【摘要】：目的：鑒定介導(dǎo)神經(jīng)母細(xì)胞瘤B104細(xì)胞株條件培養(yǎng)液(B104neuroblatoma cellsconditioned medium, B104CM)中誘導(dǎo)少突膠質(zhì)前體細(xì)胞(oligodendrocyte precursorcells, OPCs)增殖的關(guān)鍵因子，并探討B(tài)104CM誘導(dǎo)OPCs增殖的信號(hào)轉(zhuǎn)導(dǎo)機(jī)制。 方法：（1）培養(yǎng)B104神經(jīng)母細(xì)胞瘤細(xì)胞株，離心過濾收集條件培養(yǎng)液。（2）手術(shù)分離取出E16天SD大鼠胎鼠的脊髓，消化分離細(xì)胞，以免疫粘附法得到純化的OPCs，進(jìn)行原代培養(yǎng)。（3）用免疫熒光染色法標(biāo)記OPCs特異性標(biāo)志物鑒定OPCs。（4）將不同濃度的B104CM(0，10%，30%，50%，100%)加入到無(wú)生長(zhǎng)因子的少突膠質(zhì)前體細(xì)胞培養(yǎng)物中，以BrdU摻入染色法結(jié)合A2B5染色鑒定OPCs的增殖。（5）以逆轉(zhuǎn)錄RT-PCR檢測(cè)PDGF-AA、bFGF和IGF-1mRNA在B104細(xì)胞的表達(dá)，以酶聯(lián)免疫吸附測(cè)定（enzyme linked immunosorbent assay,ELISA）檢測(cè)B104CM中PDGF-AA、bFGF和IGF-1的蛋白含量，通過免疫熒光染色來(lái)驗(yàn)證PDGFR、FGFR2和IGFR在OPCs上的表達(dá)，篩選出可能促進(jìn)OPCs增殖的候選生長(zhǎng)因子。（6）應(yīng)用生長(zhǎng)因子受體的特異性抑制劑觀察B104CM中候選生長(zhǎng)因子對(duì)OPCs增殖的影響，，最終鑒定出B104CM中影響OPCs增殖的關(guān)鍵因子。（7）蛋白質(zhì)印跡法(Western Blot)檢測(cè)Erk1/2、PI3K、p38和JNK在B104CM誘導(dǎo)OPCs增殖中的活化（磷酸化）。（8）RT-PCR和Real-time-PCR法檢測(cè)B104CM誘導(dǎo)OPCs增殖過程中可能涉及的立早基因和細(xì)胞周期蛋白的表達(dá)變化。 結(jié)果：（1）成功分離、培養(yǎng)出OPCs，其純度高達(dá)95%以上。（2）BrdU摻入法結(jié)合A2B5染色證實(shí)B104CM可誘導(dǎo)OPCs的增殖。（3）RT-PCR結(jié)果證明B104細(xì)胞表達(dá)PDGF-AA、bFGF和IGF-1mRNA，ELISA檢測(cè)到PDGF-AA、bFGF和IGF-1三種生長(zhǎng)因子在無(wú)濃縮的B104CM中的濃度分別達(dá)到23.42±4.28、12.94±3.05和7.21±2.12ng/ml；免疫熒光染色發(fā)現(xiàn)OPCs表達(dá)PDGFR、FGFR和IGFR。（4）PDGFR、FGFR信號(hào)的抑制劑AG1295和PD173074顯著降低B104CM誘導(dǎo)的OPCs增殖，但I(xiàn)GFR的抑制劑無(wú)明顯影響。（5）Western Blot結(jié)果證明，B104CM誘導(dǎo)OPCs內(nèi)Erk1/2和JNK的活化（磷酸化）；Erk1/2的抑制劑U0126和JNK的抑制劑SP600125均可顯著降低B104CM誘導(dǎo)OPCs增殖。（6）RT-PCR結(jié)果證明，B104CM可刺激立早基因c-fos、c-jun、Id-2和細(xì)胞周期蛋白cyclin D1、D2、E表達(dá)的增加，且Erk抑制劑可抑制其表達(dá)。（7）B104CM刺激立早基因c-jun，c-fos，c-myc表達(dá)的增加，但降低SMAD4和ATF-2的表達(dá)，且JNK抑制劑可分別抑制和恢復(fù)其表達(dá)。 結(jié)論：1. PDGF-AA和bFGF是介導(dǎo)B104CM誘導(dǎo)OPCs增殖的關(guān)鍵因子。2. Erk1/2信號(hào)通路的激活是B104CM誘導(dǎo)OPCs增殖的關(guān)鍵分子事件，B104CM激活Erk1/2信號(hào)通路，并啟動(dòng)下游立早基因c-jun，c-fos，Id-2和細(xì)胞周期蛋白cyclin D1，cyclinD2和cyclin E等轉(zhuǎn)錄因子的表達(dá)，啟動(dòng)OPCs的增殖。3. JNK信號(hào)通路的激活也是B104CM誘導(dǎo)OPCs增殖所必需的，B104CM活化JNK信號(hào)通路，并上調(diào)下游立早基因c-jun，c-fos，c-myc的表達(dá)和下調(diào)SMAD4和ATF-2的表達(dá)，參與B104CM誘的OPCs增殖過程。
[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 鹵4.28 鹵12.94 鹵3.05 and 7.21 鹵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.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.4

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本文編號(hào)：2367375