受体相互作用蛋白激酶1对骨髓间充质干细胞生物活性的影响  

Receptor-interacting protein kinase 1 coordinates biological function of bone marrow mesenchymal stem cells

作　　者：李娜[1] 田青 刘飞飞[2] 陈松峰 李振伟[2] Li Na;Tian Qing;Liu Feifei;Chen Songfeng;Li Zhenwei(Department of Otology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Department of Orthopaedics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)

机构地区：[1]郑州大学第一附属医院耳科,450052 [2]郑州大学第一附属医院骨科,450052

出　　处：《中华实验外科杂志》2022年第2期316-320,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81902253)。

摘　　要：目的探讨受体相互作用蛋白激酶1(RIPK1)对骨髓间充质干细胞(BMSCs)死亡、增殖、分化、迁徙等生物功能的影响。方法通过RNA干扰技术降低RIPK1在BMSCs中的表达后, 光镜观察细胞形态变化, Transwell细胞迁移实验检测BMSCs迁移能力变化, 细胞计数试剂盒8(CCK-8)法检测BMSCs增殖变化, 茜素红染色检测BMSCs成骨分化能力变化, 油红O染色检测BMSCs成脂分化能力变化, 阿新蓝染染色检测BMSCs成软骨分化能力变化, 蛋白质印迹法(Western blot)分析BMSCs分化相关标志分子Runt相关基因2(Runx2)、过氧化物酶体增殖物激活受体γ(PPARγ)、性别决定区Y框蛋白9(Sox9)的表达变化以及凋亡标志性分子裂解的半胱氨酰天冬氨酸特异性蛋白酶-3(cleaved Caspase-3)、坏死性凋亡相关标志性分子p-RIPK3、RIPK3、p-MLKL的表达变化。两组间比较采用t检验, 多组间比较应用单因素方差分析。结果小干扰RNA(siRNA)干扰24 h后, 非选择性siRNA对照组吸光度值(0.46±0.00), siRIPK1组吸光度值(0.45±0.01), 差异无统计学意义(t=1.923, P>0.05);siRNA干扰48 h后, 非选择性siRNA对照组吸光度值(0.92±0.04), siRIPK1组吸光度值分别(0.63±0.03), 差异有统计学意义(t=10.910, P<0.05);siRNA干扰72 h后, 非选择性siRNA对照组吸光度值(1.52±0.01), siRIPK1组吸光度值(0.52±0.03);差异有统计学意义(t=48.960, P<0.05)。siRNA干扰RIPK1后, BMSCs内Runx2表达水平(0.41±0.08)明显低于对照组(1±0), 差异有统计学意义(t=6.628, P<0.05);BMSCs内PPARγ表达水平(0.36±0.11)明显低于对照组(1±0), 差异有统计学意义(t=6.810, P<0.05);BMSCs内Sox9表达水平(0.33±0.19)明显低于对照组(1±0), 差异有统计学意义(t=6.949, P<0.05)。茜素红染色结果显示, 与非选择性的siRNA对照组比较, RIPK1干扰显著抑制BMSCs成骨分化过程中矿化结节的形成。油红O细胞染色结果显示, 与非选择性的siRNA对照组比较, RIPK1干扰显著抑制BMSCs成脂分化过�Objective To explore the mechanism of how receptor-interacting protein kinase 1(RIPK1)regulates cell biology in bone marrow mesenchymal stem cells(BMSCs).Methods Through RNA interference to reduce the expression of RIPK1 in BMSCs,cell morphological changes were observed.Transwell was used to test the migration ability of BMSCs.The counting kit-8(CCK-8)method to test the proliferation of BMSCs.The alizarin red staining was done to test of osteogenetic differentiation ability of BMSCs.Oil red O staining was used to detect the change of lipid-forming differentiation ability of BMSCs.Assin blue staining was used to detect the change of chondrogenic differentiation ability of BMSCs.Western blotting was used to analyze the expression changes of runt related transcription factor 2(Runx2),peroxisome proliferator-activated receptor gamma(PPARγ),sex-determining region Y box protein 9(Sox9),apoptosis markers cleaved cysteinyl aspartate-specific protease-3(cleaved Caspase-3),necrotic apoptosis-related markers P-RIPk3,RIPK3,p-MLKL.T test was used for comparison between two groups,and one-way ANOVA was used for comparison between multiple groups.Results After 24 h small interfering RNA(siRNA)interference,the absorbance(A)value in the non-selective siRNA control group(0.46±0.00)and the siRIPK1 group(0.45±0.01)was not statistically significant(t=1.923,P>0.05).After 48 h siRNA interference,the A value in the non-selective siRNA control group was(0.92±0.04),and that of the siRIPK1 group was(0.63±0.03)with the difference being statistically significant(t=10.910,P<0.05).After 72 h of siRNA interference,the A value in the non-selective siRNA control group was(1.52±0.01),and that in the siRIPK1 group was(0.52±0.03)with the difference being statistically significant(t=48.960,P<0.05).After siRNA interference with RIPK1,the expression level of Runx2(0.41±0.08),PPARγ(0.36±0.11),and Sox9(0.33±0.19)in BMSCs was significantly lower than that in control group(1±0)(t=6.628,t=6.810,t=6.949,P<0.05).Alizarin red staining results showe

关 键 词：骨髓间充质干细胞 受体相互作用蛋白激酶1 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]