比較不同頻率低強度脈衝式超音波刺激對小鼠幹細胞生長與分化之影響

Comparison of Different Frequencies of Low Intensity Pulsed Ultrasound Stimulation on C3H10T1/2 Stem Cell Growth and Differentiation

宋虹瑩(Horng-Ying Song)

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/cycu201100839

摘要

幹細胞的增生分化與身體組織的再生修復有著密不可分的關聯性。而骨細胞的生成，可由骨骼中的間質幹細胞增生及分化所產生。近幾年來，低頻超音波刺激幹細胞之相關研究，已證實超音波刺激確實能夠有效促進細胞增生及分化，但卻少有研究比較不同頻率超音波對幹細胞增生與分化之影響。本研究以兩種單一頻率：1 MHz (SU1)、或3 MHz (SU3)及兩種交錯變頻頻率：先以1 MHz刺激，再以3 MHz刺激 (DU1/3)、或先以3 MHz刺激，再以1 MHz刺激 (DU3/1)的低強度脈衝式超音波(Low Intensity Pulsed Ultrasound, LIPUS)刺激小鼠間質幹細胞(C3H10T1/2)，首先以不同胎牛血清濃度百分比的細胞培養條件，找出評估不同頻率低強度超音波刺激對幹細胞增生促進的較佳實驗模式。接著，以細胞活性檢測(MTT)、蛋白質定量(Bicinchoninic Acid, BCA)、茜紅素S染色(Alizarin Red Stain)及鹼性磷酸酵素活性分析(Alkaline Phosphatase Activity Stain, ALP)來評估幹細胞增生及骨分化的細胞生理效應。最後並以聚合酶連鎖反應分析(Polymerase Chain Reaction，PCR)來深入探討不同超音波頻率刺激幹細胞於骨分化基因表現的響應。本研究結果顯示變頻組DU1/3促進幹細胞增生及分化的表現最為顯著，此頻率對於幹細胞增生及骨分化有顯著的促進作用。期望藉由本實驗結果提供更佳的超音波刺激模式應用，不論是對幹細胞的增生與骨分化促進或是應用於臨床上骨癒合的理論依據。

關鍵字

低強度脈衝超音波 ； 幹細胞 ； 細胞增生 ； 骨分化

並列摘要

Stem cell proliferation and differentiation play important roles on tissue regeneration. Osteogenesis of mesenchymal stem cells is the main process in bone formation. In recent years, there are more and more studies focusing on low intensity pulsed ultrasound (LIPUS) for mesenchymal stem cells differentiation, but few studies explore effects of ultrasound frequency on stem cell proliferation or differentiation. The purpose of this study is to check the frequency effects on stem cell growth. Two different single ultrasound frequency: 1 MHz (SU1) or 3 MHz (SU3) and two dynamic varying frequencies: 1 MHz stimulation following with 3 MHz (DU1/3) or 3 MHz stimulation following with 1 MHz (DU3/1) of low intensity ultrasound were used for mesenchymal stem cells treatments. Firstly, we found a proper condition for frequency response of cells on LIPUS treatment by using different percentages of fetal bovine serum (FBS) of culture medium. Secondly, proliferation and bone differentiation of stem cells were characterized by MTT, bicinchoninic acid, Alizarin red stain and alkaline phosphatase activity assay. Finally, osteogenetic gene expression (ALP, Con I, Runx2, OCN, GAPDH) of stem cells after LIPUS treatments will be investigated in detail using Polymerase Chain Reaction. The better FBS concentration of culture medium for LIPUS response evaluation on stem cell growth is 2.5%. LIPUS with varying frequencies (DU1/3) was found to enhance early differentiation of bone marrow mesenchymal stem cells significantly. The better varying frequency pattern of LIPUS for stem cell proliferation and osteogenesis differentiation has been proposed and it will be very helpful for future fracture healing acceleration in clinical.

並列關鍵字

low intensity pulsed ultrasound stimulation (LIPUS) ； stem cells ； proliferation ； osteogenesis

參考文獻

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