人β-防御素3转染脂肪干细胞成骨分化潜能及抗牙周炎致病菌的作用

Osteogenic differentiation potential of adipose stem cells transfected with human β-defensin 3 and its effect on anti-periodontitis pathogens

  • 摘要:
    目的 探讨人β-防御素3(hβD-3)转染脂肪干细胞(ADSCs)成骨分化潜能及体外抗牙周炎致病菌的作用。
    方法 体外提取人腹部脂肪组织,分离后用流式细胞仪鉴定人脂肪干细胞(hADSCs)免疫表型(CD29/CD44), 体外培养hADSCs, 将细胞分为对照组、空载组与实验组(按hβD-3-hADSCs接种密度分为低密度组、中密度组和高密度组),对照组不作处理,空载组转染不含hβD-3基因的慢病毒载体,实验组利用质粒构建含hβD-3基因的慢病毒载体并转染hADSCs。空载组与实验组均加入成骨分化诱导液,对照组不作处理,在诱导7、14 d后,应用实时荧光定量聚合酶链反应检测3组细胞中成骨相关基因碱性磷酸酶(ALP)、Ⅰ-型胶原蛋白(Col-Ⅰ)、骨桥蛋白(OPN)、骨钙素(OCN)的表达量,以评价hADSCs的分化能力。测量抑菌圈的直径,设置抗生素阳性对照(米诺环素组、环丙沙星组),检测转染后的hADSCs对牙周致病菌葡萄球菌、卟啉单胞菌的抑菌活性。
    结果 诱导7、14 d时,空载组与实验组的ALPCol-Ⅰ、OPNOCN mRNA相对表达量均高于对照组,差异有统计学意义(P < 0.05); 空载组与实验组诱导14 d时的ALPCol-Ⅰ、OPNOCN mRNA相对表达量高于诱导7 d时,差异有统计学意义(P < 0.05)。葡萄球菌抑菌实验中,低密度组抑菌圈小于米诺环素组与环丙沙星组,中密度组抑菌圈大于环丙沙星组,高密度组抑菌圈大于米诺环素组与环丙沙星组,差异有统计学意义(P < 0.05)。卟啉单胞菌抑菌实验中,低密度组抑菌圈小于米诺环素组与环丙沙星组,中密度组抑菌圈大于米诺环素组,高密度组抑菌圈大于米诺环素组与环丙沙星组,差异有统计学意义(P < 0.05)。
    结论 hβD-3转染可促进hADSCs成骨分化,且对牙周致病菌葡萄球菌、卟啉单胞菌均有较好的抗菌活性。

     

    Abstract:
    Objective To investigate the osteogenic differentiation potential of human β-defensin 3 (hβD-3) transfected adipose stem cells (ADSCs) and its effect on anti-periodontitis pathogenic in vitro.
    Methods The human abdominal adipose tissue was extracted in vitro, and the immunophenotype (CD29/CD44) of human adipose stem cells (hADSCs) was identified by flow cytometry after separation. The hADSCs were cultured in vitro, and the cells were divided into control group, no-load group and experimental group. The control group was untreated. The empty group was transfected with a lentiviral vector containing no hβD-3 gene. The experimental group used a plasmid to construct a lentiviral vector containing the hβD-3 gene and was transfected hADSCs. Both the no-load group and the experimental group were added with osteogenic differentiation inducer, and the control group was not treated. The expressions of osteogenic related genesalkaline phosphatase (ALP), type Ⅰ collagen (Col-Ⅰ), osteopontin (OPN), osteocalcin (OCN) of the three groups were detected by real-time fluorescent quantitative polymerase chain reaction after 7 days and 14 days of induction to evaluate the differentiation ability of hADSCs. The diameter of antibacterial zone was measured, and antibiotic positive controls (minocycline group and ciprofloxacin group) were set, and the antibacterial activity of the transfected hADSCs against the periodontal pathogens such as Staphylococcus and Porphyromonas was detected.
    Results At 7 and 14 days of induction, the relative expressions of ALP, Col-Ⅰ, OPN and OCN mRNA in the no-load group and the experimental group were significantly higher than those in the control group (P < 0.05); the relative expressions of ALP, Col-Ⅰ, OPN and OCN mRNA in the no-load group and the experimental group at 14 days induction were significantly higher than those of 7 days after induction(P < 0.05). In the Staphylococcal antibacterial experiment, the inhibition zone of the low-density group was significantly smaller than that of the minocycline group and ciprofloxacin group, the zone of inhibition in the medium-density group was significantly greater than that in the ciprofloxacin group, the zone of inhibition in the high-density group was significantly greater than that of the minocycline group and ciprofloxacin group (P < 0.05). In the bacteriostasis experiment of Porphyromonas, the inhibition zone of the low-density group was significantly smaller than that of the minocycline group and the ciprofloxacin group, the inhibition zone of medium-density group group was significantly higher than that of minocycline group, and was significantly higher in the high-density group than that of the minocycline group and the ciprofloxacin group (P < 0.05).
    Conclusion hβD-3 transfection can promote the osteogenic differentiation of hADSCs, and has good antibacterial activity against periodontal pathogens such as Staphylococcus and Porphyromonas.

     

/

返回文章
返回