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

The Physiological and Biochemical Effect of Low-level Near-Infrared Irradiation on the Mesenchymal Stem Cells Derived from Rat Bone Marrow

許泰毓(Tai-Yu Hsu)

指導教授 ： 李文婷

中原大學/工學院/生物醫學工程學系/碩士(2008年)

https://doi.org/10.6840/cycu200900334

摘要

低功率光照刺激 ( Low-level light irradiation ，LLLI ) 可引起光生物效應，如：促進細胞增生、ATP 合成量增加、粒線體膜電位改變、活化細胞週期調控蛋白等。本研究旨在探討低能量近紅外光照射對大鼠骨髓間葉幹細胞增生及分化之影響。使用光源為 850 nm 的近紅外光發光二極體，參數為功率密度 10 或 15 mW/cm2，能量劑量為 2 或 4 J/cm2，刺激頻率為單次及多次照射。為證實造成間葉幹細胞的增生或抑制是因光化學效應而非熱效應，針對光照前後磷酸鹽緩衝溶液及培養液的溫度變化進行量測，發現光照對兩種溶液的溫度並沒有很明顯的影響，溫度變化落在 26.1 至 26.3 oC 間。大鼠間葉幹細胞在10 mW/cm2，2 及 4 J/cm2 的多次光照刺激下，可促進細胞增生，且以 4 J/cm2 的多次刺激為最佳，故以此做為本研究之最佳參數。在此參數刺激下，可促進大鼠間葉幹細胞硬骨分化之鹼性磷酸酶活性及骨鈣素分泌，並抑制其脂肪分化時之三酸甘油脂累積，光照刺激對脂肪分化轉錄因子PPARγ 之基因表現亦有抑制之作用。於細胞生理生化部份，發現光照刺激一方面可抑制活性氧分子的產生、降低老化相關 β-半乳糖酶的活性；同時可促進 ATP 產生，且不會造成 LDH 釋放率增加。於基因表現部份則發現光照刺激可使熱休克蛋白 hsp70基因表現增加，但對 c-fos 的表現則無明顯差異。縱上所述，10 mW/cm2 及 4 J/cm2 近紅外光發光二極體照射可促進大鼠骨髓間葉幹細胞增生、提升其分化成硬骨細胞同時抑制其分化成脂肪細胞；且光照刺激可使胞內 ATP 含量增加、減少活性氧分子產生，具有延緩老化之作用。期望未來可建立體外光照模式，提升臨床醫學幹細胞療法之成效。

關鍵字

低功率光照刺激 ； 硬骨分化 ； 細胞增生 ； 脂肪分化 ； 發光二極體 ； 骨髓間葉幹細胞 ； 近紅外光

並列摘要

Low-level laser irradiation is a physical treatment, which can lead to cell proliferation, increase of ATP synthesis, change in mitochondrial membrane potential and the induction of the synthesis of cell cycle regulatory proteins. The propose of the study was to explore the effects on proliferation and differentiation of mesenchymal stem cells (MSCs) derived from rat bone marrow using near-infrared (850 nm) light emitting diodes (LEDs) irradiation. Four different parameters of irradiation (irradiance: 10 or 15 mW/cm2; radiant exposure: 2 or 4 J/cm2), were used to stimulate MSCs once per day or every other day. To demonstrate the effect of biomodulation was due to photochemical effect, the temperatures of PBS and culture medium during irradiation were monitored. No apparent change in temperature (26.1 to 26.3 oC) was found. The result showed that the proliferation of MSCs was promoted at the irradiance of 10 mW/cm2 and radiant exposures of 2 and 4 J/cm2, particularly at 10 mW/cm2 and 4 J/cm2. The parameter was used in the following research. It was found that alkaline phosphatase activity and osteocalcin secretion were promoted during osteoblastogenesis while triacylglycerol accumulation was inhibited during adipogenesis in the MSCs treated with LLLI. The expression level of adipogenic transcription factor, PPARγ, was down-regulated. LLLI not only decreased the production of reactive oxygen species but also the activity of senescence associated β-galactosidase. Meanwhile, the level of intracellular ATP was found to increase after LLLI. No apparent cellular damage was seen after LLLI by LDH release assay. In addition, the expression level of heat shock protein hsp70 but not c-fos was enhanced. To sum up, near infrared LED irradiation with combined irradiance of 10 mW/cm2 and the light dosage of 4 J/cm2 could enhance cell proliferation and osteogenic capability of MSCs. Adipogenic capability of MSCs was reduced after LLLI. LLLI may have anti-aging affects by increasing intracellular ATP level and decreasing ROS production. An irradiation device for LLLI used in vivo will be under development in order to improve the efficacy of stem cell therapy in clinical treatments.

並列關鍵字

lightemitting diode ； near infrared ； mesenchymal stem cell ； adipogenic differentiation ； cell proliferation ； osteogenic differentiation ； low-level light irradiation

參考文獻

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