【摘要】：目的:觀察骨髓間充質(zhì)干細(xì)胞(BM-MSCs)對(duì)氯化鈷(CoCl2)誘導(dǎo)的PC12細(xì)胞缺氧損傷及凋亡的影響并探討其作用機(jī)制。方法:將PC12細(xì)胞分為以下幾組:空白對(duì)照組、CoCl2處理組、BM-MSCs-siCTL+CoCl2處理組和BM-MSCs-siEPO+CoCl2處理組。應(yīng)用MTT、流式細(xì)胞術(shù)(FCM)及Hoechst 33258染色法檢測(cè)BM-MSCs對(duì)CoCl2誘導(dǎo)的細(xì)胞活性下降及凋亡的影響。采用逆轉(zhuǎn)錄PCR(RT-PCR)和Western blotting檢測(cè)BM-MSCs的促紅細(xì)胞生成素(EPO)表達(dá)情況。同時(shí)通過(guò)RT-PCR法檢測(cè)PC12細(xì)胞的Bcl-2與Bax表達(dá)情況。此外應(yīng)用分光光度法檢測(cè)caspase-9和-3活性。結(jié)果:MTT結(jié)果顯示BM-MSCs共培養(yǎng)能夠提高PC12細(xì)胞活力,0.6 mmol/L CoCl2單獨(dú)處理組24 h和48 h細(xì)胞存活率僅為(43.0±6.4)%和(33.8±5.7)%,1∶15細(xì)胞比BM-MSCs共培養(yǎng)24 h和48 h后細(xì)胞存活率明顯上升,分別為(77.9±3.8)%和(75.2±9.7)%(P0.01)。RT-PCR和Western blotting顯示0.6mmol/L CoCl2處理24 h和48 h明顯誘導(dǎo)BM-MSCs的EPO表達(dá)上調(diào),而EPO siRNA可完全抑制BM-MSCs的EPO表達(dá)(P0.01)。FCM及Hoechst 33258結(jié)果表明CoCl2處理能誘導(dǎo)PC12細(xì)胞損傷及凋亡,BM-MSCs-siCTL與PC12細(xì)胞共培養(yǎng)可有效抑制CoCl2的細(xì)胞毒性作用,減少細(xì)胞缺氧性損傷及凋亡,而EPO siRNA可明顯阻斷BM-MSCs的抗細(xì)胞凋亡作用(P0.01)。RT-PCR結(jié)果顯示BM-MSCs共培養(yǎng)組PC12細(xì)胞的Bcl-2表達(dá)較CoCl2處理組明顯升高,而B(niǎo)ax表達(dá)較CoCl2處理組明顯降低;EPO siRNA明顯抑制BM-MSCs介導(dǎo)的Bcl-2表達(dá)升高和Bax表達(dá)降低(P0.01)。分光光度法結(jié)果顯示BM-MSCs-siCTL共培養(yǎng)組的caspase-9和-3活性較CoCl2處理組明顯降低,而B(niǎo)M-MSCs-siEPO共培養(yǎng)組的caspase-9和-3活性較BM-MSCs-siCTL共培養(yǎng)組明顯增加(P0.01)。結(jié)論:BM-MSCs共培養(yǎng)能抑制CoCl2誘導(dǎo)的PC12細(xì)胞凋亡,其細(xì)胞保護(hù)作用的機(jī)制可能與其上調(diào)EPO的表達(dá)有關(guān)。
[Abstract]:Aim: to investigate the effects of bone marrow mesenchymal stem cells (BM-MSCs) on hypoxic injury and apoptosis of PC12 cells induced by cobalt chloride (CoCl2) and its mechanism. Methods: PC12 cells were divided into the following groups: BM-MSCs-siCTL CoCl2 treated group and BM-MSCs-siEPO CoCl2 treated group. MTT, flow cytometry (FCM) and Hoechst 33258 staining were used to detect the effect of BM-MSCs on the decrease of cell activity and apoptosis induced by CoCl2. The expression of erythropoietin (EPO) in BM-MSCs was detected by reverse transcription PCR (RT-PCR) and Western blotting. At the same time, the expression of Bcl-2 and Bax in PC12 cells was detected by RT-PCR assay. In addition, the activities of caspase-9 and -3 were detected by spectrophotometry. Results the results showed that BM-MSCs co-culture could increase the viability of PC12 cells treated with 0.6 mmol/L CoCl2 alone for 24 h and 48 h only (43.0 鹵6.4)% and (33.8 鹵5.7)%. Compared with BM-MSCs co-cultured for 24 h and 48 h, the cell survival rate increased significantly. (77.9 鹵3.8)% and (75.2 鹵9.7)% (P0.01), respectively. RT-PCR and Western blotting showed that the EPO expression of BM-MSCs was significantly up-regulated at 24 h and 48 h after 0.6mmol/L CoCl2 treatment. EPO siRNA could completely inhibit the EPO expression of BM-MSCs (P0.01). FCM and Hoechst 33258. The results showed that CoCl2 treatment could induce PC12 cell injury and apoptosis. BM-MSCs-siCTL and PC12 cell co-culture could effectively inhibit the cytotoxicity of CoCl2 and reduce the cell hypoxia injury and apoptosis. EPO siRNA significantly blocked the anti-apoptosis effect of BM-MSCs (P0.01). RT-PCR results showed that the expression of Bcl-2 in BM-MSCs co-cultured PC12 cells was significantly higher than that in CoCl2 treated group. The expression of Bax was significantly lower than that of CoCl2 (P0.01). EPO siRNA significantly inhibited the increase of Bcl-2 expression mediated by BM-MSCs and the decrease of Bax expression (P0.01). The results of spectrophotometry showed that the activities of caspase-9 and -3 in the BM-MSCs-siCTL co-culture group were significantly lower than those in the CoCl2 treatment group, while the caspase-9 and -3 activities in the BM-MSCs-siEPO co-culture group were significantly higher than those in the BM-MSCs-siCTL co-culture group (P0.01). Conclusion the co-culture of BM-MSCs can inhibit the apoptosis of PC12 cells induced by CoCl2, and its protective mechanism may be related to its up-regulation of EPO expression.
【作者單位】： 中山大學(xué)中山醫(yī)學(xué)院病理生理學(xué)教研室;中山大學(xué)附屬第一醫(yī)院血液科;中山大學(xué)腫瘤防治中心實(shí)驗(yàn)研究部;中山大學(xué)附屬第一醫(yī)院轉(zhuǎn)化醫(yī)學(xué)中心實(shí)驗(yàn)室;
【基金】：廣東省自然科學(xué)基金資助項(xiàng)目(No.9151008901000009)
【分類號(hào)】：R363

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