神经母细胞瘤B104细胞株条件培养基促进少突胶质前体细胞增殖的机制研究

发布时间：2018-12-07 15:06

【摘要】：目的：鉴定介导神经母细胞瘤B104细胞株条件培养液(B104neuroblatoma cellsconditioned medium, B104CM)中诱导少突胶质前体细胞(oligodendrocyte precursorcells, OPCs)增殖的关键因子，并探讨B104CM诱导OPCs增殖的信号转导机制。 方法：（1）培养B104神经母细胞瘤细胞株，离心过滤收集条件培养液。（2）手术分离取出E16天SD大鼠胎鼠的脊髓，消化分离细胞，以免疫粘附法得到纯化的OPCs，进行原代培养。（3）用免疫荧光染色法标记OPCs特异性标志物鉴定OPCs。（4）将不同浓度的B104CM(0，10%，30%，50%，100%)加入到无生长因子的少突胶质前体细胞培养物中，以BrdU掺入染色法结合A2B5染色鉴定OPCs的增殖。（5）以逆转录RT-PCR检测PDGF-AA、bFGF和IGF-1mRNA在B104细胞的表达，以酶联免疫吸附测定（enzyme linked immunosorbent assay,ELISA）检测B104CM中PDGF-AA、bFGF和IGF-1的蛋白含量，通过免疫荧光染色来验证PDGFR、FGFR2和IGFR在OPCs上的表达，筛选出可能促进OPCs增殖的候选生长因子。（6）应用生长因子受体的特异性抑制剂观察B104CM中候选生长因子对OPCs增殖的影响，，最终鉴定出B104CM中影响OPCs增殖的关键因子。（7）蛋白质印迹法(Western Blot)检测Erk1/2、PI3K、p38和JNK在B104CM诱导OPCs增殖中的活化（磷酸化）。（8）RT-PCR和Real-time-PCR法检测B104CM诱导OPCs增殖过程中可能涉及的立早基因和细胞周期蛋白的表达变化。 结果：（1）成功分离、培养出OPCs，其纯度高达95%以上。（2）BrdU掺入法结合A2B5染色证实B104CM可诱导OPCs的增殖。（3）RT-PCR结果证明B104细胞表达PDGF-AA、bFGF和IGF-1mRNA，ELISA检测到PDGF-AA、bFGF和IGF-1三种生长因子在无浓缩的B104CM中的浓度分别达到23.42±4.28、12.94±3.05和7.21±2.12ng/ml；免疫荧光染色发现OPCs表达PDGFR、FGFR和IGFR。（4）PDGFR、FGFR信号的抑制剂AG1295和PD173074显著降低B104CM诱导的OPCs增殖，但IGFR的抑制剂无明显影响。（5）Western Blot结果证明，B104CM诱导OPCs内Erk1/2和JNK的活化（磷酸化）；Erk1/2的抑制剂U0126和JNK的抑制剂SP600125均可显著降低B104CM诱导OPCs增殖。（6）RT-PCR结果证明，B104CM可刺激立早基因c-fos、c-jun、Id-2和细胞周期蛋白cyclin D1、D2、E表达的增加，且Erk抑制剂可抑制其表达。（7）B104CM刺激立早基因c-jun，c-fos，c-myc表达的增加，但降低SMAD4和ATF-2的表达，且JNK抑制剂可分别抑制和恢复其表达。 结论：1. PDGF-AA和bFGF是介导B104CM诱导OPCs增殖的关键因子。2. Erk1/2信号通路的激活是B104CM诱导OPCs增殖的关键分子事件，B104CM激活Erk1/2信号通路，并启动下游立早基因c-jun，c-fos，Id-2和细胞周期蛋白cyclin D1，cyclinD2和cyclin E等转录因子的表达，启动OPCs的增殖。3. JNK信号通路的激活也是B104CM诱导OPCs增殖所必需的，B104CM活化JNK信号通路，并上调下游立早基因c-jun，c-fos，c-myc的表达和下调SMAD4和ATF-2的表达，参与B104CM诱的OPCs增殖过程。
[Abstract]:Aim: to identify the key factors in inducing (oligodendrocyte precursorcells, OPCs) proliferation of oligodendrocyte precursor cells (oligodendrocyte precursorcells, OPCs) in conditioned medium (B104neuroblatoma cellsconditioned medium, B104CM) of neuroblastoma B104 cell line and to explore the signal transduction mechanism of OPCs proliferation induced by B104CM. Methods: (1) the B104 neuroblastoma cell line was cultured, and the conditioned medium was collected by centrifugation. (2) the spinal cord of the fetal rat of E16 day SD was removed from the spinal cord by surgery, the isolated cells were digested and purified OPCs, was obtained by immune adhesion method. (3) the specific marker of OPCs was labeled with immunofluorescence staining to identify OPCs. (4) different concentrations of B104CM were added to the culture of oligodendrocyte precursor cells without growth factor. BrdU incorporation and A2B5 staining were used to identify the proliferation of OPCs. (5) the expression of PDGF-AA,bFGF and IGF-1mRNA in B104 cells was detected by reverse transcriptase RT-PCR and PDGF-AA, in B104CM was detected by Elisa (enzyme linked immunosorbent assay,ELISA). The protein content of bFGF and IGF-1. The expression of PDGFR,FGFR2 and IGFR on OPCs was examined by immunofluorescence staining. (6) the effects of candidate growth factors in B104CM on the proliferation of OPCs were observed by using specific inhibitors of growth factor receptor. Finally, the key factors affecting the proliferation of OPCs in B104CM were identified. (7) Detection of Erk1/2,PI3K, by Western blot (Western Blot) Activation of p38 and JNK in B104CM induced proliferation of OPCs (phosphorylated). (8) RT-PCR and Real-time-PCR were used to detect the expression of early genes and cyclin in B104CM induced OPCs proliferation. Results: (1) the purity of OPCs, was over 95%. (2) BrdU incorporation combined with A2B5 staining confirmed that B104CM could induce the proliferation of OPCs. (3) RT-PCR showed that B104 cells expressed PDGF-AA,bFGF and IGF-1mRNA,. The concentrations of PDGF-AA,bFGF and IGF-1 in unconcentrated B104CM were 23.42 卤4.28 卤12.94 卤3.05 and 7.21 卤2.12 ng / ml, respectively, detected by ELISA. Immunofluorescence staining showed that AG1295 and PD173074, an inhibitor of OPCs expressing PDGFR,FGFR and IGFR. (4) PDGFR,FGFR signal, significantly decreased B104CM induced OPCs proliferation, but IGFR inhibitor had no significant effect on OPCs proliferation. B104CM induced the activation (phosphorylation) of Erk1/2 and JNK in OPCs. U0126, an inhibitor of Erk1/2, and SP600125, an inhibitor of JNK, could significantly reduce the proliferation of OPCs induced by B104CM. (6) the results of RT-PCR showed that B104CM could stimulate the expression of c-fos-c-junn Id-2 and the expression of cyclin D1tD2E. Erk inhibitor could inhibit its expression. (7) B104CM stimulated the increase of c-junn c-fos-c-myc expression, but decreased the expression of SMAD4 and ATF-2, and JNK inhibitor could inhibit and restore its expression respectively. Conclusion: 1. PDGF-AA and bFGF are the key factors mediating the proliferation of OPCs induced by B104CM. 2. The activation of Erk1/2 signaling pathway is the key molecular event of OPCs proliferation induced by B104CM. B104CM activates Erk1/2 signaling pathway, and activates the downstream gene c-junn c-fosi Id-2 and cyclin D1. The expression of transcription factors such as cyclinD2 and cyclin E initiated the proliferation of OPCs. 3. 3. The activation of JNK signaling pathway is also necessary for B104CM to induce OPCs proliferation. B104CM activates JNK signaling pathway and up-regulates the expression of c-junn c-fos-c-myc and down-regulates the expression of SMAD4 and ATF-2, which is involved in B104CM induced OPCs proliferation.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.4

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