基質細胞衍生因子-1alpha基因療法對骨再生及傷口癒合的作用

Effects of stromal cell-derived factor-1 alpha gene therapy on bone regeneration and wound healing

張少華(Shao-Hua Chang)

指導教授 ： 郭生興

共同指導教授 ： 林思洸(Sze-Kwan Lin)

國立臺灣大學/醫學院/臨床牙醫學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601815

摘要

牙科植體雖已被廣泛應用於缺牙區域的重建，但並非所有病患的條件都適合植牙。對於嚴重骨缺損的區域，引導骨再生術(guided bone regeneration, GBR)是一個常施行的術式，其目的為豐隆齒槽骨以利人工牙根植入，但仍有技術上之困難有待突破，其中包括如何加強缺損區骨生成能力，和確保軟組織傷口穩定癒合。過去文獻對於如何增加骨再生的能力，都是偏向於生長因子的利用，但生長因子要廣泛應用在臨床齒槽骨豐隆手術上，目前仍有其困難。另外，如何促進缺損區上方的軟組織穩定癒合也是增加引導骨再生術成功率的重要因素，但過往對於這部份的相關研究不足。
組織再生需要幹細胞（stem cells）遷移（migration）及分化（differentiation），如何吸引幹細胞以促進組織修復已成為目前再生醫學主要的發展方向。基質細胞衍生因子-1 (stromal cell-derived factor-1α, SDF-1α), 被認為是重要的幹細胞趨化素（chemokine）具有趨化特殊潛能間葉幹細胞(mesenchymal stem cell, MSC)的能力。歷年來的文獻也已證實基質細胞衍生因子-1對軟組織癒合及硬組織再生都有幫助。但過去報告多以蛋白質作為研究對象，蛋白質會有作用效期短、安全性及成本昂貴等問題。因此本研究以基因療法為策略，建構帶有基質細胞衍生因子-1α基因的質體（plasmid），以探討基質細胞衍生因子-1α基因療法的效果。
實驗中包含第一部分的聚乙烯亞胺(polyethylenimine)轉染效率測試，以此決定用於動物實驗的聚乙烯亞胺及質體DNA之最佳比例。第二部分的基質幹細胞遷移測試，檢視此質體經轉染到哺乳類細胞後是否能正常表現基質細胞衍生因子-1α蛋白質及趨化基質幹細胞。第三部分為動物實驗，利用大鼠頭蓋骨缺損模型，植入膠原蛋白膜及補骨材，並加入基因轉染試劑及質體混合液，以觀察骨骼再生與軟組織癒合之情形。
由第一部分的實驗結果得知，當聚乙烯亞胺與DNA之比例為7.5時(3uL聚乙烯亞胺及1ug DNA)，在48小時之後，可達到五成（55.68%）的轉染率。所以聚乙烯亞胺與DNA應該維持在高比例，而兩者皆應維持低濃度，在作用時間充足的條件下，仍可以達到相當的轉染效率。經第二部分的測試，證實重組之質體能在轉染至293FT細胞之後，正常表現出基質細胞衍生因子-1α蛋白質。並且在幹細胞遷移測試中，確認此蛋白質可以趨化基質幹細胞，跟對照組相比多達3.5倍。第三部分以大鼠頭蓋骨缺損為模型來評估基質細胞衍生因子-1α對於傷口癒合的療效，分為軟組織癒合及骨再生的觀察。加入基質細胞衍生因子-1α質體的組別其平均軟組織癒合速度在第14天已達100%，而對照組則只有88.7%；利用微型斷層掃描評估得知，加入基質細胞衍生因子-1α質體的組別，在術後四週其骨再生體積比率為12.9%±3.64 (n=8)比對照組7.07%±4.85 (n=6)多，兩者有統計學上顯著的差異(P<0.05)；而組織切片的結果，也同樣顯示加入基質細胞衍生因子-1α質體的組別其骨新生面積比率，與對照組相比，在四個特定時間點都是比較多的。
本實驗的結果顯示，基質細胞衍生因子-1α基因療法對於骨再生及軟組織癒合有一定的效益，將來可應用以提升齒槽脊豐隆手術之成功率。

關鍵字

基質細胞衍生因子-1 ； 間質幹細胞 ； 引導骨再生術 ； 基因療法 ； 聚乙烯亞胺

並列摘要

Although dental implantation has been widely applied for the reconstruction of edentulous ridge, implant placement may not be feasible in cases with poor bone volume. For the severely atrophic ridge, the technique of guided bone regeneration (GBR), is commonly used for alveolar ridge augmentation. However, there are still technical difficulties remain to be resolved. Such difficulties include how to increase the regenerative potential of bone tissue in defect areas and the assurance of stable soft tissue healing. Past researches mainly focused on the use of growth factors for promoting bone regenerative capacity. However, obstacles still exist in the use of growth factors for alveolar ridge augmentation. How to assure the stable healing of soft tissue overlaying the defect area is another key point for increasing the success rate of GBR, but studies focusing on this subject are rare.
Tissue regeneration requires the migration and differentiation of stem cells. Hence, recruitment of stem cells has become the main direction of development in the field of regenerative medicine. Stromal cell-derived factor-1α (SDF-1α), considered as an important stem cell chemokine, has been reported to have the capacity of attracting multipotent mesenchymal stem cells (MSCs). Studies have shown that SDF-1α is beneficial for soft tissue healing and bone regeneration, and most of the studies used SDF-1α protein as the research subject. However, protein has its limitations, such as rapid degradation leading to short lasting duration, safety concern and poor cost efficiency. Consequently, gene therapy has been the main strategy in the present study. We constructed a plasmid encoding mSDF-1α and investigated the therapeutic effect of mSDF-1α gene therapy. First, in our experiment, in-vitro transfection efficiency with polyethylenimine(PEI) was tested in order to determine the optimal ratio of PEI and plasmid DNA, which was adopted in the following animal study. Second, MSC transwell migration assay was performed to ascertain the production of SDF-1α protein by transfected mammalian cells and the protein can promote MSC migration. The third part of the experiment employed the rat calvarial defect model. Application of PEI/DNA condensates in combination with the use of collagen barrier membrane and bone substitutes was performed. The effects on soft tissue healing and bone regeneration were evaluated.
Based on the results of the first part of experiment, when the ratio of PEI and DNA was 7.5(3uL PEI and 1ug DNA), the transfection efficiency can reached 55.68% after 48 hours. The data indicated that PEI and DNA should be maintained at a high ratio with low concentration. Therefore, an acceptable transfection rate can be obtained after sufficient time. The second part of experiment verified that the SDF-1α protein can be expressed normally by mammalian cell 293FT after gene transfection. In the MSC transwell migration assay, the SDF-1α protein-added group promoted 3.5 times more MSCs migration compared to the control group.
In the third part of experiment, the rat calvarial defect model was used to evaluate the therapeutic effect of SDF-1α gene therapy on wound healing, including soft tissue healing and bone regeneration. In the aspect of assessing soft tissue healing, the SDF-1α-treated group showed an average 100% healed tissue after 14 days, while the control group was only 88.7%; In the use of micro-tomography, the data showed that SDF-1α-treated group had more new bone formation (12.9% ± 3.64) than the PEI-only group (7.07% ± 4.85) with statistically significant differences (P <0.05) ; In the histomorphometric analysis, the results demonstrated that more newly-formed bone can be detected in SDF-1α-treated group compared with the control group at four different time points.
Results of the study suggest that SDF-1α gene therapy is beneficial for bone regeneration and soft tissue healing and may have the potential to improve the success rate of alveolar ridge augmentation.

並列關鍵字

stromal cell-derived factor-1(SDF-1) ； mesenchymal stem cell(MSC) ； guided bone regeneration(GBR) ； gene therapy ； polyethylenimine(PEI)

參考文獻

1. Chiapasco, M., M. Zaniboni, and M. Boisco, Augmentation procedures for the rehabilitation of deficient edentulous ridges with oral implants. Clin Oral Implants Res, 2006. 17 Suppl 2: p. 136-59.

3. Dahlin, C., et al., Healing of bone defects by guided tissue regeneration. Plast Reconstr Surg, 1988. 81(5): p. 672-6.

5. Seibert, J. and S. Nyman, Localized ridge augmentation in dogs: a pilot study using membranes and hydroxyapatite. J Periodontol, 1990. 61(3): p. 157-65.

6. Nyman, S., et al., Bone regeneration adjacent to titanium dental implants using guided tissue regeneration: a report of two cases. Int J Oral Maxillofac Implants, 1990. 5(1): p. 9-14.

7. Buser, D., et al., Long-term stability of osseointegrated implants in bone regenerated with the membrane technique. 5-year results of a prospective study with 12 implants. Clin Oral Implants Res, 1996. 7(2): p. 175-83.