以齧齒類缺血性腦中風模式進行腦部血管相關基因之研究

Studies of Vascular Related Genes in Rodent Stroke Model

賴達明(Dar-Ming Lai)

指導教授 ： 謝豐舟 ； 杜永光 ； 李鴻

國立臺灣大學/醫學院/臨床醫學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.10064

摘要

在腦中風時，腦部組織缺血，腦血流中血紅素帶氧量降低。由於腦神經氧氣供應不足，因此腦血管產生立即反應，包括血管擴張、腦血流量增加等現象，同時腦部即誘發一連串基因或蛋白質的變化，其中許多血管相關基因會相對增加，而一些離子閥、輸送相關基因則可能降低。目前一些實驗室已成功藉調控某些血管生成蛋白的量，使中風模式後老鼠腦壞死範圍縮小。然而由於血管作用蛋白很多，彼此間又會相互作用，因此了解個別的、各種新的相關蛋白的作用為相當重要的課題。
血管生成素相似蛋白1(Angptl1)，為新近發現血管生成素相似蛋白質族中之一員 (Kim, et al., 1999b)，可調控血管增生，目前對其於生理上所扮演的角色，我們所知仍不多。我們發現在正常老鼠腦缺血後第七天，angptl1的表現量有增加的現象。
為了瞭解angptl1於腦中風所扮演的角色，我們在膠質原纖維酸性蛋白(GFAP)啟動子-angptl1基因轉殖小鼠及正常小鼠上，分別引發局部腦缺血。在未引發缺血之基因轉殖鼠中，angptl1大量於腦部表現，使得大腦皮層微小血管密度有上升之情形。雖然基因轉殖鼠及原生型小鼠在發生局部腦缺血後，其中風範圍大小相同，基因轉殖鼠腦水腫的程度則較同一胎原生型之小鼠為小。我們認為這可能是因為基因轉殖鼠的血腦障壁被破壞程度較原生型小鼠小，藉由中風後早期進行伊文斯藍滲漏實驗，我們證明基因轉殖鼠滲漏的程度較為減少。所以我們相信在局部腦缺血後，angptl1具有保護並維持血管完整性之功用。
Angptl1在胚胎發育或個體血管生成過程中所可能扮演的角色，目前我們所知的仍舊很少。為確定angptl1在胚胎生成過程中所扮演的角色以及表現的部位，我們利用基因標靶以生殖出缺乏angptl1但同時帶有nLacZ品系之小鼠。取樣自E9.5天至六個月大的小鼠，進行ß-乳糖水解酶之染色後發現，angptl1最初會在旁軸的中胚層中表現。之後會轉至肌間質之結締組織(筋膜)、關節囊及軟骨膜(喉頭-氣管，肋骨及長骨)等處表現，但肌肉則無。血管系統、中央及週邊神經系統、消化、呼吸及其他主要器官系統中，並沒有任何angptl1之表現。根據所表現的位置可推測angptl1與結締組織及軟骨的發育有關。突變型小鼠沒有明顯的表現型，有可能是因為其功能仍受許多相關的因子之影響。
Adenosine為一血管擴張、新生物質，既往文獻有提到adenosine系統在腦缺血之生理變化佔有重要的角色。為了解adenosine A1受體於腦缺血模式中所扮演的角色，我們以Wistar大鼠作為實驗動物，阻斷其兩側頸動脈與中大腦動脈的血流，造成腦缺血現象。腦缺血的大鼠中，其大腦皮層內的adenosine A1受體之基因表現量，與經過sham作為控制組，以及在暫時缺血後恢復原先的血流的大鼠相比之下，腦缺血的大鼠，其大腦皮層的adenosine A1受體於核醣核酸（RNA）表現量，有明顯上升之趨勢；而經過暫時缺血又再灌流恢復血流的大鼠，adenosine A1受體於核醣核酸中的表現量與控制組的大鼠幾乎相同，並沒有明顯之提升。另外腦缺血後大鼠其adenosine A1受體蛋白質的表現，結果幾乎與RNA表現相同。由這些實驗數據，我們推測因缺血後，腦內會有補償反應的發生，導致adenosine A1受體的基因表現量增加，而adenosine A1受體的增加，推想是受傷的組織中產生出來之一種保護性角色。

關鍵字

缺血性腦中風 ； 血管生成素相似蛋白1 ； 腺甘酸

並列摘要

Angiopoietin-like protein (Angptl) 1, a member of the angiopoietin-related protein family, modulates angiogenesis but little else is known of its physiological role. We found that angptl1 was upregulated at the 7th day after focal cerebral ischemia in normal mice. In order to understand the role of angptl1 in cerebral infarction, we induced focal cerebral ischemia in normal and glial fibrillary acidic protein promoter-angptl1 transgenic mice. In the transgenic mice without ischemia, overexpression of angptl1 in the whole brain led to a decrease in cortical microvascular density. Following focal cerebral ischemia, edema, but not infarct size, was less in transgenic mice relative to wild type littermates. This effect might be due to a reduction in the blood brain barrier breakdown, as confirmed by a decrease in Evans Blue leakage in the early post-ischemic phase. We conclude that angptl1 may have a beneficial role in the preservation of vascular integrity following focal cerebral ischemia.
Little is known of angptl1’s potential role in other processes. To identify the expression pattern and possible role of angptl1 during embryogenesis, we used gene targeting to generate angptl1 deficient, nLacZ knock-in mice. Staining for ß-galactosidase from embryonic day 9.5 to 6 months of age revealed that angptl1 was initially expressed in the paraxial mesoderm. Expression then shifted to intermuscular connective tissue (fascial plane), joint capsules and perichondrium (laryngo-trachea, ribs and long bones) but not the muscles. The vasculature, central and peripheral nervous systems, digestive, respiratory and other major organ systems did not show any angptl1 expression. This expression pattern suggests that angptl1 is related to development of the connective tissue and cartilage. Lack of phenotype in mutant mice may be due to a functional redundancy from other related factors.
In an attempt to know the role of adenosine A1 receptor in cerebral ischemia, the present study employed the ligation of bilateral carotid arteries to induce ischemia in Wistar rats. Changes of gene expression of adenosine A1 receptor in cerebral cortex of ischemic rats were compared with normal sham control and reperfusion group that received regular blood flow after a transient ischemia. The mRNA level of adenosine A1 receptor in cerebral cortex was markedly raised by this artificial ischemia. Also, reperfusion reversed this elevation to a level near the control. This change was also observed at the protein level using Western blot analysis of adenosine A1 receptor. The raised protein level of adenosine A1 receptor by ischemia was reversed to normal level after reperfusion. These data suggest that the gene expression of adenosine A1 receptor was increased by ischemia probably due to the compensative response of brain. The raised adenosine A1 receptor may play a protective role in these damaged tissues.

並列關鍵字

Stroke ； Angptl1 ； Adenosine

參考文獻

Tsao, P.N., Li, H., Wei, S.C., Ko, M.L., Chou, H.C., Hsieh, W.S. and Hsieh, F.J., 2004. Expression of angiogenic factors and their receptors in postnatal mouse developing lung. J Formos Med Assoc 103, 137-143.

Adair, T.H., 2005. Growth regulation of the vascular system: an emerging role for adenosine. Am J Physiol Regul Integr Comp Physiol 289, R283-R296.

Aden, U., Lindstrom, K., Bona, E., Hagberg, H. and Fredholm, B.B., 1994. Changes in adenosine receptors in the neonatal rat brain following hypoxic ischemia. Brain Res Mol Brain Res 23, 354-358.

Alzheimer, C., Sutor, B. and ten Bruggencate, G., 1993. Disinhibition of hippocampal CA3 neurons induced by suppression of an adenosine A1 receptor-mediated inhibitory tonus: pre- and postsynaptic components. Neuroscience 57, 565-575.

Amiry-Moghaddam, M., Otsuka, T., Hurn, P.D., Traystman, R.J., Haug, F.M., Froehner, S.C., Adams, M.E., Neely, J.D., Agre, P., Ottersen, O.P. and Bhardwaj, A., 2003. An alpha-syntrophin-dependent pool of AQP4 in astroglial end-feet confers bidirectional water flow between blood and brain. Proc Natl Acad Sci U S A 100, 2106-2111.