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

The Study of the Effect and Mechanism of Red and Near Infrared Light Illumination on the Migration of Rat Bone Marrow Mesenchymal Stem Cells

陳志威(Chih-wei Chen)

指導教授 ： 李文婷

中原大學/工學院/生物醫學工程研究所/碩士(2011年)

https://doi.org/10.6840/cycu201101103

摘要

先前本研究團隊發現低能量光照 (Low level light irradiation, LLLI) 刺激能有效促進大鼠骨髓間葉幹細胞 (rat bone marrow mesenchymal stem cells, rbMSCs) 的增生及硬骨分化能力。本研究目的在探討先前最佳刺激rbMSCs之生長參數，以釐清 LLLI 刺激是否能促進rbMSCs遷移及相關機轉。所採用的刺激光源為紅光 (630 nm) 與近紅外光 (850 nm) 之發光二極體 (Light emitting diodes, LEDs)，分別以功率密度 15 mW/ cm2、10 mW/ cm2，能量密度 4 J/ cm2進行刺激。細胞增生結果發現，近紅外光組於第 4 天的細胞數較控制組增加了 27.1 %，而紅光組則較控制組增加5.6 %；紅光與近紅外光光照刺激能促使大細胞群落 (> 0.5 cm) 的生成。細胞癒合與 Transwell 遷移分析指出，不論是紅光或近紅外光光照均可促進細胞遷移，近紅外光光照較紅光光照效果更為顯著，添加 NF-κB抑制劑會抑制紅光和近紅光光照所提升的跨膜遷移能力。分析光照後短期間細胞內生理的變化，發現光照會提生細胞色素 c 氧化酶的氧化能力，使粒腺體膜電位上升，ATP 含量上升，活性氧分子含量顯著增加。基因表現分析發現，紅光及近紅光光照刺激會促進熱休克蛋白 70表現，抑制 TIMP-1 與 TIMP-2表現。蛋白質分析發現，光照刺激後會提升 pFAK 的表現，使細胞提早分泌MMP-2 及 MMP-9。本研究證實rbMSC 經過紅光及近紅光光照刺激後，粒腺體上的光感受器細胞色素 c 氧化酶活性增加，造成粒線體膜電位上升，促進ATP生合成，同時伴隨著 ROS 的產生增加，活化 NF-κB。ATP 合成增加可活化 HSP 70 ，透過 NF-κB 訊息傳導路徑，促使細胞增生。ATP 合成增加也可促使 FAK 磷酸化，抑制 TIMPs 並提高 MMPs 的活性，提升細胞移動能力。

關鍵字

細胞遷移 ； 低能量光照 ； 發光二極體 ； 大鼠骨髓間葉幹細胞

並列摘要

Low level light irradiation (LLLI) was shown to enhance the proliferation and osteogenic capability of rat bone marrow mesenchymal stem cells (rbMSCs) previously. The purposes of the study were to understand whether LLLI can promote rbMSCs migration using the optimal parameters for cell proliferation and to elucidate the mechanism of rbMSCs migration induced by LLLI. Red (630 nm, 15 mW/ cm2) and near infrared (NIR, 850 nm, 10 mW/ cm2) light emitting diodes (LEDs) were used as light sources. Cells were irradiated at radiant exposure of 4 J/ cm2 of red and NIR light, respectively. The result of cell proliferation assay showed that the number of rbMSCs with NIR illumination increased 27 %, whereas the number of cells with red light irradiation increased 5.6%, compared to non-irradiated control group at the fourth day post-irradiation. The result of CFU-F assay showed that the number of large-size colonies (> 0.5 cm) increased after red and NIR light exposure. The results of transwell migration and wound healing assays showed that both red and NIR light irradiation could enhance cell migration, especially for near infrared. The addition of NF-κB inhibitor decreased the transmembrane migration enhanced by red and NIR light irradiation. We further observed the short term effect after LLLI and found that intracellular cytochrome c oxidase activity, mitochondrial membrane potential, ATP level and reactive oxygen species (ROS) production were enhanced immediately after illumination. Gene expression study demonstrated that red and NIR light irradiation increased HSP 70 expression, and reduced TIMP-1 and TIMP-2 expression. Protein expression study demonstrated that pFAK expression was elevated, and the secretion of MMP-2 and MMP-9 was facilitated after LLLI. The study demonstrated that red and NIR LLLI could enhance mitochondrial photoacceptor-cytochrome c oxidase activity, mitochondrial membrane potential, intracellular ATP level synthesis and ROS level in rbMSCs, thereby activating NF-κB. The increase of ATP synthesis led to HSP 70 activation and thus cell proliferation was enhanced through NF-κB signal pathway. The increase of ATP synthesis also resulted in enhanceing cell migration via phosphorylation of FAK protein, reducing TIMPs expression and activating MMPs activities.

並列關鍵字

cell migration ； light emitting diode ； rat bone marrow mesenchymal stem cell ； low level light irradiation

參考文獻

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