新型重组人血小板衍化生长因子B腺病毒载体的构建与转染牙周干细胞的研究

Construction and identification of recombinant human platelet-derived growth factor-B adenoviral vector and transfection into periodontal ligament stem cells

目的构建人血小板衍化生长因子B（platelet—derived growth factor—B，PDGF—B）重组复制缺陷型腺病毒载体，使其感染牙周韧带干细胞（periodontal ligament stem cells，PDLSC）并检测其相关生物学变化。方法采用常规分子生物学方法，构建重组穿梭载体PAdTrackCMV．PDGF—B，用PmeI酶线性化后，线性化质粒在细菌BJ5183内与腺病毒骨架载体质粒AdEasy Ⅰ同源重组，构建重组腺病毒质粒pad-PDGF—B，在HEK293细胞中包装成重组腺病毒Ad—PDGF—B。Western blotting法检测其在HEK293中的表达情况，同时利用包装好的病毒感染PDLSC，用免疫组化的方法鉴定PDGF—B在细胞中的表达；采用甲基噻唑基四唑（MTY）法检测感染病毒后PDLSC的增殖变化，用反转录聚合酶链反应（RT—PCR）检测感染后细胞分泌Ⅰ型胶原的变化。结果重组缺陷型腺病毒载体经限制性内切酶酶切分析鉴定，与预期结果一致。重组质粒转染HEK293细胞3d后即可观察到绿色荧光蛋白（green fluorescence protein，GFP）明显表达，Western blotting检测证实PDGF—B表达。重组病毒能够感染PDLSC并表达蛋白，MTT结果显示感染Ad—PDGF—B后的PDLSC增殖能力明显加强。在感染后第4天，感染Ad—PDGF-B后的PDLSC组吸光度值为（0．68±0．02），与对照组相比差异有统计学意义（P〈0．01）。同时Ⅰ型胶原表达量增高。结论利用新型腺病毒载体AdEasy系统可快速构建同时表达EGFP和PDGF-B的重组复制缺陷型腺病毒Ad-PDGF—B，此病毒能够感染PDLSC并促进其增殖，同时增强Ⅰ型胶原的表达，为牙周病患者牙周组织再生和种植体周韧带重建的基因治疗奠定基础。

Objective To construct a recombinant human platelet-derived growth factor-B （PDGF-B） adenoviral vector and to transfect it into human periodontal ligament stem cells （PDLSC）. Methods The recombinant plasmid pAd-PDGF-B was constructed by homologous recombination and confirmed by restriction endonucleases digestion. Recombinant adenovirus was packaged in HEK293 cells. PDLSC were transfected with recombinant adenovirus and PDGF-B expression was confirmed. Expression of collagen type Ⅰ gene was determined by quantitative analysis of the products of RT-PCR. The cell proliferation was determined with MTT colorimetric assay. Results The recombinant plasmid pAd-PDGF-B was confirmed by restriction endonucleases digestion. EGFP expression was observed on the third day after transfecting, and the expression of PDGF-B was detected. Immunohistochemical methods revealed that PDGF-B was expressed in PDLSC. Levels of expression of collagen type I gene were increased significantly by transfer of the exogenous PDGF-B gene to PDLSC. At the same time, findings indicated that Ad-PDGF-B stimulated PDLSC proliferation. MTI＂ assay indicated the absorbance of PDLSC by stimulating with Ad-PDGF-B was （0. 68 ＋ 0. 02）, P 〈 0. 01. Conclusions Using the AdEasy system, the human PDGF-B recombinant adenovirus can be rapidly obtained. These results indicate that recombinant adenoviruses encoding PDGF-B transgenes could modulate proliferative activity of PDLSC, enhance the high expression of collagen type Ⅰ and lay the foundation for periodontal tissue regeneration and dental implant gene therapy.