行政院國家科學委員會專題研究計畫 期中進度報告
Tamoxifen 預防腹膜纖維化之機轉及其在生體療效之研究
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計畫類別： 個別型計畫 計畫編號： NSC91-2314-B-002-339- 執行期間： 91 年 08 月 01 日至 92 年 08 月 31 日 執行單位： 國立臺灣大學醫學院內科 計畫主持人： 蔡敦仁 計畫參與人員： 洪冠予, 黃政文, 莊惠安 報告類型： 精簡報告 處理方式： 本計畫可公開查詢中 華 民 國 92 年 5 月 29 日
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行政院國家科學委員會專題研究計畫成果報告
Tamoxifen 對於治療腹膜硬化症的分子機制與動物模式研究
計畫編號：NSC 91-2314-B-002-339
執行期限：
91 年 8 月 1 日至 92 年 8 月 31 日
主持人：
蔡敦仁 台大醫院內科部腎臟內科
計畫參與人員：黃政文 台大醫院內科部腎臟內科
莊惠安 研究助理
一、中文摘要 腹膜透析是末期腎衰竭病患常用的一種腎 臟替代療法，腹膜纖維化會使腹膜功能喪 失，使病患無法繼續進行透析。本計畫第 一年是研究 Tamoxifen(TAM)對於人類腹膜 細胞以及纖維母細胞的影響。結果顯示， TAM 對於第一及第三型膠原蛋白的表現在 人類腹膜細胞有抑制效果，可是在纖維母 細胞沒有抑制效果。至於細胞生長的影 響，TAM 則對於兩種細胞都有抑制效果。證 實 TAM 對於腹膜纖維化可能有治療效果。 因此在皆下來的研究將針對 TAM 在大鼠誘 發腹膜纖維化的動物模式中的治療效果。 初步成果顯示，TAM 治療組較對照組活的較 久，而且在存活的大鼠中，TAM 治療組有較 低的纖維化指數。本計畫將繼續對此動物 模式進行更詳細深入的研究。 關鍵詞：腹膜透析，腹膜細胞，腹膜纖維 化，纖維母細胞，膠原蛋白，Tamoxifen AbstractPeritoneal dialysis (PD) is a common modality of renal replacement therapy for end-staged renal disease (ESRD) patients.
Peritoneal fibrosis (PF) will prevent a good performance of PD and lead to technique failure. In the first year of this study, the effect of tamoxifen (TAM) on human peritoneal mesothelial cells (HPMCs) and peritoneal fibroblasts was studied. The TAM inhibit the production collagen I and III in HPMCs but not in fibroblasts.
Proliferation of both cells are inhibited by TAM. In the second year, we extend to in vivo study in rat PF model induced by bleach. The results showed that TAM will improve survival and reduce PF. More experiments of in vivo studies will be performed in the next two years.
Keywords: Collagen, Fibroblast, Mesothelial
Cell, Peritoneal Dialysis, Peritoneal Fibrosis, Tamoxifen
二、緣由與目的
連續可攜式腹膜透析(簡稱 CAPD)是 國人常用的透析方式之一，其最嚴重的併 發 症 之 一 是 腹 膜 纖 維 化 (peritoneal fibrosis，簡稱 PF)。發生 PF 的機轉主要為 腹膜表面細胞(human peritoneal mesothelial
cell，簡稱 HPMC)過度增生及細胞外間質 堆積[1]。目前對於 PF 還缺乏有效的治療或 預防方法。Tamoxifen(TAM)是臨床上廣泛 用於癌症輔助治療的藥物，近年來，已有 臨床個案報告指出: TAM 對於 CAPD 病人 發生 PF 可能也有療效[2]。進一步查尋文獻 發現: TAM 對於其他一些人體纖維硬化相 關 病 症 ， 如 : 後 腹 膜 腔 纖 維 化 (retroperitoneal fibrosis) [3, 4]、腸繫膜硬化 症(sclerosing mesenteritis) [5]、縱隔纖維化 ( fibrosing mediastinitis)和子宮頸硬化症[6] 等，都曾有治療成功的零星相關報告。既 然發生 PF 的機轉與 HPMC 過度增生及細 胞外間質堆積有關，而 TAM 對 HPMC 生 長是否有抑制之作用，從未有人報告過。 本研究計畫第一年已發現 TAM 對於第一及 第三型膠原蛋白的表現在人類腹膜細胞有 抑制效果。本計畫第二年是利用漂白水誘 發大鼠產生腹膜纖維化，並以 TAM 加以治 療。 三、結果與討論
The rat about 450 g were used for this model. They were divided into three groups
including vehicle groups (injecting DMSO solution, 2 ul/ml in distilled water), TAM group (injecting TAM 2.5 mg/kg/day), and control group (injecting DMSO, 2 ul/ml in distilled water). The treatments were started two days prior to bleach injection. The bleach was diluted with 2:100 with PBS and injected into the peritoneal cavity, 5ml/100 g, in the vehicle and TAM groups. The treatment solution were injected daily from left lower quadrant of the abdominal wall in all three groups. One week after treatment, we sacrifice the rats and obtained the 1. PF scores. 2. Frozen tissues from parietal peritoneum, omentum, and adhesion bands for mRNA. 3. Tissues from liver of right lobe, intestine, and omentum fixed in
formaldehyde for HE and PAS staining. Fig. 1. Kaplan-Meier survival analysis of rats with PF induced by bleach between TAM treated (－, n=8) and vehicle only (---, n=7) (p=0.06).
In the preliminary experiment, the bleach will induce PF 7 days after instillation. The rats will die in the following weeks. TAM could prevent mortality in Kaplan-Meier survival analysis (Fig. 1). The rats were rarely dead in the first week. So the following experiments were conducted one week after instillation of bleach.
Gross evaluation of the fibrosis was according to fibrosis scoring. In brief, we counted the number of adhesion bands between liver-omentum, intestine-abdominal wall, intestine-intestine, intestine-omentum after laparotomy. The score was 0 if there was no adhesion bands, 1 for 1~3 bands, 2 for more than 3 bands, and 3 for sheet like adhesion. The full score was 12. The cacoon formation was 12 points.
Fig. 2 shows the distribution of fibrosis scroe among the three groups. The fibrosis scores in control group (0.3 0.5) was less than the bleach vehicle group (10.4 1.8) and tamoxifen treated group (3.6 2.0). In addition, tamoxifen treated group had lower fibrosis score.
Peritoneal Fibrosis Scores
0 5 10 15
0.0 1.0 2.0 3.0 4.0
Fig. 2: The PF scores in three groups.(C: control; T: bleach with tamoxifen treatment; D: bleach with vehicle)
Visceral peritoneum around liver was measured under PAS stain. Five
measurements were done in each specimen under high power fields. Fig. 3 shows the peritoneum thickness around liver in three groups.
The exact thickeness was presented in the
C u m m u l a t i v e S u r v i v a l 1.0 .9 .8 .7 .6 .5 .4 .3 .2 C T D
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Fig. 4. The peritoneal thickness was 0.28 0.06 for control group, 1.17 0.45 for tamoxifen group and 3.03 0.11 for bleach with vehicle group. The thickness increased after bleach instillation, and tamoxifen can reduce this effect.
Control
Tamoxfen
DMSO
Fig. 3: The thickness of visceral peritoneum of liver. The thickness of tamoxifen treated group was less than bleach with vehicle group (DMSO)
Peritoneum thickness around liver surface
0.28 1.17 3.03 0.00 1.00 2.00 3.00 4.00 C T D Th ic kne ss 數列1
Fig. 4: The visceral peritoneum thickness around the liver surface. The thickness of control group was significantly less than the tamoxifen (T) and bleach vehicle (D) groups
(P was less than 0.0001 in both groups.) The thickness was also significantly reduced in tamoxifen group (P< 0.0001).
Control
Tamoxifen
DMSO
Fig. 5: Border of omentum was thickened after bleach instillation.
Control
DMSO
Fig. 6: The border of mesentery thickened with neovasclarization after bleach
instillation.
Although it was difficult to exactly measure the peritoneal thickness around intestine and omentum, the borders of the omentum and intestine mesentery were also thickened (Fig. 5, 6). In addition, the neovascularization was also noted in the subperitoneal area.
四計畫成果自評
In the past two years, we found that tamoxifen was effective in reduce collagen production in vitro and reduce peritoneal fibrosis in vivo. The subperitoneal area neovascularization was also noted in our animal model. The tamoxifen had been noted to reduce angiogenesis in cancer. The relationship between tamoxifen and bleach induce angiogenesis is worth our further investigation.
五、參考文獻
1. Dobbie JW. Pathogenesis of peritoneal fibrosing syndrome (scelerosing peritonitis) in peritoneal dialysis. Perit Dial Int 1992; 12: 14-27
2. Allaria PM, Giangrande A, Gandini E, Pisoni IB. Continuous ambulatory peritoneal dialysis and sclerosing
encapsulating peritonitis: tamoxifen as a new therapeutic agent. J Nephrol 1999; 12: 395-7
3. al-Musawi D. Mitchenere P. al-Akraa M. Idiopathic retroperitoneal fibrosis treated with tamoxifen only. Br J Urol 1998; 82: 442-3
4. Frankart L. Lorge F. Donckier J. Tamoxifen for retroperitoneal fibrosis.
Postgrad Med J 1997; 73: 653-4
5. Venkataramani A. Behling CA. Lyche KD. Sclerosing mesenteritis: an unusual cause of abdominal pain in an
HIV-positive patient. Am J Gastroenterol 1997; 92: 1059-60
6. Savelli BA. Parshley M. Morganroth ML. Successful treatment of sclerosing
cervicitis and fibrosing mediastinitis with tamoxifen. Chest 1997; 111: 1137-40
附件：封面格式
行政院國家科學委員會補助專題研究計畫成果
報告
Tamoxifen 對於治療腹膜硬化症的分子機制與動物模式研究
計畫類別：█個別型計畫 □整合型計畫
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