不同工作週期之低能量超音波刺激在體外對纖維母細胞的影響

In vitro study on the effect of fibroblasts stimulated by low intensity ultrasounds at different duty cycles

紀玉霜(Yu- Shuang Ji)

指導教授 ： 李文婷 ； 王士豪

中原大學/工學院/醫學工程研究所/碩士(2003年)

https://doi.org/10.6840/cycu200300620

摘要

以往的研究顯示超音波刺激對於組織修補和傷口癒合有良好的效應，但對超音波刺激機制及不同刺激環境的影響等因子仍缺乏完整的探討。故本研究嘗試使用不同刺激環境及不同工作週期的超音波來探討細胞受超音波刺激後的增長效應。
實驗是先針對第13代及第23代的人類包皮纖維母細胞，施以1 MHz，強度7.5 mW/cm2（ISATA），脈衝重覆週期1 ms，20％ 工作週期的超音波刺激。將相同代數的人類包皮纖維母細胞植入6-well（直徑35mm）培養皿（細胞數為104 cells/well），細胞培養和刺激皆在37oC溫度下進行。並使用血球計數盤來對細胞進行計數。實驗結果顯示未經刺激的控制組細胞在經過5天培養後接近飽和狀態。第13代的纖維母細胞在超音波刺激3分鐘後的增長速率最快（刺激組斜率/控制組斜率 = 1.34），第23代的纖維母細胞以5分鐘刺激的增長較快（刺激組斜率/控制組斜率 = 1.26）。推測是因為第23代的纖維母細胞比第13代的纖維母細胞生長機能差，導致接受刺激的時間需較長。
在不同環境下刺激的結果顯示，細胞在37oC下的刺激效果（刺激組斜率/控制組斜率 = 1.16）大於室溫下（約20oC ± 2oC）刺激的細胞增長速率（刺激組斜率/控制組斜率 = 1.08）。細胞行完整細胞週期後（24小時）進行超音波刺激的效果（刺激組斜率/控制組斜率 = 1.06）也大於未行完整細胞週期的細胞（12小時）增長速率（刺激組斜率/控制組斜率 = 0.77）。在日光燈下進行超音波刺激的效果（刺激組斜率/控制組斜率 = 1.12）大於無開日光燈下的刺激（刺激組斜率/控制組斜率 = 1.05）。可知在不同環境下，超音波刺激細胞會影響細胞的增長速率。
為進一步探討不同工作週期的超音波刺激對細胞的增長影響，我們使用第13代的纖維母細胞進行超音波刺激，工作週期為5至100％，刺激時間為每日3分鐘。結果顯示在以10％、15％及20％工作週期的超音波刺激下，細胞的增生速率皆大於控制組之細胞。但其它工作週期的超音波刺激卻反而使纖維母細胞的增生速率小於控制組的細胞，此結果可能是因較長工作週期及瞬間強度較大的超音波對細胞產生較大的損害作用。
超音波刺激前後培養皿及水箱內的溫度變化皆不顯著（< 1 oC），所以推論細胞增生不是因為超音波的熱效應，而可能是超音波非熱效應造成細胞膜的通透性質改變，使細胞內外的離子濃度產生變化。使較多的鈣及鈉離子進入細胞內，促進了細胞分裂與增長速率加快。本研究證實適當的超音波刺激加上良好的工作環境可促進人類包皮纖維母細胞的增生。

關鍵字

工作週期 ； 超音波 ； 細胞增生 ； 人類包皮纖維母細胞

並列摘要

Previous studies have shown that ultrasonic stimulation has an effect to improve tissue repairs and wound healings. However, factors such as mechanisms and operations for the ultrasonic stimulation are still lack of exploration completely. For these reasons, this research tried to investigate cell proliferation after ultrasonic stimulations under different conditions and different duty cycles.
The pilot experiment was carried out using the 13th and 23th passages of human foreskin fibroblasts. 1 MHz of ultrasound with intensity of 7.5 mW/cm2 (SATA), duty cycle of 20%, and pulse repetition period of 1 ms was applied. Fibroblasts of the same passage were cultured in a six-well plate（cell numbers is 104 cells/well）, in which cells were both incubated and stimulated at 37oC temperature. Cell numbers were counted using the hemocytometer. Results of all experiments showed that cell proliferation from the control groups tend to be saturated after the fifth cultured day. The cell proliferation rate from the 13th passage fibroblasts was increased, slope (S) of stimulated groups/slope of control groups = 1.34, after received ultrasound stimulation for three minutes per day, and that from the 23th passage fibroblasts was increased, S = 1.26. It could be due to that the 13th passage fibroblast has a better growth functions than that of the 23th passage fibroblasts.
Under different stimulation conditions, the cell proliferation rate corresponding to stimulation under 37oC temperature, S = 1.16, was better than that under the room temperature, S = 1.08. The cell proliferation rate associated with stimulations at a period of 24 hours full cell cycle, S = 1.06, was better than that of 12 hours half cell cycle S = 0.77. The stimulation under light of fluorescent lamp produced S = 1.12 that is better than that without light, S = 1.05. These results suggest that cell proliferation rates are also affected by different ambient conditions.
To further explore factors of ultrasonic stimulations of different duty cycles on the fibroblast cells proliferation, we applied the 13th passage fibroblasts that were stimulated by ultrasound of different duty cycles ranged from 5 to 100%. The time of stimulation was three minutes per day. The results showed that proliferation rates of fibroblasts associated with 10%, 15% and 20% ultrasonic stimulations are all larger than those of the duty cycles those and that of the control group. These suggest that ultrasound of only certain duty cycles may be benign to the cell proliferation and ultrasound of higher intensity on the other hand tends to damage cells.
The variation of temperature in the water tank throughout all experiments were not large apparently suggesting that the actual mechanism of fibroblast proliferation might be due to ultrasound of the non-thermal bio-effect in which the permeability of cell membrane was modified. Therefore, the concentrations of those ions within and outside the cell were varied that moreover caused movement of more calcium or sodium ions to be entering into cells for accelerating cell division and proliferation. This study validated that the proliferation of fibroblasts could be greatly enhanced under applying ultrasonic stimulations under proper operational conditions.

並列關鍵字

Ultrasound ； Duty cycle ； Human foreskin fibroblast ； Cell proliferation

參考文獻

[1] Gail ter, Haar, “Therapeutic ultrasound,” European Journal of ultrasound, Vol. 9, No. 1, pp. 3-9, 1999.

[2] P. Reher, E. N. I. Elbeshir, W. Harvey, S. Meghji and M. Harris, “The stimulation of bone formation in vitro by therapeutic ultrasound,” Ultrasound in Medicine & Biology, Vol. 23, No. 8, pp. 1251-1258, 1997.

[6] “Other nonthermal bioeffects: organs, cells and tissues,” Ultrasound in Medicine & Biology, Vol. 24, Supplement 1, pp. S35-S39, 1998.

[8] S. R. Young and M. Dyson , “Effect of therapeutic ultrasound on healing of full-thickness excised skin lesions,” Ultrasonics, Vol. 28, No. 3, pp. 175-180, 1990.

[9] M. Schafer, S. Dubin, A. Geshury, and F. Ko, “Experimental methods for ultrasonically enhanced wound healing,” IEEE Ultrasonics Symposium, Vol. 3, No. 5, pp. 1853-1856, 1994.