积分吸光度值):刺激前E1A+细胞分别为79.3±4.6和80.3±3.8,在LPS和TNF-α刺激后分别为81.8±3.9～89.9±1.6和94.1±1.9～99.8±1.6,均明显高于对照组(刺激前分别为68.3±3.8和69.4±4.3,刺激后分别为70.1±2.8～80.8±3.6和73.4±4.9～83.2±6.7)。给予BSO预处理后再用LPS和TNF-α刺激,E1A-细胞NF-κB蛋白表达的积分吸光度值(1.22±0.16和1.75±0.13)与LPS或TNF-α单独作用组(1.25±0.18和1.69±0.19)无明显差别;E1A+细胞NF-κB蛋白表达的积分吸光度值(1.75±0.10和2.26±0.21)明显高于LPS或TNF-α单独作用组(1.35±0.12和1.80±0.14)。结论:腺病毒潜伏感染持续表达E1A蛋白可以影响细胞GSH,降低细胞对氧化应激的耐受性,并可能通过此机制造成NF-κB异常转录活化,而GSH的降低又进一步放大了E1A介导的NF-κB转录活化作用。     

胶原合成介导的软骨细胞的光生物调节作用

目的:了解低强度激光照射对软骨细胞增殖的影响及其机制。方法:选取3周龄新西兰白兔分离培养软骨细胞,在2.5%新生牛血清中培养,用半导体激光(650 nm,2.96 mW/cm2)(sem iconductor laser irrad iation,SLI)照第4代软骨细胞,每天分别照射1 m in、3 m in、5 m in、7 m in、10 m in、20 m in,共6 d。收集激光照射后第2 d、4 d、6 d、8 d、10 d和12 d的细胞培养液,用氯胺T消化法检测羟脯氨酸(H rp)的含量。在培养至第13 d时,用XTT法检测细胞的活性,了解细胞的增殖情况。结果:在2.5%新生牛血清中,SLI对软骨细胞具有明显的光生物调节作用:(1)在培养至第13 d时,所有剂量组在照射后XTT吸光度值均有不同程度的增高,其中3 m in、5 m in、7 m in和10 m in组的增高较为明显(P<0.01);(2)两因素重复测定资料的方差分析结果显示,SLI照射后软骨细胞合成胶原的能力在逐步增加,而对照组在培养至第2周开始H rp含量明显下降。结论:SLI照射可促进2.5%新生牛血清中兔软骨细胞增殖,这个过程可能是通过促进胶原合成实现的。     

水飞蓟宾诱导卵巢癌HO-8910细胞凋亡增殖及其机制探讨

目的:探讨水飞蓟宾对人卵巢癌HO-8910细胞增殖的抑制作用及其作用机制。方法:HO-8910细胞分为四组:(1)对照组;(2)水飞蓟宾低浓度组;(3)水飞蓟宾中浓度组;(4)水飞蓟宾高浓度组。通过MTT法测定细胞增值率,流式细胞技术检测细胞凋亡情况,Hoechst染色观查细胞核凋亡,Western-blot检测bax及bcl-2表达。结果:细胞增殖检测结果显示,水飞蓟宾低、中、高浓度组的抑制率(83.00±5.51%、65.33±3.48%、56.67±4.37%)与对照组(97.33±4.25%)比较差异具有统计学意义(P0.05)。流式细胞技术结果显示,水飞蓟宾低、中、高浓度组凋亡细胞比例分别为16.93±2.34%、26.20±2.21%和37.93±1.98%,与对照组(1.43±0.72%)相比差异均有统计学意义(P0.05)。Hoechst染色结果显示,水飞蓟宾低、中、高浓度组凋亡细胞比例分别为12.56±2.55%、25.73±2.05%和39.14±3.69%,与对照组(0.54±0.67%)相比差异均有统计学意义(P0.05)。此外,水飞蓟宾可以升高bax基因表达水平,降低bcl-2基因表达平。结论:水飞蓟宾能明显抑制HO-8910细胞增殖,促进细胞凋亡,通过改变凋亡因子表达诱导卵巢癌HO-8910细胞凋亡。     

mirRNA-17-5p抑制物对骨肉瘤细胞系SOSP_9607增殖和凋亡的影响

目的:探讨miR-17-5p抑制物对于骨肉瘤细胞系SOSP_9607细胞增殖和凋亡的影响.方法:四甲基偶氮唑蓝(MTT)法测定细胞增殖,进一步计算抑制率,流式细胞仪测定细胞凋亡.将SOSP_9607细胞分为对照组和实验组,对照组分为阴性对照和正常细胞对照组.实验组采用miR-17-5p抑制物(hsa-miR-17-5p inhibitors)抑制SOSP_9607细胞内miR-17-5p的活性.结果:与对照组相比,实验组显著抑制SOSP_9607细胞的增殖,有明显的剂量依赖性(P<0.01).随着浓度从50 nmol/L逐渐增加至200 nmol/L,抑制率逐渐增高(P<0.01).实验组凋亡率(9.6±1.8)%与阴性对照组凋亡率(3.5±0.4)%相比明显增高(P<0.01).结论:miR-17-5p抑制物通过抑制SOSP_9607细胞中miR-17-5p的活性对SOSP_9607细胞的增殖和凋亡发挥重要作用.     

急性低氧和腺苷对大鼠脾脏T淋巴细胞增殖的影响

目的:观察急性低氧和腺苷对大鼠脾脏T淋巴细胞增殖的影响,以探讨急进高原时机体免疫功能改变的规律和机制.方法:大鼠在5 000 m模拟高原减压低氧3 d后,用3H-TdR掺入法检测脾脏T淋巴细胞增殖功能,增加细胞培养液中腺苷浓度观察对T淋巴细胞增殖的影响.结果:在5.0 mg/L 和2.5 mg/L刀豆素A(concanavalin A,ConA)的刺激下,对照组大鼠脾脏T淋巴细胞的刺激指数分别为64.0±23.7和43.5±21.7;急性低氧组大鼠显著降低至29.4±11.3 和20.2±16.1;10 μmol/L和100 μmol/L腺苷能够显著抑制大鼠脾脏T淋巴细胞的增殖反应,且这种抑制作用具有明显的浓度依赖性.结论:急性低氧可在一定程度上抑制T淋巴细胞功能,这种抑制作用可能与低氧引起的腺苷含量增加有关.     

MicroRNA-411a-3p过表达抑制Ca~(2+)信号转导与羊驼黑色素细胞的增殖和迁移（英文）

黑色素细胞中产生的黑色素转移及黑色素细胞的增殖和迁移均与色素沉积有关。黑色素细胞的增殖需要有丝分裂原协同进行。黑色素细胞的增殖和分化受组织环境以及多种毛色基因的调控。miRNA-411a-3p在不同毛色羊驼皮肤中呈差异表达,且通过靶向IGF1R调控黑色素生成。但miRNA-411a-3p是否与黑色素颗粒迁移、黑色素细胞的增殖和迁移相关未见报道。本研究通过miRNA-411a-3p转染羊驼黑色素细胞后发现,与对照组相比,钙离子信号转导水平下降了(91.73±1.53)%(P0.01),与黑色素转移有关的Rab27a和肌球蛋白Va在蛋白质水平的表达均被下调,同时与细胞增殖有关的整联蛋白β1和β5相关基因在转录水平分别下降了(44.67±13.67)%(P0.01)和(30.72±6.23)%(P0.01),在蛋白质水平表达下降了(45.18±1.96)%(P0.001)和(11.52±1.09)%(P0.001)。综上所述,miRNA-411a-3p过表达后会抑制钙离子信号转导,以及羊驼黑色素细胞的增殖和迁移。     

The Effect of 58S Bioactive Glass Coating on Polyethylene Terephthalates in Graft-Bone Healing

In this study the effects of surface modification of Polyethylene Terephthalates (PET) fibers with 58S bioactive glasses on osteoblasts proliferation and osseointegration in the tibia-articular tendon-bone healing model were investigated.PET sheets were coated with 58S bioactive glass and uncoated PET sheets were used as a control.Scanning Electron Microscope (SEM) and X-ray photoelectron spectrometer were adopted to analyze the surface characteristics of the fibers.MT3T3-E 1 cells were cultured with the PET fibers and the MTT and ALP were tested at 1,3,5 days.Twenty-four skeletally mature male New Zealand white rabbits were randomly divided into two groups,the 58S-PET group and the PET group.Both groups underwent a surgical procedure to establish a tibia-articular tendon-bone healing model.Mechanical examinations and histological assays were taken to verify the coating effects in vivo.Results of both MTT and ALP tests show significant differences (P ＜ 0.01) between the 58S-PET group and the PET group.At 6 weeks and 12 weeks,the max load-to-failure was significantly higher in the 58S-PET group.In the histological assays,distinct new bone formation was observed only in the 58S-PET group and stronger osseointegration was seen in the 58S-PET group than that in the control group.The 58S-coating on PET could enhance the proliferation and activity of the osteoblasts and therefore promote the new bone formation and tendon-bone healing.     

力生长因子E肽对成骨细胞增殖及基因表达的影响

为了探索力生长因子羧基端E结构域的后24个氨基酸组成的短肽(MGF-Ct24E)对成骨细胞生物学活性的影响,通过组织块培养法获得大鼠原代成骨细胞,采用MTT法和流式细胞仪检测细胞的增殖及细胞周期分布情况,基因芯片技术检测细胞基因表达谱,并用定量PCR实验验证芯片检测结果。结果显示MGF-Ct24E组的细胞增殖活性明显高于对照组,且在培养第一天促增殖效果最为显著。细胞周期结果显示MGF-Ct24E显著提高了S期和G2/M期的细胞所占比例。基因芯片检测发现差异表达基因共1397个,其中上调922,下调475,且差异表达的基因主要是关于细胞的增殖分化调节,生长因子结合和活性调节等方面。MGF-Ct24E对成骨细胞的这种增殖分化调控提示MGF-Ct24E在促进骨修复方面有着潜在的应用价值。     

Beta-catenin is not activated by downregulation of PTEN in osteoblasts

Selective knockdown of phosphatase and tensin homolog (PTEN) has been recently shown to increase life long accumulation of bone and its ability to increase osteoblast lifespan. In order to determine how loss of PTEN function affects osteoblast differentiation, we created cell lines with stable knockdown of PTEN expression using short hairpin RNA vectors and characterized several clones. The effect of deregulated PTEN in osteoblasts was studied in relationship to cell proliferation and differentiation. Downregulation of PTEN initially affected the cell’s attachment and spreading on plastic but cells recovered after a brief period of time. When cell proliferation was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, we noticed a small but significant increase in growth rates with PTEN reduction. The size of individual cells appeared larger when compared to control cells. Differentiation properties of these osteoblasts were increased as evidenced by higher expression of several of the bone markers tested (alkaline phosphatase, osteocalcin, osterix, bone morphogenetic protein 2, Cbfa1, osteoprotegerin, and receptor activator of NF-kappaB ligand) and their mineralization capacity in culture. As stabilization of beta-catenin is known to be responsible for growth deregulation with PTEN loss in other cell types, we investigated the activation of the canonical Wnt pathway in our cell lines. Immunofluorescence staining, protein expression in subcellular fractions for beta-catenin, and assays for activation of the canonical Wnt/beta-catenin signaling were studied in the PTEN downregulated cells. There was an overall decrease in β-catenin expression in cells with PTEN knockdown. The distribution of β-catenin was more diffuse within the cell in the PTEN-reduced clones when compared to controls where they were mostly present in cell borders. Signaling through the canonical pathway was also reduced in the PTEN knockdown cells when compared to control. The results of this study suggest that while decreased PTEN augments cell proliferation and positively affects differentiation, there is a decrease in β-catenin levels and activity in osteoblasts. Therefore, at least in osteoblasts, β-catenin is not responsible for mediating the activation of osteoblast differentiation with reduction in PTEN function.     

Human osteoblasts from younger normal and osteoporotic donors show differences in proliferation and TGFβ-release in response to cyclic strain

Mechanical stimulation of bone tissue by physical activity stimulates bone formation in normal bone and may attenuate bone loss of osteoporotic patients. However, altered responsiveness of osteoblasts in osteoporotic bone to mechanical stimuli may contribute to osteoporotic bone involution. The purpose of the present study was to investigate whether osteoblasts from osteoporotic patients and normal donors show differences in proliferation and TGFβ production in responses to cyclic strain. Human osteoblasts isolated from collagenase-treated bone explants of 10 osteoporotic patients (average age 70 ± 6 yr) and 8 normal donors (average age 54 ± 10 yr) were plated into elastic rectangular silicone dishes. Subconfluent cultures were stimulated by cyclic strain (1%, 1 Hz) in an electromechanical cell stretching apparatus at three consecutive days for each 30 min. The cultures were assayed for proliferation, alkaline phosphatase activity and TGFβ release in each three parallel cultures. In all experiments, osteoblasts grown in the same elastic dishes but without mechanical stimulation served as controls. Significant differences between stimulated cultures and unstimulated controls were determined by a paired two-tailed Wilcoxon test. In comparison to the unstimulated controls, osteoblasts from normal donors significantly increased proliferation (p = 0.025) and TGFβ secretion (p = 0.009) into the conditioned culture medium. In contrast, osteoblasts from osteoporotic donors failed to increase both proliferation (p > 0.05) and TGFβ release (p > 0.05) in response to cyclic strain. Alkaline phosphatase activity was not significantly affected (p > 0.05) in normal as well as osteoporotic bone derived osteoblasts.

These findings suggest a different responsiveness to 1% cyclic strain of osteoblasts isolated from normal and osteoporotic bone that could be influenced by both the disease of osteoporosis and the higher average age of the osteoporotic patient group. While osteoblasts from osteoporotic donors failed to increase proliferation and TGFβ release under the chosen mechanical strain regimen that stimulated both parameters in normal osteoblasts, it is possible that some other strain regimen would provide more effective stimulation of osteoporotic cells.     

Effects of mechanical vibration on cell morphology,proliferation, apoptosis,and cytokine expression/secretion in osteocyte‐like MLO‐Y4 cells exposed to high glucose

Diabetic patients exhibit significant bone deterioration. Our recent findings demonstrate that mechanical vibration is capable of resisting diabetic bone loss, whereas the relevant mechanism remains unclear. We herein examined the effects of mechanical vibration on the activities and functions of osteocytes (the most abundant and well‐recognized mechanosensitive cells in the bone) exposed to high glucose (HG). The osteocytic MLO‐Y4 cells were incubated with 50 mM HG for 24 h, and then stimulated with 1 h/day mechanical vibration (0.5 g, 45 Hz) for 3 days. We found that mechanical vibration significantly increased the proliferation and viability of MLO‐Y4 cells under the HG environment via the MTT, BrdU, and Cell Viability Analyzer assays. The apoptosis detection showed that HG‐induced apoptosis in MLO‐Y4 cells was inhibited by mechanical vibration. Moreover, increased cellular area, microfilament density, and anisotropy in HG‐incubated MLO‐Y4 cells were observed after mechanical vibration via the F‐actin fluorescence staining. The real‐time polymerase chain reaction and western blotting results demonstrated that mechanical vibration significantly upregulated the gene and protein expression of Wnt3a, β‐catenin, and osteoprotegerin (OPG) and decreased the sclerostin, DKK1, and receptor activator for nuclear factor‐κB ligand (RANKL) expression in osteocytes exposed to HG. The enzyme‐linked immunosorbent assay assays showed that mechanical vibration promoted the secretion of prostaglandin E₂ and OPG, and inhibited the secretion of tumor necrosis factor‐α and RANKL in the supernatant of HG‐treated MLO‐Y4 cells. Together, this study demonstrates that mechanical vibration improves osteocytic architecture and viability, and regulates cytokine expression and secretion in the HG environment, and implies the potential great contribution of the modulation of osteocytic activities in resisting diabetic osteopenia/osteoporosis by mechanical vibration.     

<Emphasis Type="Italic">In vitro</Emphasis> behaviour of osteoblast cells seeded into a COL/β-TCP composite scaffold

The purpose of the present study was to investigate the effect of a collagen/β-tricalcium phosphate (COL/β-TCP) composite on osteoblast growth and proliferation. The COL/β-TCP composite was prepared by mixing COL type I with β-TCP, in 1:1 (w/w) ratio and conditioned as sponge by freeze-drying. The osteoblast culture was obtained from rat calvaria bones by enzymatic digestion and cells were seeded in the COL/β-TCP composite. The cell morphology and viability, alkaline phosphatase and osteocalcin, as markers of osteoblast proliferation were evaluated at 3, 7 and 25 days of culture. Histological sections revealed that cell colonization progressively increased inside the COL/β-TCP scaffold, and osteoblasts had a random distribution throughout the scaffold. Cells cultured into the COL/β-TCP scaffold presented osteoblast phenotype, intense staining of alkaline phosphatase and increased production of osteocalcin. Transmission electron micrographs revealed intimate contacts between osteoblasts and the scaffold. MTT test indicated that the viability of the cells cultivated in the presence of COL/β-TCP scaffold was similar to that of the control. All these results show that our COL/β-TCP composite act as a good substrate for rat osteoblast proliferation and migration and could be a promising substitute for bone repair.     

Growth and differentiation of alveolar bone cells in tissue-engineered constructs and monolayer cultures

The ability to enhance bone regeneration by implanting autologous osteoblasts in combination with an appropriate scaffold would be of great clinical interest. The aim of our study was to compare the growth and differentiation of alveolar bone cells in tissue-engineered constructs and in monolayer cultures, as the basis for developing procedures for routine preparation of bone-like tissue constructs. Alveolar bone tissue was obtained from four human donors and explant cultures of the cells were established. Expanded cells were seeded on macroporous hydroxyapatite granules, and cultured in medium supplemented with osteogenic differentiation factors for up to 3 weeks. Control monolayer cultures were established in parallel, and cultured in media with or without osteogenic supplements. Cell proliferation, alkaline phosphatase (AP) activity and gene expression of AP, osteopontin and osteocalcin were determined under different culture conditions at weekly intervals. Cells in tissue constructs exhibited growth patterns similar to those in control monolayer cultures: enhanced proliferation was noted during the first 2 weeks of cultivation, followed by a decrease in cell numbers. AP activity at 3 weeks was higher in all cultures in osteogenic medium than in control medium. Gene expression levels were stable in monolayer cultures in both types of media whereas, in tissue constructs, they exhibited patterns of osteogenic differentiation. Light and scanning electron microscopy examination of the cell-seeded constructs showed uniform cell distribution, as well as cell attachment and growth into the interior region of the hydroxyapatite granules. Our results show that bone-like constructs with viable cells exhibiting differentiated phenotype can be prepared by cultivation of alveolar-bone cells on the tested hydroxyapatite granules.     

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.     

Osteoblasts proliferation and differentiation stimulating activities of the main components of Fructus Psoraleae corylifoliae

Osteoporosis is a disease of bones that leads to an increased risk of fracture. Fructus of Psoralea corylifolia L. (scurfpea fruit) is commonly utilized for treating bone fractures and joint diseases for thousands of years in China. This study was aimed to screen active principles, which might have the potency to stimulate osteoblasts proliferation and differentiation from scurfpea fruit. A HPLC method was established to analyze the main components in scurfpea fruit. Totally 11 compounds have been identified by comparing their retention time with correspondent standard substances. The MTT and ALP methods were utilized for the assay of osteoblasts proliferation and differentiation activity. Icariin, a prenylated flavonoid glycoside was treated as the positive control. Bavachin and isobavachin significantly stimulated cell proliferation, while bakuchiol exhibited stronger effect to enhance osteoblasts differentiation. All these compounds were found with a characterized structure that in each of their molecule backbones, a prenylated side chain was attached. These results lead to a hypothesis that prenyl group might be crucial to exhibit the activity. The structure–effect relationship of these compounds with prenyl group in mouse primary calvarial osteoblasts needs to be explored in further research.