低能量光照刺激促進大鼠骨髓間葉幹細胞之遷移能力

Low Level Light Therapy Enhances the Migration of Mesenchymal Stem Cells Derived from Rat Bone Marrow

黃柏雅(Po-Ya Huang)

指導教授 ： 李文婷

中原大學/理學院/奈米科技碩士學位學程/碩士(2013年)

https://doi.org/10.6840/cycu201301119

摘要

摘要

間葉幹細胞 (mesenchymal stem cells, MSCs) 能透過血流跨越血管內皮屏障由骨髓遷移至受傷組織，並在受損部位分化成具功能的細胞，因此在再生醫學上扮演重要的角色。趨化素基質衍生因子 (Stroma-derived factor, SDF) -1α在幹細胞遷移至受損或缺氧組織亦具有加乘的效果。本研究目的在探討添加 SDF-1α (SDF) 以及4 J/cm2紅光 (Red) 與近紅外光 (NIR) 低能量光照刺激 (Low level light irradiation, LLLI) 提升大鼠骨髓 MSCs 遷移能力之機制。跨膜遷移試驗得知Red 和 NIR 組的移動細胞數高於控制組。而 SDF+Red 和 SDF+NIR 組與Red 和 NIR 組相比更能顯著提升細胞的遷移能力。此外，C-X-C chemokine receptor type 4 (CXCR4) 的基因和蛋白質表現以及焦點黏附激酶 (Focal adhesion kinase, FAK) 蛋白質磷酸化的表現也明顯提高。利用小干擾 RNA (small interfering RNA, siRNA)轉染大鼠骨髓 MSCs抑制FAK和 CXCR4表現，結果發現轉染 FAK siRNA 和CXCR4 siRNA分別可使細胞遷移降低 41 % 和58 %，無論是LLLI 或 SDF 處理仍無法使細胞遷移回復到與未轉染控制組相同的數目。其中，轉染 FAK siRNA 和CXCR4 siRNA分別可使 pFAK 表現分別降低36 % 和78 %；Red、NIR 和 SDF + Red、SDF + NIR 組的pFAK 表現全部被抑制。然而轉染 FAK siRNA 以及LLLI 或 SDF 處理對CXCR4的表現並無顯著影響。西方墨點法偵測發現 Red、NIR、SDF、SDF + Red 及SDF + NIR 組的phospho-Nuclear factor-κappa B (pNF-κB) p65表現皆明顯提升且會轉位至細胞核內，尤以SDF + NIR 組最為明顯。轉染 FAK siRNA後，無論是 LLLI 或 SDF 處理組的 pNF-κB p65表現均與控制組無差異；而轉染 CXCR4 siRNA 則會抑制所有組別之 pNF-κB p65表現。在基質金屬蛋白酶 (Matrix metalloprotease, MMP)分析方面，明膠分解酶圖譜分析和即時定量聚合酶鏈鎖反應 (Quantitative real-time polymerase chain reaction, QPCR) 結果得知 Red、NIR、SDF、SDF + Red 及 SDF + NIR 組的 MMP-2 活性和基因表現量同樣明顯提高，SDF、SDF + Red 及 SDF + NIR 組的MMP-9 及MMP-14的表現皆高於控制組；而基質金屬蛋白酶組織抑制劑 (Tissue inhibitor of matrix metalloproteinase, TIMP)-2 的基因表現則低於控制組。轉染FAK 和 CXCR4 siRNA 會使 MSCs的 MMP-2活性分別降低 23 及 25 %，而LLLI或SDF處理則會使其活性明顯降低，尤以SDF + NIR 組為甚。在 β1-integrin 及細胞骨架 F-actin表現影響方面，LLLI刺激及SDF誘導會提升β1-integrin 蛋白質的表現。CXCR4 siRNA 轉染 MSCs，會抑制各組之 β1-integrin 表現；轉染 FAK siRNA 則對各組之 β1-integrin表現無顯著影響。螢光染色發現Red、NIR、SDF、SDF + Red 及 SDF + NIR 組的細胞皮層有大量F-actin 聚集情形，亦偵測到其Rac/Cdc42及Rho 蛋白表現增加。在熱休克蛋白方面，QPCR 分析發現 SDF、SDF + Red 及 SDF + NIR 組的HSP27 基因表現分別降低37、25及22 %，Red、NIR、SDF、SDF + Red 及SDF + NIR 組的HSP90 基因表現分別降低62、58、58、71 及 79 %。綜上所述，紅光與近紅外光LLLI刺激和SDF-1α 處理能透過活化CXCR4、FAK及NF-κB的磷酸化、提升MMPs的分泌，進而使F-actin 聚合、增加Rac/Cdc42及Rho 蛋白表現，進而促進大鼠骨髓 MSCs 的遷移。

關鍵字

近紅外光 ； 紅光 ； 細胞遷移 ； 骨髓間葉幹細胞 ； 低能量光照 ； 基質衍生因子

並列摘要

Abstract

Mesenchymal stem cells (MSCs) play an important role in tissue regeneration which involves the processes of mobilization of stem cells from the bone marrow, homing of these cells to the site of injury, and differentiation of the stem cell into a functional cell of the injured tissue. Stroma-derived factor-1α (SDF-1α) plays a critical role in stem cell migration towards areas of tissue injury and hypoxia. The aim of this study was to examine the influence of low level light irradiation (LLLI) using red and near infrared (NIR) at energy dose on 4 J/cm2 on migration of rat bone marrow MSCs in the absence and presence of SDF-1α (SDF). Results of transwell migration assay showed that the migration of MSCs after Red and NIR light irradiation was greater than control. The migration was further enhanced with SDF-1α treatment. The gene and protein expression of C-X-C chemokine receptor type 4 (CXCR4) and phosphorylation of focal adhesion kinase (FAK) were also enhanced. The expression of FAK and CXCR4 was inhibited by transfecting MSCs with FAK and CXCR4 small interfering RNA (siRNA). MSCs transfected with FAK siRNA and CXCR4 siRNA exhibited decreased cell migration by 41% and 58%, respectively. LLLI or SDF treatment could not recover cell migration capability to the level comparable to that of untransfected control group. MSCs transfected with FAK siRNA and CXCR4 siRNA exhibited decreased pFAK expression by 36% and 78%, respectively. The expression of pFAK was also inhibited in the groups of Red, NIR, SDF, SDF + Red, and SDF + NIR. However, the level of CXCR4 expression was not affected by FAK siRNA transfection, LLLI or SDF treatment. The level of phospho-Nuclear factor-κappa B (pNF-κB) p65 expression was found to increase as shown by western blotting, and nuclear translocation of pNF-κB was observed in the groups of Red, NIR, SDF, SDF + Red, and SDF + NIR, especially in SDF + NIR group. LLLI or SDF treatment did not influence pNF-κB expression in FAK siRNA transfected MSCs. But pNF-κB expression was inhibited in all treatment groups of CXCR4 siRNA transfected cells. As demonstrated by gelatin zymography and quantitative real-time polymerase chain reaction (QPCR) analysis, the activity and gene expression matrix metalloproteinase (MMP)-2 were both elevated in the groups of Red, NIR, SDF, SDF + Red, and SDF + NIR. The levels of MMP-9 and MMP-14 expression of SDF, SDF + Red, and SDF + NIR groups were higher than that of control group, whereas the level of tissue inhibitor of matrix metalloproteinase (TIMP)-2 was lower than that of control group. MSCs transfected with FAK siRNA and CXCR4 siRNA exhibited decreased MMP-2 activity by 36% and 78%, respectively. LLLI or SDF treatment further suppressed MMP-2 activity, especially in SDF + NIR group. β1-integrin expression was upregulated in LLLI and SDF treatment but inhibited by CXCR4 siRNA transfection. There was no significant difference of β1-integrin expression in FAK siRNA transfected cells. The results of fluorescence staining showed that F-actin polymerization accumulated in the cortex of MSCs as well as Rac/Cdc42 and Rho protein expression in Red, NIR, SDF, SDF + Red and SDF + NIR groups. Furthermore, F-actin polymerization was inhibited after transfection of FAK siRNA and CXCR4 siRNA. QPCR analysis found that the expression of HSP27 gene of SDF, SDF + Red and SDF + NIR group decreased by 37, 25 and 22 %, respectively. The expression of HSP90 gene of Red, NIR, SDF, SDF + Red and SDF + NIR group decreased by 62, 58, 58, 71 and 79 %, respectively, compared to that of control group. This suggests that decreased levels of HSP27 and HSP90 might help LLLI and SDF-1α induced cell migration. In summary, LLLI with red and NIR light and SDF-1α treatment could stimulate MSCs motility in vitro through CXCR4 activation, FAK and NF-κB phosphorylation, increasing MMPs secretion, and upregulating F-actin polymerization as well as Rac/Cdc42 and Rho protein expression.

並列關鍵字

red light ； cell migration ； low level light irradiation ； bone marrow mesenchymal stem cell ； stroma-derived factor ； near infrared light

參考文獻

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