摘要
目的探讨小RNA干扰靶向抑制Par-4基因表达对人骨髓间充质干细胞凋亡的影响。方法原代培养人骨髓间充质干细胞，谷氨酸诱导凋亡。设计和合成针对Par-4基因mRNA的siRNA（Par-4-siRNA），构建真核细胞表达载体，用脂质体介导转染人骨髓间充质干细胞，利用G418筛选稳定表达细胞株。实时荧光定量PCR（real-timePCR）法检测Par-4mRNA的表达量。流式细胞术测定细胞凋亡百分率。Western blot测定磷酸化（Thr308）的蛋白激酶Aktl蛋白表达量。比色法测定Caspase-3酶活性。结果筛选出的Par4-SiRNA-1、2显著抑制人骨髓间充质干细胞中Par-4基因的mRNA表达，mRNA水平分别降低88％、67％。在谷氨酸诱导后24h，人骨髓间充质干细胞发生凋亡，百分率为60．30％±6．82％。Par-4-SiRNA-1、2都显著抑制谷氨酸的诱导作用，凋亡百分率降为38．80％±3．97％（P〈0．01）和45．49％±4．32％（P〈0．01）。谷氨酸诱导后24h人骨髓间充质干细胞中磷酸化Aktl蛋白表达下调（89．074-6．42和28．30±5．65，P〈0．叭），而Par-4-SiRNA-1、2对之下调都有显著抑制作用（63．564-6．75和45．59±4．88，均有P〈0．01）。谷氨酸诱导后24h人骨髓间充质干细胞中Caspase-3酶活性上调（0．1428±0．0495和0．8616±0．1051，P〈0．01），而Par-4-SiRNA-1、2对之上调都有显著抑制作用（0．6581±0．0555和0．7041±0．0401，均有P〈0．01）。结论小RNA干扰可靶向抑制Par-4基因的表达，拮抗谷氨酸诱导的人骨髓间充质干细胞的凋亡。
Objective The prostate apoptosis response factor-4 （Par-4） gene was originally identified by differential screening for genes that are up-regulated when prostate cells are induced to undergo apoptosis. Par-4 was found to possess potent apoptotic activity in various cellular systems in response to numerous stimuli. The aim of this study was to explore the effects of small interfering RNA （siRNA） against Par-4 gene on the apoptosis of human bone marrow mesenchymal stem cells （hBMSCs） exposed to glutamate. Methods Primary culture of hBMSCs was carried out and siRNAs targeted Par-4 gene （ Par-4- SiRNA） were chemically synthesized. Eukaryocytic expression vector was built and were transfected into hBMSCs with liposome. After selecting with G418, the stable cell clones were treated with glutamate . The expression of Par-4 mRNA was determined by real-time PCR. The apoptosis of hBMSCs was quantified by flow cytometry. Western blotting was used to detect the protein levels of phosphorylated Aktl （Thr308）. Relative Caspase-3 activity was determined by colorimetric assay. Results The Par-4-SiRNA-1 and Par-4- siRNA-2 could markedly down-regulate the mRNA levels of Par-4 gene in hBMSCs. With the transfections of Par-4-SiRNA-1 and Par-4-SiRNA-2, the levels of Par-4 mRNA were respectively decreased by 88% and 67%. Both Par-4-SiRNA-1 and Par-4-SiRNA-2 inhibited significantly the apoptosis of hBMSCs induced by glutamate, in which the percentages of apoptotic cells were respectively decreased to 38. 80% ± 3.97% （P 〈 0. O1 ） and 45.49% ±4. 32% （P 〈 O. 01 ） from 60. 30% ± 6. 82%. Western blot assays demonstrated that, glutamate down-regulated the expression of phosphorylated Aktl proteins in hBMSCs （ 89. 07 ±6. 42 and 28. 30 ±5. 65, respectively, P 〈 O. 01 ）. However, Par-4-SiRNA-1 and Par-4-SiRNA-2 could markedly recover the down-regulation of Aktl proteins induced by glutamate （ 63.56± 6. 75 and 45.59 ± 4. 88,respectively, P 〈 0. 01 ）. And the relative Caspase-3 activity which was enhanced by the treatment with glutamate（0. 1428±0. 0495 and 0. 8616±0. 1051, P 〈0. 01 ）, was suppressed by Par4-SiRNA-1 and Par- d-SiRNA-2 （0. 8616 ±0. 1051 and 0. 6581 ± 0. 0555, respectively, P 〈 0. 01 ）. Conclusion SiRNA against Par4 gene could inhibit the apoptosis of hBMSCs induced by glutamate, and its inhibitory effects may be mediated by the up-regulation of phosphorylated Aktl and the suppression of the relative Caspase-3 activity.
出处
《中华儿科杂志》
CAS
CSCD
北大核心
2008年第11期836-841,共6页
Chinese Journal of Pediatrics
基金
国家自然科学基金资助项目（30570863）
江苏省卫生厅医学科技发展基金重大项目（K200504）
