摘要
目的探讨脊椎蛋白2(R-spondin 2,Rspo2)对BMSCs成骨分化及卵巢去势(ovariectomy,OVX)小鼠骨密度和骨矿含量的影响。方法取7只4周龄雌性C57BL/6小鼠长骨骨髓,采用全骨髓培养法分离培养BMSCs并传代,经流式细胞仪鉴定细胞表型后,取第3代细胞与10、20、40、80和100 nmol/L Rspo2共培养,采用细胞计数试剂盒8(cell counting kit 8,CCK-8)检测细胞增殖,评价Rspo2对BMSCs活性影响并筛选不影响细胞活性的Rspo2干预浓度;与不同筛选浓度Rspo2培养后更换为成骨诱导培养基诱导培养,行ALP及实时荧光定量PCR(real time fluorescence quantitative PCR,RT-qPCR)检测RUNX家族转录因子2(RUNX family transcription factor 2,Runx2)、Ⅰ型胶原(collagen typeⅠalpha 1,Col1)、骨钙素(osteocalcin,OCN)基因表达,评价Rspo2对BMSCs成骨能力的影响;以上检测均以正常培养BMSCs作为对照。另取18只10周龄雌性C57BL/6小鼠,随机分为假手术组、OVX组和OVX+Rspo2组,每组6只。假手术组仅行双侧背部切开缝合,其余两组通过双侧OVX建立骨质疏松模型;术后OVX+Rspo2组每周腹腔注射Rspo2(1 mg/kg),其余两组接受相同剂量生理盐水。干预12周后,3组称重小鼠体质量及子宫质量,评估OVX模型制备是否成功;取胫骨行HE染色观察骨量变化、免疫组织化学染色观察骨组织中Runx2蛋白表达水平;ELISA法检测血清中骨代谢标记物[ALP、OCN、Ⅰ型前胶原氨基端肽(typeⅠprocollagen amino-terminal peptide,PINP)]和骨吸收标记物[β胶原降解产物(β-isomerized C-telopeptide,β-CTX)]表达水平;Micro-CT检测胫骨微结构变化,并对骨小梁厚度(trabeculae thickness,Tb.Th)、骨小梁数量(trabeculae number,Tb.N)、骨小梁分离度(trabeculae separation,Tb.Sp)和骨体积分数(bone volume fraction,BV/TV)进行三维组织形态计量分析。结果CCK-8检测示80 nmol/L以下浓度的Rspo2不会产生细胞毒性(P>0.05),且20 nmol/L Rspo2组细胞活力明显高于对照组(P<0.05)。基于上述结果,�
Objective To investigate the effects of R-spondin 2(Rspo2)on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and bone mineral content in ovariectomized mice.Methods BMSCs were extracted from the bone marrow of the long bones of 74-week-old female C57BL/6 mice using whole bone marrow culture and passaged.After the cell phenotype was identified by flow cytometry,the 3rd generation cells were co-cultured with 10,20,40,80,and 100 nmol/L Rspo2.Then,the cell activity and proliferative capacity were determined by cell counting kit 8(CCK-8),and the intervention concentration of Rspo2 was screened for the subsequent experiments.The osteogenic differentiation ability of BMSCs was detected by alkaline phosphatase(ALP)staining,and the mRNA levels of osteogenesis-related genes[RUNX family transcription factor 2(Runx2),collagen typeⅠalpha 1(Col1),osteocalcin(OCN)]were detected by real-time fluorescence quantitative PCR(RT-qPCR).In addition,1810-week-old female C57BL/6 mice were randomly divided into sham operation group(sham group),ovariectomy group(OVX group),and OVX+Rspo2-intervention group(OVX+Rspo2 group),with 6 mice in each group.The sham group only underwent bilateral back incision and suturing,while the other two groups established osteoporosis mouse models by bilateral ovarian castration.Then,the mice were given a weekly intraperitoneal Rspo2(1 mg/kg)treatment in OVX+Rspo2 group and saline at the same dosage in sham group and OVX group.After 12 weeks of treatment,the body mass and uterus mass of the mice were weighed in the 3 groups to assess whether the OVX model was successfully prepared;the tibia bones were stained with HE and immunohistochemistry staining to observe the changes in tibial bone mass and the expression level of Runx2 protein in the bone tissues.Blood was collected to detect the expressions of bone metabolism markers[ALP,OCN,typeⅠprocollagen amino-terminal peptide(PINP)]and bone resorption marker[β-collagen degradation product(β-CTX)]by ELISA assay.Micro-CT was used to
作者
柳鑫
石博文
蔡成阔
王昊天
贾鹏
LIU Xin;SHI Bowen;CAI Chengkuo;WANG Haotian;JIA Peng(Department of Orthopedics,Tianjin Hospital,Tianjin,300211,P.R.China)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2024年第11期1399-1407,共9页
Chinese Journal of Reparative and Reconstructive Surgery
基金
天津市卫生健康科技项目(TJWJ2024QN058)
天津市天津医院科技基金(TJYYQ2401)。
关键词
脊椎蛋白2
BMSCS
骨质疏松
骨形成
小鼠
R-spondin 2
bone marrow mesenchymal stem cells
osteoporosis
bone formation
mouse
