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| 孕哺期维生素D补充对子代大鼠软骨内成骨过程及mTOR通路的影响 |
| The effect of vitamin D supplementation during pregnancy and lactation on the endochondral osteogenesis process and mTOR pathway in offspring rats |
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| DOI：10.3969/j.issn.1006-7108.2024.11.008 |
| 中文关键词: 维生素D 软骨内成骨 雷帕霉素靶蛋白 信号通路 自噬 |
| 英文关键词:Vitamin D intrachondral osteogenesis mechanistic target of rapamycin signaling pathway autophagy |
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| 中文摘要: |
| 目的 探究孕哺期维生素D（VD）补充对子代大鼠软骨内成骨过程及哺乳动物雷帕霉素靶蛋白（mTOR）通路的影响。方法 24只雌性Wistar大鼠与12只雄性大鼠合笼后，随机分为对照组（Control组）、VD低剂量组（VD-Low组）、VD高剂量组（VD-High组）和mTOR抑制剂雷帕霉素组（RAPA组），每组各6只。孕哺期[妊娠第0天（GD 0）到子鼠出生后第21天（PND 21）]给予相应的VD（10、20 ng/kg）和雷帕霉素（1 mg/kg）。子鼠喂养4周后，测量子鼠体重和体长；分离子鼠胫骨并进行micro-CT扫描和重建；全身及局部骨架染色观察子鼠骨骼发育；HE染色观察胫骨生长板软骨细胞形态；免疫组化染色检测胫骨组织中软骨细胞增殖相关蛋白[Ki67、增殖细胞核抗原（PCNA）]、分化相关蛋白[Ⅱ型胶原α1链（Col2A1）、性别决定区Y框转录因子9（SOX9）]、肥大相关蛋白[X型胶原α1链（Col10A1）、基质金属蛋白酶13（MMP-13）、骨桥蛋白（OPN）]、成骨相关蛋白[Ⅰ型胶原（Col Ⅰ）、骨钙素（OCN）、碱性磷酸酶（ALP）]水平；RT-qPCR检测胫骨组织中mTOR通路相关mRNA[mTOR、p70S6核糖体蛋白激酶（p70S6K）、elF4E-结合蛋白1（4E-BP1）]水平；Western blot检测胫骨组织中mTOR通路相关蛋白（mTOR、p-mTOR、p70S6K、p-p70S6K、4E-BP1、p-4E-BP1）及自噬相关蛋白[微管相关蛋白1轻链3（LC3）、Beclin1、p62]水平。结果 与Control组相比，VD-Low组、VD-High组、RAPA组子鼠体重、体长增加；胫骨组织骨小梁厚度（Tb.Th）增大；胫骨组织生长板软骨细胞排列整齐，生长板厚度、增殖区和肥大区厚度均增加；胫骨组织中Ki67、PCNA、Col2A1、SOX9、Col10A1、MMP-13、OPN、Col I、OCN、ALP、LC3 Ⅱ/Ⅰ、Beclin1蛋白水平升高，mTOR、p70S6K、4E-BP1 mRNA水平、p62、p-mTOR/mTOR、p-p70S6K/p70S6K、p-4E-BP1/4E-BP1蛋白水平降低（P均＜0.05）；且呈VD剂量依赖性（P均＜0.05）。结论 孕哺期VD补充能够促进子鼠软骨内成骨，其可能通过抑制mTOR信号通路，并激活自噬发挥作用。 |
| 英文摘要: |
| Objective To explore the effect of vitamin D (VD) supplementation during pregnancy and lactation on the endochondral osteogenesis process and mechanistic target of rapamycin (mTOR) pathway in offspring rats. Methods After twenty-four female Wistar rats and twelve male rats were housed together，female rats were randomly divided into Control group，ow-dose VD group (VD-Low group)，high-dose VD group (VD-High group)，and mTOR inhibitor rapamycin group (RAPA group)，with six rats in each group. VD (10，20 ng/kg) and rapamycin (1 mg/kg) were administered during the pregnancy and lactation period [from day 0 of pregnancy (GD 0) to day 21 after birth (PND 21)]. After feeding the offspring for 4 weeks，The weight and length of offspring mice were measured；The tibia of the offspring mice were separated and subjected to micro CT scanning and reconstruction；Whole body and local skeletal staining were used to observe the skeletal development of offspring mice；HE staining was used to observe the morphology of chondrocytes in tibial growth plate；Immunohistochemical staining was used to detect proliferation related protein [Ki67，proliferating cell nuclear antigen (PCNA)]，differentiation related protein [collagen II α1 chain (Col2A1)，SRY-box transcription factor 9 (SOX9)]，hypertrophy related protein [collagen X α1 chain (Col10A1)，matrix metalloproteinase 13 (MMP-13)，osteopontin (OPN)] and osteogenic related protein [collagen I (Col I)，osteocalcin (OCN)，alkaline phosphatase (ALP)] levels in chondrocyte of tibial tissue；RT-qPCR was used to detect mTOR，ribosomal protein S6 kinase (p70S6K) and eIF4E-binding protein (4E-BP1) mRNA levels in tibial tissue；Western blot was used to detect microtubule-associated protein 1 light chain 3 (LC3)，Beclin1，p62，mTOR，p-mTOR，p70S6K, p-p70S6K，4E-BP1 and p-4E-BP1 levels in tibial tissue. Results Compared with Control group，body weight and length of offspring mice in VD Low group，VD High group and RAPA group were increased，trabecular thickness (Tb.Th) in tibial tissue were increased，The arrangement of chondrocytes in tibial tissue growth plate was neat，the thickness of the growth plate, proliferative zone and hypertrophic zone were increased，Ki67，PCNA，Col2A1，SOX9，Col10A1，MMP-13，OPN，Col I，OCN，ALP，LC3 II/I，Beclin1 protein levels were increased，mTOR，p70S6K，4E-BP1 mRNA levels，p62，p-mTOR/mTOR，p-p70S6K/p70S6K and p-4E-BP1/4E-BP1 protein levels were decreased (all P＜0.05). ConclusionVD supplementation during pregnancy and lactation could promote endochondral osteogenesis in offspring mice，which might play a role by inhibiting mTOR signaling pathway and activating autophagy. |
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