Physiological responses of osteoblasts to cyclic stretching and the change of intracellular calcium concentration

The development of bone tissues is regulated by mechanical stimulation.Cyclic stretching was applied to the osteoblasts that were delivered from rat calvarie,The results showed that stretching at 500με increased the cell proliferation while loading at 1000με and 1500με inhabited cell growth ,Loading also increased the adhesive force between cells and substrate as well as spreading areas of osteobalsts.Furthermore,the fluorescence probe Fluo-3/AM was used to investigate the effect of stretching stimulation on the intracellular calcium concentration of osteoblasts.The intracellular calcium concentration of osteoblasts that were stretched at 500 με for 5 min was 92.9% higher than the control ,After being treated with the panax ontoginseng saponins,the streteched osteoblasts still expressed 28.6% higher intracellular calcium concentration than that of the control ,which proved that both the influx of extracellualr calcium and the release of intracellular calcium store were involved in the increase of intracellular calcium concentration when osteoblasts responded to the cyclic stretching And the influx of extracellular calcium through transmembrance channel played a main role.     

Relationship between the partitioning properties of motoneuron recruitment and the needle feeling propagation

Histological and physiological studies indicated that most skeletal muscles can be divided into a series of relatively independent sub-volumes: "neuromuscular compartments" and the partitioning property of the muscle result in the localization of muscle reflex. In the present experiment we studied the recruitment properties of medial gastracnemius (MG) muscle motoneuron pool of decerebrate cat with two kinds of local mechanical stimulation: local stretch and acupuncture-like stimuli. The results indicate that: (ⅰ) there is an obvious property of localization of recruitment activity, only the MNs which innervate the stimulated compartment were recruited by weaker and shorter stimuli; (ⅱ) recruitment activity spread to those MNs which supply the adjacent and distal compartments during the strength of stimulation or duration of the stimulation was increased; and (ⅲ) the recruitment property of muscle activity elicited by the local mechanical stimulation is thought similar to that of "needle feeling" along the meridian pathway during stimulation of acupuncture point.     

Nicotine-induced upregulation of antioxidant protein Prx 1 in oral squamous cell carcinoma

Nicotine is a source of exogenous oxidative stress, which is associated with the pathogenesis of numerous diseases including oral squamous cell carcinoma (OSCC), whereas an antioxidant protein, peroxiredoxin 1 (Prx 1), plays an important role in the modulation of this condition. This study was to investigate the association between Prx 1 and tobacco-induced oxidative stress. The expression of Prx 1 and GST in OSCC Tca8113 cells, which were pre-treated with nicotine, was determined. In the present study, MTT assay, reactive oxygen species (ROS) assay, RT-PCR and Western blot analyses, respectively, were conducted to assess cell viability, ROS level, and expression level of Prx 1 and GST in nicotine-treated Tca8113 cells. Nuclear factor kappa B (NF- B) expression was detected by immuno-fluorescence. Our results showed the growth of Tca8113 cells was increased in a dose-dependent manner when cells were treated with nicotine at concentrations from 0.1 to 10 mol/L, but the proliferation of the cells decreased at 100 mol/L. ROS levels increased in all groups treated with nicotine at concentrations of 0.1, 1, 10, or 100 mol/L for 24h. Prx 1 and GST mRNA and protein expression were up-regulated in cells treated with nicotine for the same time at different concentrations or at the same concentration for different times (P<0.05). NF-B was translocated from cytoplasm to nucleus, the expression of NF- B was increased in nucleus. These results suggest that up-regulation of Prx1 expression appears to be associated with tobacco-induced oxidative stress, which may play an important role in the pathogenesis of OSCC.     

The Effect of Cyclic Stretching on Matrix Production, Mineralization and Differentiation of Osteoblasts

A four-point bending apparatus is used to investigate the effects of stretching on collagen synthesis, mineralization and differentiation of osteoblasts. Cells are stretched at 1500με for 24 hours. The responses of osteoblasts to mechanical signal of physiological stretching are evaluatedfrom three aspects: collagen production, extracellular inorganic calcium secretion and ALP activity. The results show that osteoblasts decrease the collagen synthesis, calcium secretion and ALP activity compared to the control cells (65.82 %, 73.51%, 48.10 96 respectively ), confirming that cyclic stretching at 1500με inhabits the ohvsiological activity of osteoblasts.     

Inducible nitric oxide synthase expression is related to angiogenesis,bcl—2 and cell proliferation in hepatocellular carcinoma

In this study,we examined the expression of inducible nitric oxide synthase(iNOS) and vascular endothelial growth factor(VEGF) by immunohistochemical staining in 76 tissue sections collected from bepa-tocellular carcinoma(HCC) patients undergoing hepatectomy.Microvascular density (MVD) was determined by counting endothelial cells immunostained using anti-CD34 antibody.We performed DNA-flow cytometric analyses to elucidate the impact of iNOS and VEGF expression on the cell cycle of HCC.Most of the HCC cells that invaded stroma were markedly immunostained by iNOS antibody.The iNOS stain intensity of the liv-er tissue close to the tumor edge was stronger than that of HCC tissue,and the strongest was the hepatocytes colser to the tumor tissue.However,iNOS expression in 10 normal hepatic samples was undetectable.VEGF positive expression ratio was 84.8% in iNOS positive expression cases,and the ratio was 35.3% in negative cases.There was significant correlation(P=0.000) between iNOS and VEGF expression.Moreover,iNOS expression was significantly associated with bcl-2 and MVD,but without p53 expression.DNA-flow cytometric analyses showed that combined expression of iNOS and VEGF had significant impact on the cell cycle in HCC.PI(Proliferating Index) and SPF(S-phase fraction)in the combined positive expression of iNOS and VEGF group was significantly higher than that in the combined negative group.The present findings suggested that iNOS expression was significantly associated with angiogenesis,bcl-2 and cell proliferation of HCC.     

Expression and function of a new angiogenic factor AA98 target molecule at the maternal-embryonic boundary of rhesus monkey

The target molecule of monoclonal antibody AA98 (AA for short) is a new vascular endothelial cell related factor and plays a role in angiogenesis as indicated by the previous data. To investigate its role in angiogenesis and placentation in primate, we examined its expression in the implantation sites on D17, 19, 28 and 34 of gestation in rhesus monkey by immunohistochemistry and Western immunoblot. Western blot analysis showed that the primary antibody used in this study was specific for its epitope. AA protein was mainly expressed in small blood vessels and in some cytotrophoblast cells. The AA staining was found mainly in the endothelial cells and vascular small muscle. This observation supported the AA‘s role in angiogenesis. AA was spatio-temporarily expressed in cytotrophoblasts: weak in proliferating trophoblast within cell column and endovascular trophoblast, strong in trophoblastic subpopulation within the basal plate and vascular trophoblast; AA staining within the basal plate was down-regulated during early placentation. The shift of AA98 expression in extravillous trophoblasts suggestes a role of this new factor during the course of cytotrophoblast metastasis and spiral artery remodeling. The spatio-temporarily expression indicats that AA98 could be also used as a trophoblast cellular marker to characterize the acquisition of a vascular endothelial and invasive phenotype.     

Homoharringtonine induces apoptosis of endothelium and down－regulates VEGF expression of K562 cells

Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry, The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner, Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.     

Partial inhibition of CDK4 gene expression leads to the extension of G1 phase of V79-8 cells

V79-8 is an abnormal cell line which does not have detectable G1 and G2 phases in its cell cycle. This cell line is derived from V79 cell line which has Gl phase but lacks G2 phase. By using an anti-sense approach, CDK4 gene expression was partially inhibited to find whether CDK4 might contribute to the lack of Gl phase in V79-8 cells. Anti-CDK4 anti-sense plasmid was constructed and used to transfect V79-8 cells. Clones of transfected cells (V79-8-asCDK4) were examined, in comparison with V79-8 cells, to determine its growth curve, cell doubling-time (GT), the level of CDK4 gene expression and the levels of expression of some other growth related genes. V79-8-asCDK4 cells showed a slower growth rate with a doubling time 2.5-h longer than that of V79-8 cells. A flow cytometry (FCM) analysis demonstrated that the 2.5 h increase of the doubling time of V79-8-asCDK4 cells was mainly due to the appearance of Gl phase because its G2 + M phase was not significantly different from that of V79-8 cells. The decrease of CDK4 gene expression in V79-8-asCDK4 cells was shown by Northern-blot. Changes in the expression levels of the growth-related genes TGF-β, cyclin D1 and Rb were also detected in V79-8-asCDK4 cells. CDK4 functions mainly in G1 and at the transition between G1 and S phases. Expression of an anti-sense CDK4 gene fragment reduces the levels of endogenous CDK4, CDK4/cyclinD kinase activity and the phosphorylation of Rb. These events may postpone the inactivation of the check-point leading to the delay of entry into S phase and the reappearance of G1 phase in V79-8-asCDK4 cells.     

IGF-1对绵羊皮肤中GHR、IGF-1、IGF-1R、kAP3.2和KAP6-1基因表达的影响

选取36只健康、体况一致的新疆细毛羊随机分成6组。在每只羊的左侧肩胛部皮下局部注射0．5mL（10ng／mL）的IGF-1,右侧肩胛部皮肤为未注射IGF-1的对照组,然后分别于第0、3、6、9、12、50d采集各皮肤样品,用反转录多聚酶链式反应（RT-PCR）方法,定量分析绵羊皮肤中生长激素受体（GHR）、胰岛素样生长因子1（IGF-1）和胰岛素样生长因子1型受体（IGF-1R）、角蛋白关联蛋白KAP3．2和KAP6-1mRNA的相对丰度。试验结果表明,IGF-1对GHR基因的表达有下调的作用,对IGF-1、IGF-1R基因的表达没有显著的影响,而对KAP3．2、KAP6—1基因的表达具有显著的促进作用。说明IGF-1促进绵羊角蛋白关联蛋白基因的表达可能是通过生长轴以外的其他途径实现的。     

力生长因子与成骨细胞

骨组织对所处的力学环境有极强的适应能力.力学因素及非力学因素共同参与调节骨组织的生长、发育与适应性变化,但是关于两者之间的关联调控目前知之甚少.在骨骼肌肉系统中,IGF-I是一种与力刺激密切相关的骨生长因子.在力刺激作用下,IGF-I的pre-mRNA能选择性的剪接成为力生长因子（Mechano-growth factor,MGF）,MGF具有促进成肌干细胞和成骨细胞增殖,并抑制其终末分化的作用.在力刺激下,MGF参与骨骼肌肉的重建和修复过程.目前初步研究显示MGF主要分布在细胞核,其信号传导不通过IGF-IR介导.但关于MGF的细胞受体,作用靶点、作用机制和信号传导还未得到有效阐明;本文就MGF与骨骼的力学调控之间的关系进行综述.     

IGF-Ⅱ在原核生物中的表达

胰岛素生长因子IGF-Ⅰ和IGF-Ⅱ在机体的许多组织中都有表达,具有促进细胞生长和分裂等的多种生物学功能。在本研究中,我们采用RT-PCR法克隆和扩增到了IGF-Ⅱ基因,通过双酶切、胶回收纯化、连接后得到了pET21b-IGF-Ⅱ重组质粒载体,然后将其转化DH5α中,经IPTG诱导后表达的重组蛋白经纯化,并用Western blot检测,显示重组质粒pET21b-IGF-Ⅱ编码一个约25kD的外源蛋白。     

蜣螂醇提物抗良性前列腺增生研究

比较蜣螂不同浓度醇提物抗良性前列腺增生的作用,并探讨相关机制,为蜣螂的开发利用奠定基础。采用皮下注射丙酸睾酮制造大鼠良性前列腺增生模型,以前列腺湿重、前列腺指数、血清酸性磷酸酶质量浓度（ρ_PAP）、雌二醇质量浓度（ρ_E2）、双氢睾酮浓度（DHT）(c_DHT)、睾酮质量浓度（ρ_T）、表皮细胞生长因子（EGF）、转化生长因子-β1（TGF-β1）、碱性成纤维细胞生长因子（bFGF）及胰岛素样生长因子-1（IGF-1）为指标,考察蜣螂醇提物对良性前列腺增生的影响。结果表明良性前列腺模型造模成功,与模型组相比,85%乙醇提取物组能明显降低大鼠前列腺湿重与前列腺指数,明显降低ρ_PAP,ρ_E2,c_DHT及ρ_T水平,明显降低EGF,bFGF及IGF-1的表达,提高TGF-β1的表达,可显著降低前列腺体密度,显著增加比表面及比膜面值。蜣螂85%乙醇提物具有良好的抗良性前列腺增生的作用,其机制与调节EGF,TGF-β1,bFGF及IGF-1的表达有关。     

蜣螂醇提物抗良性前列腺增生研究

比较蜣螂不同浓度醇提物抗良性前列腺增生的作用,并探讨相关机制,为蜣螂的开发利用奠定基础。采用皮下注射丙酸睾酮制造大鼠良性前列腺增生模型,以前列腺湿重、前列腺指数、血清酸性磷酸酶质量浓度（ρ_PAP）、雌二醇质量浓度（ρ_E2）、双氢睾酮浓度（DHT）(c_DHT)、睾酮质量浓度（ρ_T）、表皮细胞生长因子（EGF）、转化生长因子-β1（TGF-β1）、碱性成纤维细胞生长因子（bFGF）及胰岛素样生长因子-1（IGF-1）为指标,考察蜣螂醇提物对良性前列腺增生的影响。结果表明良性前列腺模型造模成功,与模型组相比,85%乙醇提取物组能明显降低大鼠前列腺湿重与前列腺指数,明显降低ρ_PAP,ρ_E2,c_DHT及ρ_T水平,明显降低EGF,bFGF及IGF-1的表达,提高TGF-β1的表达,可显著降低前列腺体密度,显著增加比表面及比膜面值。蜣螂85%乙醇提物具有良好的抗良性前列腺增生的作用,其机制与调节EGF,TGF-β1,bFGF及IGF-1的表达有关。     

IGF-Ⅱ and IGFBP-1 reversely regulate blastocyst implantation in mouse

Insulin-like growth factor (IGF)-Ⅱ and IGF binding protein (IGFBP)-1, members of IGF family, are important in the cyclic development of endometrium and the blastocyst implantation. In the present study, the indirect immunofluorescence showed that IGF-Ⅱ and IGFBP-1 were specifically expressed at the maternal-fetal interface. In a co-culture system, IGF-Ⅱ significantly enhanced the attachment and outgrowth of the blastocyst on monolayer of uterine epithelial cells, while IGFBP-1 did not affect the blastocyst attachment, but markedly inhibited the blastocyst outgrowth. The results of zymography showed that IGF-Ⅱ enhanced the activities of MMP-2 and MMP-9, while IGFBP-1 did not affect the activities of MMP-2 and MMP-9. In conclusion, the equilibrium between the invasion of trophoblast and the inhibition of deciduas may be regulated by the interaction between the IGF-Ⅱ-expressing invading cytotrophoblast and maternal deciduas-derived IGFBP-1.