碱性成纤维细胞生长因子基因转染对犬牙龈成纤维细胞的影响

Effects of basic fibroblast growth factor transfection on canine gingival fibroblasts

背景：研究发现，局部应用外源性碱性成纤维细胞生长因子可明显促进体外培养牙龈成纤维绌胞的增殖，口内局部应用可加速牙龈组织伤口的愈合。目的：观察碱性成纤维细胞生长因子基因转染对Beagle犬牙龈成纤维细胞生物学特性的影响。设计、时间、地点：观察对比体外细胞学实验，于2008-04／09在福建医科大学细胞与发育工程中心及解放军南京军区福州总医院比较医学科完成。材料：Beagle犬4只，12月龄，体质量10-13kg，雄性；含有人全长碱性成纤维细胞生长因子cDNA的plRES2．EGFP．bFGF质粒为自行构建。方法：切取Beagle犬左上颌第2，3，4前磨牙的游离龈，无菌磷酸盐缓冲液冲洗4遍，将组织块剪碎后用2．5g／L的胰酶37℃消化2h，离心后弃上清，加入含体积分数为10％胎牛血清的DMEM，接种于6孔板并覆盖玻片，置丁37℃、体积分数为5％C02的培养箱培养，取对数生长期细胞消化传代。利用脂质体介导法将plRES2．EGFP．bFGF质粒转染Beagle犬牙龈成纤维细胞，以空质粒转染组及未转染组作为对照。主要观察指标：MTT法检测牙龈成纤维细胞增殖状况；AO／EB双染色检测牙龈成纤维细胞凋亡：化学比色法检测牙龈成纤维细胞碱性磷酸酶活性。结果：3组细胞均在转染后第3天开始进入对数生长期。MTT检测结果显示，转染后随着时间改变，与空质粒转染组及未转染组相比，实验组牙龈成纤维细胞的增殖能力明显增强（P〈0．05），而空质粒转染组及未转染组差异无显著性意义（P〉0．05）。AO／EB双染色结果显示，实验组牙龈成纤维细胞凋亡率低于空质粒转染组及未转染组（P〈0．05）。转染碱性成纤维细胞生长因子基因后，牙龈成纤维细胞碱性磷酸酶活性未见明显改变，与未转染细胞差异无显著性意义。结论：碱性成纤维细胞生长因子基因转染可以加速牙龈成纤维细胞的增殖，抑制其凋亡，无促使牙龈成纤维细胞骨向分化的作用。

BACKGROUND： Studies have demonstrated that exogenous basic fibroblast growth factor （bFGF） has intensive effects to promote proliferation of gingival fibroblasts （GFs） cultured in vitro and the healing of gingival wounds. OBJECTIVE： To investigate the effects of bFGF gene transfection on the biological performance of Beagle canine GFs. DESIGN, TIME AND SETTING： An observation and comparison in vitro experiment regarding cells was accomplished in Centre of Cell Biology and Development of Fujian Medical University and Department of Comparative Medicine in Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA from April to September of 2008. MATERIALS： Four beagle dogs, male, 12 months old, weighing 10-13 kg were used in this experiment, plRES2-EGFP-bFGF plasmid containing full-length human bFGF gene cDNA was constructed and conserved by our institution. METHODS： Free gingiva of the 2nd, 3rd and 4th premolars were excised from left upper jaw of Beagle dogs, rinsed with aseptic phosphate buffer four times, then cut into pieces and digested with 2.5 g/L pancreatin for 2 hours at 37 ℃. After the centrifugation and supernatant removal, DMEM containing 10% fetal bovine serum was added to incubate on 6-well plate with coverlips in 5% CO2 incubator at 37 ℃. Logarithmically growing cells were digested and passaged. GFs were transfected with plRES2-EGFP-bFGF plasmid using liposome mediated method, while vacant piasmid transfection and un-transfection group served as controls. MAIN OUTCOME MEASURES： Proliferation and apoptosis feature of the GFs were evaluated by MTT and AOEB, respectively. The activity of alkaline phosphatase was assayed by chemical colorimetry. RESULTS： All of three groups cells entered log phrase on three days after transfection. MTT results showed that the proliferation of GFs transfected with bFGF was greater than cells transfected with vacant vector and untransfected cells （P 〈 0.05）. AO/EB dyeing showed the apoptosis rate of GFs transfected with bFGF was reduced compared with other two groups （P 〈 0.05）. After bGFG gene transfection, the ALP activity remained unchanged and there was no significant difference compared with untransfected cells. CONCLUSION： The transfection of bFGF gene to GFs can promote the proliferation of GFs and depress the apoptosis. No promotion is present with regard to the GFs differentiation.