嗅鞘细胞培养液对神经样细胞RhoA活性和轴突生长的影响
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摘要
研究目的:
观察嗅鞘细胞培养液(olfactory ensheathing cells culture medium,OECCM)对神经样细胞PC12细胞活性RhoA的表达和轴突生长的影响,探讨嗅鞘细胞促进脊髓轴突生长与RhoA/ROCK信号通路的关系。
研究方法:
(1)原代嗅鞘细胞培养并纯化,鉴定,收集培养上清制备成OECCM。
(2)PC12细胞株培养,经OECCM和或RhoA激活剂LPA作用后,用Western blotting方法检测活性RhoA水平。
(3)PC12细胞株培养,加入OECCM后观察细胞形态学变化。
(4)PC12细胞株培养,经OECCM和或RhoA激活剂LPA作用后,用免疫荧光法观测RhoA吸光度(A)值的变化。
(5)PC12细胞株培养,经OECCM和或RhoA激活剂LPA作用后,细胞用鬼笔环肽进行染色,并在荧光显微镜下观察应激纤维的形成变化。
(6)PC12细胞株培养,经OECCM作用后,用免疫荧光法检测神经丝蛋白(neurofilament protein , NF)的表达及分布。
(7)PC12细胞株培养,经OECCM作用后,用Western blotting方法检测神经丝蛋白、突触蛋白和突触素蛋白的相对含量。
研究结果:
(1)培养的嗅鞘细胞呈双极、三极和多极,胞体较小,立体感强,折光性好,细胞突起相互交织成网状结构,嗅鞘细胞的纯度在85%以上。
(2)嗅鞘细胞培养液可抑制RhoA活性,并能拮抗LPA引起的活性RhoA增高。
(3)在PC12细胞株中,经OECCM作用后,细胞长有长短不一突起,并随着时间的延长交织成网。
(4)在PC12细胞株中,经OECCM和或RhoA激活剂LPA作用后,免疫荧光显示OECCM作用组RhoA吸光度(A)值明显低于对照组,而LPA组中则明显增高。
(5)嗅鞘细胞培养液对LPA引起的应激纤维形成有抑制作用。
(6)在PC12细胞株中,经OECCM作用后,神经丝蛋白的分布范围逐渐增加,其分布面积与β-tubulin分布面积的比值较对照组显著增高。
(7)在PC12细胞株中,经OECCM作用后,神经丝蛋白、突触蛋白和突触素蛋白的表达显著高于对照组。
结论:
嗅鞘细胞培养液可促进神经样细胞PC12细胞轴突生长,在结构和功能上向神经元分化,并在此过程中抑制了RhoA活性,表明嗅鞘细胞促神经轴突生长机制可能通过RhoA/ROCK信号通路。
Objective
To observe the expression of olfactory ensheathing cells culture medium(OECCM)on RhoA activity in nerve-like cells PC12 cells, and investigate the relationship between Olfactory ensheathing cells promote axon growth and RhoA / ROCK signaling pathway. Methods
(1) Primary olfactory ensheathing cells were cultured, purified and identified, and the purified cell conditioned medium was collected into the OECCM.
(2) PC12 cells were cultured and treated with OECCM and/or LPA, and then the amount of GTP-RhoA was detected by Western Blotting.
(3) PC12 cells were cultured and treated with OECCM, and then the morphological changes were observed .
(4) PC12 cells were cultured and treated with OECCM and/or LPA, and then the absorbance value changes of RhoA were observed by immunofluorescence.
(5) PC12 cells were cultured and treated with OECCM and/or LPA, and then the cells were stained with phalloidin and the formation of stress fiber was visualized under fluorescent microscope.
(6) PC12 cells were cultured and treated with OECCM, and then the expression and distribution of neurofilament protein was detected by immunofluorescence.
(7) PC12 cells were cultured and treated with OECCM, and then the relative content of neurofilament proteins, synapsin, synaptophysin were detected by Western Blotting.
Results
(1) Cultured olfactory ensheathing cells were bipolar, tripolar and multipolar , and the cell bodies were smaller,stereoscopic, and good refraction. The cell processes are intertwined into a network structure, and the olfactory ensheathing cells in more than 85% purity.
(2) Olfactory ensheathing cell culture medium could inhibit the activation of RhoA, and could antagonize RhoA activity caused by LPA.
(3) In the PC12 cell line, by the role of olfactory ensheathing cell culture medium, the cells have long processes of varying lengths, and with time woven into nets.
(4) In the PC12 cell line , by the role of olfactory ensheathing cell culture medium and/or LPA, the immunofluorescence staining showed the absorbance value of RhoA in olfactory ensheathing cell culture medium group was significantly lower than the control group, while the LPA group was significantly higher.
(5) Olfactory ensheathing cell culture medium was able to antagonize the LPA-induced stress fiber formation.
(6) In the PC12 cell line, by the role of olfactory ensheathing cell culture medium, immunofluorescence staining showed the distribution of neurofilament protein gradually increased and the distribution of size and size distribution ofβ-tubulin ratio was significantly higher than the control group.
(7) In the PC12 cell line, by the role of olfactory ensheathing cell culture medium, the Western blotting showed the expression level of NF, synapsin and synaptophysin in olfactory ensheathing cell culture medium group was significantly higher than the control group.
Conclusion
Olfactory ensheathing cell culture medium could promote the axonal growth of PC12 cells, the structural and functional differentiation into neurons, and in the process inhibit RhoA activity, which makes olfactory ensheathing cells promote neurite growth mechanism through the RhoA / ROCK signaling pathway may exist.
观察嗅鞘细胞培养液(olfactory ensheathing cells culture medium,OECCM)对神经样细胞PC12细胞活性RhoA的表达和轴突生长的影响,探讨嗅鞘细胞促进脊髓轴突生长与RhoA/ROCK信号通路的关系。
研究方法:
(1)原代嗅鞘细胞培养并纯化,鉴定,收集培养上清制备成OECCM。
(2)PC12细胞株培养,经OECCM和或RhoA激活剂LPA作用后,用Western blotting方法检测活性RhoA水平。
(3)PC12细胞株培养,加入OECCM后观察细胞形态学变化。
(4)PC12细胞株培养,经OECCM和或RhoA激活剂LPA作用后,用免疫荧光法观测RhoA吸光度(A)值的变化。
(5)PC12细胞株培养,经OECCM和或RhoA激活剂LPA作用后,细胞用鬼笔环肽进行染色,并在荧光显微镜下观察应激纤维的形成变化。
(6)PC12细胞株培养,经OECCM作用后,用免疫荧光法检测神经丝蛋白(neurofilament protein , NF)的表达及分布。
(7)PC12细胞株培养,经OECCM作用后,用Western blotting方法检测神经丝蛋白、突触蛋白和突触素蛋白的相对含量。
研究结果:
(1)培养的嗅鞘细胞呈双极、三极和多极,胞体较小,立体感强,折光性好,细胞突起相互交织成网状结构,嗅鞘细胞的纯度在85%以上。
(2)嗅鞘细胞培养液可抑制RhoA活性,并能拮抗LPA引起的活性RhoA增高。
(3)在PC12细胞株中,经OECCM作用后,细胞长有长短不一突起,并随着时间的延长交织成网。
(4)在PC12细胞株中,经OECCM和或RhoA激活剂LPA作用后,免疫荧光显示OECCM作用组RhoA吸光度(A)值明显低于对照组,而LPA组中则明显增高。
(5)嗅鞘细胞培养液对LPA引起的应激纤维形成有抑制作用。
(6)在PC12细胞株中,经OECCM作用后,神经丝蛋白的分布范围逐渐增加,其分布面积与β-tubulin分布面积的比值较对照组显著增高。
(7)在PC12细胞株中,经OECCM作用后,神经丝蛋白、突触蛋白和突触素蛋白的表达显著高于对照组。
结论:
嗅鞘细胞培养液可促进神经样细胞PC12细胞轴突生长,在结构和功能上向神经元分化,并在此过程中抑制了RhoA活性,表明嗅鞘细胞促神经轴突生长机制可能通过RhoA/ROCK信号通路。
Objective
To observe the expression of olfactory ensheathing cells culture medium(OECCM)on RhoA activity in nerve-like cells PC12 cells, and investigate the relationship between Olfactory ensheathing cells promote axon growth and RhoA / ROCK signaling pathway. Methods
(1) Primary olfactory ensheathing cells were cultured, purified and identified, and the purified cell conditioned medium was collected into the OECCM.
(2) PC12 cells were cultured and treated with OECCM and/or LPA, and then the amount of GTP-RhoA was detected by Western Blotting.
(3) PC12 cells were cultured and treated with OECCM, and then the morphological changes were observed .
(4) PC12 cells were cultured and treated with OECCM and/or LPA, and then the absorbance value changes of RhoA were observed by immunofluorescence.
(5) PC12 cells were cultured and treated with OECCM and/or LPA, and then the cells were stained with phalloidin and the formation of stress fiber was visualized under fluorescent microscope.
(6) PC12 cells were cultured and treated with OECCM, and then the expression and distribution of neurofilament protein was detected by immunofluorescence.
(7) PC12 cells were cultured and treated with OECCM, and then the relative content of neurofilament proteins, synapsin, synaptophysin were detected by Western Blotting.
Results
(1) Cultured olfactory ensheathing cells were bipolar, tripolar and multipolar , and the cell bodies were smaller,stereoscopic, and good refraction. The cell processes are intertwined into a network structure, and the olfactory ensheathing cells in more than 85% purity.
(2) Olfactory ensheathing cell culture medium could inhibit the activation of RhoA, and could antagonize RhoA activity caused by LPA.
(3) In the PC12 cell line, by the role of olfactory ensheathing cell culture medium, the cells have long processes of varying lengths, and with time woven into nets.
(4) In the PC12 cell line , by the role of olfactory ensheathing cell culture medium and/or LPA, the immunofluorescence staining showed the absorbance value of RhoA in olfactory ensheathing cell culture medium group was significantly lower than the control group, while the LPA group was significantly higher.
(5) Olfactory ensheathing cell culture medium was able to antagonize the LPA-induced stress fiber formation.
(6) In the PC12 cell line, by the role of olfactory ensheathing cell culture medium, immunofluorescence staining showed the distribution of neurofilament protein gradually increased and the distribution of size and size distribution ofβ-tubulin ratio was significantly higher than the control group.
(7) In the PC12 cell line, by the role of olfactory ensheathing cell culture medium, the Western blotting showed the expression level of NF, synapsin and synaptophysin in olfactory ensheathing cell culture medium group was significantly higher than the control group.
Conclusion
Olfactory ensheathing cell culture medium could promote the axonal growth of PC12 cells, the structural and functional differentiation into neurons, and in the process inhibit RhoA activity, which makes olfactory ensheathing cells promote neurite growth mechanism through the RhoA / ROCK signaling pathway may exist.
引文
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