| 摘要: |
| 目的 研究通过声诺维（SonoVue）转染骨形态发生蛋白2（BMP2）进入人牙周膜成纤维细胞（HPDLFs）的条件及其对HPDLFs成骨向诱导的作用。方法 原代培养HPDLFs，按不同声诺维浓度、超声强度和辐照时间转染细胞，筛选获得最佳转染参数。将细胞随机分为4组：声诺维+质粒+超声组、脂质体+质粒组、超声组和空白对照组，按所筛选条件进行转染，分别于转染后3、5、7、9 d时检测碱性磷酸酶活性，21 d时通过茜素红染色检查钙化情况；7、10、14 d时行RT-PCR对骨桥素（OPN）、骨涎蛋白（BSP）、Ⅰ型胶原蛋白（ColⅠ）、骨钙素（OCN）、成骨特异性转录因子（Runx2）的mRNA进行半定量检测。结果 超声功率为0.8 W/cm2、辐照时间为90 s、声诺维浓度为20%时细胞转染率与存活率均较佳，以该参数条件为转染参数。各时间点声诺维+质粒+超声组细胞的碱性磷酸酶活性及钙化结节形成量均高于空白对照组和超声组（P<0.05）；RT-PCR结果显示各时间点声诺维+质粒+超声组细胞OPN、BSP、ColⅠ、OCN、Runx2的mRNA表达量均高于超声组与空白对照组（P<0.05），但与脂质体+质粒组相比差异无统计学意义。结论 通过声诺维能成功将BMP2转染进入HPDLFs并诱导其成骨向分化，为声诺维在牙周组织工程的应用奠定了基础。 |
| 关键词: 声诺维 骨形态发生蛋白2 牙周膜 成纤维细胞 骨生成 |
| DOI：10.16781/j.0258-879x.2016.09.1108 |
| 投稿时间：2016-02-14修订日期：2016-05-09 |
| 基金项目:重庆市卫生局医学科研面上项目（20122138），重庆高校创新团队建设计划资助项目，重庆市高校市级口腔生物医学工程重点实验室资助项目. |
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| Bone morphogenetic protein 2 transfection by SonoVue promotes osteogenesis differentiation of human periodontal ligament fibroblasts |
| WANG Xue-man,LUO Shu-mei*,ZHONG Xiao-bo,HE Bin,TANG Yu-ying |
(Department of Conservation Dentistry and Endodontics, the Affiliated Stomatology Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 401147, China
*Corresponding author) |
| Abstract: |
| Objective To explore the conditions of transfecting bone morphogenetic protein 2 (BMP2) gene into human periodontal ligament fibroblasts (HPDLFs) by SonoVue and its effects on osteogenesis differentiation of HPDLFs. Methods The HPDLFs were primary cultured and transfected under different conditions (concentrations of SonoVue, different ultrasound exposure intensity and time) to screening for the optimum parameters. Cells were randomly divided into four groups:SonoVue+plasmid+ultrasound group, liposome+plasmid group, ultrasound group, and blank control group. After transfection with the corresponding strategies, the alkaline phosphatase activity was detected on day 3, 5, 7 and 9 d, and alizarin red staining was applied to check the calcium nodus on day 21 d, respectively. The mRNA expression of osteopontin (OPN), bone sialoprotein (BSP), typeⅠcollagen (ColⅠ), osteocalcin (OCN), and runt-related transcription factor-2 (Runx2) was detected by RT-PCR on day 7, 10 and 14 d of transfection. Results The transfection rate and the cell survival rate were relatively high under the following condition:0.8 W/cm2, 90 s and 20% SonoVue solution, and they were selected as the final transfection parameters. Both alkaline phosphatase activity and matrix mineralization content in SonoVue+plasmid+ultrasound group were significantly higher than those in blank control group and ultrasound group (P<0.05) at all time points. RT-PCR results revealed that the expression of OPN, BSP, ColⅠ, OCN and Runx2 mRNA in SonoVue+plasmid+ultrasound group was significantly higher than that in the ultrasound group and blank control group (P<0.05), but was similar to that in the liposome+plasmid group (P>0.05). Conclusion The BMP2 gene can be successfully transfected into HPDLFs by SonoVue and can induce osteogenesis differentiation of HPDLFs, which lays a foundation for its application in periodontal tissue engineering. |
| Key words: SonoVue bone morphogenetic protein 2 periodontal ligament fibroblasts osteogenesis |
