以變頻低強度脈衝式超音波調控小鼠胚胎幹細胞增生與骨分化作用

Modulation of Proliferation and Osteogenesis of Murine Embryonic Stem Cells using Low Intensity Pulsed Ultrasound (LIPUS) with Varying Frequencies

簡霈熏(Pei-Hsun Chien)

指導教授 ： 張恆雄

中原大學/工學院/生物醫學工程研究所/碩士(2010年)

https://doi.org/10.6840/cycu201000821

摘要

過去的臨床與動物實驗研究證實，特定參數超音波能促進骨折癒合，且在體外細胞培養實驗中顯示，多種細胞(包括幹細胞)能在適當的超音波刺激下會促進其增生或分化。文獻指出，不同頻率超音波對骨折癒合的促進效應不同；在骨折癒合不同階段施予同一頻率超音波刺激對於骨折癒合有不同的促進效果；不同頻率的超音波對於不同細胞的刺激作用不同。幹細胞在組織再生過程中扮演重要角色，近年來，超音波已被證實在特定參數下能有效促進幹細胞的增生與分化。因此，超音波調控幹細胞生長可能是促進骨折癒合的關鍵因素之一，且不同頻率的超音波對於不同生長時期的幹細胞應有不同程度的作用。
本實驗使用一般與骨分化兩種培養基來模擬不同小鼠幹細胞(C3H10T1/2)生長環境，探討不同頻率低強度脈衝式超音波(low intensity pulsed ultrasound，LIPUS)在這兩種環境下，促進細胞增生與骨分化的最佳頻率，提出不同頻率超音波於不同幹細胞生長環境與生長階段的刺激模式，進而驗證變頻低強度脈衝式超音波調控幹細胞增生與骨分化作用之可行性，期能作為將來變頻低強度超音波促進骨折癒合刺激模式的理論依據。實驗中細胞的增生情形以Trypan blue定量評估；細胞的骨分化表現則以鹼性磷酸酵素活性分析。而定性方面則是以Alizarin Red S與ALP染色法來判定幹細胞進行骨分化之特性；由實驗結果來看，較高頻率(3MHz)之低強度脈衝式超音波刺激在細胞增生時期具有較佳的促進細胞增生作用；而較低頻率(1MHz)之低強度脈衝式超音波刺激則在細胞分化時期具有較佳的促進細胞分化作用。初步表明了變頻超音波促進癒合之可行性。

關鍵字

細胞增生 ； 幹細胞 ； 變頻式低強度脈衝超音波 ； 骨分化作用

並列摘要

Ultrasound had been shown to be a potent method for bone fracture healing acceleration and induction in vitro, in vivo and in clinical for many years. Different frequency of ultrasound has different effects on different fracture healing period and cell types. In our previous data, it showed that varying frequencies pattern of ultrasound would have better effects on bone fracture healing acceleration than the single frequency stimulation. Stem cells are the key factors in tissue regeneration process. Recently, the concept of ultrasound stimulation for the stem cell differentiation and proliferation has also been proved.
Two kinds of mediums (basal and bone differentiation medium) were used to mimic different growth environments and stages of stem cells (C3H10T1/2) under different frequencies of low-intensity pulsed ultrasound (LIPUS) treatments. The optimum stimulation frequency of C3H10T1/2 proliferation and osteogenesis in different growth stages were evaluated by trypan blue exclusion method, alkaline phosphotase activity, Alizarin Red stain and ALP stain.
The results showed that higher frequency (3 MHz) low-intensity pulsed ultrasound had better stimulating effects on C3H10T1/2 cell proliferation than lower frequency treatments. On the other hand, lower frequency (1 MHz) ultrasound had better stimulating effects on C3H10T1/2 cell differentiation than higher frequency treatments. It means that choosing a suitable frequency at different periods for stem cell proliferation or differentiation stimulating might be a new feasible way to accelerate fracture healing.

並列關鍵字

Stem cells ； Low intensity pulsed ultrasound stimulation (LIPUS) ； Varying frequency ； Proliferation ； Osteogenesis

參考文獻

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