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標題: | 低能量紅光照射對小鼠骨髓間葉幹細胞之影響 The Effect of Low Energy Red Light Irradiation on Mouse Bone Marrow Mesenchymal Stem Cell Fate |
作者: | Yen-Hua Lee 李妍樺 |
指導教授: | 吳信志 |
關鍵字: | 光療法,小鼠骨髓間葉幹細胞,細胞老化,骨質疏鬆症, phototherapy,mouse bone marrow mesenchymal stem cells,cell senescence,osteoporosis, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 間葉幹細胞(mesenchymal stem cell) 位於骨髓、脂肪等成體組織中,具有複能性分化潛能 (multipotentcy) 和自我更新(self-renew)的能力,因此可做為治療某些先天性疾病或退化性疾病的細胞來源。其在骨髓內所佔比例約為0.003%,要應用於臨床治療必須透過體外培養擴增,然而,間葉幹細胞之增殖 (proliferation)、分化 (differentiation) 及遷移 (migration) 能力隨體外培養繼代次數而下降,且其治療效果會受細胞移入體內後增生、遷移及分化之能力影響。目前已有研究證實,特定波長的低能量光照可影響細胞增生、移動、貼附等能力。為探討低能量光照對小鼠骨髓間葉幹細胞之影響,以提升其臨床應用之療效,本試驗採用波長650 nm之紅光磷化銦鎵鋁 (AlGaInP) 發光二極體為光源,以功率密度 (power density) 5.09 mW/cm2,不同能量密度 (包括:0, 0.3, 0.9或1.5 J/cm2) 照射,分別觀察其照射後細胞之增生及遷移與脂肪、硬骨分化及細胞衰老等情形。以噻唑藍(3-(4, 5-dimethylithiazol-2-yl)-2, 5-diphenyl tetrazolium bromide, MTT)比色法進行細胞增生之分析,傷口癒合測試 (wound healing assay) 測量其遷移能力,並以油紅染色及茜素紅染色定量脂肪及硬骨分化情形。結果顯示經0.3~1.5 J/cm2之紅光照射之處理組,其增殖能力皆顯著高於未照光之控制組。經紅光照射後以硬骨分化液誘導分化三天,經能量密度0.9 J/cm2之間葉幹細胞,其硬骨分化形態較其他處理組成熟,其骨橋蛋白 (osteopontin) mRNA表現量上升40%,但經茜素紅色定量結果顯示,其鈣堆積情形和其他處理組間並無顯著差異。光照後以脂肪分化液誘導分化九天後,其油紅色定量結果顯示,細胞經紅光照射者其油滴堆積情形與控制組無顯著差異 (p > 0.05)。經能量密度0, 0.3, 0.9或1.5 J/cm2之低能量紅光照射十二小時後,其細胞遷移區域依序分別為28.2±5.9, 37.6±5.3, 44±3.2, 41.4±3.0%,但各處理組間無顯著差異 (p > 0.05)。測量端粒長度、端粒酶活性、細胞週期抑制因子p16及p21,以評估間葉幹細胞經低能量紅光照射後之老化情形。實驗結果顯示,經低能量紅光照射之間葉幹細胞,其老化情形和未照射之控制組間無顯著差異 (p > 0.05)。活體實驗部分,將經紅光照射之小鼠骨髓間葉幹細胞以脛骨髓內注射方式注入骨質疏鬆症小鼠骨髓,四週後注射未照射紅光間葉幹細胞之小鼠其骨體積及骨小樑數目顯著高於注射經紅光照射間葉幹細胞之處理組 (p < 0.05)。由此可知,經紅光照射後小鼠骨髓間葉幹細胞可能會抑制骨小樑之再生。
綜合上述,於體外培養時,低能量紅光照射可顯著促進小鼠骨髓間葉幹細胞增生,且有提升細胞遷移、硬骨及脂肪分化能力之趨勢。此外,以此條件照射小鼠骨髓間葉幹細胞並不會增加其老化速度。然而將已照射之小鼠骨髓間葉幹細胞做為治療骨質疏鬆症之細胞來源,則會削減其對於骨再生之功能,此結果提示低能量紅光療法可能不適合應用於骨質疏鬆症之間葉幹細胞治療。 Due to their differentiation potential and self-renewal ability, mesenchymal stem cells (MSCs) can be applied for the treatment of some congenital or degenerative diseases. The efficacy of grafted MSCs is limited in proliferation, migration and differentiation ability. In addition, it is rare, only one MSC in 3.4 × 104 bone marrow cells. It is essential to culture and expand MSCs in vitro before putting them to therapeutic use. However, these abilities will decrease during in vitro culture process. Low energy red light irradiation was proved to be able to stimulate cell growth, migration, and adhesion. In order to validate the effect of low energy red light irradiation on mouse bone marrow MSCs proliferation, migration, adipogenic and osteogenic differentiation. MSCs were exposed to aluminum gallium indium phosphide light emitting diode at wavelength of 650 nm with different energy density (0, 0.3, 0.9 or 1.5 J/cm2, respectively). Cell proliferation was evaluated by using 3-(4, 5-dimethylithiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cell migration ability was tested by wound healing assay. Adipogenic and osteogenic differentiation were analyzed by Oil Red O staining and Alizarin red staining and quantified by soluble dye measurement. The results showed that the cell number was significantly increased at day 4 in all irradiated groups (p < 0.05). In the wound healing assay, 12 hours after irradiation at the energy density of 0, 0.3, 0.9 or 1.5 J/cm2, the percentage of migration area was 28.2 ± 5.9, 37.6 ± 5.3, 44 ± 3.2, 41.4 ± 3.0%, respectively. To further investigate the differentiation ability, MSCs were incubated with adipogenic induction medium after low energy red light irradiation for 9 days. The oil droplets accumulation in irradiated groups were no significant different than control groups. After 3 days of osteogenic induction, the osteopontin mRNA expression was 40 % higher than control. However, there was no significant discrepancy (p>0.05) in calcium accumulation. To elucidate the cell senescence condition after low energy red light irradiation. We test the telomere length, telomerase activity, cell cycle inhibitor p16 and p21 gene expression. The results showed that there was no significant aging found in these cells after they had exposed to red light irradiation. To further confirm the above investigated results, we transplanted the low energy red light irradiated mouse bone marrow MSCs into osteoporosis model mouse to validate the effect in vivo. The treatment group that injecting MSCs could increase the bone volume and trabecular bone number. Surprisingly, the bone density in the group of injecting the irradiated MSCs could not be increased. The reason why the low energy red light irradiation result in significant decrease of the MSCs function needs further investigation to clarify. In summary, low energy red light irradiation can stimulate mouse bone marrow MSCs proliferation, and slightly increase their differentiation and migration abilities in vitro. Moreover, the low energy red light irradiation won’t accelerate cell senescence. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10400 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 動物科學技術學系 |
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