幹細胞萃取物促進人類牙齦纖維母細胞的再程序化

僅藉由四個轉錄因子體細胞產生的誘導性多能幹細胞（iPS），已將細胞的再程式化(reprogramming)帶領進入另一領域。由於傳統iPS需要載體，但載體基因對於母體的嵌入等影響無法完全預測，已有源源不絕的研究，報導包括利用蛋白質而不直接參與DNA等方法來改善此問題。這種方法需要大量蛋白質萃取物，但目前的研究多利用老鼠胚胎幹細胞或畸胎瘤細胞株等作為來源，較難在日後應用於臨床。臍帶血已廣為認可為一穩定的具潛能性的間質幹細胞來源;人類牙齦纖維母細胞有極高的增生能力，且對於牙醫師為一方便收集的體細胞。儘管人類牙齦纖維母細胞在最近已被證實為傳統iPS方法進行再程式化的可行細胞來源，臍帶血間質幹細胞和人類牙齦纖維母細胞目前都未曾被使用於此種蛋白萃取方式進行的再程式化。鏈球菌溶血素O (SLO)為一種會造成細胞膜穿孔的細菌內毒素，已被廣泛使用於此種蛋白萃取方式進行的再程式化。然而，鏈球菌溶血素O在非致命劑量下對牙齦纖維母細胞的細胞毒性與訊息傳遞等影響目前仍未知。因此，本實驗研究目的為探討臍帶血間質幹細胞蛋白質萃取的方法使人類牙齦細胞進行再程式化及其表現。研究假設人類牙齦纖維母細胞可被幹細胞萃取再程式化。根據上述問題進行以下研究方法：利用MTT和螢光染色評估SLO對牙齦纖維母細胞生存率和細胞膜穿孔效率的影響，利用西方墨點法分別SLO對牙齦纖維母細胞的訊息傳遞影響。加入臍帶血間質幹細胞萃取物的細胞，則利用反轉錄-聚合酶酵素鏈鎖反應和DNA微陣列，測量再程式化造成的基因的改變。此外，也透過多種細胞分化的方式確認再程式化的表現。研究結果:低濃度SLO 對於牙齦和皮膚纖維母細胞的生存程度影響不大，但仍會達成一定程度的細胞膜開孔，且會誘導ERK1/2與P38的磷酸化表現。加入臍帶血間質幹細胞萃取物的牙齦纖維母細胞，會表現OCT4和NANOG等幹細胞基因; 且在後續成脂、成骨、神經等誘導分化上有較理想的表現。結論:臍帶血間質幹細胞萃取物可使牙齦纖維母細胞在適當SLO濃度作用下進行再程式化，造成基因與分化能力的改變。

關鍵字

人類牙齦纖維母細胞；再程式化；鏈球菌溶血素O

並列摘要

Induced pluripotent stem cell (iPS), generated from somatic cells by the four transcription factors, has been startling development of reprogramming technology. Since the integration of vectors is not fully predictable, a flood of studies have been publishing, including protein based-DNA free method. This method required abundant protein extracts, which were often collected by embryonic stem cells or teratocarcinoma, were harder to be applied clinically. Human cord blood has been established as a stable and potential source of mesenchymal stromal cells (cbMSC). Human gingival fibroblasts (GF) characterize as high proliferation rate and friendly collected by dentists. Although GF has recently been proved as a potential source by the traditional four-factor reprogramming via retrovirus, both cbMSC and GF have never been used in any DNA free reprogramming methods. Streptolysin O (SLO), a pore forming endotoxin, is widely used in protein delivery for DNA free reprograming. However, the response of GF treated with low sublytic dose has never been studied as well. Thus, we hypothesize that GF could be reprogrammed by protein extracs, and the research goal of the study is to reprogram GF by cbMSC’s protein extracts. Materials and methods: The questions remained were evaluated as follows: SLO caused cell viability change was evaluated by MTT assay, the membrane permeabiliting efficiency was evaluated by fluorescence stain, and phosphorylation of signal pathways were evaluated by Western Blot. After adding stem cell extracts, the reprogramming effect was examined by RT-PCR and DNA microarray for mRNA level. The differentiation ability was also checked as well. Results: Though SLO showed little effect at the concentration below 1000ng/mL, it caused membrane permeabilization in both GF and SF. Phosphorylation of ERK1/2 and P38 were also induced when SLO was treated. Adding cbMSC extracts in SLO treated GF increased the mRNA expression of OCT4 and NANOG, which were regarded as stem cell markers. Furthermore, these treated cells differentiated better in adipogenic, osteogenic, and neurogenic induction. In conclusion, cbMSC extracts could reprogram SLO treated GF causing changes in genetic level and differentiation ability.

並列關鍵字

human gingival fibroblast ； reprogramming ； streptolysin O

參考文獻

Chapter 7 References

1. Chen, F.M., et al., A review on endogenous regenerative technology in periodontal regenerative medicine. Biomaterials, 2010. 31(31): p. 7892-927.

2. Chen, F.M. and Y. Jin, Periodontal tissue engineering and regeneration: current approaches and expanding opportunities. Tissue Eng Part B Rev, 2010. 16(2): p. 219-55.

3. Williams, D.F., To engineer is to create: the link between engineering and regeneration. Trends in Biotechnology, 2006. 24(1): p. 4-8.

4. Morrison, S.J. and J. Kimble, Asymmetric and symmetric stem-cell divisions in development and cancer. Nature, 2006. 441(7097): p. 1068-74.

國際替代計量

幹細胞萃取物促進人類牙齦纖維母細胞的再程序化

全文下載

主題瀏覽