臺灣博碩士論文加值系統

牙周炎是一種慢性發炎的疾病，會導致牙齒的支持組織受到破壞，進而造成牙齒的喪失，而齒槽骨吸收是一項牙周炎的重要病徵。目前已知Porphyromonas gingivalis (P. gingivalis) 是牙周炎的致病菌種之一，而且在動物實驗中可以造成骨吸收。根據相關研究顯示，目前對骨質疏鬆症與牙周炎之關連性仍有爭議。因此本研究的目的在於研究罹患停經後骨質疏鬆症之動物模型受P. gingivalis之脂多醣 (lipopolysaccharide，P. gingivalis LPS) 侵襲後，其血清中與骨表現有關的osteoprotegerin (OPG)、receptor activator of nuclear factor ĸB ligand (RANKL)、Interleukin-6 (IL-6) 的濃度變化，並以注入Escherichia coli的LPS (E. coli LPS) 作為對照。
實驗是選擇90隻10週大的ICR雌性小白鼠 (由國立台灣大學醫學院實驗動物中心提供)，45隻切除卵巢的老鼠作為實驗組，45隻假性切除卵巢的老鼠作為對照組。手術4週後從眼窩抽血離心取得血清以作為分析IL-6、OPG及RANKL的基準點。分別將100 µg的P. gingivalis LPS、E. coli LPS注入兩組老鼠 (每組各30隻) 的腹膜腔，1小時、3小時、6小時、24小時、48小時後抽血，比較各時間點與注射前OPG、RANKL、OPG/RANKL ratio、IL-6的差異。並在48小時後犧牲動物，取下右側股骨作tartrate-resistant acid phosphatase (TRAP) 染色。剩下各15隻的實驗組及對照組老鼠在手術後4週犧牲，取下右側股骨作TRAP染色的對照。OPG、RANKL、IL-6的濃度測定是以螢光免疫分析法 (enzyme linked immunosorbent assay，ELISA) 檢測。以 Wilcoxon signed rank test檢驗各組組內注入脂多醣後1、3、6、24、48小時與未注入脂多醣前血清中的OPG、RANKL、OPG/RANKL ratio、IL-6是否有差異，Mann-Whitney U test分別檢定實驗組與對照組注射P. gingivalis LPS、E. coli LPS後血清中的OPG、RANKL、OPG/RANKL ratio、IL-6有無差異，以Spearman rank correlation coefficients檢定各組在各個時間點的IL-6與OPG、RANKL、OPG/RANKL ratio間的相關性。以Kruskal-Wallis test檢定各組股骨骨髓中TRAP染色呈陽性的細胞數是否有差異。於本實驗設定p < 0.05有統計學上之差異。
實驗組及對照組在P. gingivalis LPS的刺激下，代表噬骨細胞生成 (osteoclastogenesis) 的OPG/RANKL ratio在注入後3小時會先下降 (p< 0.05)，之後呈現上升的趨勢。實驗組及對照組在注入P. gingivalis LPS後1、3小時，血清中IL-6的濃度比注入前高 (p< 0.05)。而不論是實驗組或是對照組，注入E. coli LPS後，血清中的OPG、RANKL、OPG/RANKL ratio的變化比注入P. gingivalis LPS大，而IL-6除了濃度更高之外，作用時間也比較久。各組的IL-6表現和OPG、RANKL、OPG/RANKL ratio沒有相關。而在TRAP染色方面，實驗組老鼠會比對照組有較多的TRAP染色呈陽性的細胞數目 (p< 0.05)，而注入脂多醣並不影響細胞的數目。
在切除卵巢的老鼠腹膜腔內只注入一次脂多醣的本實驗中，我們觀察到代表噬骨細胞生成的OPG/RANKL系統在P. gingivalis LPS的刺激下，除了受到P. gingivalis LPS的影響之外，應該還有其他的因子會共同調控OPG、RANKL的表現。IL-6的表現是直接受到P. gingivalis LPS的影響，血清中的濃度會先上升，之後回到正常的範圍，而不論有無切除卵巢都不影響IL-6的表現，並且IL-6的表現和OPG、RANKL、OPG/RANKL ratio沒有相關性。此外，我們也發現對血清中的OPG、RANKL、OPG/RANKL ratio、IL-6變化，E. coli LPS的效能比P. gingivalis LPS強。

Periodontitis is a chronic inflammatory disease which results in the breakdown of tooth supporting structures, especially alveolar bone destruction. Deep periodontal pocket, attachment level loss, and alveolar bone loss are found under clinical examination. Porphyromonas gingivalis (P. gingivalis), a black-pigmented gram-negative anaerobic bacterial rods, is a vital pathogen for adult periodontitis. P. gingivalis has been proved to contribute to bone resorption in many vivo studies. However, unlike the clear relationship between osteoporosis and tooth loss, controversy still exists concerning the association between osteopenia/opteoporosis and the periodontal pathogen. The purpose of the present study is to clarify the etiologic relationship of P. gingivalis lipopolysaccharide (P. gingivalis LPS) and the expression of OPG, RANKL, IL-6 in the serum of postmenopausal osteoporosis mice. Injection of Escherichia coli LPS (E. coli LPS) was need as the control.
Ninty 10-week old ICR female mice were divided into ovariectomized group (experimental group) and sham-operation group (control group). After operation 4 weeks, we collect the serum as baseline. 100 µg P. gingivalis LPS and E. coli LPS were separately injected into the peritoneum of experimental group (30 mice) and control group (30 mice). Subsequently, we collected the serum 1,3,6,24 and 48 hours after the injection of both LPS. Then the concentrations of IL-6, OPG and RANKL of serum will be estimated by using sandwich ELISA. The femur bone was dissected for TRAP stain analysis. Besides, 4 weeks after operation, 15 experimental mice and 15 control mice was sacrificed for TRAP stain to compare lipopolysaccharide effect. Mann-Whitney U test was applied to study the difference of OPG, RANKL, OPG/RANKL ratio, IL-6 between P. gingivalis LPS and E. coli LPS. Wilcoxon signed rank test was used to study the difference of OPG, RANKL, OPG/RANKL ratio, IL-6 between each time interval and baseline in group. The strength of correlations between IL-6 and OPG, RANKL, OPG/RANKL ratio at each time interval in group were determined by Spearman rank correlation coefficients. The difference of TRAP positive cell count in the specimen among was compared by using Kruskal-Wallis test. Results with p< 0.05 were defined statistically different.
In the injection of P. gingivalis LPS of experimental and control group, OPG/RANKL ratio decreased at 3 hours (p< 0.05), then trended to rising tendency. IL-6 rose at 1、3 hours (p< 0.05). Besides, the changes of OPG, RANKL, OPG/RANKL ratio, IL-6 were greater in injection of E. coli LPS than P. gingivalis LPS. There is stronger TRAP positive cells distribution in experimental group than control group (p< 0.05). No matter single booster of P. gingivalis LPS or E. coli LPS did not effect the expression of TRAP positive cells.
It should be noticed that only single shot of bacterial LPS was applied in this animal model. P. gingivalis LPS directly effected the expression of IL-6, but not OPG/RANKL system. It indicated that OPG/RANKL ratio of experimental group injected with P. gingivalis LPS might also be regulated by a more complex system. Removal of ovary did not effect the expression of IL-6; our data also indicated there were no correlation of IL-6, expression of OPG, RANKL, and OPG/RANKL ratio. In addition, the stimulatory effect of E. coli LPS was stronger than P. gingivalis LPS.

目 錄
目錄----------------------------------------------------------I
圖目錄------------------------------------------------------------------------------------II
表目錄-----------------------------------------------------------------------------------III
中文摘要--------------------------------------------------------------------------------IV
Abstract--------------------------------------------------------------------------------VII


第一章 緒論
第一節 骨質疏鬆症--------------------------------------------------------------1
第二節 研究目的-----------------------------------------------------------------5
第二章 文獻查證
第一節 OPG/RANKL 系統---------------------------------------------------7
第二節 Interleukin-6 (IL-6)--------------------------------------------------10
第三節 Tartrate-resistant acid phosphatase (TRAP)-------------------11
第三章 研究材料與方法
第一節 停經後骨質疏鬆症動物模型及分組-------------------------------12
第二節 研究方法----------------------------------------------------------------13
第三章 實驗結果
第一節 存活率--------------------------------------------19
第二節 切除卵巢對OPG、RANKL、OPG/RANKL ratio
及 IL-6的影響--------------------------------------------------------20
第三節 P. gingivalis LPS、E. coli LPS對OPG、RANKL
、OPG/RANKL ratio及 IL-6的影響------------------------------21
第四節 組織切片-TRAP染色------------------------------------------------26
第五章 討論
第一節 切卵巢手術對老鼠的影響-------------------------------------------27
第二節 注射P. gingivalis LPS、E. coli LPS 對切除卵巢老鼠
、假性切除卵巢的老鼠的影響--------------------------------------29
第六章 結論----------------------------------------------------------------------------34

參考文獻--------------------------------------------------------------------------------35
圖目錄
圖1. 實驗組及對照組手術後4週的OPG值-------------------------------------43
圖2. 實驗組及對照組手術後4週的RANKL值-------------------------------43
圖3. 實驗組及對照組手術後4週的OPG/RANKL ratio值-----------------44
圖4. 實驗組及對照組手術後4週的IL-6值------------------------------------44
圖5. 實驗組及對照組老鼠注入P. gingivalis LPS的OPG值變化---------45
圖6. 實驗組及對照組老鼠注入E. coli LPS的OPG值變化-----------------46
圖7. 實驗組及對照組老鼠注入P. gingivalis LPS
的RANKL值變化-------------------------------------------------------------47
圖8. 實驗組及對照組老鼠注入E. coli LPS的RANKL值變化-------------48
圖9. 實驗組及對照組老鼠注入P. gingivalis LPS
的OPG/RANKL ratio值變化-----------------------------------------------49
圖10. 實驗組及對照組老鼠注入E. coli LPS
的OPG/RANKL ratio 值變化---------------------------------------------50
圖11. 實驗組及對照組老鼠注入P. gingivalis LPS的IL-6值變化----------51
圖12. 實驗組及對照組老鼠注入E. coli LPS的IL-6值變化----------------52
圖13. 實驗組老鼠注入P. gingivalis LPS、E. coli LPS
的OPG值比較----------------------------------------------------------------53
圖14. 對照組老鼠注入P. gingivalis LPS、E. coli LPS
的OPG值比較----------------------------------------------------------------54
圖15. 實驗組老鼠注入P. gingivalis LPS、E. coli LPS
的RANKL值比較------------------------------------------------------------55
圖16. 對照組老鼠注入P. gingivalis LPS、E. coli LPS
的RANKL值比較------------------------------------------------------------56
圖17. 實驗組老鼠注入P. gingivalis LPS、E. coli LPS
的OPG/RANKL ratio值比較----------------------------------------------57
圖18. 對照組老鼠注入P. gingivalis LPS、E. coli LPS
的OPG/RANKL ratio值比較----------------------------------------------58
圖19. 實驗組老鼠注入P. gingivalis LPS、E. coli LPS
的IL-6值比較-----------------------------------------------------------------59
圖20. 對照組老鼠注入P. gingivalis LPS、E. coli LPS
的IL-6值比較-----------------------------------------------------------------60
圖21. 實驗組老鼠注入P. gingivalis LPS的TRAP染色----------------------61
圖22. 對照組老鼠注入P. gingivalis LPS的TRAP染色----------------------62
圖23. 實驗組老鼠注入E. coli LPS的TRAP染色------------------------------63
圖24. 對照組老鼠注入E. coli LPS的TRAP染色------------------------------64
圖25. 實驗組老鼠切除卵巢後4週的TRAP染色------------------------------65
圖26. 對照組老鼠假性切除卵巢後4週的TRAP染色-------------------------66
圖27. 實驗組老鼠注入P. gingivalis LPS、E. coli LPS
的TRAP陽性細胞數量-----------------------------------------------------67


表目錄
表1. 實驗組老鼠注入P. gingivalis LPS在各個時間點時IL-6
與OPG RANKL、OPG/RANKL ratio的相關性------------------------68
表2. 對照組老鼠注入P. gingivalis LPS在各個時間點時IL-6
與OPG、RANKL、OPG/RANKL ratio的相關性---------------------69
表3. 實驗組老鼠注入E. coli LPS在各個時間點時IL-6
與OPG、RANKL、OPG/RANKL ratio的相關性---------------------70
表4. 對照組老鼠注入E. coli LPS在各個時間點時IL-6
與OPG、RANKL、OPG/RANKL ratio的相關性---------------------71

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