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目的 探讨内质网与线粒体偶联及线粒体融合蛋白(mitofusion 2,Mfn2)对牙周膜干细胞(periodontal ligament stem cells,PDLSC)成骨分化能力的影响,为促进牙周炎患者牙周组织再生提供理论基础和治疗靶点.方法 刮取正常及牙周炎牙齿(由第四军医大学口腔医学院口腔颌面外科门诊收集)的牙周膜组织,用原代和有限稀释法单克隆化培养健康来源的(health,H)PDLSC (H-PDLSC)及炎症来源的(periodontitis,P)PDLSC(P-PDLSC),透射电镜及细胞器特异性荧光染色检测内质网-线粒体偶联水平;实时荧光定量PCR(quantitative real-time PCR,qPCR)检测两种来源PDLSC中Mfn2的表达差异;用肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)模拟炎症微环境,分别应用正常培养基及含5、10 mg/L TNF-α的培养基培养H-PDLSC,设为H-PDLSC组、H-PDLSC+TNF-α (5 mg/L)组和H-PDLSC+TNF-α(10 mg/L)组,培养7 d后qPCR检测Mfn2表达水平;通过小干扰RNA Mfn2(siMfn2)下调P-PDLSC中Mfn2的表达,成骨诱导液培养7 d后qPCR检测成骨因子的表达差异,培养28 d后茜素红染色及氯化十六烷基吡啶定量检测各组矿化结节形成差异.结果 透射电镜下观察内质网与线粒体偶联结构,与H-PDLSC组(偶联长度/线粒体周长为0.23±0.07、偶联长度/内质网周长为0.18±0.05)相比,P-PDLSC组中内质网线粒体偶联水平显著增加(偶联长度/线粒体周长为0.55± 0.10、偶联长度/内质网周长为0.44±0.08)(P<0.01);特异性荧光染色检测内质网与线粒体共定位显示,P-PDLSC组内质网与线粒体共定位系数(0.71±0.09)显著高于H-PDLSC组(0.40±0.10)(P<0.01).P-PDLSC组Mfn2表达量(1.46±0.10)显著高于H-PDLSC组(0.99±0.08)(P<0.01);H-PDLSC+TNF-α (5 mg/L)组及H-PDLSC+TNF-α(10 mg/L)组Mfn2表达量均显著高于H-PDLSC组(P<0.01).成骨诱导7 d qPCR结果显示,P-PDLSC+siMfn2组碱性磷酸酶、Runt相关转录因子2及骨钙蛋白mRNA表达量均显著高于对照组(P<0.01),28 d茜素红染色及氯化十六烷基吡啶定量结果与qPCR结果一致.结论 炎症微环境下PDLSC的内质网-线粒体偶联增加,线粒体融合蛋白Mfn2表达量升高导致PDLSC成骨分化能力下降,其具体机制还需进一步研究.

Objective To investigate the effect of endoplasmic reticulum (ER)-mitochondria coupling and the expression of mitofusion 2 (Mfn2) in periodontal ligament stem cells (PDLSC), so as to provide a theoretical basis and therapeutic target for promoting periodontal regeneration in treatment of periodontitis. Methods The periodontal ligament tissue was scraped from extracted intact human teeth and teeth with periodontitis collected in the Department of Oral and Maxillofacial Surgery, the Fourth Military Medical University. The health PDLSC (H-PDLSC) and inflammatory PDLSC (P-PDLSC) were obtained respectively from the primary culture of the human teeth and cloned using 1imited diluted method. The level of ER-mitochondrial coupling was observed by transmission electron microscopy and organelle-specific fluorescence staining. Quantitative real-time PCR (qPCR) was used to detect the expression of Mfn2 in H-PDLSC and P-PDLSC. Tumor necrosis factor alpha (TNF-α) was used to simulate the inflammatory microenvironment. H-PDLSC was cultured in normal medium and media containing 5 and 10 mg/L TNF-α, named as H-PDLSC group, H-PDLSC+TNF-α (5 mg/L) group and H-PDLSC+TNF-α (10 mg/L) group, respectively. At the 7th day, qPCR was applied to detect the mRNA level of Mfn2. The expression of Mfn2 in P-PDLSC was down-regulated by small interfering RNA siMfn2. The osteogenic differentiation of P-PDLSC and P-PDLSC+siMfn2 were examined by qPCR at the 7th day, and alizarin red staining and cetyl pyridine chloride quantitative analysis at the 28th day after osteogenic induction. Results The level of ER-mitochondrial coupling in the P-PDLSC group (the length of the coupling structure/mitochondrial perimeter was 0.55±0.10, the length of the coupling structure/endoplasmic reticulum perimeter was 0.44± 0.08) was significantly higher than that in the H-PDLSC group (P<0.01). The co-localization of endoplasmic reticulum and mitochondria of P-PDLSC group was 0.71±0.09, which was significantly higher than that of H-PDLSC group (P<0.01). The expression level of Mfn2 in P-PDLSC (1.46 ± 0.10) was higher than that in H-PDLSC (0.99±0.08). The expression levels of Mfn2 in H-PDLSC+TNF-α (5 mg/L) and H-PDLSC+TNF-α (10 mg/L) groups were 1.28±0.19, 1.54±0.43, respectively, which were both significantly higher than that in H-PDLSC (0.82±0.14) (P<0.01). P-PDLSC transfected with siMfn2 down-regulated the expression of Mfn2, and the osteogenic differentiation ability of P-PDLSC was restored. The results showed that the expression of alkaline phosphatase, Runt-related transcription factor-2 (RUNX2) and osteocalcin mRNA in P-PDLSC+siMfn2 group were significantly higher than that of the control group (P<0.01). The alizarin red staining and quantitative results of cetyl pyridinium chloride were consistent with the qPCR results. Conclusions In the microenvironment of inflammation, ER-mitochondrial coupling and the expression of Mfn2 of PDLSC increased, which might lead to a decrease in osteogenic differentiation of PDLSC. The specific mechanism needs to be further studied.