高醣腹膜透析液及發炎細胞激素對腹膜間皮細胞前列腺素、血管內皮細胞生長因子的作用:COX抑制劑的影響

The effect of high glucose peritoneal dialysis solution and proinflammatory cytokines on prostaglandin and vascular endothelial growth factor (VEGF) production in human peritoneal mesothelial cells : effect of cyclooxygenase (COX) inhibitor

吳文中(Wen-chung Wu)

指導教授 ： 蔡敦仁

共同指導教授 ： 郭明良

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00832

摘要

一､中文摘要

背景：長期腹膜透析會導致腹膜結構與弁鄐W的變化甚至發生嚴重併發症，使腹膜透析無法持續。因此，如何保持腹膜完整性是一項重要課題。臨床研究發現，腹膜透析患者發生脫水不良(ultrafiltration failure)時，其微血管的增生(已知與VEGF-A最有關)促使溶質吸收加速及淋巴吸收增加(原因目前未明)是兩項最重要的因素。因此我們設計實驗，探討高醣透析液及發炎細胞激素對腹膜間皮細胞的PGE2及VEGF(VEGF-A或VEGF -C,D)的影響﹔PGE2對腹膜間皮細胞VEGF的影響﹔與COX抑制劑對經高醣透析液及發炎細胞激素處理時腹膜間皮細胞的VEGF的影響。

方法：本研究以培養的腹膜間皮細胞 (human peritoneal mesothelial cell , HPMC)作實驗：(一)、高醣腹膜透析液及IL-1β加入培養中的腹膜間皮細胞,以VEGF-A ELISA ; VEGF-A,C,D RT-PCR方法來偵測所增加的VEGF是何種type [A,C,D?] 並以COX 的Western blot, PGE2 ELISA來偵測是否增加？(二)、以PGE2加入培養中的腹膜間皮細胞, 以VEGF-A ELISA ; VEGF-A,C,D RT-PCR方法來偵測其變化。 (三)、高醣腹膜透析液及IL-1β加入培養的腹膜間皮細胞中,再加入COX抑制劑,以VEGF-A ELISA; VEGF-A,C,D RT-PCR方法 來偵測其變化。

結果：將高醣腹膜透析液 (Dianeal 1.5％, 2.5％, 4.25％)或IL-1β(1, 5 ng/ml)加入培養中的腹膜間皮細胞時，VEGF-A、 COX-2、及PGE2分泌皆增加﹔但VEGF-C,或D分泌未增加。高醣腹膜透析液及IL-1β加入培養的腹膜間皮細胞中,再加入COX抑制劑時，PGE2降低;但VEGF-A 並未減低。PGE2加入腹膜間皮細胞,並不會引起VEGF-A之增加。腹膜間皮細胞所增加的VEGF-A,並不會被COX 抑制劑所抑制。於是我們探討腹膜間皮細胞上PGE2 的EP 受體主要是EP3,EP4受體 ，異於大腸癌細胞上EP受體(主要是EP1,EP2受體) 。如此可以解釋COX 抑制劑對腹膜間皮細胞並不像對大腸癌細胞可以抑制VEGF-A的生成。此外，我們發現高醣透析液及PGE2加入培養中的腹膜纖維母細胞 (fibroblast)可以引起VEGF-C之增加，並且經高醣腹膜透析液刺激之腹膜纖維母細胞所增加的VEGF-C,會被COX 抑制劑(NS398)所抑制。

結論：高醣腹膜透析液及IL-1β對腹膜間皮細胞所增加的VEGF是VEGF-A，而VEGF-C或D表現 並未增加﹔高醣腹膜透析液及IL1-β對腹膜間皮細胞所增加的VEGF-A，並不會被COX 抑制劑所抑制。PGE2並不會促進腹膜間皮細胞分泌VEGF-A增加，此與腹膜間皮細胞上PGE2的EP受體種類可能有關。但經高醣腹膜透析液及PGE2刺激之腹膜纖維母細胞，VEGF-C卻是增加，並且經高醣腹膜透析液刺激之腹膜纖維母細胞所增加的VEGF-C，會被COX 抑制劑(NS398)所抑制。腹膜纖維母細胞所分泌VEGF-C是否與腹膜脫水不良、腹膜淋巴吸收增加相關？是我們未來要努力的目標。

關鍵字

間皮細胞 ； 血管內皮細胞生長因子 ； 前列腺素

並列摘要

八､論文英文簡述（Summary）

Background. The structural and functional changes in peritoneum always bothered the patients reveiving long-term continuous ambulatory peritoneal dialysis (CAPD). It was suggested that human peritoneal mesothelial cells (HPMCs) contribute to the production of vascular endothelial growth factor (VEGF),which may enhance vasodilation, vascular permeability, and neoangiogenesis in peritoneal membrane. The present study was designed to evaluate the influence of high glucose dialysis solution and porinflammatory cytokine on prostaglandin and VEGF production in HPMC and the influence of cyclooxygenase (COX) inhibitor.

Methods. HPMCs were cultured from human omentum by an enzyme digestion method. High glucose dialysis solution (Dianeal 1.5%, 2.5%, 4.25%) , IL-1β(1, 5 ng/ml) , PGE2 ,and High glucose dialysis solution /IL-1β with COX inhibitor were tested in cultured HPMCs respectively. Expression of COX-2 and PGE2 proteins were dteremined by Western blot and EIISA. VEGF-A was measured in cell supernatant by ELISA and VEGF-C were measured by Western blot and VEGF-A,C,D mRNA expressions were evaluated by RT-PCR.

Results. High glucose dialysis solution (Dianeal 1.5%, 2.5%, 4.25%) and IL-1β( 1, 5 ng/ml) enhanced the VEGF-A, COX-2, PGE2 expression in HPMC culture; but were not able to up-regulate the VEGF-C or D. High glucose dialysis solution (Dianeal 1.5%, 2.5%, 4.25%) and IL-1β(1, 5 ng/ml) in HPMC culture with COX inhibitor was not able to down-regulate the VEGF-A even with the reduction of PGE2 level. PGE2 was not able to up-regulate the VEGF-A in HPMC culture. COX inhibitor failed to inhibit the VEGF-A produced by the high glucose dialysis solution and IL-1β in HPMC culture. We studied the PGE2 receptors distributed on the surface of HPMC. EP3 and EP4 receptors predominate on HPMC surface. In the present study, the VEGF-C production increased while peritoneal fibroblasts cultured with high glucose dialysis solution and the increment of VEGF-C could be inhibit by COX inhibitor (NS398).

Conclusion. The VEGF up-regulated by the high glucose dialysis solution and IL-1β in HPMC culture is VEGF-A, not VEGF-C or VEGF-D. VEGF-C production increased while peritoneal fibroblasts cultured with high glucose dialysis solution and the increment of VEGF-C could be inhibited by COX inhibitor (NS398). COX inhibitor failed to inhibit the VEGF-A expression by HPMCs cultured with high glucose dialysis solution and IL-1β. PGE2 was not able to up-regulate the VEGF-A production in HPMC culture. In the future studies, we will try to elucidate the relationship between the VEGF-C produced by peritoneal fibroblasts and the peritoneal ultrafiltration failure or peritoneal lymphatic absorption .

並列關鍵字

human peritoneal mesothelial cells ； vascular endothelial growth factor (VEGF) ； prostaglandin

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