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研究生:張悅昕
研究生(外文):Yueh-xin Chang
論文名稱:週期性張力與低能量雷射對骨母細胞基因的表現
論文名稱(外文):Study on Gene Expression of Cyclic Mechanical Tension and Low Level Laser Therapy of Osteoblast Cell
指導教授:高喜澤
指導教授(外文):Chia Tze Kao
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:牙醫學系碩士班
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:75
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實驗目的

研究週期性張力與低能量雷射對老鼠骨細胞的相互影響。

材料與方法

本實驗以MC3T3-E1的骨細胞照射低能量雷射,能量為0 J/cm2 、5 J/cm2 、 10 J/cm2後放入Flexercell Strain Unit 以 12%張力,去觀察24hrs , 48hrs的Collagen-1(Col-1)、 Osteopontin(OPN)、osteoprotegerin (OPG)、receptor activator of nuclear factor-jB ligand (RANKL)、 bone morphogenetic proteins ( BMP-2、BMP-4) 、等等mRNA的表現。

實驗結果

結果顯示Col-1 mRNA經過24小時12%週期性張力表現上升,24小時後有下降的情況,張力去除表現也下降。BMP-2 mRNA經過24小時12%週期性張力表現上升。OPN mRNA經過照射低能量雷射 5 J/cm2 10 J/cm2 都能夠刺激表現。另外照射低能量雷射 5 J/cm2 加上週期性張力12% 經過24小時則可以明顯OPG mRNA表現上升,並降低RANKL mRNA 產生。BMP-4 mRNA經過照射 5 J/cm2 低能量雷射加上週期性張力12% 經過24小時表現會上升,24小時後有下降的情況

結語

週期性張力刺激可以對骨細胞產生生物性變化,而讓Col-1 mRNA, OPN mRNA , OPG mRNA , BMP-2 mRNA 和 BMP-4 mRNA表現上升,而讓RANKL mRNA表現下降。這樣有助於影響bone remodeling ,當配合5 J/cm2 低能量雷射可以增加整體效果。

Purpose
The purpose of our study was to investigate interaction of cyclic mechanical tension and low level laser therapy of osteoblast cell.

Materials and Methods
MC3T3-E1 osteoblastic cells were stretched by 12% at a frequency of 6 cycles/min using a Flexercell Strain Unit after exposed to low-level laser treatment at 0, 5 and 10 J/cm2 using a 920nm diode laser for 24 hrs to 48 hrs, After stretching, expression of mRNAs encoding collagen type I (Col-I), osteopontin (OPN) , bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-4 (BMP-4),osteoprotegerin (OPG) and receptor activator of nuclear factor-jB ligand (RANKL) were investigated.

Result
The highest levels of Col-I and BMP-2 mRNA expression occurred at 24 hrs without LLLT , while those of BMP-4 occurred at 24 hrs with 5 J/cm2 LLLT. Col-I and BMP-4 mRNA expression decreased after 24hrs . LLLT at 5 and 10 J/cm2 significantly increased the expression of OPN mRNA. 12 % Cyclic tensile strain with 5 J/cm2 LLLT induced in OPG synthesis and a concomitant decrease in RANKL mRNA expression.

Conclusion
These results indicate that cyclic tensile strain influence the biological behavior of osteoblasts, Increased the expression of mRNAs encoding Col-1 mRNA, OPN mRNA , OPG mRNA , BMP-2 mRNA and BMP-4 mRNA , while those decrease in RANKL mRNA expression. Mechanical strain plays an important role in bone remodeling during growth and development
In Addition to the stretching groups profoundly affects bone remodeling,the stretching groups with 5 J/cm2 had a better results.

目錄 Contents
誌謝 .............2
中文摘要 .............3
英文摘要 .............4
目錄 .............5~7
第 壹 章 前言............. 8
壹、一 雷射............. 8
壹、一、(一) 雷射............. 8
壹、一、(二) 雷射在醫學上的應用............. 10
壹、一、(三) 二極體雷射............. 11
壹、一、(四) 低能量雷射............. 12
壹、一、(五) 雷射對細胞的生理功能之影響............. 13
壹、一、(六) 雷射刺激細胞可能之分子機制............. 14
壹、一、(七) 雷射對矯正治療的影響 16.............
壹、二 週期性張力............. 17
壹、三 研究目的............. 20
第 貳 章 材料與方法............. 21
貳、一 二極體雷射............. 21
貳、二 週期性張力............. 22
貳、三 細胞增生能力分析 I............. 23
貳、四 細胞增生能力分析 II............. 24
貳、五 實驗設計............. 26
貳、六 所使用細胞株與培養條件............. 27
貳、七 RT-PCR............. 28
貳、八 Total RNA 的萃取與定.............量 29
貳、九 Von Kossa Stain染色............. 30
貳、十 影像分析............. 31
貳、十一 Statistics Analysis............. 32
第 參 章 結果............. 33
參、一 細胞形態的觀察 33
參、二 細胞增生能力分析(I)............. 34
參、三 細胞增生能力分析(II)............. 35
參、四 RT-PCR............. 36
第肆章 討論............. 37
第伍章 結論............. 42
參考文獻 .............43~48
第陸章 圖表 .............49
圖一 雷射照射方式圖 .............50
圖二 細胞形態圖( Control / 5J / 10J ) .............51~52
圖三 細胞增生能力分析I .............53
圖四 細胞增生能力分析II .............54
圖四、(一) Day 10 .............54
圖四、(二) Day 12............. 55
圖四、(三) Day 16 .............56
圖四、(四) Day 20 .............57
圖四、(五) 外觀照 .............58
圖四、(六) Von Kossa Stain............. 59
圖五 Col-1 mRNA 定量柱狀圖 .............60
圖六 Osteopontin mRNA 定量柱狀圖............. 61
圖七 OPG mRNA 定量柱狀圖 .............62
圖八 RANKL mRNA 定量柱狀圖............. 63
圖九 RANKL/OPG 關係示意圖.............64
圖十 BMP-2 mRNA 定量柱狀圖.............65
圖十一 BMP-4 mRNA 定量柱狀圖............. 66
第柒章 附錄............. 67
柒、一 實驗儀器............. 67
柒、二 實驗材料............. 68
柒、三 低能量雷射操作說明............. 69
柒、四 骨母細胞表型的發展示意圖............. 73
柒、五 造骨細胞生長分化過程基因表現變化示意圖............. 74
柒、六 RT-PCR Probe 設計圖............. 75


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