摘要
目的探究多发性骨髓瘤外泌体调控破骨细胞分化相关基因表达的功能及机制。方法为了探究多发性骨髓瘤细胞对破骨细胞分化的影响,实验分为control组(THP-1细胞)、OPM2组(THP-1+OPM2细胞)、U266组(THP-1+U266细胞)、OPM2+GW4869组(THP-1+OPM2细胞+GW4869)和U266+GW4869组(THP-1+U266细胞+GW4869)。提取OPM2和U266细胞分泌的外泌体(OPM2 exo和U266 exo),透射电镜观察外泌体结构,Western blot鉴定外泌体标志蛋白TSG101、Hsp70、CD9。为了探究外泌体对破骨细胞分化的影响,实验分为OPM2 exo组(THP-1细胞+OPM2 exo)、U266 exo组(THP-1细胞+U266 exo)和PBS组(THP-1细胞+PBS)。qRT-PCR检测所有组细胞中活化T细胞核因子1(nuclear factor of activated T-cells 1,NFATc1)、细胞原癌基因c-Fos、组织蛋白酶K(Cathepsin K,CTSK)mRNA相对表达量,Western blot检测所有组THP-1细胞NFATc1、c-Fos、CTSK、微管相关蛋白轻链3Ⅱ(microtubule-associated protein light chain 3Ⅱ,LC3Ⅱ)、动力蛋白激活蛋白4(dynactin 4,P62)蛋白表达量。结果OPM2 exo和U266 exo粒径在100 nm左右,“杯托”样结构,并且表达标志蛋白TSG101、Hsp70、CD9。相比于control组,OPM2组和U266组THP-1细胞NFATc1、c-Fos、CTSK mRNA和蛋白表达显著上调(P<0.001),LC3Ⅱ蛋白表达显著上调(P<0.001),P62蛋白表达显著下调(P<0.001)。OPM2+GW4869组细胞NFATc1、c-Fos、CTSK mRNA和蛋白表达显著低于OPM2组(P<0.001),U266+GW4869组细胞NFATc1、c-Fos、CTSK mRNA和蛋白表达显著低于U266组(P<0.001)。相比于PBS组,OPM2 exo组和U266 exo组NFATc1、c-Fos、CTSK mRNA和蛋白表达显著上调(均P<0.001),LC3Ⅱ蛋白表达显著上调(P<0.001),P62蛋白表达显著下调(P<0.001)。结论多发性骨髓瘤细胞外泌体可能通过促进细胞自噬诱导破骨细胞分化。
Objective To investigate the role and mechanism of multiple myeloma exosomes in regulating the expression of genes related to osteoclast differentiation.Methods To investigate the effect of multiple myeloma cells on osteoclast differentiation,the experiments were divided into control group(THP-1 cells),OPM2 group(THP-1+OPM2),U266 group(THP-1+U266),OPM2+GW4869 group(THP-1+OPM2+GW4869)and U266+GW4869 group(THP-1+U266+GW486).Exosomes secreted by OPM2 and U266 cells(OPM2 exo and U266 exo)were extracted,and then the exosomal structure was observed under transmission electron microscopy,and the exosomal marker proteins TSG101,Hsp70,and CD9 were identified by Western blot.To investigate the effect of exosomes on osteoclast differentiation,the experiments were divided into OPM2 exo group(THP-1 cell+OPM2 exo),U266 exo group(THP-1 cell+U266 exo)and PBS group(THP-1 cell+PBS).RT-qPCR was used to detect the relative expression levels of NFATc1,oncogene c-Fos,and CTSK mRNA in all groups.Western blot was used to detect the relative expression levels of NFATc1,c-Fos,CTSK,LC3Ⅱand P62 in all groups.Results The particle sizes of OPM2 exo and U266 exo were around 100 nm,with a“cupholder”like structure,and OPM2 exo and U266 exo expressed marker proteins TSG101,Hsp70,and CD9.Compared with control group,the mRNA and protein expressions of NFATc1,c-Fos and CTSK,and the protein expression of LC3Ⅱwere significantly upregulated in OPM2 group and U266 group(P<0.001),while the expression of P62 protein was significantly downregulated(P<0.001).The mRNA and protein expressions of NFATc1,c-Fos,CTSK in OPM2+GW4869 group were significantly lower than those in OPM2 group(P<0.001),and the mRNA and protein expressions of NFATc1,c-Fos and CTSK in U266+GW4869 group were significantly lower than those in U266 group(P<0.001).Compared with PBS group,the mRNA and protein expressions of NFATc1,c-Fos,CTSK were significantly upregulated in OPM2 exo group and U266 exo group(P<0.001),LC3Ⅱprotein expression was significantly upregulated(P<0.001),and P62 protein expression was significantly downregulated(P<0.001).Conclusion Multiple myeloma cell exosomes can induce osteoclast differentiation by promoting the cellular autophagy.
作者
吴洋
吴镜
罗晓慧
张哲梅
侯金霞
WU Yang;WU Jing;LUO Xiaohui;ZHANG Zhemei;HOU Jinxia(Clinical Laboratory Center,Gansu Provincial Hospital,Lanzhou 730000,China)
出处
《山西医科大学学报》
CAS
2023年第11期1470-1476,共7页
Journal of Shanxi Medical University
基金
兰州市科技计划项目(2020-ZD-25)。
关键词
多发性骨髓瘤
外泌体
破骨细胞分化
破骨细胞分化相关基因
自噬
multiple myeloma
exosomes
osteoclast differentiation
osteoclast differentiation-related gene
autophagy
