中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2013年
41期
7188-7198
,共11页
王昱翔%张宏其%郭超峰%唐明星%刘少华%邓盎%高琪乐%邓展生%陈静%刘金洋%吴建煌
王昱翔%張宏其%郭超峰%唐明星%劉少華%鄧盎%高琪樂%鄧展生%陳靜%劉金洋%吳建煌
왕욱상%장굉기%곽초봉%당명성%류소화%산앙%고기악%산전생%진정%류금양%오건황
组织构建%骨组织构建%雌激素受体β%RNA干扰%人成骨样细胞%反转录病毒载体%骨保护素%RANKL%国家自然科学基金
組織構建%骨組織構建%雌激素受體β%RNA榦擾%人成骨樣細胞%反轉錄病毒載體%骨保護素%RANKL%國傢自然科學基金
조직구건%골조직구건%자격소수체β%RNA간우%인성골양세포%반전록병독재체%골보호소%RANKL%국가자연과학기금
estrogen receptor beta%osteoprotegrin%receptor activator of nuclear factor kappa B%osteoblasts
背景：目前对雌激素β受体基因如何参与骨代谢的研究较少。目的：研究雌激素受体β对人成骨样细胞骨保护素、核因子kB受体活化因子配体(receptor activator of NF-κB ligand，RANKL)表达的影响。方法：将先前含有最有效干扰序列及非特异性shRNA的反转录病毒分别感染人成骨样MG63细胞株后，筛选稳定克隆并扩大培养，以空白及非特异性shRNA作为对照，检测稳定抑制雌激素受体β的效率。分别向3组细胞即MG63细胞、雌激素受体βshRNA反转录病毒感染的MG63细胞、阴性对照shRNAnc反转录病毒感染的MG63细胞加入17β-雌二醇(E2)干预，检测人成骨样细胞株MG63的骨保护素、RANKL mRNA和蛋白表达情况。结果与结论：用pRNAT-H1.4/Retro-雌激素受体β-shRNA3进一步稳转人成骨样MG63细胞株，与空白及阴性病毒对照组相比，筛选出雌激素受体β表达稳定抑制的人成骨样细胞株，雌激素受体βmRNA 抑制率为(88.17±1.17)%(P<0.05)，蛋白抑制率为(89.01±1.22)%(P<0.05)，证实实验成功建立了人成骨样细胞ERβ亚型基因敲低细胞模型。雌激素干预48 h后，显示雌激素受体β稳定抑制的MG63细胞较空白组及阴性对照组骨保护素 mRNA及蛋白表达上调(P<0.05)，RANKL mRNA和蛋白表达下调(P<0.05)，骨保护素RANKL表达上调(P<0.05)，提示雌激素受体β可能通过调节骨保护素/RANKL在骨代谢中发挥作用。
揹景：目前對雌激素β受體基因如何參與骨代謝的研究較少。目的：研究雌激素受體β對人成骨樣細胞骨保護素、覈因子kB受體活化因子配體(receptor activator of NF-κB ligand，RANKL)錶達的影響。方法：將先前含有最有效榦擾序列及非特異性shRNA的反轉錄病毒分彆感染人成骨樣MG63細胞株後，篩選穩定剋隆併擴大培養，以空白及非特異性shRNA作為對照，檢測穩定抑製雌激素受體β的效率。分彆嚮3組細胞即MG63細胞、雌激素受體βshRNA反轉錄病毒感染的MG63細胞、陰性對照shRNAnc反轉錄病毒感染的MG63細胞加入17β-雌二醇(E2)榦預，檢測人成骨樣細胞株MG63的骨保護素、RANKL mRNA和蛋白錶達情況。結果與結論：用pRNAT-H1.4/Retro-雌激素受體β-shRNA3進一步穩轉人成骨樣MG63細胞株，與空白及陰性病毒對照組相比，篩選齣雌激素受體β錶達穩定抑製的人成骨樣細胞株，雌激素受體βmRNA 抑製率為(88.17±1.17)%(P<0.05)，蛋白抑製率為(89.01±1.22)%(P<0.05)，證實實驗成功建立瞭人成骨樣細胞ERβ亞型基因敲低細胞模型。雌激素榦預48 h後，顯示雌激素受體β穩定抑製的MG63細胞較空白組及陰性對照組骨保護素 mRNA及蛋白錶達上調(P<0.05)，RANKL mRNA和蛋白錶達下調(P<0.05)，骨保護素RANKL錶達上調(P<0.05)，提示雌激素受體β可能通過調節骨保護素/RANKL在骨代謝中髮揮作用。
배경：목전대자격소β수체기인여하삼여골대사적연구교소。목적：연구자격소수체β대인성골양세포골보호소、핵인자kB수체활화인자배체(receptor activator of NF-κB ligand，RANKL)표체적영향。방법：장선전함유최유효간우서렬급비특이성shRNA적반전록병독분별감염인성골양MG63세포주후，사선은정극륭병확대배양，이공백급비특이성shRNA작위대조，검측은정억제자격소수체β적효솔。분별향3조세포즉MG63세포、자격소수체βshRNA반전록병독감염적MG63세포、음성대조shRNAnc반전록병독감염적MG63세포가입17β-자이순(E2)간예，검측인성골양세포주MG63적골보호소、RANKL mRNA화단백표체정황。결과여결론：용pRNAT-H1.4/Retro-자격소수체β-shRNA3진일보은전인성골양MG63세포주，여공백급음성병독대조조상비，사선출자격소수체β표체은정억제적인성골양세포주，자격소수체βmRNA 억제솔위(88.17±1.17)%(P<0.05)，단백억제솔위(89.01±1.22)%(P<0.05)，증실실험성공건립료인성골양세포ERβ아형기인고저세포모형。자격소간예48 h후，현시자격소수체β은정억제적MG63세포교공백조급음성대조조골보호소 mRNA급단백표체상조(P<0.05)，RANKL mRNA화단백표체하조(P<0.05)，골보호소RANKL표체상조(P<0.05)，제시자격소수체β가능통과조절골보호소/RANKL재골대사중발휘작용。
BACKGROUND:Studies concerning how estrogen receptorβparticipates in bone metabolism are few now. OBJECTIVE:To investigate the effect of estrogen receptorβon the expression of osteoprotegerin and receptor activator of nuclear factor-κB ligand in human osteblast-like cells. METHODS:The retrovirus with the most effective interference sequence and non-specific short hairpin RNA was used to transfect human osteoblast-like cellMG63 in order to screen out the stable colon, and then amplified and cultured. The blank control and non-specific short hairpin RNA were used as control, and the stable inhibition rate of estrogen receptorβwas detected. The 17β-estradiol was added into the cells in three groups, that were MG63 cells, short hairpin RNA retrovirus estrogen receptorβ-mediated MG63 cells and negative control short hairpin RNA retrovirus-medicated MG63 cells, in order to detect the expressions of osteoprotegerin and receptor activator of nuclear factor-κB ligand mRNA in human osteoblast-like cells. RESULTS AND CONCLUSION: The human osteoblast-like MG63 cellline was further stably transfected with pRNAT-H1.4/Retro-estrogen receptorβshort hairpin RNA3, and then compared with the blank control and negative control, and found that estrogen receptorβcould express the stable inhibited human osteoblast-like cellline. The inhibition rate of estrogen receptorβmRNA was (88.17±1.17)%(P<0.05), and the inhibition rate of estrogen receptorβprotein was (89.01±1.22)%(P<0.05), indicating that estrogen receptorβgene knockdown human osteoblast-like cellmodels were constructed successful y. After estrogen intervention for 48 hours, the inhibition of MG63 cells with estrogen receptorβcould up-regulate the osteoprotegerin mRNA and protein expression in the blank control group and the negative control group (P<0.05), down-regulate the receptor activator of nuclear factor-κB ligand mRNA and protein expression (P<0.05), and up-regulate the osteoprotegerin receptor activator of nuclear factor-κB ligand expression. The results indicate that estrogen receptorβmay play an important role in bone metabolism through regulating osteoprotegerin/receptor activator of nuclear factor-κB ligand ratio.
