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  • 黄美能,李博,蔚一博,韩煦,杨欣谕,全知怎,方奥,蓝雨吟.降钙素促进人牙周膜干细胞的胶原合成和成骨作用[J].第二军医大学学报,2019,40(9):954-962    [点击复制]
  • HUANG Mei-neng,LI Bo,WEI Yi-bo,HAN Xu,YANG Xin-yu,QUAN Zhi-zen,FANG Ao,LAN Yu-yin.Calcitonin promotes collagen synthesis and osteogenesis in human periodontal ligament stem cells[J].Acad J Sec Mil Med Univ,2019,40(9):954-962   [点击复制]
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降钙素促进人牙周膜干细胞的胶原合成和成骨作用
黄美能1△,李博2△,蔚一博1*,韩煦1,杨欣谕1,全知怎1,方奥1,蓝雨吟1
0
(1. 海军军医大学(第二军医大学)长海医院口腔科, 上海 200433;
2. 海军军医大学(第二军医大学)长海医院骨科, 上海 200433
共同第一作者
*通信作者)
摘要:
目的 考察降钙素(CT)促进人牙周膜干细胞(hPDLSC)的胶原合成和成骨作用。方法 将50名成人受试者分为慢性牙周炎(CP)组(n=25)和对照组(n=25)。CP组中,选择探诊深度≥5 mm的上颌前部和有骨丢失影像学证据的部位,从每例患者的6个上颌位点收集龈沟液(GCF)样本。对照组中,对没有炎症的多个部位(每名受试者10~12个)进行取样以确保收集足够量的GCF。采用酶联免疫吸附试验(ELISA)检测GCF中CT、转化生长因子β1(TGF-β1)和骨形态发生蛋白(BMP)2/4/7的表达,通过Spearman相关分析考察CT表达与临床参数牙周袋探诊深度(PD)、临床附着丧失(CAL)和牙龈指数(GI)及上述指标的相关性。用携带CT基因的重组腺病毒(Ad.CT)感染hPDLSC,并通过实时定量PCR和蛋白质印迹法分别检测TGF-β1、BMP2/4/7、碱性磷酸酶(ALP)、骨钙蛋白(OCN)和Ⅰ/Ⅲ型胶原蛋白(ColⅠ/Ⅲ)的mRNA和蛋白质表达。结果 CP组患者GCF中CT表达水平高于对照组[(32.62±1.46)ng/mL vs(17.70±0.76)ng/mL,P<0.01)]。CP组患者CT表达与临床参数PD、CAL、GI呈负相关(P<0.01、P<0.05)。CP组患者GCF中BMP2/4/7和TGF-β1的表达水平均高于对照组[BMP2:(138.67±4.04)ng/mL vs(103.96±2.78)ng/mL;BMP4:(155.53±3.55)ng/mL vs(133.15±2.92)ng/mL;BMP7:(106.59±2.85)ng/mL vs(90.22±1.56)ng/mL;TGF-β1:(105.92±3.40)ng/mL vs(89.85±2.42)ng/mL;P均<0.01],且CP患者上述指标均与CT表达呈正相关(P<0.01、P<0.05)。腺病毒感染的CT过表达使hPDLSC中TGF-β1、ColⅠ/Ⅲ及成骨细胞标志物BMP2/4、ALP和OCN表达增加(P均<0.01)。与Ad.CT和空载腺病毒共同感染的细胞相比,用Ad.CT和特异性阻断TGF-β1小干扰RNA(siRNA)的重组腺病毒(Ad.TGF-β1 siRNA)共同感染的细胞胶原蛋白表达水平更低(ColⅠ:0.16±0.02 vs 0.22±0.03;ColⅢ:0.11±0.01 vs 0.15±0.02;P均<0.01)。与Ad.CT感染的细胞相比,Ad.CT和头蛋白共处理细胞中ALP和OCN的蛋白质表达水平更低(ALP:0.19±0.02 vs 0.25±0.03;OCN:0.13±0.01 vs 0.19±0.02;P均<0.01)。结论 CT通过TGF-β1和BMP信号转导途径促进hPDLSC的胶原合成和成骨作用。
关键词:  降钙素  人牙周膜干细胞  胶原  骨生成
DOI:10.16781/j.0258-879x.2019.09.0954
投稿时间:2019-05-18修订日期:2019-06-24
基金项目:国家自然科学基金青年科学基金(8180041799),上海市科研计划项目(18ZR1438300).
Calcitonin promotes collagen synthesis and osteogenesis in human periodontal ligament stem cells
HUANG Mei-neng1△,LI Bo2△,WEI Yi-bo1*,HAN Xu1,YANG Xin-yu1,QUAN Zhi-zen1,FANG Ao1,LAN Yu-yin1
(1. Department of Stomatology, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. Department of Orthopaedics, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China
Co-first authors.
* Corresponding author)
Abstract:
Objective To investigate the effect of calcitonin (CT) on promoting collagen synthesis and osteogenesis of human periodontal ligament stem cells (hPDLSCs). Methods Fifty adult participants were divided into chronic periodontitis (CP) group (n=25) and control group (n=25). In the CP group, the anterior maxilla with probing depth ≥ 5 mm and the sites with imaging evidence of bone loss were selected. The gingival crevicular fluid (GCF) samples were collected from 6 maxillary sites in each patient. In the control group, multiple sites without inflammation (10 to 12 per subject) were sampled to ensure that a sufficient amount of GCF was collected. The expression of CT, transforming growth factor β1 (TGF-β1) and bone morphogenetic protein (BMP) 2/4/7 in GCF was detected by enzyme linked immunosorbent assay (ELISA), and the correlation between CT expression and clinical parameters such as periodontal pocket probing depth (PD), clinical attachment loss (CAL) and gingival index (GI), and the above-mentioned indicators was investigated with Spearman correlation analysis. hPDLSCs were infected with the adenoviruses carrying CT gene (Ad.CT) and the expression of mRNA and protein of TGF-β1, BMP2/4/7, alkaline phosphatase (ALP), osteocalcin (OCN) and collagen type Ⅰ/Ⅲ (ColⅠ/Ⅲ) were detected by quantitative real-time PCR and Western blotting. Results The expression level of CT in GCF of the CP group was significantly higher than that of the control group ([32.62±1.46] ng/mL vs[17.70±0.76] ng/mL, P<0.01). The expression of CT was positively correlated with clinical parameters such as PD, CAL and GI (P<0.01, P<0.05). The expression levels of BMP2/4/7 and TGF-β1 in GCF of the CP group were significantly higher than those of the control group (BMP2:[138.67±4.04] ng/mL vs[103.96±2.78] ng/mL, BMP4:[155.53±3.55] ng/mL vs[133.15±2.92] ng/mL; BMP7:[106.59±2.85] ng/mL vs[90.22±1.56] ng/mL; TGF-β1:[105.92±3.40] ng/mL vs[89.85±2.42] ng/mL; all P<0.01). The expression of BMP2/4/7 and TGF-β1 was negatively correlated with CT expression (P<0.01, P<0.05). The overexpression of CT significantly increased the expression of TGF-β1, ColⅠ/Ⅲ and osteoblast markers BMP2/4, ALP and OCN in GCF (all P<0.01). Compared with the cells co-infected with Ad.CT and Ad.Null, the cells co-infected with Ad.CT and small interfering RNA specifically blocking TGF-β1 (Ad.TGF-β1 siRNA) had significantly lower collagen expression (ColⅠ:0.16±0.02 vs 0.22±0.03; ColⅢ:0.11±0.01 vs 0.15±0.02; both P<0.01). Compared with Ad.CT infected cells, the protein expression levels of ALP and OCN were significantly decreased in Ad.CT and noggin co-treated cells (ALP:0.19±0.02 vs 0.25±0.03; OCN:0.13±0.01 vs 0.19±0.02; both P<0.01). Conclusion CT can promote collagen synthesis and osteogenesis in hPDLSCs through TGF-β1 and BMP signaling transduction pathways.
Key words:  calcitonin  human periodontal ligament stem cells  collagen  osteogenesis