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。

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.

目錄

摘要 I
Abstract III
致謝 VI
目錄…………………………………………………………………………..VII
圖索引 XIII
表索引 XVII
縮寫表 XVIII
第一章 緒論 1
1.1. 前言 1
1.2. 研究動機及目的 2
第二章 文獻回顧 4
2.1. 低能量光照治療之起源 4
2.2. 低能量光照所引發細胞內的訊息傳遞 6
2.2.1. PI3K/Akt 訊息傳導路徑 8
2.2.2. Src/PKC 訊息傳導路徑 9
2.2.3. MAPK/ERK 訊息傳導路徑 10
2.2.4. GSK-3β/Bax 訊息傳導路徑 11
2.3. 間葉幹細胞的治療與應用 12
2.3.1. 趨化素和受器 13
2.3.2. 內源性電場影響 14
2.4. MSCs 遷移引發細胞內的訊息傳遞 14
2.4.1. 細胞遷移機制 14
2.4.2. FAK 訊息傳導路徑 15
2.4.3. MMPs 及 TIMPs 18
2.4.4. proMMPs 的活化 20
2.4.5. HSP27/HSP70/HSP90 20
2.4.6. 骨橋蛋白 22
2.5. 低能量光照促進細胞遷移之作用機制 23
第三章 材料與方法 25
3.1. 紅光與近紅外光 LED 光板組裝 25
3.2. 實驗設計 26
3.3. 低能量光照刺激參數 27
3.4. 細胞分離與培養技術 28
3.4.1. 大鼠來源 28
3.4.2. 麻醉藥配製 28
3.4.3. 紅血球裂解溶液配製 29
3.4.4. 抗凝血劑配製 29
3.4.5. MSCs 分離過程 29
3.4.6. 磷酸鹽緩衝溶液配製 31
3.4.7. MSCs 長期培養基 31
3.4.8. 胰蛋白酶配製 32
3.4.9. 台盼藍染劑配製 32
3.4.10. MSCs 繼代過程 33
3.4.11. 細胞計數 33
3.5. 細胞跨膜遷移能力分析 34
3.5.1. SDF-1α 操作溶液配製 34
3.5.2. 不同濃度 SDF-1α 對 MSCs 遷移能力之影響 35
3.5.3. LLLI 刺激添加 SDF-1α 對 MSCs 遷移能力之影響 .35
3.6. 明膠分解酶圖譜分析 36
3.6.1 焦集膠緩衝溶液製備 36
3.6.2. 分離膠緩衝溶液製備 36
3.6.3. 10 % SDS 溶液製備 36
3.6.4. 0.8 % 明膠溶液製備 37
3.6.5. 10 % APS 製備 37
3.6.6. 7.5 % 聚丙烯胺凝膠製備 37
3.6.6.1. 含 0.1 % 明膠分離膠製備 37
3.6.6.2. 4 % 焦集膠製備 37
3.6.7. 蛋白質電泳緩衝溶液製備 37
3.6.7.1. 3 X 非還原樣本緩衝溶液 37
3.6.7.2. 1 X 電泳緩衝溶液 38
3.6.8. 明膠分解酶圖譜分析呈色藥物配製 38
3.6.8.1. Triton-X100 緩衝溶液 38
3.6.8.2. Tris-HCl 緩衝溶液 38
3.6.8.3. 呈色緩衝溶液 38
3.6.8.4. Coomassie blue 染劑 38
3.6.8.5. 退染溶液 39
3.6.9. 明膠分解酶圖譜分析步驟 39
3.7. 西方墨點法分析 40
3.7.1. 西方墨點法相關藥物配製 40
3.7.1.1. 緩衝溶液 40
3.7.1.2. 1 X TBST 溶液 41
3.7.1.3. 細胞裂解溶液 41
3.7.1.4. 5 % 脫脂奶粉溶液 41
3.7.2. 西方墨點法步驟 41
3.8. 蛋白質定量分析 44
3.9. 免疫螢光染色分析 45
3.10. 螢光染色分析 47
3.10.1. 螢光染色步驟 47
3.10.2. 螢光光譜儀分析 48
3.11. LLLI 刺激添加趨化素 SDF-1α，對相關基因表現之影響 48
3.11.1. 反轉錄聚合酶鏈鎖反應 49
3.11.2. mRNA 的萃取及純化 49
3.11.3. Total RNA 定量 50
3.11.4. 反轉錄作用步驟 50
3.11.5. 聚合酶鏈鎖反應 51
3.11.6. DNA 瓊膠電泳 51
3.11.7. ImageJ 半定量化 52
3.11.8. 即時定量聚合酶鏈鎖反應 52
3.12. 小干擾 RNA 原理及技術 56
3.12.1. 設計雙股 FAK siRNA 和 CXCR4 siRNA 序列 56
3.12.2. 螢光顯微鏡觀察 MSCs 轉染 siRNA 後之轉染效率 58
3.12.3. 流式細胞儀分析siRNA 轉染 MSCs 後之轉染效率 59
3.12.3.1. FAK siRNA 和 CXCR4 siRNA 藥品前處理 59
3.12.3.2. 轉染作用和流式細胞儀分析 59
3.12.4. 西方墨點法分析 siRNA 轉染 MSCs 之蛋白質表現 61
3.12.5. 螢光染色分析 siRNA 轉染 MSCs 之 F-actin 表現 62
3.12.6. 免疫沉澱法 63
3.12.7. ELISA 定量分析 65
3.13. 統計分析 66
第四章 結果 67
4.1. LLLI 刺激及添加 SDF-1α 對大鼠骨髓 MSCs 遷移能力之影響 67
4.2. LLLI 刺激及添加 SDF-1α 對 CXCR4 和 pFAK 表現之影響 70
4.3. FAK siRNA 和 CXCR4 siRNA 轉染大鼠骨髓 MSCs 74
4.4. LLLI 刺激及添加 SDF-1α 對FAK siRNA 和 CXCR4 siRNA 轉
染大鼠骨髓 MSCs 的遷移能力影響 77
4.5. LLLI 刺激及添加 SDF-1α 對大鼠骨髓 MSCs 之 MMPs 及 TIMPs 表現的影響 81
4.6. LLLI 刺激及添加 SDF-1α 對 FAK siRNA 和 CXCR4 siRNA 轉
染之大鼠骨髓 MSCs FAK 磷酸化及 CXCR4 的影響 102
4.7. LLLI 刺激及添加 SDF-1α 對促進大鼠骨髓 MSCs pNF-κB 活化
並轉位至核內的表現 109
4.8. LLLI 刺激及添加 SDF-1α 對大鼠骨髓 MSCs β1-integrin 表現之
影響 114
4.9. LLLI 刺激及添加 SDF-1α 對大鼠骨髓 MSCs 的細胞骨架、形態
以及 Rho GTPase 活化之影響 119
4.10. LLLI 刺激及添加 SDF-1α 對大鼠骨髓 MSCs 熱休克蛋白基因
表現之影響 140
第五章 討論 144
5.1. LLLI 刺激添加 SDF-1α 對大鼠骨髓 MSCs 遷移能力之影響 144
5.2. LLLI 刺激添加 SDF-1α 誘導會活化大鼠骨髓 MSCs 之 pFAK
和 CXCR4 的表現 144
5.3. FAK siRNA 和 CXCR4 siRNA 轉染大鼠骨髓 MSCs 145
5.4. LLLI 刺激及添加 SDF-1α 能雙向誘導大鼠骨髓 MSCs 遷移 145
5.5. LLLI 刺激及添加 SDF-1α 誘導大鼠骨髓 MSCs 分泌 MMPs 146
5.6. FAK siRNA 和 CXCR4 siRNA 轉染大鼠骨髓 MSCs 經 LLLI 刺激及 SDF-1α 處理對 FAK 磷酸化及 CXCR4 表現之影響 149
5.7. LLLI 刺激及添加 SDF-1α 促進大鼠骨髓 MSCs 之 pNF-κB 表現並轉位至核內 150
5.8. LLLI 刺激及添加 SDF-1α 會活化 CXCR4 並經由 NF-κB 磷酸化來調控 β1-integrin 的表現 152
5.9. LLLI 刺激及添加 SDF-1α 可促進細胞骨架 F-actin 聚合、改變細胞形態和活化 Rho GTPase 蛋白 153
5.10. LLLI 刺激及添加 SDF-1α 對大鼠骨髓 MSCs 熱休克蛋白基因表現之影響 155
第六章 結論與未來展望 157
參考文獻 166
附錄表 178
A.儀器表 178
B.藥品資料表 180
C.附圖 184









圖索引

圖1-1研究架構 3
圖3-1 LED光板 26
圖 3-2 初代大鼠骨髓 MSCs 的群落形態 34
圖 4-1不同濃度之 SDF-1α 對 MSCs 遷移能力之影響 68
圖 4-2 LLLI 處理及 SDF-1α 添加對大鼠骨髓 MSCs 遷移能力之影響 69
圖 4-3 LLLI 及添加 SDF-1α 對 pFAK 表現之影響 71
圖 4-4 LLLI 及添加 SDF-1α 對 CXCR4 表現之影響 72
圖 4-5 LLLI 及添加 SDF-1α 對 CXCR4 基因表現之影響 73
圖 4-6螢光顯微鏡觀察 FAK siRNA 和 CXCR4 siRNA 轉染大鼠骨髓MSCs 細胞轉染情形 75
圖 4-7流式細胞儀分析 FAK siRNA 和 CXCR4 siRNA 轉染大鼠骨髓MSCs 之轉染效率 76
圖 4-8 LLLI 刺激及添加 SDF-1α 對 FAK siRNA 轉染之大鼠骨髓 MSCs 的細胞遷移能力影響 78
圖 4-9 LLLI 刺激及添加 SDF-1α 對 CXCR4 siRNA 轉染大鼠骨髓 MSCs 的細胞遷移能力影響 80
圖 4-10 LLLI 刺激及添加 SDF-1α 對大鼠骨髓 MSCs 分泌 MMP-2 活性之影響 85
圖 4-11 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs 的 MMP-2 基因表現之影響 87
圖 4-12 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs 分泌 MMP-9 活性之影響 89
圖 4-13 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs 的 MMP-9 基因表現之影響 90
圖 4-14 LLLI 刺激及添加 SDF-1α 對 FAK siRNA 轉染大鼠骨髓 MSCs 所分泌之 MMP-2 活性之影響 92
圖 4-15 LLLI 刺激及添加 SDF-1α 對 FAK siRNA 轉染大鼠骨髓 MSCs 所分泌之 MMP-9 活性之影響 94
圖 4-16 LLLI 刺激及添加 SDF-1α 對 CXCR4 siRNA 轉染大鼠骨髓 MSCs 所分泌之 MMP-2 活性之影響 95
圖 4-17 LLLI 刺激及添加 SDF-1α 對CXCR4 siRNA 轉染大鼠骨髓 MSCs 所分泌之 MMP-9 活性之影響 97
圖 4-18 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs TIMP-1 基因表現之影響
98
圖 4-19 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs TIMP-2 基因表現之影響 100
圖 4-20 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs MMP-14 基因表現之影響 102
圖 4-21 LLLI 刺激及 SDF-1α 處理對 FAK siRNA 轉染之大鼠骨髓 MSCs 的 pFAK 和 CXCR4 表現之影響 104
圖 4-22 LLLI 刺激及 SDF-1α 處理對 CXCR4 siRNA 轉染之大鼠骨髓 MSCs 的 pFAK 和 CXCR4 表現之影響 107
圖 4-23 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs pNF-κB 表現之影響 111
圖 4-24 LLLI 處理及添加 SDF-1α 可增強大鼠骨髓 MSCs pNF-κB 的活化並轉位至核內 112
圖 4-25 LLLI 刺激及添加 SDF-1α 對 FAK siRNA 轉染之大鼠骨髓 MSCs pNF-κB 表現之影響 113
圖 4-26 LLLI 處理及添加 SDF-1α 對 CXCR4 siRNA 轉染之大鼠骨髓 MSCs 的 pNF-κB 表現之影響 114
圖 4-27 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs β1-integrin 表現之影響
117
圖 4-28 LLLI 刺激及 SDF-1α 處理 FAK siRNA 轉染之大鼠骨髓 MSCs 的 β1-integrin 表現之影響 118
圖 4-29 LLLI 刺激及 SDF-1α 處理對 CXCR4 siRNA 轉染之大鼠骨髓 MSCs 的 β1-integrin 表現之影響 119
圖 4-30 LLLI 及 SDF-1α 處理 5 min 後，大鼠骨髓 MSCs F-actin 表現之變化情形 122
圖 4-31 LLLI 及 SDF-1α 處理 15 min 後大鼠骨髓 MSCs 形態之變化情形 124
圖 4-32 LLLI 及 SDF-1α 培養 15 min 後，大鼠骨髓 MSCs 之 Cdc42/Rac 之變化情形..................................................................................128
圖 4-33 LLLI 及 SDF-1α 培養 15 min 後，大鼠骨髓 MSCs 之 Rho 蛋白質之變化情形 132
圖 4-34 LLLI 及 SDF-1α 處理 FAK siRNA 轉染之大鼠骨髓 MSCs 5 min 後 F-actin 表現之變化情形 136
圖 4-35 LLLI 及 SDF-1α 處理 CXCR4 siRNA 轉染之大鼠骨髓 MSCs 5 min 後 F-actin 表現之變化情形 140
圖 4-36 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs HSP27 基因表現之影響
145
圖 4-37 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs HSP70 基因表現之影響
146
圖 4-38 LLLI 處理及添加 SDF-1α 對大鼠骨髓 MSCs HSP90 基因表現之影響 147
圖 6-1 LLLI 處理添加 SDF-1α 雙向誘導 rbMSCs 遷移之機制 163


















表索引

表 3-1 實驗設計及量測時間 27
表 3-2 西方墨點法之抗體濃度稀釋參考表 43
表 3-3 標準曲線之 BSA 濃度稀釋表 44
表 3-4 QPCR 反應組成分配製 53
表 3-5 QPCR 機型 ABI7300 Two-step cycling protocol 53
表 3-6 MSCs 之 PCR 所使用的引子核酸序列及反應參數 54

參考文獻

[1]Huang YY, Sharma SK, Carroll J, Hamblin MR, (2011) Biphasic dose response in low level light therapy - an update. Dose-response. 9(4):602-618.
[2]Hou JF, Zhang H, Yuan X, Li J, Wei YJ, Hu SS, (2008) In vitro effects of low-level laser irradiation for bone marrow mesenchymal stem cells: proliferation, growth factors secretion and myogenic differentiation. Lasers Surg Med. 40(10):726-733.
[3]Tsai WC, Hsu CC, Pang JH, Lin MS, Chen YH, Liang FC, (2012) Low-level laser irradiation stimulates tenocyte migration with up-regulation of dynamin II expression. PloS one. 7(5):e38235.
[4]Caplan AI, Dennis JE, (2006) Mesenchymal stem cells as trophic mediators. J Cell Biochem. 98(5):1076-1084.
[5]Yagi H, Soto-Gutierrez A, Parekkadan B, Kitagawa Y, Tompkins RG, Kobayashi N, Yarmush ML, (2010) Mesenchymal stem cells: mechanisms of immunomodulation and homing. Cell transplant. 19(6):667-679.
[6]Li WT, Leu YC, Wu JL, (2010) Red-light light-emitting diode irradiation increases the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells. Photomed Laser Surg. Suppl 1:S157-65.
[7] 許泰毓 (2008) 低能量近紅外光刺激對大鼠骨髓間葉幹細胞生理生化之影響. 碩士論文: 中原大學生物醫學工程研究所.
[8] 陳志威 (2011) 紅光與近紅外光光照刺激大鼠骨髓間葉幹細胞遷移影響及其機制探討. 碩士論文: 中原大學生物醫學工程研究所.
[9]Murdoch C, (2000) CXCR4: chemokine receptor extraordinaire. Immunol rev. 177:175-184.

[10]Wear JO, Ark LR, Williams J, Ala H, (1969) The laster and its biomedical application. South Med J. 62:588-592.
[11]Baratto L, Calza L, Capra R, Gallamini M, Giardino L, Giuliani A, Lorenzini L, Traverso S, (2011) Ultra-low-level laser therapy. Lasers Med Sci. 26(1):103-112.
[12]Posten W, Wrone DA, Dover JS, Arndt KA, Silapunt S, Alam M, (2005) Low-level laser therapy for wound healing mechanism and efficacy. Dermatol Surg. 31(3):334-340.
[13]Prabhu V, Rao SB, Rao NB, Aithal KB, Kumar P, Mahato KK, (2010) Development and evaluation of fiber optic probe-based helium-neon low-level laser therapy system for tissue regeneration--an in vivo experimental study. Photochem Photobiol. 86:1364-1372.
[14]Shukla S, Sahu K, Verma Y, Rao KD, Dube A, Gupta PK, (2010) Effect of helium-neon laser irradiation on hair follicle growth cycle of Swiss albino mice. Skin Pharmacol Physiol. 23(2):79-85.
[15]Houreld N, Abrahamse H, (2010) Low-intensity laser irradiation stimulates wound healing in diabetic wounded fibroblast cells (WS1). Diabetes Technol Ther. 12(12):971-978.
[16]Moriyama Y, Moriyama EH, Blackmore K, Akens MK, Lilge L, (2005) In vivo study of the inflammatory modulating effects of low-level laser therapy on iNOS expression using bioluminescence imaging. Photochem Photobiol. 81(6):1351-1355.
[17]Toomarian L, Fekrazad R, Tadayon N, Ramezani J, Tuner J, (2012) Stimulatory effect of low-level laser therapy on root development of rat molars: a preliminary study. Lasers Med Sci. 27(3):537-542.
[18]Bouvet-Gerbettaz S, Merigo E, Rocca JP, Carle GF, Rochet N, (2009) Effects of low-level laser therapy on proliferation and differentiation of murine bone marrow cells into osteoblasts and osteoclasts. Lasers Surg Med. 41(4):291-297.

[19]Eichler M, Lavi R, Shainberg A, Lubart R, (2005) Flavins are source of visible-light-induced free radical formation in cells. Lasers Surg Med. 37(4):314-319.
[20]Chen AC, Arany PR, Huang YY, Tomkinson EM, Sharma SK, Kharkwal GB, Saleem T, Mooney D, Yull FE, Blackwell TS, Hamblin MR, (2011) Low-level laser therapy activates NF-κB via generation of reactive oxygen species in mouse embryonic fibroblasts. PloS one. 6(7):e22453.
[21] Karu T, (1999) Primary and secondary mechanisms of action of visible to near-IR radiation on cells. J Photochem Photobiol B. 49(1):1-17.
[22]Cantrell DA, (2001) Phosphoinositide 3-kinase signalling pathways. J Cell Sci. 114(8):1439-1445.
[23]Chen CH, Hung HS, Hsu SH, (2008) Low-energy laser irradiation increases endothelial cell proliferation, migration, and eNOS gene expression possibly via PI3K signal pathway. Lasers Surg Med. 40(1):46-54.
[24]Zhang L, Xing D, Gao X, Wu S, (2009) Low-power laser irradiation promotes cell proliferation by activating PI3K/Akt pathway. J Cell Physiol. 219(3):553-562.
[25]Zhang J, Xing D, Gao X, (2008) Low-power laser irradiation activates Src tyrosine kinase through reactive oxygen species-mediated signaling pathway. J Cell Physiol. 217(2):518-528.
[26]Gao X, Chen T, Xing D, Wang F, Pei Y, Wei X, (2006) Single cell analysis of PKC activation during proliferation and apoptosis induced by laser irradiation. J Cell Physiol. 206(2):441-448.
[27]Miyata H, Genma T, Ohshima M, Yamaguchi Y, Hayashi M, Takeichi O, Ogiso B, Otsuka K, (2006) Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation of cultured human dental pulp cells by low-power gallium-aluminium-arsenic laser irradiation. Int Endod J. 39(3):238-244.

[28]Zhang L, Zhang Y, Xing D, (2010) LPLI inhibits apoptosis upstream of Bax translocation via a GSK-3beta-inactivation mechanism. J Cell Physiol. 224(1):218-228.
[29]Liu ZJ, Zhuge Y, Velazquez OC, (2009) Trafficking and differentiation of mesenchymal stem cells. J Cell Biochem. 106(6):984-991.
[30]Zhang D, Huang W, Dai B, Zhao T, Ashraf A, Millard RW, Ashraf M, Wang Y, (2010) Genetically manipulated progenitor cell sheet with diprotin A improves myocardial function and repair of infarcted hearts. Am J Physiol Heart Circ Physiol. 299(5):H1339-H1347.
[31]Wagner J, Kean T, Young R, Dennis JE, Caplan AI, (2009) Optimizing mesenchymal stem cell-based therapeutics. Curr Opin Biotechnol. 20(5):531-536.
[32]Ito H, (2011) Chemokines in mesenchymal stem cell therapy for bone repair: a novel concept of recruiting mesenchymal stem cells and the possible cell sources. Mod Rheumatol. 21(2):113-121.
[33]Li L, Jiang J, (2011) Regulatory factors of mesenchymal stem cell migration into injured tissues and their signal transduction mechanisms. Front Med. 5(1):33-9.
[34]Ganju RK, Brubaker SA, Meyer J, Dutt P, Yang Y, Qin S, Newman W, Groopman JE, (1998) The alpha-chemokine, stromal cell-derived factor-1alpha, binds to the transmembrane G-protein-coupled CXCR-4 receptor and activates multiple signal transduction pathways. J Biol Chem. 273(36):23169-23175.
[35]Zlotnik A, Yoshie O, (2000) Chemokines a new classification system and their role in immunity. Immunity. 12:121–127.
[36]Shirozu M, Nakano T, Inazawa J, Tashiro K, Tada H, Shinohara T, Honjo T, (1995) Structure and Chromosomal Localization of the Human Stromal Cell Derived Factor 1 (SDF1) Gene. Genomics. 28:495-500.


[37]Ahr B, Denizot M, Robert-Hebmann V, Brelot A, Biard-Piechaczyk M, (2005) Identification of the cytoplasmic domains of CXCR4 involved in Jak2 and STAT3 phosphorylation. J Biol Chem. 280(8):6692-6700.
[38]Sharma M, Afrin F, Satija N, Tripathi RP, Gangenahalli GU, (2011) Stromal-derived factor-1/CXCR4 signaling: indispensable role in homing and engraftment of hematopoietic stem cells in bone marrow. Stem Cells Dev. 20(6):933-946.
[39] Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P, (2008) Molecular Biology of the Cell, 5th Edition. New York:Garland Science.
[40]Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR, (2003) Cell migration: integrating signals from front to back. Science. 302(5651):1704-1709.
[41]Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT, (1992) pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci. 89(11):5192-5196.
[42]Mitra SK, Hanson DA, Schlaepfer DD, (2005) Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol. 6(1):56-68.
[43]Yun SP, Ryu JM, Han HJ, (2011) Involvement of beta1-integrin via PIP complex and FAK/paxillin in dexamethasone-induced human mesenchymal stem cells migration. J Cell Physiol. 226(3):683-692.
[44]Cox BD, Natarajan M, Stettner MR, Gladson CL, (2006) New concepts regarding focal adhesion kinase promotion of cell migration and proliferation. J Cell Biochem. 99(1):35-52.
[45]Wu X, Suetsugu S, Cooper LA, Takenawa T, Guan JL, (2004) Focal adhesion kinase regulation of N-WASP subcellular localization and function. J Biol Chem. 279(10):9565-9576.



[46] Meenderink LM, Ryzhova LM, Donato DM, Gochberg DF, Kaverina I, Hanks SK. (2010) P130Cas Src-binding and substrate domains have distinct roles in sustaining focal adhesion disassembly and promoting cell migration. PLoS One. 5(10):e13412.
[47]Esparza J, Vilardell C, Calvo J, Juan M, Vives J, Urbano-Márquez A, Yagüe J, Cid MC, (1999) Fibronectin upregulates gelatinase B (MMP-9) and induces coordinated expression of gelatinase A (MMP-2) and its activator MT1-MMP (MMP-14) by human T lymphocyte cell lines. A process repressed through RAS/MAP kinase signaling pathways. Blood. 94(8):2754-66.
[48]Visse R, Nagase H, (2003) Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry.
Circ Res. 92(8):827-839.
[49]Das A, Yaqoob U, Mehta D, Shah VH, (2009) FXR promotes endothelial cell motility through coordinated regulation of FAK and MMP-9. Arterioscler Thromb Vasc Biol. 29(4):562-570
[50]Cheung PY, Sawicki G, Wozniak M, Wang W, Radomski MW, Schulz R, (2000) Matrix Metalloproteinase-2 Contributes to ischemia-reperfusion injury in the heart. Circulation. 101:1833-1839..
[51]Itoh Y, (2006) MT1-MMP: a key regulator of cell migration in tissue. IUBMB life. 58(10):589-596.
[52]Ries C, Egea V, Karow M, Kolb H, Jochum M, Neth P, (2007) MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines. Blood. 109(9):4055-4063.
[53]Ferns G, Shams S, Shafi S, (2006) Heat shock protein 27: its potential role in vascular disease. Int J Exp Pathol. 87(4):253-274.



[54]Lee JW, Kwak HJ, Lee JJ, Kim YN, Park MJ, Jung SE, Hong SI, Lee JH, Lee JS, (2008) HSP27 regulates cell adhesion and invasion via modulation of focal adhesion kinase and MMP-2 expression. Eur J Cell Biol. 87(6):377-387.
[55]Wegele H, Muller L, Buchner J, (2004) Hsp70 and Hsp90--a relay team for protein folding. Rev Physiol Biochem Pharmacol. 151:1-44.
[56]Koyasu S, Nishida E, Kadowaki T, Matsuzaki F, Iida K, Harada F, Kasuga M, Sakai H, Yahara I, (1986) Two mammalian heat shock proteins, HSP90 and HSP100, are actin-binding proteins. Proc Natl Acad Sci U S A. 83(21):8054-8058.
[57]Simard JP, Reynolds DN, Kraguljac AP, Smith GS, Mosser DD, (2011) Overexpression of HSP70 inhibits cofilin phosphorylation and promotes lymphocyte migration in heat-stressed cells. J Cell Sci. 124(14):2367-2374.
[58] Picard D, (2004) Hsp90 invades the outside. Nat Cell Biol. 6(6) :479-480.
[59] Walsh N, Larkin AM, Swan N, Conlon K, Dowling P, Mcdermott R,
Clynes M, (2011) RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation. Cancer Lett.
306:180-189.
[60]Zou C, Luo Q, Qin J, Shi Y, Yang L, Ju B, Song G, (2012) Osteopontin promotes mesenchymal stem cell migration and lessens cell stiffness via integrin beta1, FAK, and ERK pathways. Cell Biochem Biophys. 65(3):455-462.
[61] Song X, Wang X, Zhuo W, Shi H, Feng D, Sun Y, Liang Y, Fu Y, Zhou D, Luo Y, (2010) The regulatory mechanism of extracellular Hsp90 on matrix metalloproteinase-2 processing and tumor angiogenesis. J Biol Chem. 285(51):40039-40049.
[62] Picard D, (2004) Hsp90 invades the outside. Nat Cell Biol. 6(6):479-480.
[63] Tang CH, Tan TW, Fu WM, Yang RS, (2008) Involvement of matrix metalloproteinase-9 in stromal cell-derived factor-1/CXCR4 pathway of lung cancer metastasis. Carcinogenesis. 29(1):35-43.
[64] Rehman AO, Wang CY, (2009) CXCL12/SDF-1α Activates NF-κB and promotes oral cancer invasion through the Carma3/Bcl10/Malt1 complex. Int J Oral Sci. 1(3):105-118.
[65] Annunziata CM, Stavnes HT, Kleinberg L, Berner A, Hernandez LF, Birrer MJ, Steinberg SM, Davidson B, Kohn EC, (2010) NF-κB transcription factors are co-expressed and convey poor outcome in ovarian cancer. Cancer. 116(13):3276-3284.
[66] Lu DY, Tang CH, Yeh WL, Wong KL, Lin CP, Chen YH, Lai CH, Chen YF, Leung YM, Fu WM, (2009) SDF-1alpha up-regulates interleukin-6 through CXCR4, PI3K/Akt, ERK, and NF-kappaB-dependent pathway in microglia. Eur J Pharmacol. 613:146-154.
[67] Oron U, Maltz L, Tuby H, Sorin V, Czerniak A, (2010) Enhanced liver regeneration following acute hepatectomy by low-level laser therapy. Photomed Laser Surg. 28(5):675-678.
[68] Tuby H, Maltz L, Oron U, (2011) Induction of autologous mesenchymal stem cells in the bone marrow by low-level laser therapy has profound beneficial effects on the infarcted rat heart. Laser Surg Med. 43:401-409.



[69] Takino T, Sato H, Shinagawa A, Seiki M, (1995) Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library. MT-MMPs form a unique membrane-type subclass in the MMP family. J Biol Chem. 270(39):23013-23020.
[70]Sen R, Baltimore D, (1986) Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism. Cell. 47(6):921-8.
[71]Hoffmann A, Levchenko A, Scott ML, Baltimore D, (2002) The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation. Science. 298(5596):1241-1245.
[72]Pujalte I, Passagne I, Brouillaud B, Treguer M, Durand E, Ohayon-Courtes C, L'Azou B, (2011) Cytotoxicity and oxidative stress induced by different metallic nanoparticles on human kidney cells. Part Fibre Toxicol. 8:10.
[73]Ryu CH, Park SA, Kim SM, Lim JY, Jeong CH, Jun JA, Oh JH, Park SH, Oh WI, Jeun SS, (2010) Migration of human umbilical cord blood mesenchymal stem cells mediated by stromal cell-derived factor-1/CXCR4 axis via Akt, ERK, and p38 signal transduction pathways. Biochem Biophys Res Commun. 398(1):105-110.
[74]Rosazza C, Escoffre JM, Zumbusch A, Rols MP, (2011) The actin cytoskeleton has an active role in the electrotransfer of plasmid DNA in mammalian cells. Mol Ther. 19(5):913-921.
[75]Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC, (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391(6669):806-811.
[76]Hammond SM, (2005) Dicing and slicing: the core machinery of the RNA interference pathway. FEBS Lett. 579(26):5822-5829.


[77] Huang YC, Hsiao YC, Chen YJ, Wei YY, Lai TH, Tang CH, (2007) Stromal cell-derived factor-1 enhances motility and integrin up-regulation through CXCR4, ERK and NF-κB-dependent pathway in human lung cancer cells. Biochem Pharmacol. 74:1702-1712.
[78] Huang D, Khoe M, Befekadu M, Chung S, Takata Y, Ilic D, Bryer-Ash M, (2007) Focal adhesion kinase mediates cell survival via NF-κB and ERK signaling pathways. Am J Physiol Cell Physiol. 292:C1339-C1352.
[79] Patricia AM, Snoek-van B, Von den Hoff JW, (2005) Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. BioTechniques. 38:73-83.
[80]Xie TX, Wei D, Liu M, Gao AC, Ali-Osman F, Sawaya R, Huang S, (2004) Stat3 activation regulates the expression of matrix metalloproteinase-2 and tumor invasion and metastasis. Oncogene. 23(20):3550-3560.
[81] Gilmore AP, Romer LH, (1996) Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Mol. Biol. Cell. 7:1209-1224.
[82]Wang K, Zhao X, Kuang C, Qian D, Wang H, Jiang H, Deng M, Huang L, (2012) Overexpression of SDF-1alpha Enhanced Migration and Engraftment of Cardiac Stem Cells and Reduced Infarcted Size via CXCR4/PI3K Pathway. PloS one. 7(9):e43922.
[83] Tang CH, Chuang JY, Fong YC, Maa MC, Way TD, Huang CH, (2008) Bone-derived SDF-1 stimulates IL-6 release via CXCR4, ERK and NF-κB pathways and promotes osteoclastogenesis in human oral cancer cells. Carcinogenesis. 29(8):1483-1492.

[84]Yu X, Chen D, Zhang Y, Wu X, Huang Z, Zhou H, Zhang Y, Zhang Z, (2012) Overexpression of CXCR4 in mesenchymal stem cells promotes migration, neuroprotection and angiogenesis in a rat model of stroke. J Neurol Sci. 316(1-2):141-149.
[85]Wynn RF, Hart CA, Corradi-Perini C, O'Neill L, Evans CA, Wraith JE, Fairbairn LJ, Bellantuono I, (2004) A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood. 104(9):2643-2645.
[86] Shen Y, Ma Y, Gao M, Lai Y, Wang G, Yu Q, Cui F, Liu X, (2013) Integrins-FAK-Rho GTPases pathway in endothelial cells sense and
response to surface wettability of plasma nanocoatings. ACS Appl. Mater. Interfaces. 5:5112-5121.
[87] Hanna S, El-Sibai M, (2013) Signaling networks of Rho GTPases in cell motility. Cell Signal. 25(10):1955-1961.
[88]Ilić D, Furuta Y, Kanazawa S, Takeda N, Sobue K, Nakatsuji N, Nomura S, Fujimoto J, Okada M, Yamamoto T, (1995) Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature. 377:539-544.
[89] Moyer RA, Wendt MK, Johanesen PA, Turner JR, Dwinell MB, (2007) Rho activation regulates CXCL12 chemokine stimulated actin rearrangement and restitution in model intestinal epithelia. Lab Invest. 87(8):807-817.
[90] Adiguzel E, Hou G, Sabatini PJB, Bendeck MP, (2013) Type VIII collagen signals via β1 integrin and RhoA to regulate MMP-2 expression and smooth muscle cell migration. Matrix Biol. 32(6):332-341.

[91] 呂曜竹 (2007) 紅光發光二極體光照對大鼠骨髓間葉幹細胞生長及分化之影響. 碩士論文: 中原大學生物醫學工程研究所.
[92] Liu X, Zhou C, Li Y, Ji Y, Xu G, Wang X, Yan J, (2013) SDF-1 promotes endochondral bone repair during fracture healing at the traumatic brain injury condition. PloS one. 8(1):e54077.
[93] Ferrand J, Lehours P, Schmid-Alliana A, Me´graud F, Varon C, (2011) Helicobacter pylori Infection of gastrointestinal epithelial cells in vitro induces mesenchymal stem cell migration through an NF-κB-dependent Pathway. PloS one. 6(12):e29007.
[94] Kawai K, Xue F, Takahara T, Kudo H, Yata Y, Zhang W, Sugiyama T, (2012) Matrix metalloproteinase-9 contributes to the mobilization of bone marrow cells in the injured liver. Cell Transplant. 21:453-464.
[95] Cao J, Wang L, Du ZJ, Liu P, Zhang YB, Sui JF, Liu YP, Lei DL, (2013) Recruitment of exogenous mesenchymal stem cells inmandibular distraction osteogenesis by the stromalcell-derived factor-1/chemokine receptor-4 pathway in rats. Brit J Oral Max Surg. 51:1-5.
[96] Guang LG, Boskey AL, Zhu W, (2013) Age-related CXC chemokine receptor-4-deficiency impairs osteogenicdifferentiation potency of mouse bone marrow mesenchymal stromalstem cells. Int J Biochem Cell Biol. 46:1-8.
[97] Liu N, Tian J, Cheng J, Zhang J, (2013) Directional migration of CXCR4 gene-modified bone marrow-derived mesenchymal stem cells to the kidney area after acute kidney injury. J Cell Biochem. 114(9):1-39.