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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、9d时检测碱性磷酸酶活性,21d时通过茜素红染色检查钙化情况;7、10、14d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/cm~2、辐照时间为90s、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。
Objective To study the conditions of human dermal fibroblasts (HPDLFs) transfected with bone morphogenetic protein 2 (BMP2) by SonoVue and their effect on the osteogenesis of HPDLFs. Methods Primary cultured HPDLFs were transfected into cells at different Sonovic concentrations, intensity of ultrasound and irradiation time, and the optimal transfection parameters were screened. The cells were randomly divided into 4 groups: Sonovitz + plasmid + ultrasound group, liposome + plasmid group, ultrasound group and blank control group, according to the screening conditions were transfected, respectively, after transfection 3, 5, The alkaline phosphatase activity was detected on the 9th day and the calcification on the 21st day by alizarin red staining. The osteocalcin (OPN), bone sialoprotein (BSP), collagen type Ⅰ ColⅠ, OCN and Runx2 mRNA. Results The ultrasound transfection efficiency and survival rate were both 0.8 W / cm ~ 2 and 90 s when the concentration of Sonoglu was 20%. The parameters were the transfection parameters. The alkaline phosphatase activity and calcified nodule formation in sonic + plasmid + ultrasound group at each time point were higher than those in blank control group and ultrasound group (P <0.05). The results of RT-PCR showed that at each time point, + MRNA expression of OPN, BSP, ColⅠ, OCN and Runx2 in plasmid + ultrasound group were higher than that in ultrasound group and blank control group (P <0.05), but there was no significant difference compared with liposome + plasmid group. CONCLUSION BMP2 can be transfected into HPDLFs successfully through sonic nociception and induce its osteogenic differentiation, which lays a foundation for the application of sodovigeny in periodontal tissue engineering.