以 Porphyromonas gingivalis 之脂多醣和白介質-1b 刺激受尼菲迪平 誘導增生牙齦細胞觀察男性荷爾蒙接受體、白介質-6 與 Th1 / Th2 細
胞激素之表現
Expression of androgen receptor, interleukin-6, and Th1 / Th2 cytokines in the cells derived from nifedipine induced gingival overgrowth tissue stimulated with Porphyromonas gingivalis lipopolysaccharide and interleukin-1b
中文摘要
我們於2001 年針對因尼菲迪平（nifedipine）引發牙齦増生患者之増生牙齦
（nifedipine induced gingival overgrowth, NIGO）的組織以及其他的牙周組織進行 Th1 / Th2 的免疫染色，結果在 NIGO 病人的牙齦中，可以看到明顯的男性荷爾 蒙接受體（androgen receptor, AR）與 Th1 的表現，在此 in vivo 的研究我們提出 AR 的增強與 Th1 cytokine 的表現可能是導致 NIGO 致病機轉的假說。
因此本實驗延續上述in vivo 的臨床實驗，我們在牙周手術區域取下健康 H 組（n=4, age: 30-39 y/o）和 NIGO 組（n=4, age: 48-65 y/o）的牙齦纖維母細胞，在體外培 養到第三代後，將牙周致病菌Porphyromonas gingivalis 萃取出之脂多醣內毒素
（lipopolysaccharide, LPS），以濃度10 μg/ml 和 IL-1b 10 ng/ml 刺激 48 小時後，
以不加任何藥物作為控制組。其後分離上清液來做酵素連結免疫吸附分析
（enzyme-linked immunosorbent assay, ELISA），以測量細胞激素IL-2、IL-4、IL-6 的表現，而細胞層則透過聚合酶連鎖反應（polymerase chain reaction, PCR）探測 IL-2、IL-4、IL-6 和 AR 的 cDNA 基因表現。結果發現在 IL-2 和 IL-4 不論是在 上清液或是細胞層的表現都低於可偵測值，而AR 和 IL-6 則在 cDNA 和 ELISA
的表現都有可偵測的增加。我們將PCR 產物在紫外光下連接 Gel Doc 的應用程
式做半定量，得到之數值以Mann-Whitney U Test 做組間的統計，發現這兩種（H、
NIGO）細胞在 AR 和 IL-6 的表現上，經過 IL-1b 刺激 48 小時後，都有顯著的增 加，其p<0.05；而經過 LPS 刺激之組別與控制組則在 AR 和 IL-6 表現上，並沒
有統計上之差異；而IL-6 的 ELISA 表現以相同統計方式所得結果亦是無統計上
之差異。另外若以Spearman Rank Correlation Coefficient 來統計 IL-6 和 AR 的相 關性，則發現不論是何種藥物刺激，彼此之間並無顯著的相關。牙周病致病機轉 除了細菌之外，其所引發之炎性反應，也會引發許多細胞激素的參予。因此我們 透過上述實驗可知，炎前細胞激素（IL-1b）對 NIGO 細胞所引起的反應也較細
菌內毒素的影響為大，而NIGO 的細胞對於 IL-1b 的刺激感受性亦強於正常的牙
齦纖維母細胞。因此對於NIGO 的病人，我們除了以傳統的牙周治療以控制牙周
炎性反應之外，也可併用抗發炎用藥或生物製劑，以減緩因牙周發炎而引起
NIGO 之牙齦腫大現象。
英文摘要
From immunostaining results of Th1/Th2 of nifedipine induced gingival overgrowth
（NIGO）tissues and other periodontal tissues in 2001, a significant expression of androgen receptor（AR）could be observed. Using these results in vivo, we addressed the hypothesis that AR’s improvement and Th1 cytokine’s expression might be the pathogenic factors to NIGO.The present study extended above clinical trials in vivo.
Gingival fibroblast from healthy, H（n=4, age: 30-39 y/o）tissues and NIGO（n=4, age:
48-65 y/o）tissues were handled from periodontal surgery area and put into cell culture.
After third generation, pre-determined IL-1β and LPS of Porphyromonas gingivalis were used to stimulated sample cells for 48 hours. The concentration of the
pre-determined extract was 10 μg/ml for LPS endotoxins and 10 ng/ml for IL-1β, respectively. These gingival fibroblasts were used as our control group in this experiment. Expression of IL-2、IL-4 and IL-6 extracted from supernatant were measured by ELISA. On the other hand, Cell layer extracted from pellet were analyzed by PCR to detect expression level of mRNA of IL-2、IL-4、IL-6 and AR.
After 48 hours induction, the expression of IL-2 and IL-4 is under detetable value in this study either in supernatant or cell layer. Increased expression was detectable in the part of AR and IL-6 through mRNA level and ELISA. We used Gel Doc software to semi-quantify our PCR product under ultraviolet and studied the statistic difference between groups by Mann-Whitney U software. Results with p<0.05 showed that the amount of AR and IL-6 form these two groups（H and NIGO）were increased after IL-1β induction for 48 hours. However, the difference of AR and IL-6 between control group and experimental group with LPS stimulated was not remarkable. The information from using Spearman Rank Correlation Coeffienct for IL-6 and AR studies also showed no significant results while stimulated by different drugs.Besides bacteria, pathogenic mechanism of periodontal disease could not only induced
inflammation but also evoke lots of cytokines to participate in. Through above
experiments, we discovered NIGO cells were more sensitive to the induction of IL-1b than by P. gingivalis - LPS. It implies that in the treatment of NIGO, traditional
periodontal treatment adjusted with anti-inflammatory drugs and biological agents can be conducted to inhibit both the effects of periodontal pathogen and pre-inflammatory cytokine on NIGO gingival fibroblast
