骨髓间充质干细胞通过上调EPO表达减轻缺氧损伤引起的PC12细胞凋亡

发布时间：2018-09-04 15:36

【摘要】：目的:观察骨髓间充质干细胞(BM-MSCs)对氯化钴(CoCl2)诱导的PC12细胞缺氧损伤及凋亡的影响并探讨其作用机制。方法:将PC12细胞分为以下几组:空白对照组、CoCl2处理组、BM-MSCs-siCTL+CoCl2处理组和BM-MSCs-siEPO+CoCl2处理组。应用MTT、流式细胞术(FCM)及Hoechst 33258染色法检测BM-MSCs对CoCl2诱导的细胞活性下降及凋亡的影响。采用逆转录PCR(RT-PCR)和Western blotting检测BM-MSCs的促红细胞生成素(EPO)表达情况。同时通过RT-PCR法检测PC12细胞的Bcl-2与Bax表达情况。此外应用分光光度法检测caspase-9和-3活性。结果:MTT结果显示BM-MSCs共培养能够提高PC12细胞活力,0.6 mmol/L CoCl2单独处理组24 h和48 h细胞存活率仅为(43.0±6.4)%和(33.8±5.7)%,1∶15细胞比BM-MSCs共培养24 h和48 h后细胞存活率明显上升,分别为(77.9±3.8)%和(75.2±9.7)%(P0.01)。RT-PCR和Western blotting显示0.6mmol/L CoCl2处理24 h和48 h明显诱导BM-MSCs的EPO表达上调,而EPO siRNA可完全抑制BM-MSCs的EPO表达(P0.01)。FCM及Hoechst 33258结果表明CoCl2处理能诱导PC12细胞损伤及凋亡,BM-MSCs-siCTL与PC12细胞共培养可有效抑制CoCl2的细胞毒性作用,减少细胞缺氧性损伤及凋亡,而EPO siRNA可明显阻断BM-MSCs的抗细胞凋亡作用(P0.01)。RT-PCR结果显示BM-MSCs共培养组PC12细胞的Bcl-2表达较CoCl2处理组明显升高,而Bax表达较CoCl2处理组明显降低;EPO siRNA明显抑制BM-MSCs介导的Bcl-2表达升高和Bax表达降低(P0.01)。分光光度法结果显示BM-MSCs-siCTL共培养组的caspase-9和-3活性较CoCl2处理组明显降低,而BM-MSCs-siEPO共培养组的caspase-9和-3活性较BM-MSCs-siCTL共培养组明显增加(P0.01)。结论:BM-MSCs共培养能抑制CoCl2诱导的PC12细胞凋亡,其细胞保护作用的机制可能与其上调EPO的表达有关。
[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 卤6.4)% and (33.8 卤5.7)%. Compared with BM-MSCs co-cultured for 24 h and 48 h, the cell survival rate increased significantly. (77.9 卤3.8)% and (75.2 卤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.
【作者单位】：中山大学中山医学院病理生理学教研室;中山大学附属第一医院血液科;中山大学肿瘤防治中心实验研究部;中山大学附属第一医院转化医学中心实验室;
【基金】：广东省自然科学基金资助项目(No.9151008901000009)
【分类号】：R363

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