Background To explore the effect of human osteopontin (hOPN) on the proliferation, transmigration and expression of matrix metallproteinase-2 (MMP-2) and matrix metallproteinase-9 (MMP-9) in osteosarcoma (OS) cells in...Background To explore the effect of human osteopontin (hOPN) on the proliferation, transmigration and expression of matrix metallproteinase-2 (MMP-2) and matrix metallproteinase-9 (MMP-9) in osteosarcoma (OS) cells in vitro.Methods The prokaryotic-expression vector of hOPN was produced. hOPN was then subcloned into E coli BL21 (DE3) cells and purified with ProBondTM Columns. The proliferation, cell cycle and the expression of cyclin A in OS cells were investigated by using MTT assay, flow cytometry and Western blot respectively. The transmigration of OS cells was checked by using transwell cell culture chamber. The micro-pore-filter-membrane system was used to study the chemiotaxis of hOPN to OS cells. The levels of total protein were examined according to Coomassie Brilliant Blue manuals. The expression of MMP-2 and MMP-9 were evaluated by detecting the volume of degradation of gelatin on SDS-PAGE gel.Results The prokaryotic-expression vector of hOPN and purified hOPN protein were achieved hOPN promoted OS cells proliferation in a dose-dependent manner, and stimulated cyclin A expression in OS cells to accelerate cell division cycle. hOPN facilitated the trans-membrane migration of OS cells. hOPN also enhanced the secretion of MMP-2 and MMP-9 in OS cells.Conclusion hOPN could stimulate cyclin A expression in OS cells. hOPN has chemiotaxis to OS cells and increases their transmigration. hOPN enhances the secretion of MMP-2 and MMP-9 in OS cells.展开更多
<span style="font-family:Verdana;">Neutrophils are the most numerous leukocyte in mammals and normally they are the first phagocyte observed in recently damaged or infected tissues. They play a key rol...<span style="font-family:Verdana;">Neutrophils are the most numerous leukocyte in mammals and normally they are the first phagocyte observed in recently damaged or infected tissues. They play a key role </span><span style="font-family:Verdana;">in</span><span style="font-family:;" "=""><span><span style="font-family:Verdana;"> the innate immune responses to </span><i></i></span><i><i><span style="font-family:Verdana;">Leishmania</span></i><span></span></i><span style="font-family:Verdana;"> and several other microorganisms, nonetheless an exacerbated neutrophils activity can generate a harmful response to the host, therefore its turnover rate is very important to maintain the homeostasis and averts the host tissue damage. Both apoptosis followed by phagocytosis by mononuclear phagocytes (eferocytosis) and reverse transmigration have been considered the main processes for the clearance of neutrophils from injured or infected tissues. However, the interaction with </span><i></i></span><i><i><span style="font-family:Verdana;">Leishmania</span></i><span></span></i><span style="font-family:Verdana;"> and other microbes, as well as molecules produced by arthropod vectors such as sandflies saliva can modify the behavior of neutrophils, causing immediate lysis to prolong their life. In fact, as a result of a long course of coevolution, several microorganisms have developed skills to avoid neutrophil effector mechanisms and take advantage of neutrophil clearance pathways to promote their spread in the host’s body. </span><i><i><span style="font-family:Verdana;">Leishmania</span></i><span></span></i><span style="font-family:Verdana;">, </span><i><i><span style="font-family:Verdana;">Chlamydia pneumoniae</span></i><span></span></i><span style="font-family:Verdana;"> and </span><i><i><span style="font-family:Verdana;">Yersinia pestis</span></i><span></span></i><span style="font-family:Verdana;"> for example use the efferocytic Trojan horse process for their dissemination and immune protection, in a different way vaccinia A展开更多
为研究人骨形态发生蛋白(human bone morphogenetic proteins,hBMP)12对人骨肉瘤细胞株MG63和U2OS的作用,分别用hBMP12重组腺病毒(AdBMP12)以及含重组hBMP12(recombinant hBMP12,rhBMP12)的条件培养液干预人骨肉瘤细胞MG63和U2OS,利用...为研究人骨形态发生蛋白(human bone morphogenetic proteins,hBMP)12对人骨肉瘤细胞株MG63和U2OS的作用,分别用hBMP12重组腺病毒(AdBMP12)以及含重组hBMP12(recombinant hBMP12,rhBMP12)的条件培养液干预人骨肉瘤细胞MG63和U2OS,利用台盼蓝拒染法、TUNEL法、吖啶橙/溴乙啶(AO/EB)荧光双染法、Transwell小室和碱性磷酸酶活性测定法分别检测细胞增殖、凋亡、迁移以及成骨分化能力的变化.与相应对照组相比,AdBMP12和含rhBMP12的条件培养液的干预致两种骨肉瘤细胞株细胞存活率降低,并呈一定的时间依赖性;凋亡率均随时间延长而增加,并且两种检测方法的结果一致;不同时间点的细胞穿膜数均明显减低;碱性磷酸酶活性在干预3d后开始逐渐增加,至第9d仍可观测到.以上差异均有统计学意义(P<0.01).提示无论是以腺病毒介导基因转入还是重组蛋白直接作用方式,hBMP12都可以抑制人骨肉瘤细胞株MG63和U2OS的增殖和迁移,并诱导其凋亡和向成骨细胞分化.展开更多
文摘Background To explore the effect of human osteopontin (hOPN) on the proliferation, transmigration and expression of matrix metallproteinase-2 (MMP-2) and matrix metallproteinase-9 (MMP-9) in osteosarcoma (OS) cells in vitro.Methods The prokaryotic-expression vector of hOPN was produced. hOPN was then subcloned into E coli BL21 (DE3) cells and purified with ProBondTM Columns. The proliferation, cell cycle and the expression of cyclin A in OS cells were investigated by using MTT assay, flow cytometry and Western blot respectively. The transmigration of OS cells was checked by using transwell cell culture chamber. The micro-pore-filter-membrane system was used to study the chemiotaxis of hOPN to OS cells. The levels of total protein were examined according to Coomassie Brilliant Blue manuals. The expression of MMP-2 and MMP-9 were evaluated by detecting the volume of degradation of gelatin on SDS-PAGE gel.Results The prokaryotic-expression vector of hOPN and purified hOPN protein were achieved hOPN promoted OS cells proliferation in a dose-dependent manner, and stimulated cyclin A expression in OS cells to accelerate cell division cycle. hOPN facilitated the trans-membrane migration of OS cells. hOPN also enhanced the secretion of MMP-2 and MMP-9 in OS cells.Conclusion hOPN could stimulate cyclin A expression in OS cells. hOPN has chemiotaxis to OS cells and increases their transmigration. hOPN enhances the secretion of MMP-2 and MMP-9 in OS cells.
文摘<span style="font-family:Verdana;">Neutrophils are the most numerous leukocyte in mammals and normally they are the first phagocyte observed in recently damaged or infected tissues. They play a key role </span><span style="font-family:Verdana;">in</span><span style="font-family:;" "=""><span><span style="font-family:Verdana;"> the innate immune responses to </span><i></i></span><i><i><span style="font-family:Verdana;">Leishmania</span></i><span></span></i><span style="font-family:Verdana;"> and several other microorganisms, nonetheless an exacerbated neutrophils activity can generate a harmful response to the host, therefore its turnover rate is very important to maintain the homeostasis and averts the host tissue damage. Both apoptosis followed by phagocytosis by mononuclear phagocytes (eferocytosis) and reverse transmigration have been considered the main processes for the clearance of neutrophils from injured or infected tissues. However, the interaction with </span><i></i></span><i><i><span style="font-family:Verdana;">Leishmania</span></i><span></span></i><span style="font-family:Verdana;"> and other microbes, as well as molecules produced by arthropod vectors such as sandflies saliva can modify the behavior of neutrophils, causing immediate lysis to prolong their life. In fact, as a result of a long course of coevolution, several microorganisms have developed skills to avoid neutrophil effector mechanisms and take advantage of neutrophil clearance pathways to promote their spread in the host’s body. </span><i><i><span style="font-family:Verdana;">Leishmania</span></i><span></span></i><span style="font-family:Verdana;">, </span><i><i><span style="font-family:Verdana;">Chlamydia pneumoniae</span></i><span></span></i><span style="font-family:Verdana;"> and </span><i><i><span style="font-family:Verdana;">Yersinia pestis</span></i><span></span></i><span style="font-family:Verdana;"> for example use the efferocytic Trojan horse process for their dissemination and immune protection, in a different way vaccinia A
文摘为研究人骨形态发生蛋白(human bone morphogenetic proteins,hBMP)12对人骨肉瘤细胞株MG63和U2OS的作用,分别用hBMP12重组腺病毒(AdBMP12)以及含重组hBMP12(recombinant hBMP12,rhBMP12)的条件培养液干预人骨肉瘤细胞MG63和U2OS,利用台盼蓝拒染法、TUNEL法、吖啶橙/溴乙啶(AO/EB)荧光双染法、Transwell小室和碱性磷酸酶活性测定法分别检测细胞增殖、凋亡、迁移以及成骨分化能力的变化.与相应对照组相比,AdBMP12和含rhBMP12的条件培养液的干预致两种骨肉瘤细胞株细胞存活率降低,并呈一定的时间依赖性;凋亡率均随时间延长而增加,并且两种检测方法的结果一致;不同时间点的细胞穿膜数均明显减低;碱性磷酸酶活性在干预3d后开始逐渐增加,至第9d仍可观测到.以上差异均有统计学意义(P<0.01).提示无论是以腺病毒介导基因转入还是重组蛋白直接作用方式,hBMP12都可以抑制人骨肉瘤细胞株MG63和U2OS的增殖和迁移,并诱导其凋亡和向成骨细胞分化.
