摘要
目的探讨防御素α4(DEFA4)在老年骨质疏松症中对成骨细胞衰老的作用机制。方法应用不同浓度过氧化氢刺激成骨前体细胞系MC3T3后通过细胞计数试剂盒(CCK-8)检测成骨细胞活性,选取活性抑制率约为50%的浓度用于诱导MC3T3细胞衰老。依据不同处理措施,将细胞分为对照组、衰老组和DEFA4组。正常组加入完全培养基,衰老组加入含1.0μmol/L H_(2)O_(2)的完全培养基,DEFA4组加入含有过表达DEFA4质粒慢病毒的完全培养基。CCK-8用于检测成骨细胞活性。实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质免疫印迹法(Western blot)用于检测经不同处理后MC3T3细胞中衰老相关基因p53、p16INK4a、p21的表达。采用β-半乳糖苷酶染色检测成骨细胞β-半乳糖苷酶阳性表达。两组之间采用t检验进行统计分析。结果加入不同浓度H_(2)O_(2)后,H_(2)O_(2)组比正常组成骨细胞活性明显下降,0.1μmol/L[(13.66±1.74)%比(0±2.56)%,t=8.072,P<0.05],0.5μmol/L[(31.42±1.67)%比(0±1.78)%,t=13.723,P<0.05],1.0μmol/L[(42.75±1.99)%比(0±1.64)%,t=31.663,P<0.05],5.0μmol/L[(69.44±2.67)%比(0±1.42)%,t=38.431,P<0.05],以上结果表示H_(2)O_(2)诱导成骨细胞衰老模型有效。DEFA4对成骨细胞具有抑制生长作用,DEFA4组成骨细胞活性低于正常组,差异有统计学意义[(41.44±1.22)%比(0±1.34)%,t=29.663,P<0.05],相较于1.0μmol/L H_(2)O_(2)诱导的衰老组,成骨细胞活性受抑制程度相当,差异无统计学意义[(41.44±1.22)%比(42.33±2.56)%,t=2.013,P>0.05]。DEFA4可以显著促进衰老相关基因表达:使用RT-qPCR检测衰老相关基因p53、p16INK4a、p21的表达,结果显示衰老组衰老相关基因表达高于对照组,差异有统计学意义[p53(2.00±0.06比1.00±0.02,t=27.392,P<0.05),p16INK4a(1.65±0.07比1.00±0.01,t=15.923,P<0.05),p21(2.58±0.14比1.00±0.01,t=19.502,P<0.05);而DEFA4组衰老相关基因表达与衰老组相当,差异无统计学意义[p53(2.17±0.10比2.00±0.06,t=2.522,P>0.05),p16INK4a(1.77±0.05比1.65±0.07,t=2.413,P>0.05),p21(2.81±0.11比2.58±0.14,t=2.232,P>0.05)。DEFA4对成骨细胞衰老相关蛋白表达的影响:衰老组衰老相关蛋白相对表达量显著高于正常组,差异有统计学意义[p53(5.12±0.33比1.00±0.33,t=15.922,P<0.05),p16INK4a(4.80±0.42比1.00±0.23,t=13.743,P<0.05),p21(3.48±0.08比1.00±0.26,t=15.792,P<0.05);衰老相关蛋白相对表达量DEFA4组与衰老组相当,差异无统计学意义[p53(4.78±0.05比5.12±0.33,t=1.762,P>0.05),p16INK4a(4.63±0.04比4.80±0.42,t=0.691,P>0.05),p21(3.64±0.12比3.48±0.08,t=1.923,P>0.05)。结论DEFA4通过提升衰老相关蛋白的表达来诱导成骨细胞衰老,导致细胞形态和功能发生衰老特征性的变化。
Objective To investigate the mechanism of action of defensinα4(DEFA4)on osteoblast senescence in senile osteoporosis.Methods After stimulating the osteogenic precursor cell line MC3T3 with different concentrations of hydrogen peroxide,the osteoblast activity was detected using a cell counting kit(CCK-8),and a concentration with an activity inhibition rate of about 50%was selected to induce aging of MC3T3 cells.According to different treatment measures,cells were divided into control group,aging group,and DEFA4 group.The normal group was added to complete culture medium,the aging group was added to complete culture medium containing 1μmol/L H_(2)O_(2),and the DEFA4 group was added to complete culture medium containing overexpressing DEFA4 plasmid lentivirus.CCK-8 assay was used to detect osteoblast activity.Real time quantitative reverse transcription polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the expression of aging related genes p53,p16INK4a,and p21 in MC3T3 cells after different treatments.β-galactosidase staining was used to detect the positive expression ofβ-galactosidase in osteoblasts.Statistical analysis was conducted between the two groups using t-test.Results Compared to the normal group,osteoblast viability significantly decreased after the addition of H_(2)O_(2)at concentrations of 0.1μmol/L[(13.66±1.74)%vs.(0±2.56)%,t=8.07,P<0.05],0.5μmol/L[(31.42±1.67)%vs.(0±1.78)%,t=13.72,P<0.05],1.0μmol/L[(42.75±1.99)%vs.(0±1.64)%,t=31.66,P<0.05],and 5.0μmol/L[(69.44±2.67)%vs.(0±1.42)%,t=38.43,P<0.05],indicating the effectiveness of the H_(2)O_(2)-induced osteoblast senescence model.DEFA4 exhibited an inhibitory effect on osteoblast growth.Compared to the normal group,osteoblast viability was significantly suppressed in the DEFA4 group[(41.44±1.22)%vs.(0±1.34)%,t=29.66,P<0.05].Compared to the 1.0μmol/L H_(2)O_(2)-induced senescence group,the degree of osteoblast viability inhibition was comparable in the DEFA4 group,with no statistically significant difference[(41.44±1.22)%vs.(42.33±2.56)%,t=2.01,P>0.05].DEFA4 significantly promoted the expression of senescence-associated genes(p53,p16INK4a,and p21).The results showed that the expression of these genes was significantly higher in the senescence group than in the control group[p53(2.00±0.06 vs.1.00±0.02,t=27.39,P<0.05),p16INK4a(1.65±0.07 vs.1.00±0.01,t=15.92,P<0.05),p21(2.58±0.14 vs.1.00±0.01,t=19.50,P<0.05)].However,the expression of senescence-associated genes in the DEFA4 group was comparable to that in the senescence group,with no statistically significant difference[p53(2.17±0.10 vs.2.00±0.06,t=2.52,P>0.05),p16INK4a(1.77±0.05 vs.1.65±0.07,t=2.41,P>0.05),p21(2.81±0.11 vs.2.58±0.14,t=2.23,P>0.05)].The relative expression levels of senescence-associated proteins were significantly higher in the senescence group than in the normal group[p53(5.12±0.33 vs.1.00±0.33,t=15.92,P<0.05),p16INK4a(4.80±0.42 vs.1.00±0.23,t=13.74,P<0.05),p21(3.48±0.08 vs.1.00±0.26,t=15.79,P<0.05)].The relative expression levels of senescence-associated proteins in the DEFA4 group were comparable to those in the senescence group,with no statistically significant difference[p53(4.78±0.05 vs.5.12±0.33,t=1.76,P>0.05),p16INK4a(4.63±0.04 vs.4.80±0.42,t=0.69,P>0.05),p21(3.64±0.12 vs.3.48±0.08,t=1.92,P>0.05)].Cells in the normal group showed lowβ-galactosidase expression levels and normal cell morphology.Cells in the senescence group had an increased proportion ofβ-galactosidase-positive cells,with enlarged nuclear volumes and typical senescence characteristics.Cells in the DEFA4 group showed little difference inβ-galactosidase expression compared to the senescence group,and senescence morphology was also present.Conclusion DEFA4 induces osteoblast senescence by enhancing the expression of senescence-associated proteins,leading to characteristic morphological and functional changes associated with cellular aging.This discovery provides significant insights into understanding the impact of DEFA4 on bone health and the development of osteoporosis.
作者
袁亮
屈彩龙
湛杰
冯征源
刘威
戴珏
Yuan Liang;Qu Cailong;Zhan Jie;Feng Zhengyuan;Liu Wei;Dai Yu(Second Orthopedic Department of Liuyang People’s Hospital,Liuyang 410300,China;Department of Breast nail Surgery,Liuyang People’s Hospital,Liuyang 410300,China)
出处
《中华实验外科杂志》
2025年第4期683-687,共5页
Chinese Journal of Experimental Surgery
基金
长沙市自然科学基金(kq2502353)。
