Dihydropyridine是在臨床上常用於治療高血壓藥物中的一種，長期服用的患者特別是男性，有一定的比例會產生牙齦增生的現象。我們的團隊先前的研究中發現這類患者牙齦纖維母細胞中睪固酮受器的表現量是一般人的兩倍。而IL-1β 和睪固酮受器之間的作用似乎和牙齦增生有相關性。此外在文獻回顧中發現氧化氮合成酶的其中一種,誘導性氧化氮合成酶(iNOS)則是會被IL-1β活化，在短時間內釋出大量的氧化氮。氧化氮在許多組織裡扮演多重的角色，例如調控結締組織生長因子、血管擴張、神經訊息傳遞、抑制血小板附著凝集以及宿主對細菌、真菌和寄生蟲的免疫反應等等。但是dihydropyridine類的藥物對和類巨噬細胞共同培養且以多脂多醣刺激的健康牙齦纖維母細胞反而抑制了誘導性氧化氮合成酶活性和氧化氮的產生。為了了解在藥物引起的牙齦增生中，誘導性氧化氮合成酶是否正是影響睪固酮受器表現的原因之ㄧ，我們將牙周手術期間取下牙齦標本，培養成的牙齦母細胞分類成,健康(4個樣本)、服用dihydropyridine造成牙齦增生(DIGO cells)(6個樣本)共兩個組別，分別以滿盤前48小時以不加刺激的對照組，加入IL-1β (10 ng/ml)刺激模擬牙周發炎，加入nifedipine (0.34μM)模擬服藥病人血清中藥物濃度以及合併IL-1β (10 ng/ml)和nifedipine (0.34μM)模擬服藥且有牙周發炎產生牙齦增生的情形，在四種不同的刺激條件下，分析兩個組別real time PCR的結果去檢測iNOS和睪固酮受器mRNA的表現，western blot的結果去找出睪固酮受器的蛋白質表現，以及細胞上清液中氧化氮代謝物-亞硝酸鹽的濃度。以Mann-Whitney U-test、Student t test 和 Spearman correlation coefficient去檢視其差異程度和相關性。結果發現，無論健康、服用dihydropyridine造成牙齦增生兩個組別的細胞，受到IL-1β刺激後，iNOS mRNA表現和上清液亞硝酸鹽濃度均顯著增加，而睪固酮受器 mRNA、睪固酮受器蛋白質反而顯著減少。只受到nifedipine刺激時，兩組細胞各種表現和對照組沒有顯著差異。當IL-1β和nifedipine合併刺激時，兩個組別的 iNOS mRNA表現和上清液亞硝酸鹽濃度反而都顯著減少，睪固酮受器 mRNA、睪固酮受器蛋白質卻顯著增加，特別是服用dihydropyridine造成牙齦增生組別睪固酮受器蛋白質表現比健康組別細胞增加的多。這意味著對於因為高血壓長期服用dihydropyridine造成牙齦增生的病人來說，牙周發炎因子扮演了重要的角色。

Object: Dihydropyridine is one of the common medicine for hypertension with gingival overgrowth in some patients. Our previous studies found that there was two fold of androgen receptor (AR) in these patients to normal people. Interaction between interleukin-1β (IL-1β) and AR were related to gingival overgrowth. In addition, one kind of isoforms of NO synthases, inducible nitric oxide synthase (iNOS) , which is typically induced by inflammatory and immune stimuli such as interleukin-1β (IL-1β) and produces large amount of nitric oxide (NO). NO plays multiple important roles in many tissues , for example, regulation for the connective tissue growth factor (CTGF), homeostatic functions, including vasodilatation; neurotransmission; inhibition of platelet adhesion and aggregation; host defense against infectious agents like bacteria, fungi and parasites, and tumor cell killing. Nifedipine is a special calcium channel blocker which dose-dependently inhibits iNOS expression and NO production in normal human gingival fibroblasts which were co-cultured with RAW264 cells (macrophage-like cells) and stimulated with lipopolysaccharide (LPS). In order to understand weather iNOS effects AR expression in gingival overgrowth. Material and method: Two types of periodontal tissue: healthy (n = 4) and dihydropyridine induced gingival overgrowth (n = 6) in periodontal surgery. Each of these groups was stimulated with the control, IL-1β (10ng/ml), nifedipine (0.34μM) and IL-1β (10 ng/ml) combined with nifedipine (0.34μM) at 48 hrs before confluent state to intimate the condition of patients who took dihydropyridines and had DIGO. The data of real time PCR analysis of iNOS, AR mRNA and western bolt expression of AR protein were compared in these two groups. Besides, Griess reagent was used to detect the nitrite concentration respectively in the supernatant of all samples. Mann-Whitney U test、Student t test and Spearman correlation coefficient were used to examine the difference and correlation. Result: Under the stimulation of IL-1β, mRNA expression of iNOS and nitrite concentration increases significantly while mRNA appearance of AR、 AR protein content decrease conspicuously. When nifedipine was added, no significant difference was indicated. Significant decrease of mRNA expression of iNOS and nitrite concentration were shown when IL-1β combined with nifedipine were administered. However, the AR mRNA and AR protein expression conversely raised, especially in the DIGO cells. Conclusion: The activation of iNOS as stimulated by IL-1?? is reversely related to the expression of AR mRNA in DIGO cells. While DIGO cells stimulated with IL-1β in conjunction with nifedipine, AR expression is significantly activated. It implies that inflammatory cytokine plays an important role in the anabolic overgrowth of gingival tissue when patients with hypertension are undertaking long term dihydropyridine therapy.

中文摘要………………………………………………………….P.1~2
Abstract.......................................................................................... P.3~4
Keywords……………………………………………….………...P.5
Chapter1 Introduction…...……………………………….…….... P.6
Section 1 NO & nitric oxide synthase..………………………...P.7
Section 2 NO & Periodontal disease….……………...…….......P.8
Section 3 Dihydropyridines & our previous studies…..…....….P.9~10
Section 4 Specific aim………………………………………….P.10
Chapter 2 Materials & Methods…………………………………..P.11
Section1 Participant Selection……………………………….....P.12
Section2 Fibroblast cell culture………………………………...P.13
Section 3 Measurement of nitric oxide concentration………….P.14
Section 4 Quantization of iNOS and AR mRNA………………P.15~16
Section 5 Western Blot Procedure……………………………...P.17~18
Section 6 Statistical Procedure……….……………………...…P.19
Chapter 3 Results…………………………………………………P.20
Section 1 mRNA expression of iNOS and AR ………………..P.21
Section2 Mean nitrite concentration……………………………P.22
Section 3 AR protein expression……………………………….P.23
Section 4 Spearman correlation coefficient…………………….P. 23
Chapter 4 Discussion……………………………………………...P.24
Section 1 Dihydropyridine suppresses the effect of IL-1β to iNOS
activity in DIGO cells………………………………………….P.25~26
Section 2 NO inhibits AR mRNA/protein expression in DIGO
cells…………………………………………………………….P.27~28
Section 3 IL-1β and nifedipine are both synergistic factors in
AR regulation..............................................................................P.29
Section 4 IL-1β and nifedipine may regulate AR through NO or
calcium ion……………………………………………..............P.30~31
Section 5 Future study: genetic difference between healthy and DIGO
cells………………………………………………………….…P.32
Chapter 5 Conclusion…………………………………………….P.33
References………………………………………………………. P.34~39
Tables…………………………………………………………….P.40~42
Figures……………………………………………………………P.43~47

1.Moncada S, Martin JF. Evolution of nitric oxide. Lancet ; 1993 ; 341:1511.
2.Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA ; 1987 ; 84 : 9265-9269.
3.Furchgott R.F. Studies on relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that the acid-activatable inhibitory factor from retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide. In Vasodilation: Vascular Smooth Muscle, Peptides, and Endothelium (P.M. Vanhoutte, ed.); Raven Press, New York, pp. ; 1998 ; 401-414.
4.Shikano K, Berkowitz BA. Endothelium-derived relaxing factor is a selective relaxant of vascular smooth muscle ; J Pharmacol Exp Ther ; 1987 ; 243 : 55-60.
5.Moncada S, Palmer RM, Higgs E. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev ; 1991 ; 43 : 109–142.
6.Luss H, Li RK, Shapiro RA, Tzeng E. Dedifferentiated human ventricular cardiac myocytes express inducible nitric oxide synthase mRNA but not protein in response to IL-1, TNF, IFN gamma, and LPS. J Mol Cell Cardiol ; 1997 ; 29 : 1153-65.
7.Bruch GD, Ruzicka T, Kolb BV. Nitric oxide in human skin: current status and future prospects ; J Invest Dermatol ; 1998 ; 110 : 1–7.
8.Robert M, Ashok RA, Steven BA. The role of nitric oxide in inflammation and immunity ; arthritis & rheumatism ; 1998 ; 41 : 1111-1151.
9.Forstermann U, Closs EI, Pollock JS. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions ; Hypertension 1994 ; 23 : 1121– 31.
10.Shogo T, Koji N. Perspective of cytokine regulation for periodontal treatment fibroblast biology. J periodontal ; 2003 ; 74 : 103-110
11.Graves DT. The potential role of chemokines and inflammatory cytokines in periodontal disease progression. Clin Infect Dis. ; 1999 ; 28 : 482-90.
12.Hirose M, Ishihara K, Saito A, Nakagawa T, Yamada S, Okuda K. Expression of cytokines and inducible nitric oxide synthase in inflamed gingival tissue. J Periodontal ; 2001 ; 72 : 590– 7.
13.Kendall HK, Haase HR, Li H, Xiao Y, Bartold PM. Nitric oxide synthase type-II is syhthesized by human gingival tissue and cultured human gingival fibroblasts. J Periodontal Res ; 2000 ; 35 :194– 200.
14.Daghigh F, Borghaei RC, Thornton RD, Bee JH. Human gingival fibroblasts produce nitric oxide in response to proinflammatory cytokines. J Periodontal ; 2002 ; 73 : 392– 400.
15.Hung WT, Lu HK, Chou HH, Kuo MYP. Immunohistochemical analysis of Th1/Th2 cytokine profiles and androgen receptor expression in the pathogenesis of nifedipine-induced gingival overgrowth. J Periodontal Res. ; 2003 ; 38 : 422-7.
16.Lu HK, Chou HP, Li CL, Wang MY, Wang LF. Stimulation of cells derived from nifedipine-induced gingival overgrowth with Prphyromonas gingivalis , lipopolysaccharide ,and interleukin-1β; Journal of Dental Research. 2007 ; 86 : 1100-1104.
17.Lu HK, Chang HC, Wang LF. Inhibition of the expression of androgen receptor and IL-6 may suppress the activity of CTGF mRNA and collagen production. Mater thesis, 2007, Taipei Medical University Library.
18.Zuber JP, Spertini F. Immunological basis of systemic sclerosis. Rheumatology ; 2006 ; 45 : iii23–iii25.
19.Takagi K, Kawaguchi Y, Hara M, Sugiura T, Harigai M, Kamatani N. Serum nitric oxide (NO) levels in systemic sclerosis patients: correlation between NO levels and clinical features. Clin Exp Immunol ; 2003 ; 134 : 538–544.
20.Dooley A, Gao B, Bradley N, Abraham1 DJ, Black1 CM, Jacobs M, Bruckdorfer KR. Abnormal nitric oxide metabolism in systemic sclerosis: increased levels of nitrated proteins and asymmetric dimethylarginine. Rheumatology ; 2006 ; 45 : 676–684.
21.Hsu YC, Hsiao M, Chien YW, Lee WR. Exogenous nitric oxide stimulated collagen type I expression and TGF-1 production in keloid fibroblasts by a cGMP-dependent manner. Nitric Oxide ; 2007 ; 16 : 258–265.
22.Hsu YC, Hsiao M, Chien YW, Wang LF. Nitric oxide produced by iNOS is associated with collagen synthesis in keloid scar formation. Nitric Oxide ; 2006 ;14 : 327–334.
23.Zhu H, Bian K, Murad F. Nitric oxide accelerates the recovery from burn wounds. World J Surg ; 2007 ; 31 : 624–631.
24.Wei X, Zoltan S, Wang Y, George AC. Nitric oxide enhances collagen synthesis in cultured human tendon cells. J of orthopaedic research ; 2006 :159~171.
25.Hsu YC, Wang LF , Chien YW. Nitric oxide in the pathogenesis of diffuse pulmonary fibrosis. Free Radical Biology & Medicine ; 2007 ; 42 : 599–607.
26.Betocchi S, Bonow RO, Cannon RO 3rd, Lesko LJ, Ostrow HG, Watson RM, Rosing DR. Relation between serum nifedipine concentration and hemodynamic effects in nonobstructive hypertrophic cardiomyopathy. Am J Cardiol. ; 1988 ; 1 ; 61: 830-5.
27.Gutierrez LM, Lesko LJ, Whipps R, Carliner N, Fisher M. Pharmacokinetics and pharmacodynamics of nifedipine in patients at steady state. J Clin Pharmacol ; 1986 ; 26 : 587-92.
28.Phillips BG, Bauman JL, Schoen MD, Hoon TJ, Schrader BJ, Stuart R. Serum nifedipine concentrations and response of patients with pulmonary hypertension. Am J Cardiol ; 1996 ; 77 : 996-9.
29.Cronauer MV, Ince Y, Engers R, Rinnab L. Nitric oxide-mediated inhibition of androgen receptor activity: possible implications for prostate cancer progression. Oncogene ; 2007 ; 26 : 1875–1884.
30.Yasushi F, Sadaaki M, Makio S. Inhibition by nifedipine of adherence- and activated macrophage-induced death of human gingival fibroblasts. European Journal of Pharmacology ; 2001 ; 415 : 95–103.
31.Csaba s, Jane AM. Nifedipine inhibits the induction of nitric oxide synthase by bacterial lipopolysaccharide ; J of pharmacology and experimental therapeutics ; 1993 ; 265 : 674-680.
32.Gong Y, Blok LJ, Perry JE, Lindzey JK, Tindall DJ. Calcium regulation of androgen receptor expression in the human prostate cancer line LNCaP. Endocrinology ; 1995 ; 136 : 2172-2178.
33.Cabeza M, Flores M, Bratoeff E, Pena A, Mendez E, Ceballos G. Intracellular Ca2+ stimulates the binding to androgen receptors in platelets. Steroids ; 2004 ; 69 : 767-772.
34.Ikeda M, Ikeda U, Kano S Interleukin-1 suppresses mesangial cell growth via inhibition of Ca2+ entry. Cytokine ; 1991 ; 3 :131-133.
35.Morita K, Miyasako T, Kitayama S, Dohi T Interleukin-1 inhibits voltage-dependent P/Q-type Ca2+ channel associated with the inhibition of the rise of intracellular free Ca2+ concentration and catecholamine release in adrenal chromaffin cells. Biochim Biophys Acta ; 2004 ; 1673:160-169.
36.Hayashi K, Wakino S, Sugano N, Ozawa Y, Homma K, Saruta T. Ca2+ Channel Subtypes and Pharmacology in the Kidney. Circulation Research. 2007 ; 100 : 342.
37.Triggle DJ. Calcium channel antagonists: clinical uses--past, present and future.Biochem, Pharmacol ; 2007 ; 74 : 1-9.
38.Kass RS, Arena JP, Chin S. Block of L-type calcium channels by charged dihydropyridines. Sensitivity to side of application and calcium. J Gen Physiol. 1991 ; 98 : 63-75.
39.Shu HF, Wang BR, Wang SR, Yao W. IL-1beta inhibits IK and increases [Ca2+] in the carotid body glomus cells and increases carotid sinus nerve firings in the rat. Eur J Neurosci ; 2007 ; 25 : 3638-47.
40.Zhou C, Ye HH, Wang SQ, Chai Z. Interleukin-1beta regulation of N-type Ca2+ channels in cortical neurons ; Neurosci Lett ; 2006 ; 31 ;403:181-5.
41.Zhou C, Ye HH, Wang SQ, Chai Z. Interleukin-1beta downregulates the L-type Ca2+ channel activity by depressing the expression of channel protein in cortical neurons. J Cell Physiol ; 2006 ; 206 : 799-806.
42.Sitmo M, Rehn M, Diener M. Stimulation of voltage-dependent Ca2+ channels by NO at rat myenteric neurons. Am J Physiol Gastrointest Liver Physiol ; 2007 ; 293: G886–G893.
43.Bullon P, Gallardo I, Goteri G, Rubini C, Battino M, Ribas J, Newman HN. Nifedipine and cyclosporin affect fibroblast calcium and gingiva. J Dent Res ; 2007 ; 86 : 357-362.