探討HMG CoA還原酶抑制劑對於視網膜色素上皮細胞分泌血管內皮生長因子的影響

Effect of HMG CoA reductase inhibitor on VEGF secretion of human retinal pigment epithelium

張集武(Chi-Wu Chang)

指導教授 ： 王惠珠

共同指導教授 ： 吳文權(Wen-Chuan Wu)

高雄醫學大學/醫學院/醫學研究所/碩士(2007年)

摘要

研究目的：本實驗之研究目的，在探討降血脂藥物HMG CoA reductase inhibitor對人類視網膜色素上皮細胞分泌血管內皮生長因子之影響。

研究材料及方法：本實驗以培養之人類視網膜色素上細胞為對象，加入HMG CoA reductase inhibitor後，先以MTT ASSAY測試是否產生細胞毒性並觀察細胞型態上的變化，再以固定濃度之HMG CoA reductase inhibitor，Atorvastatin 1.0 μM與視網膜色素上細胞作用。以RT-PCR及electrophoresis偵測VEGF mRNA的表現，並以ELISA的方法偵測VEGF proteins濃度的變化，觀察VEGF的濃度是否會受到HMG CoA reductase inhibitor的影響。

研究結果：HMG CoA reductase inhibitor在較低濃度0.1μM和1.0μM時，24小時和48小時之後並未對視網膜色素上皮細胞產生毒性反應。10 μM濃度在24小時的時間，MTT assay的數值顯示，並沒有產生明顯細胞死亡的情形。在48小時細胞存活度明顯下降(P < 0.01)，表示10 μM濃度的HMG CoA reductase inhibitor對視網膜色素細胞，隨時間增加而產生毒性作用。加入1.0 μM濃度的HMG CoA reductase inhibitor之後，視網膜色素細胞合成VEGF mRNA 的表現有顯著下降的情形(P < 0.05)。此外，加入1.0 μM濃度的HMG CoA reductase inhibitor也會讓視網膜色素細胞分泌VEGF proteins出現顯著下降的情形(P < 0.05)。

結論：HMG CoA reductase inhibitor在體外的實驗顯示10 μM濃度時，對視網膜色素上皮細胞會產生毒性反應。在1.0 μM濃度時，會對視網膜色素上皮細胞VEGF轉錄及轉譯作用產生抑制現象，而使VEGF蛋白質合成的濃度減少。

關鍵字

血管內皮生長因子 ； HMG CoA還原酶抑制劑 ； 視網膜色素上皮細胞

並列摘要

Purpose: To determinate whether HMG CoA reductase inhibitor affects vascular endothelial growth factor secretion of human retinal pigment epithelium cells.

Material and method: Cultured human pigment epithelium cell line (R50) was applied in our study. To determine cytotoxicity of HMG CoA reductase inhibitor, MTT assay and morphological observation under microscope were undergone. We added the HMG CoA reductase inhibitor to retinal pigment epithelium cells in safe concentration of 1.0 μM to evaluate VEGF mRNA expression at certain time point by RT-PCR and electrophoresis. VEGF proteins were also detected by ELISA method after retinal pigment epithelium cells incubated with HMG CoA reductase inhibitor.

Result: At lower concentration of 0.1 and 1.0 μM HMG CoA reductase inhibitor, MTT assay showed no cytotoxicity to retinal pigment epithelium cells at time of 24 and 48 hours. MTT assay showed no cytotoxicity for 10 μM HMG CoA reductase inhibitor to RPE cells at time of 24 hours. However, markedly decreased of RPE cells survival after 48 hours of incubation (P < 0.05).
After 1.0 μM HMG CoA reductase inhibitor was added to RPE cells, VEGF mRNA expression was suppressed ( P < 0.05 ) at 30, 60 and 360 minutes compared with control. 1.0μM HMG CoA reductase inhibitor also demonstrate reduction of VEGF protein after incubated with RPE cells.

Conclusion: In vitro, HMG CoA reductase inhibitor showed marledly cytotoxicity to RPE cells at concentration of 10 μM after 48 hours. Under concentration without cytotoxicity, HMG CoA reductase inhibitor suppressed the transcription of VEGF mRNA and translation of VEGF protein from RPE cells.

並列關鍵字

Vascular endothelial gorwth factor ( VEGF ) ； HMG CoA reductase inhibitor ； retinal pigment epithelium ( RPE )

參考文獻

1. Russell WR, Lawrence PC, Narsing AR, et at al. Occlusive retinal vasculitis associated with systemic lupus erythematous. Arch Ophthalmol 2000; 118(4): 588-9.

2. Unilateral retinal vasculitis, branch retinal artery occlusion and subsequent retinal neovascularization in Crohn’s disease. International Ophthalmol 2001; 24(2): 89-92.

3. Aiello LP, Avery RL, Arrigg PG, Keyt BA, Jampel HD. VEGF in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med 1994; 331:1480-1487.

4. Peter Carmeliet, Rakesh KJ. Angiogenesis in cancer and other diseases. Nature 2000; 407: 249-257.

5. Peter Carmeliet. Mechanism of angiogenesis and arterogenesis. Nature Med 2000; 6: 389-395.