臺灣博碩士論文加值系統

牙周韌帶細胞參與了牙周組織的修復與再生，其具有多分化潛能，在特殊的情況下可以朝向成骨細胞的方向分化，而在骨質代謝中，藉由活化NF-κB 的訊息傳導途徑可以使蝕骨細胞前體分化成蝕骨細胞。微型核醣核酸 (microRNA, miR) 為一小分子RNA，可以在轉譯後調節基因的表現量，其在正常細胞裡參與了許多細胞運轉的過程，其中包括了細胞的分化。在微型核醣核酸中，miR-146a 會受到NF-κB的調控，它也會透過刪減標靶而間接減少NF-κB之活化。因此本研究探討miR-146a與牙周韌帶細胞分化間的關係及探討miR-146a是否經由 NF-κB 的訊息傳導途徑促使牙周韌帶細胞的分化。首先將體外培養的人類牙周韌帶細胞感染攜帶hTERT基因之反轉錄病毒將其不朽化，次則誘導其分化並觀察其骨分化標誌之變化與miR-146a的表現量；另外，以慢病毒攜帶miR-146a 基因，感染人類不朽化牙周韌帶細胞，探討其對分化的影響。實驗結果顯示，被誘導及感染miR-146a的人類不朽化牙周韌帶細胞其骨分化標誌及ALP 的活性皆有增加，表示miR-146a 參與了人類不朽化牙周韌帶細胞的分化；抗壞血酸誘導調高了miR-146a 之表現，而感染miR-146a 的人類不朽化牙周韌帶細胞其細胞核內NF-κB的表現量下降，表示miR-146a 抑制了NF-κB的訊息傳導途徑。本研究初步結論miR-146a 之表現可促進牙周韌帶細胞之分化，經miR-146a 基因工程改造之牙周韌帶細胞或許具牙周修復之應用價值。

Periodontal ligament (PDL) cells exhibit potentials in hard and soft tissue regeneration because of their mutipotential properties. Osteoblastic line of differentiation of PDL cells will occur under special condition. In bone metabolism, pre-osteoclasts differentiate into osteoclasts via activation of NF-κB signaling pathway. MicroRNAs, small RNAs, regulating the gene expression at the post-transcription level, are associated with the cell process, including cell differentiation. miR-146a, one of microRNAs, was reported involved in the regulation of NF-κB. The purpose of this study was to investigate the regulatory role of miR-146a in the differentiation of PDL cells through NF-κB signaling pathway. The PDL cells were first infected by retrovirus carrying hTERT gene to achieve immortalized PDL cells (I-PDL cells). Treatment of ascorbic acid induced the genesis of bone differentiation markers and ALP activity. Interestingly, the treated I-PDL cells also had the up-regulation of miR-146a. Furthermore, I-PDL cells infected with lentivirus carrying miR-146a had the high enforced miR-146a expression and also exhibited higher bone differentiation potential in relation to control cells. In addition, nuclear NF-κB was also decreased in such cells implicating that miR-146a expression inactivated NF-κB signaling. This preliminary study concludes that miR-146a can enhance the differentiation of PDL cells. The PDL cells genetically modified by miR-146a could have clinical potential for periodontal repair.

目錄
頁次
中文摘要………………………………………………………………………………1
英文摘要………………………………………………………………………………2
壹、 緒論………………………………..………………………...……………..…4
一、 微型核醣核酸…………………………………………………………….4
二、 微型核醣核酸146a (miR-146a) .......................……………………...8
三、 牙周韌帶細胞……………………………………………………….……9
四、 核轉錄因子 kappa B (NF-κB).........................................................13
貳、 研究目標…………………………………………………………………….16
參、 研究材料與方法………….………………………………………………....17
一、 牙周韌帶細胞………….………………………………………………..17
二、 細胞培養……………….………………………………….…………….17
三、 冷凍保存細胞及解凍細胞….……………………………………….….19
四、 細胞生長曲線………………..………………………………………….19
五、 不朽化牙周韌帶細胞之建立…..……………………………………….19
六、 誘導不朽化牙周韌帶細胞之分化…..………………………………….25
七、 反轉錄聚合酶連鎖反應………………..……………………………….25
八、 酵素免疫分析法…………………………..…………………………….26
九、 免疫螢光染色………………………………...…………………………27
十、 免疫細胞染色…………………………………...………………………27
十一、 西方點墨法…………………………………...……………………..28
十二、 核蛋白及質蛋白之萃取………………………...…………………..30
十三、 感染miR-146a及GFP至不朽化牙周韌帶細胞………......….....31
十四、 微型核醣核酸之定量反轉錄聚合酶連鎖反應…………….…..…..32
十五、 茜素紅染色…………………………………………………..……...33
十六、 統計分析……………………………………………………...……..34
肆、 研究結果…………………………………………………………………….35
一、 以攜帶hTERT基因的反轉錄病毒感染牙周韌帶細胞使其不朽化....35
二、 誘導不朽化牙周韌帶細胞之分化…………………………………..….36
三、 以攜帶miR-146a之慢病毒感染不朽化牙周韌帶細胞….…......38
四、 miR-146a 與NF-κB 之間的關係……………….…………………...40
伍、 討論…………………………………………………………………………43
陸、 圖例…………………………………………………………………………50
圖一、不朽化牙周韌帶細胞及親代牙周韌帶細胞其細胞內hTERT基因的表現量………………………………………………………………50
圖二、抗壞血酸與β-Glycerophosphate誘導不朽化牙周韌帶細胞分化後骨細胞分化標誌的表現量增加 (反轉錄聚合酶連鎖反應).............51
圖三、抗壞血酸與β-Glycerophosphate誘導不朽化牙周韌帶細胞分化　　 後ALP蛋白質的表現量增加 (西方點墨法)...……………..........52
圖四、抗壞血酸與β-Glycerophosphate誘導不朽化牙周韌帶細胞分化後ALP蛋白質的表現量增加 (免疫螢光染色)……………….……..53
圖五、抗壞血酸與β-Glycerophosphate誘導不朽化牙周韌帶細胞分化後ALP蛋白質的表現量增加 (免疫細胞染色)……….……………..54
圖六、抗壞血酸與β-Glycerophosphate誘導不朽化牙周韌帶細胞分化後ALP的活性增加……….………………...………………………...55
圖七、抗壞血酸與β-Glycerophosphate誘導不朽化牙周韌帶細胞分化後NF-κB的表現量 (免疫細胞染色)……………….………………..56
圖八、抗壞血酸與β-Glycerophosphate誘導不朽化牙周韌帶細胞分化後miR-146a的表現量增加 (定量反轉錄聚合酶連鎖反應)..……...57
圖九、慢病毒對不朽化牙周韌帶細胞的感染效率…….………………..58
圖十、以miR-146a慢病毒感染不朽化牙周韌帶細胞後miR-146a的表現
量增加 (定量反轉錄聚合酶連鎖反應)…………..………………..59
圖十一、大量表現miR-146a之不朽化牙周韌帶細胞其骨細胞分化標誌的
　　　　表現量 (反轉錄聚合酶連鎖反應)………….…………………..60
圖十二、大量表現miR-146a之不朽化牙周韌帶細胞ALP蛋白質的表
　　　　現量增加 (免疫細胞染色)……………………………………..61
圖十三、大量表現miR-146a之不朽化牙周韌帶細胞其細胞內外OPN蛋白質的表現量及其在細胞內分佈的情形 (西方點墨法)……..62
圖十四、大量表現miR-146a之不朽化牙周韌帶細胞具有較高之ALP活
　　　　性………………………………………………………………..64
圖十五、大量表現miR-146a之不朽化牙周韌帶細胞具有較高之礦化活
　　　　性………………………………………………………………..65
圖十六、大量表現miR-146a之不朽化牙周韌帶細胞NF-κB的表現量
(免疫細胞染色)……..……….…………………………………66
圖十七、大量表現miR-146a之不朽化牙周韌帶細胞NF-κB的表現量
　　　　及其在細胞內分佈的情形 (西方點墨法)……….……………67
柒、 附圖………………………………………………………………………..68
附圖一、p-BABE-puro質體之輿圖…………………………………….68
附圖二、p-BABE-puro-hTERT質體之輿圖.………………….…….....69
附圖三、miR-146a-GFP之慢病毒質體之輿圖……..…………………70
捌、 附表………………………………………………………………………..71
附表一、哺乳類之微型核醣核酸的生物功能…………...……………..71
附表二、與癌症有關的微型核醣核酸…………...……………………..72
附表三、實驗使用之引子序列及黏合溫度…………………………….74
附表四、實驗使用之抗體……………………………………………….75
附表五、各種溶液的配方……………………………………………….76
玖、 參考文獻………………………………………………………………….79

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