黃體激素對老鼠動脈平滑肌細胞的生長抑制作用

The antiproliferation effect of progesterone in rat arterial smooth muscle cells

劉兆偉(Chao-Wei Liu)

指導教授 ： 李文森

臺北醫學大學/醫學院/醫學科學研究所/碩士(2001年)

摘要

中文摘要 本篇論文主要是在探討黃體激素(progesterone)對於老鼠的主動脈平滑肌細胞(Rat aortic smooth muscle cells)生長的抑制情形及其相關的作用機轉。由動脈粥狀硬化等心血管疾病所造成的死亡人數的研究發現，停經前的婦女要比同年齡的男性得病機會少(Gordon et al., 1978)，而停經後的婦女如果同時服用適量的雌性素(estrogen)以及助孕素(progestin)，也會比只有單獨服用estrogen的人較不易得心血管疾病。已經發表的文獻中對於estrogen的效用探討頗多，但對於progestin的單獨作用效果則較少探究。Progesterone 已被發現對於老鼠動脈平滑肌細胞的生長具有抑制的效果。利用北方墨點法(Northern blotting)發現和細胞週期相關的細胞週期素Cyclin A及Cyclin E的mRNA在老鼠的動脈平滑肌細胞內表現有受到progesterone的抑制。因此，推測progesterone可能是影響細胞在由G1 phase進入到S phase的過程。本研究即以西方墨點法（Western blotting)的方法來進一步的探討progesterone對於老鼠的動脈平滑肌細胞的細胞週期之影響。 由我們的研究結果發現，黃體激素(progesterone)存在時(500 nM)，會使得老鼠的主動脈平滑肌細胞(Rat aortic smooth muscle cells)的生長受到抑制。而進一步去探討其作用機轉時，發現隨著progesterone濃度的增加(0~500 nM)，一些和細胞週期調控相關的蛋白質如：Cyclin A、Cyclin E、Cdk 2、Cdk 4的表現量都有逐漸減少的情形。除此之外，一些已知會抑制細胞週期進行的cyclin dependent kinase(CDK) inhibitor如p21、p27蛋白質的表現量則隨著progesterone濃度的增加(0~500 nM)而增加。而由免疫沉澱法的結果顯示，和Cdk 2結合的cyclin dependent kinase inhibitor p21及p27的蛋白質表現量隨著progesterone濃度的增加(0~500 nM)而有增加的情形出現。而kinase assay的研究結果發現，Cdk 2的酵素活性也隨著progesterone濃度的增加(0~500 nM)而受到抑制，但是Cdk 4的酵素活性卻沒有受到影響。以p21的antisense oligonucleotide預處理細胞(5 nM, 10 nM, 20 nM/1 hr)，發現可以避免由progesterone所造成的細胞週期抑制現象，而單獨加入p21 antisense oligonucleotide則對DNA合成能力沒有影響，推測p21這種cyclin dependent kinase inhibitor在progesterone對RASMCs所造成的細胞週期抑制現象中扮演了一種關鍵的角色。 本篇論文的研究結果顯示，黃體激素(progesterone)的存在會造成老鼠的主動脈平滑肌細胞的生長受到抑制，而了解其相關的作用機轉之後，相信對於研究這種荷爾蒙在預防心血管疾病發生的保護作用方面能夠有所助益。

關鍵字

黃體激素 ； 心血管疾病

並列摘要

Abstract The objective of this thesis research is to investigate the anti-proliferation effect of progesterone on the cell line Rat aortic smooth muscle cell and its underlying molecular mechanisms. Cardiovascular diseases, the principal cause of death in the developed world, affect men far more frequently than premenopausal women of the same age (Gordon et al., 1978). Estrogens or progestins appear to have a protective effect in premenopausal women. Resent reports show that the risk of coronary heart disease is lower in women who take estrogens and progestins together rather than estrogens alone. However, there has been little evidence of an independent effect of progestins in animal studies Progesterone has been shown to have a direct and inhibitory effect on the proliferation of subcultured arterial smooth muscle cells. The levels of Cyclin A and Cyclin E mRNA decline in the prescene of progesterone in rat aortic smooth muscle cells (RASMCs), suggesting that progesterone interrupts the cell cycle at the G1/S transition. This cell cycle-dependent inhibition of arterial smooth muscle cell proliferation by progesterone may represent a mechanism for the hormone’s protective effect against cardiovascular diseases. In vitro studies demonstrated that progesterone (500 nM) induced inhibition of Rat aortic smooth muscle cell growth. Further more, western blot analysis demonstrated that progesterone (5~500 nM) induced a dose-dependent decreased expression of protein which mediated cell cycle progression：Cyclin A、Cyclin E、Cdk 2、Cdk 4. On the other hand, western blot also demonstrated an increased expression of cyclin-dependent kinase(CDK) inhibitor, p21、p27. Immunoprecipitation demonstrated that progesterone (5~500 nM) induced a dose-dependent increased expression of cyclin dependent kinase inhibitor, p21 and p27 which bind to Cdk 2. Kinase assay demonstrated that progesterone (5~500 nM) induced a dose-dependent decrease of Cdk 2 enzyme activity, but it has no effect on the enzyme activity of Cdk 4. Pretreating cells with p21 antisense oligonucleotide (5 nM, 10 nM, 20 nM/1 hr) prevents the effect of cell cycle inhibition due to progesterone, p21 antisense oligonucleotide alone in RASMC has no effect on DNA synthesis, we suggest that p21, the cyclin dependent kinase inhibitor plays a key role in RASMC cell cycle inhibition results from progesterone. In conclusion, our data demonstrated that progesterone induced an anti-proliferation effect in rat aortic smooth muscle cells. Understanding the mechanism of its effect may represent an advance to study the hormone’s protective effect against cardiovascular diseases

並列關鍵字

progesterone ； cardiovascular disease

參考文獻

L., Heinrich M.S., Thomas L. Progesterone Receptor Isoform, PR-B and PR-A, in Breast Cancer: Correlations with Clinicopathologic Tumor Parameters and Expression of AP-1 Factors. Horm. Res., 54, 32-37, 2000 Biggs, J.

L., Barret-Connor, E. and Cowan, L.D. Cardiovascular mortality and noncontraceptive use of estrogen in women: results from Lipid Research Clinics Program Follow up Study. Cir., 75, 1102-1109, 1987 Challis,J.

R.G.,Workewych,J.V.&Patrick,J.E. Diurnal changes in the concentration of progesterone in the plasma of women at 34-35weeks of gestation.J. Endocr. 89, 337-341, 1981 Chen, D.

H., Kevin, K., Chen, D.F, Amy, S., Chen, J., Perry, D.N., and Vicente, A. Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27KIP1, an inhibitor of neointima formation in the rat carotid artery. J. Clin. Invest. 99, 2334-2341, 1997 Clarke, C.

L., Sutherland, R.L.: Progestin regulation of cellular proliferation. Endocr Rev. 11, 266-301, 1991 Colditz, G.