摘要
目的探讨碱性成纤维细胞生长因子(bFGF)基因修饰的牙龈成纤维细胞(GFs )在牙周组织再生中的作用及其与骨形成蛋白-7(hBMP-7)基因修饰的骨髓基质细胞(BMSCs)联合应用的效果,为基因治疗与组织工程联合应用于牙周组织缺损再生治疗提供依据。
方法1.利用DNA重组技术,将hbFGF片段克隆到真核表达载体pIRES2-EGFP中,HindⅢ单酶切及序列分析鉴定获得的重组质粒;2.半消化法培养Beagle犬GFs,体外应用脂质体进行基因转染,RT-PCR、免疫细胞化学染色及ELISA检测目的基因的转录和表达;3.体外基因转染后,观察细胞形态变化,MTT法描绘细胞的生长曲线,AO/EB双染色和流式细胞技术检测细胞的凋亡,检测碱性磷酸酶(ALP)活性;4.通过荧光显微镜、激光共聚焦扫描显微镜、透射电镜及组织学进行观察表达目的基因的细胞与脱细胞真皮基质ADM膜复合;5.人工构建6只Beagle犬慢性Ⅱ度根分叉病变模型,随机分为A、B2组。每组3只犬的18颗病变牙又随机分成3组,分别命名为用BMP-7、BMP-7/GFs和BMP-7/hbFGF治疗,测量治疗前后附着丧失程度,角化龈宽度变化,龈沟液中NO含量,6周、12周后取材作病理切片,H.E染色观察牙周组织再生情况,计算新生牙骨质长度百分比和新生牙槽骨面积百分比,结果进行统计学分析。
结果1.bFGF被定向插入到真核表达载体pIRES2-EGFP中;2.通过脂质体介导成功地对Beagle犬GFs进行了pIRES2-EGFP-hbFGF瞬时转染,24h后即可见到转染的部分细胞发出较强的绿色荧光,转染效率为15%-25%。RT-PCR、免疫组织化学染色和ELISA检测表明,bFGF在GFs中得到了有效表达;3.目的基因转染后细胞形态略有变化,增殖能力明显增强,凋亡率下降,碱性磷酸酶活性无明显改变;4.转染细胞复合ADM膜后生长良好,24h细胞已贴附、伸展。透射电镜可见细胞能够在支架材料中生长,并与支架材料紧密结合;5.各组附着丧失的情况都有所改善,6周时各组之间差异无显著性;12周时BMP-7/GFs组和BMP-7/hbFGF组的改善更明显。治疗后各组角化龈宽度均有恢复,6周时,BMP-7/hbFGF组相比其它两组治疗效果更好;而12周时中BMP-7/GFs组和BMP-7/hbFGF组结果无差异,与BMP-7组相比有明显改善。两个时间点中各组之间治疗后NO含量无明显差异,都比治疗前明显降低。各实验组和对照组均可见不同程度的牙周组织再生,6周时组之间差异无显著性;12周时,BMP-7/hbFGF组的新生牙槽骨面积百分比和新生牙骨质长度百分比与其它两组相比有明显增加(P<0.05)。
结论1.利用基因工程原理成功构建了真核表达质粒pIRES2-EGFP-hbFGF。
2.hbFGF基因可在GFs中有效表达。
3. hbFGF基因转染可以促进GFs的增值,抑制其凋亡,hbFGF基因修饰的GFs有望成为牙周组织工程理想的种子细胞。
4. hbFGF基因修饰GFs在ADM膜上生长良好,hbFGF基因修饰的GFs与ADM膜复合物有望应用于牙周组织工程。
5. hbFGF基因修饰的GFs能更好地促进牙周组织再生,特别是牙槽骨及牙骨质的再生。hbFGF基因强化的组织工程技术是修复牙周组织缺损的一种较好的方法,有很好的临床应用前景。
Objective To investigate the effects of bFGF gene modified GFs alone or together with BMSCs modified by hBMP-7 gene on periodontal regeneration and to provide a new approach to repair the periodontal defects.
Methods①Complete sequence of human bFGF gene was cloned into pIRES2-EGFP with recombination DNA technique, named pIRES2-EGFP-bFGF. The recombinant plasmid was identified by restriction enzymolysis(HindⅢ) and DNA sequencing.②Recombinant plasmid pIRES2-EGFP-bFGF and vector pIRES2-EGFP were transfected into GFs of Beagle dogs respectively. Expression of bFGF in GFs was identified by RT–PCR, ELISA and immunohistochemistry.③Proliferation and apoptosis feature of the transfected cells were evaluated by MTT and AO/EB.The activity of cell alkaline phosphatase(ALP) was assayed to evaluated the ability of bone formation.④GFs expressed hbFGF gene were cultured with ADM in vitro. Histological examination was performed with light microscopy. Adhesion situation was analyzed by fluorescence microscope, confocal laser scanning microscope and transmission electron microscope.⑤A total 36 experimental classⅡfurcation defects were created surgically in six Beagle dogs, and randomly divided into two groups under therapy of six weeks and twelve weeks. Each group was divided into three subgroups, named BMP-7, BMP-7/GFs and BMP-7/hbFGF. 6wk or 12wk later, CAL(clinical attachment level), KG(width of keratinized gingival) and content of NO in gingival crevicular fluid were detected. After animals were sacrificed, periodontal regeneration was evaluated by histological and morphmetric analysis to calculate the percentage of new alveolar area and new cementum length.
Results①Human bFGF gene was inserted into vector pIRES2-EGFP correctly.②24hr after transfection, green fluorescence can be detected in GFs, the efficiency of transfection was nearly 15-25%. RT-PCR, ELISA and immunohistochemistry showed that bFGF was only expressed in GFs which transfected pIRES2-EGFP-bFGF. Transfected GFs had some morphological changes, and the proliferation was promoted, while apoptosis was impressed (P< 0.05). ALP activity remained unchanged (P>0.05).④The transfected cells adhered to ADM and stretched well after 24h of culture. Investigation under transmission electron microscope found that cells and material grew together, forming semi-desmosome like structure.⑤Improvement of CAL was observed in each group. Although there was no difference between each subgroup on 6wk (P>0.05), CAL were significantly improved in BMP-7/GFs and BMP-7/hbFGF on 12wk, comparing with BMP-7 subgroup (P<0.05). In all groups, there were different extent of KG recoveries. BMP-7/hbFGF subgroup surpassed the other subgroup on 6wk (P<0.05); meanwhile, both of BMP-7/GFs and BMP-7/hbFGF subgroups showed more recoveries than BMP-7 on 12wk (P<0.05). NO level in every subgroup was elevated after therapy. However, no difference was observed among groups on 6wk as well as 12wk (P>0.05). In both the experimental groups and the control group, there were different extents of regeneration. There was no significant difference on percentage of new alveolar area and new cementum length between three subgroups on 6wk(P>0.05),BMP-7/hbFGF subgroup increased apparently compared with others(P<0.05).
Conclusion①Human bFGF gene recombinant eukaryotic expression plasmid was constructed successfully.②bFGF gene can express in GFs of Beagle dogs by transfeting recombinant plasmid into cells.③The introduction of bFGF gene to GFs promoted the proliferation of cells as well as depressed the apoptosis. GFs expressed bFGF gene may be served as an ideal cell source for periodontal tissue engineering.④The AMD has a good biocompatibility with hbFGF transfected GFs. It shows that the compound caould be used in periodontal tissue engineering.⑤Introduction of bFGF gene to GFs promoted the regeneration of periodontium, especially the regeneration of alveolar bone and cementum. hbFGF gene enhanced tissue engineering could successfully repair the periodontal defects.
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