阻斷睪固酮接受器與

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阻斷睪固酮接受器與 IL-6 的活性可抑制結締組織生長因子的表現與

膠原蛋白的生成

Inhibition of the activities of androgen receptor and IL-6 may suppressed

CTGF expression and collagen production

中文摘要

Nifedipine 為一種鈣離子阻斷劑，為廣泛運用於治療心血管疾病及挾心症之藥物。長期服用之病患在牙周出現牙齦增生(nifedipine induced gingival overgrowth：NIGO)的現象，其增生的組織往往造成牙周囊袋的加深並造成長期慢性發炎的病症，加重牙周病的嚴重度與其治療、維持的難度。

對於此藥物產生增生的機轉目前尚未明朗。於我們研究小組最近之發表的結果，發現此增生的機轉可能與性荷爾蒙以及發炎因子之細胞內機轉有關。此研究是比較 nifedipine 以及 IL-1β刺激臨床增生牙齦與健康牙齦之牙齦母纖維細胞 (gingival fibroblast) 後, 利用睪固酮接受器阻斷劑以及 IL-6 抗體, 進一步抑制細胞產生結締組織生長因子(Connective tissue growth factor：CTGF)之 m-RNA 以及第一型膠原蛋白(type Ⅰcollagen：ColⅠ) 之 m-RNA 與第一型膠原蛋白質之分泌, 以釐清 AR 與 IL-6 於 NIGO 生成中所扮演的角色。

牙齦纖維母細胞來源於台北醫學大學附設醫院牙周病科之病患。凡服用 Nifedipine 介於 6~36 周者並造成牙齦增生之牙齦組織，並依照 Ingle＇s overgrowth Index (Ingle et al.2000)分類之第二級與第三級增生為反應組 (Responder group, R)。另外未服用 Nifedipine 也未有牙齦增生現象的病患之牙齦組織為健康組(Healthy group, H)。兩組病人皆接受過牙周第一階段治療(牙根平整術及洗牙)，並於第二階段牙周手術期間取下牙齦標本，培養牙齦母細胞； R 與 H 兩組各有四位病人之細胞。每單一標本分別給予 Nifedipine(10μM)與 IL-1β(10ng/ml) 之投藥分為 control(C)、Nifedipine(Ni)、IL-1β(IL)與 Nifedipine＋IL-1β(Ni+IL)四組。使用 Q-PCR 進行 responder 與 healthy 兩組間之比較以及組內 CTGF 與 ColⅠ之 m-RNA 濃度比較；而培養組織之上清液蒐集用於總膠原蛋白(Tatal collagen realesed to medium, Sircol Assay)的偵測。另外我們投予 AR antagonist (Flutamide)以及 IL-6 antibody (IL-6 blocker), 阻斷 AR 與 IL-6，比較阻斷前後 CTGF 與 ColⅠ之 m-RNA 表現之變化，以及比較斷前後上清液內膠原蛋白生成總量。

實驗結果：CTGF m-RNA 各組的表現(圖 1-1)，各組內(C、Ni、IL、Ni+IL)CTGF m-RNA 表現的差異(圖 1-1)，(C≦Ni<IL≦Ni+IL)。H 組與 R 組的比較(圖 1-2)，各組中皆有 healthy<responder 的趨勢，而在 IL 組與 Ni+IL 組中 healthy 與 responder 組的比較有統計上顯著差異。阻斷前與阻斷後的差異：(圖 1-3)阻斷 AR 與 IL-6 後 CTGF m-RNA 皆有下降趨勢，而阻斷 AR 相較於阻斷 IL-6 下降的趨勢更為強烈。

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ColⅠ m-RNA 的表現：各組內(C、Ni、IL、Ni+IL) ColⅠm-RNA 表現的差異(圖 2-1)，(C≦Ni<IL≦Ni+IL)。Healthy 與 responder 組的比較(圖 2-2)，各組中皆有 healthy<responder 組的趨勢，而在 IL 組與 Ni+IL 組中 healthy 與 responder 組的比較上有統計上顯著差異。阻斷前與阻斷後的差異：(圖 2-3)阻斷 AR 與 IL-6 後 ColⅠ m-RNA 皆有下降趨勢，而阻斷 IL-6 相較於阻斷 AR 下降的趨勢更為強烈。上清液中 collagen 的表現：各組內(C、Ni、IL、Ni+IL) 上清液中 collagen 表現的差異(圖 3-1)，(C≦Ni<IL≦Ni+IL)。H 組與 R 組的比較(圖 3-2)，各組中皆有 Healthy<Responder 的趨勢，而在 IL 組與 Ni+IL 組中 healthy 與 responder 組的比較有統計上顯著差異。阻斷前與阻斷後的差異：(圖 3-3)阻斷 AR 與 IL-6 後上清液中 collagen 皆有下降趨勢，而阻斷 IL-6 相較於阻斷 AR 下降的趨勢更為強烈。

結論: 阻斷 AR 與 IL-6 會抑制 NIGO cells 之 CTGF m-RNA 與 collagen 蛋白生成的活性，可直接抑制牙齦增生。NIGO 的纖維母細胞相較健康的纖維母細胞表現出較為強烈 CTGF m-RNA 的表現與 collagen 生成的趨勢。AR 可能是上游調控 CTGF m-RNA 表現的重要因子，而 IL-6 可能是上游調控 collagen 生成的重要因子。 Nifedipine 並未直接影響 CTGF m-RNA 與 collagen 的生成，在整個牙齦增生的機轉中可能並非直接影響增生的角色。

英文摘要

Nifedipine is most frequently reported to induce gingival overgrowth during long-term treatment of cardiovascular disorders such as hypertension and angina pectoris. Nifedipine induced gingival overgrowth (NIGO) will increase periodontal pocket depth and chronic periodontal inflammation to promote severity of periodontal disease.

However the mechanism of nifedipine-induced gingival overgrowth is still not quite clear. According to our recent resarch reports, we found the mechanism may have relationships to androgen receptor (AR) and inflammatory factors. The aim of the present study is to verify the regulatory relation of AR, IL-6 and their down stream expression of connective tissue growth factor (CTGF), type I collagen (Col I) mRNA, and type I collagen produciton in NIGO cell culture.The cell line (gingival fibroblast) was derived from the patients visiting the periodontal department of Taipei Medical University Hospital. The patients who were taking the nifedipine for 6~36 weeks an being found gingival overgrowth at grade 2 and grade 3 (Ingle’s gingival overgrowth Index .2000) were included as responder group. The patients without taking

nifedipine and gingival overgrowth were included as healthy group. Both group were treated with phase I periodontal treatment (root planing and scaling), and the gingical samples were havested during periodontal surgical phase to culture the gingival fibroblasts. Each sample was given Nifedipine(Ni)(10μM)、IL-1β(IL)(10ng/ml)、

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non or both and divided to 4 sub-groups (C、Ni、IL、Ni+IL). Using Q-PCR to dectect the m-RNA levels of connective tissur growth factor(CTGF) and typeⅠcollagen(Col Ⅰ) than comparing the m-RNA levels between reponder and healthy groups.We uesed Sircol Assay to quantify collagen protein released in culture medium by fibroblast during in vitro culture. In addtion, we treated each sample with AR

antagonist (flutamide) and IL-6 antibody (IL-6 blocker) and compared the expression of m-RNA levels of CTGF and ColⅠ, and total collagen in supernatant medium. Results: In the expression of CTGF m-RNA (Fig 1-1)： Fig 1-1shows that C≦Ni<IL ≦Ni+IL。Comparing the healthy and responder groups (Fig 1-2)，results show healthy group<responder group；in C and Ni groups there was no significantly different between healthy and responder group，but there was significantly different in IL and Ni+IL groups. Between before and after blockage：(Fig 1-3) In all groups showd blocking AR or IL-6 will degrade the expression of CTGF m-RNA ，and blocking AR showed more highly degradation of CTGF m-RNA than blocking IL-6. The

expression of ColⅠm-RNA (Fig 2-1)：Comparing C、Ni、IL、Ni+IL groups(Fig 2-1)， the results show C≦Ni<IL≦Ni+IL. Comparing the H to R groups (Fig 2-2)，in all groups the results show Healthy<Responder；in C and Ni groups there was no

significantly different between H and R groups，but there was significantly different in IL and Ni+IL groups between H and R groups. Between before and after blockage： (Fig 2-3) In all groups showd blocking AR or IL-6 will degrade the expression of Col Ⅰ m-RNA，and blocking IL-6 showed more highly degradation of ColⅠ m-RNA than blocking AR. The expression of collagen in medium (Fig 3-1)：Comparing C、 Ni、IL、Ni+IL groups(Fig 3-1)，the results show C≦Ni<IL≦Ni+IL. Comparing the H to R groups (Fig 3-2)，in all groups the results show Healthy<Responder；in C and Ni groups there was no significantly different between H and R groups，but there was significantly different in IL and Ni+IL groups between H and R groups. Between before and after blockage：(Fig 3-3) In all groups showd blocking AR or IL-6 will reduce the expression of collagen in medium，and blocking IL-6 shows more highly reduction of collagen in medium than blocking AR.

Conclusion：Inhibition of activity of androgen receptor and IL-6 may suppress the expression of CTGF mRNA and collagen production，and may indirectly suppress gingival enlargement. The NIGO fibroblasts show more highly expression of CTGF m-RNA and collagen than healthy fibroblasts. AR may directly upregulate the expression of CTGF m-RNA, but in a less affect on Col I mRNA and collagen produciton. IL-6 may directly upregulates both the expression of CTGF mRNA and production of collagen, and in a less degree on AR mRNA.Nifedipine may not

directly upregulate the expression of CTGF m-RNA and collagen production in NIGO cells