Objective: To select a target molecule associated with invasive potential in PC-3M cell. Methods: Cell subclones were isolated from PC-3M cell line with the method of limited dilution and their invasive ability charac...Objective: To select a target molecule associated with invasive potential in PC-3M cell. Methods: Cell subclones were isolated from PC-3M cell line with the method of limited dilution and their invasive ability characterized by monolayer invasion assay. The expression of u-PAR in the cell subclones at mRNA and protein levels was assayed respectively by non-competitive quantitative RT-PCR and immunohistochemical assay. Results: The expression level of u-PAR in highly invasive cell subclones was higher than that of lower invasive subclones. Conclusion: The higher expression level of u-PAR is associated with the relative strong invasive ability of PC-3M subclones. It is indicated that the u-PAR might be a promising target molecule for inhibiting invasion of highly invasive PC-3M cell subclones.展开更多
Objective： To investigate the role of urokinase-type plasminogen activator/urokinase-type plasminogen receptor（u-PA/u-PAR） system in glioma angiogenesis under hypoxic conditions, we studied the effect of glioma-con...Objective： To investigate the role of urokinase-type plasminogen activator/urokinase-type plasminogen receptor（u-PA/u-PAR） system in glioma angiogenesis under hypoxic conditions, we studied the effect of glioma-conditioned medium on the hypoxia induced changes in human endothelial-like ECV304 cells proliferation, apoptosis, cord formation in vitro and u-PA/u-PAR expression. Methods： MTT assay was used to examine the changes in cell proliferation. Cell apoptosis was analyzed by Hoechst 33258 staining. Matrigel cord-like formation assay was used to evaluate the angiogenesis ability of ECV304 cells in vitro. Expressions of u-PA/u-PAR mRNA were detected by quantitative real-time RT-PCR. Results： Hypoxia inhibited ECV304 cells proliferation and induced cell apoptosis. Hypoxic conditioned medium（H-CM） while not normoxic conditioned medium（N-CM） of U251 glioma ceils partially blocked the effect of hypoxia on ECV304 cells proliferation and apoptosis. H-CM of U251 glioma ceils also promoted the cord formation of ECV304 cells seeded on matrigel. When u-PA or u-PAR monoclonal antibodies were added into ECV304 cells culturing medium, cord formation ability was partially inhibited. H-CM of U251 glioma cells induced uPA and uPAR expression in ECV304 cells. Conclusion： These suggest that u-PA/u-PAR system is involved in glioma angiogenesis trigged by hypoxic microenviroment.展开更多
To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were ...To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79%and 60% , respectively. Although the anti-sense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P<0.01). The anti-sense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.展开更多
To evaluate the specific inhibition of antisense u PAR on the u PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u PAR ob...To evaluate the specific inhibition of antisense u PAR on the u PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u PAR obtained by RT PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u PAR recombinant was transfected into highly invasive cell subclones. The u PAR expression in neo resistant cells was examined by RT PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u PAR in transfected cells decreased sharply, and the rate of inhibition was 53 % and 73 %, respectively, indicating that an antisense u PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u PAR on invasion in highly invasive cell subclones of human prostate carcinoma.展开更多
文摘Objective: To select a target molecule associated with invasive potential in PC-3M cell. Methods: Cell subclones were isolated from PC-3M cell line with the method of limited dilution and their invasive ability characterized by monolayer invasion assay. The expression of u-PAR in the cell subclones at mRNA and protein levels was assayed respectively by non-competitive quantitative RT-PCR and immunohistochemical assay. Results: The expression level of u-PAR in highly invasive cell subclones was higher than that of lower invasive subclones. Conclusion: The higher expression level of u-PAR is associated with the relative strong invasive ability of PC-3M subclones. It is indicated that the u-PAR might be a promising target molecule for inhibiting invasion of highly invasive PC-3M cell subclones.
基金supported by the Foundation from the Health Department of Hubei Province(No.JX1B019)
文摘Objective： To investigate the role of urokinase-type plasminogen activator/urokinase-type plasminogen receptor（u-PA/u-PAR） system in glioma angiogenesis under hypoxic conditions, we studied the effect of glioma-conditioned medium on the hypoxia induced changes in human endothelial-like ECV304 cells proliferation, apoptosis, cord formation in vitro and u-PA/u-PAR expression. Methods： MTT assay was used to examine the changes in cell proliferation. Cell apoptosis was analyzed by Hoechst 33258 staining. Matrigel cord-like formation assay was used to evaluate the angiogenesis ability of ECV304 cells in vitro. Expressions of u-PA/u-PAR mRNA were detected by quantitative real-time RT-PCR. Results： Hypoxia inhibited ECV304 cells proliferation and induced cell apoptosis. Hypoxic conditioned medium（H-CM） while not normoxic conditioned medium（N-CM） of U251 glioma ceils partially blocked the effect of hypoxia on ECV304 cells proliferation and apoptosis. H-CM of U251 glioma ceils also promoted the cord formation of ECV304 cells seeded on matrigel. When u-PA or u-PAR monoclonal antibodies were added into ECV304 cells culturing medium, cord formation ability was partially inhibited. H-CM of U251 glioma cells induced uPA and uPAR expression in ECV304 cells. Conclusion： These suggest that u-PA/u-PAR system is involved in glioma angiogenesis trigged by hypoxic microenviroment.
文摘To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79%and 60% , respectively. Although the anti-sense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P<0.01). The anti-sense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.
基金ThisworkprojectsupportedbyagrantfromNaturalSciencesFoundationofHubeiProvince (No .2 0 0 0J0 77)
文摘To evaluate the specific inhibition of antisense u PAR on the u PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u PAR obtained by RT PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u PAR recombinant was transfected into highly invasive cell subclones. The u PAR expression in neo resistant cells was examined by RT PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u PAR in transfected cells decreased sharply, and the rate of inhibition was 53 % and 73 %, respectively, indicating that an antisense u PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u PAR on invasion in highly invasive cell subclones of human prostate carcinoma.
