摘要
目的通过观察分析胸腺基质促淋巴细胞生成素(thymic stromal lymphopoietin,TSLP)作用于小鼠腰椎髓核细胞,明确胸腺基质促淋巴细胞生成素对小鼠髓核细胞生存活动的影响及机制。方法从小鼠腰椎间盘分离获得髓核细胞,培养获得小鼠髓核细胞株,通过免疫荧光学染色鉴定细胞类型。实验组分为:①对照组,添加等量DMEM培养液;②TNF-α组(阳性对照组),加入500 ng/mL TNF-α诱导;③TSLP组,加入10 ng/mL TSLP诱导;④TSLP+LY94002组(阻断PI3K),加入10 ng/mL TSLP+10 μmol LY294002;⑤TSLP+Embelin组(阻断XIAP),加入10 ng/mL TSLP+ 50 μmol Embelin。均于恒温孵箱培养24 h,。蛋白免疫印迹分析细胞内Akt/PKB、XIAP、caspase-3的表达变化。流式细胞术检测观察小鼠髓核细胞凋亡率的变化。结果荧光显微镜观察见所培养细胞Ⅱ型胶原蛋白染色阳性,证实所培养细胞为髓核细胞。蛋白表达及细胞凋亡率检测结果显示,与对照组相比,TSLP组小鼠髓核细胞内Akt磷酸化明显增强,差异有统计学意义(t=9.510,P=0.001),XIAP表达增加,差异有统计学意义(t=8.851,P=0.001),caspase-3变化不明显,小鼠髓核细胞凋亡率变化不明显。与TSLP组相比,TSLP+LY94002组的Akt磷酸化减弱(t=8.798,P=0.001),XIAP表达下降(t=7.032,P=0.002),caspase-3活化明显增强(t=5.908,P=0.004)。小鼠髓核细胞凋亡率明显升高,差异有统计学意义(t=21.268,P=0.001)。TSLP+Embelin组caspase-3活化明显增强(t=7.990,P=0.001)。小鼠髓核细胞凋亡率也明显升高(t=21.279,P=0.001)。结论TSLP具有通过激活小鼠髓核细胞内PI3K/Akt通路上调XIAP的表达,抑制caspase-3的活化,最终抑制髓核细胞的凋亡的作用,其可通过激活PI3K/Akt通路抑制髓核细胞凋亡的作用。
Objective The aim of current study is to determine the effect and mechanism of thymic stromal lymphopoietin on apoptosis of mouse nucleus pulposus cells by investigating the apoptotic activity and variation of intracellular phosphorylated protein kinase B (p-Akt), X-linkedinhibitor of apoptosis protein (XIAP), cysteinyl aspartate specific proteinase-3 (caspase-3), with the treatment of thymic stromal lymphopoietin. Methods Mouse lumbar nucleus pulposus cells were cultured and identified under a fluorescence microscope. Second or third passage cells maintained in monolayers were used for the following experiments. The groups were divided randomly into normal group, TNF-α treated group, TSLP treated group, TSLP+LY94002 treated group and TSLP+Embelin treated group. As a control, normal group was treated with PBS. TNF-α treated group was treated with 500 ng/ml TNF-α as a positive control. TSLP treated group was treated with 10 ng/ml rhTSLP. TSLP+LY94002 treated group and TSLP+Embelin treated group were treated with 10 ng/ml TSLP with the pretreatment of different pathway inhibitors for 30 min in different corresponding experiments, for which 10 μmol LY294002 or 50 LY294002 responding experimentsreatment of different pathway inhibitors formouse nucleus pulposus cells was detected by FACS.The expression levels of the intracellular p-Akt, XIAP, caspase-3 were investigated by Western blot analysis. Results As the culture cell type II collagen staining was positive observed by fluorescence microscopy, we confirmed that the cultured cells were nucleus pulposus cells. In comparison with negative control, the levels of p-Akt, XIAP in TSLP treated group were elevated (t=9.510, P=0.001;t=8.851, P=0.001). Thecaspase-3 activity were slightly enhanced and the rate of cells apoptosis was no significance. Compared with TSLP treated group, downregulated level of p-Akt and XIAPand upregulatedcaspase-3 activity in TSLP+LY294002 treated group were observed (t=8.798, P=0.001;t=7.032, P=0.002;t=5.908, P=0.004). Upregulated caspase-3
作者
郑文凯
黄智
达逸峰
邢文华
李峰
辛大奇
刘胜祥
杨学军
祝勇
Zheng Wenkai;Huang Zhi;Da Yifeng;Xing Wenhua;Li Feng;Xin Daqi;Liu Shengxiang;Yang Xuejun;Zhu Yong(The Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, China;Inner Mongolia Medical University, Hohhot 010030, China)
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2019年第6期346-353,共8页
Chinese Journal of Orthopaedics
基金
国家自然科学基金(81460332).
