臺灣博碩士論文加值系統

人類高血壓的形成與血管壁之生理學上及生化學上的改變有關。這些改變中包括了血管平滑肌的過度收縮所導致的周邊阻力的增加。血管平滑肌收縮的程度取決於肌凝蛋白輕鏈(myosin light chain, MLC)磷酸化的程度，而其磷酸化程度又受MLC激酶(myosin light chain kinase, MLCK)與MLC磷酸酶(myosin light chain phosphatase, MLCP)兩者活性的調控。MLCP的活性不屬於鈣離子從屬性(calcium dependent manner)，其所調節的血管張力稱為鈣離子敏感化(calcium sensitization)。
本論文包含兩個部分，第一部分有關血管平滑肌細胞(vascular smooth muscle cells, VSMCs)內的細胞生理, 探討血管張力素II (angiotensin II, Ang II)對於鈣離子敏感化訊息路徑的作用。而第二部分比較了高血壓個體和正常血壓個體, 其VSMCs中此鈣離子敏感化訊息路徑的差異。
在第一部分中，我們找尋確認在VSMCs中，Ang II刺激之後，會接合到單體G蛋白(小G蛋白)(monomeric G protein, small G protein) Rho A以及活化Rho A/Rho kinase/MYPT1訊息軸線的專一蛋白分子。將Sprague-Dawley大鼠的初級培養之血管平滑肌細胞處理以Leukemia-associated RhoGEF (LARG)的小分子干擾核糖核酸(Small Interfering RNA, siRNA)，接著處理以Ang II 加上 PD123319(Ang II第二型受體拮抗劑，AT2 antagonist), Ang II加上losartan (Ang II第一型受體拮抗劑，AT1 antagonist), 或者 Val5-Ang II (Ang II第一型受體促進劑，AT1 agonist)。隨後以定量及時聚合酶連鎖反應(quantitative real-time PCR, qPCR)來測定mRNA含量，以西方墨點法(Western blot analysis)測定蛋白含量。另外使用等長收縮力量紀錄法(an isometric force recording method)來測量主動脈環收縮力。結果顯示，在Ang II刺激之後的0.5小時，leukemia-associated RhoGEF (LARG) mRNA增加達顯著差異。以Ang II加上PD123319刺激，或以Val5-Ang II刺激後的3、6、和9小時，其LARG蛋白量、Rho A蛋白活性、以及myosin phosphatase target subunit 1 (MYPT1)的磷酸化程度皆有顯著增加。此外，用siRNA將LARG基因減弱(knock down)之後，降低了VSMCs對於AT1受體活化後所造成的收縮效應。主動脈環的收縮力研究也證實了LARG siRNA會降低Rho A/MYPT1對Ang II刺激的活化效應。
這部分的結果提供了一個直接證據，就是Ang II透過AT1受體, 向上調節LARG的基因表達，並且會活化LARG/Rho A/MYPT1訊息軸線，因而維持血管張力。
在第二部分，我們比較高血壓和正常血壓大鼠其血管平滑肌中，三個RhoGEF包括LARG，p115-RGEF，以及PSD-95/Disc-large/ZO-1 homology (PDZ)-RhoGEF表現的不同特性。自發性高血壓鼠(spontaneously hypertensive rats, SHR)和Wistar-Kyoto rats (WKY)的初級培養血管平滑肌細胞(primary cultured VSMCs)處理以Ang II加上PD123319或者Val5-Ang II。之後，三種RhoGEFs的mRNA含量以qPCR測量。以西方墨點法測定三種RhoGEFs蛋白的含量。主動脈環則先以LARG siRNA或者scramble siRNA處理48小時，之後進行收縮力紀錄實驗。實驗結果發現，WKY的VSMCs中，p115-RhoGEF, PDZ-RhoGEF，和LARG其mRNA的基礎表現量並未隨年齡而增加。然而，在12週大的成年SHR，這三種RhoGEF的mRNA表現量，相較於5週大的未成年SHR有顯著增加。而在AT1活化之後，只有WKY(包括5週和12週)、5週大SHR的LARG的mRNA分別在0.5小時和6小時有顯著增加。蛋白方面, 12週大WKY，在AT1活化之後6小時，觀察到LARG蛋白增加，但在12週大的SHR其LARG蛋白表現在AT1活化之後仍然沒有改變。再者，在12週大的SHR，主動脈環LARG knockdown可以修正其主動脈環過度的收縮。
我們這部分的研究結果顯示了，這三種RhoGEF基因的基礎表現，以及其對於AT1促進劑刺激的反應，而這些表現在SHR和WKY之間也有年齡從屬性(age-dependent manner)的差異。這些結果顯示LARG的基因表現情形與或許與SHR高血壓的發生有某些關聯。
綜合言之，Ang II透過AT1來活化Rho A的上游分子LARG，因此活化Rho A/Rho kinase/MYPT1訊息軸線，而維持了血管基本張力；自發性高血壓個體可能因為其VSMCs的AT1數量較多，導致Ang II透過AT1調控LARG的基因表現增多，因而過度加強了此軸線的活性，而導致血管過度收縮，周邊阻力增加，而引起高血壓。

Human hypertension is associated with physiological and biochemical changes in vascular walls. These changes include excessive contractions of vascular smooth muscles that lead to an increase in peripheral resistance. The extent of vascular smooth muscle contraction depends on the phosphorylated level of myosin light chain (MLC), regulated by the balance between the activity of MLC kinase (MLCK) and MLC phosphatase (MLCP). MLCP activity is independent of Ca2+, the MLCP-mediated regulation of vascular tone is known as Ca2+ sensitization. This dissertation contains two parts. The first part studied the cell physiology of VSMCs including the effect of Ang II on calcium sensitization pathway. And the second part studied the comparison of RhoGEFs properties difference between hypertensive (SHR) and normotensive (WKY) subject.
In the first part, we identify a specific molecule(s) that binds monomeric G protein Rho A and activates the Rho A/Rho kinase/MYPT1 axis, by stimulation of Angiotensin II (Ang II). Primary cultured vascular smooth muscle cells (VSMCs) from Sprague-Dawley rats were treated with or without siRNAs against leukemia-associated RhoGEF (LARG) and then treated with Ang II plus PD123319, Ang II plus losartan, or Val5-Ang II. Subsequently, mRNA and protein levels were determined by quantitative real-time PCR and Western blot analysis. An aortic ring contractile tension experiment was carried out using an isometric force recording method. The results indicated that after Ang II stimulation, LARG mRNA was significantly increased at 0.5hours. The amounts of LARG protein, Rho A activity, and phosphorylation of myosin phosphatase target subunit 1 (MYPT1), were increased at 3, 6, and 9 hours after treatment with Ang II plus PD123319 or Val5-Ang II. Moreover, knockdown of LARG by siRNA reduced the effects of AT1 receptors activation in VSMCs. The ex vivo contractile force study using aortic rings, confirmed the LARG siRNA diminishing effect on Rho A/MYPT1 activity in response to Ang II. Our results provide direct evidence that Ang II upregulates LARG gene expression and activates the LARG/Rho A/MYPT1 axis via AT1, thereby maintaining vascular tone.
In the second part, the different properties of the three RhoGEF including leukemia-associated RhoGEF (LARG), p115-RhoGEF, and PDZ-RhoGEF expression in vascular smooth muscle between hypertensive and normotensive rats were compared. Primary cultured VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were treated with angiotensin II (Ang II) plus Ang II type 2 receptor antagonists PD123319, or Ang II type 1 receptors (AT1) agonist Val5-Ang II. The mRNA levels of RhoGEFs were measured by qPCR. The protein levels of RhoGEFs were measured by Western blot analysis. Aortic rings were pretreated with LARG siRNA or scrambled siRNA for 48 h; and then, the contractile force was recorded. The results show that the baseline levels of p115-RhoGEF, PSD-95/Disc-large/ZO-1 homology (PDZ)-RhoGEF, and LARG mRNA in VSMCs of WKY does not increase with age. However, there is a significant elevation of these RhoGEFs’ mRNA in the 12-week old SHR (SHR-12W) compared to the 5-week old SHR (SHR-5W). After AT1 activation by agonists, only LARG mRNA of WKY-5W, WKY-12W, and SHR-5W significantly increased at 0.5 and 6 h. The LARG protein increases at 6 h after AT1 activation in WKY-12W, but it remains unchanged in SHR-12W. Moreover, the ex vivo LARG-knockdown can correct excessive contraction of the aortic ring of SHR-12W. We show the differences of age-dependent RhoGEF expression and in response to Ang II stimulation between SHR and WKY rats. The LARG is differentially regulated and knockdown of LARG reduces more contraction of VSMCs in SHR. The findings support that LARG gene expression maybe related to the genesis of hypertension in SHR.
In summary, Ang II activate LARG protein, the upstream molecule of Rho A, by AT1 and activate Rho A/Rho kinase/MYPT1 axis to maintain the vascular basal tone. The spontaneously hypertensive subject may be because of the large amount of AT1 on VSMC, leading to the increased expression of LARG regulated by Ang II. These excessively enhance the activity of the Rho A/Rho kinase/MYPT1 axis and lead to excessive vasoconstriction and hypertension via increased peripheral resistance.

口試委員會審定書 ii
誌謝 iii
目錄 iv
中文摘要 1
Abstract 5
導論 9
第一部分 15
血管張力素II調節大白鼠血管平滑肌中LARG/Rho A/MYPT1訊息軸線 15
Angiotensin II Regulates LARG/Rho A/MYPT1 axis in Rat Vascular Smooth Muscle 15
材料方法 16
細胞培養 16
RNA萃取以及反轉錄步驟 16
定量即時聚合酶連鎖反應(quantitative real-time PCR, qPCR) 16
細胞萃取蛋白以及西方墨點法 18
第一種全細胞蛋白萃取 18
第二種膜質分開萃取蛋白(for Rho A translocation detection) 18
Rho GTPase 活性檢測 19
siRNA 試驗 20
主動脈環張力測量 20
統計分析 22
Manufacturer Reference Guide 22
實驗結果 24
大鼠主動脈平滑肌細胞在Ang II刺激之後 RhoGEFs的基因表現 24
Ang II透過AT1 受器來誘發LARG蛋白表現 24
AT1受器活化可以刺激Rho A蛋白的活性增加並位移至細胞膜 24
LARG的基因減弱(gene knockdown)會降低Rho A蛋白的活性 25
AT1 活化會增加 MYPT1 磷酸化 26
LARG的基因減弱降低 MYPT1 磷酸化程度 26
預先處理LARG siRNA會減少因Ang II所引起的血管收縮 27
討論 28
第二部分 34
自發性高血壓大白鼠血管平滑肌LARG基因基礎表現以及受血管張力素II刺激之後的基因表現 34
Differential Baseline Expression and Angiotensin II Stimulation of LARG in Vascular Smooth Muscles of Spontaneously Hypertensive Rats 34
材料方法 35
初級細胞培養 35
RNA萃取以及反轉錄步驟 36
定量及時聚合酶連鎖反應(quantitative real-time PCR, qPCR) 36
蛋白萃取以及西方墨點法 37
細胞蛋白萃取 37
組織蛋白萃取 38
主動脈環張力測量 39
統計分析 40
實驗結果 41
RhoGEF mRNA在SHR和WKY中的表現 41
RhoGEF mRNA基礎表現量 41
RhoGEF mRNA在Ang II刺激之後的表現 41
在WKY和SHR，LARG蛋白的表現 42
SHR主動脈肌肉環以LARG siRNA預處理，對其血管自發性張力及收縮的影響 43
討論 45
總結 50
未來方向 51
圖1. 平滑肌細胞內鈣離子敏感化訊息路徑示意圖 54
圖2. 血管平滑肌細胞以血管張力素II (Ang II)刺激之後，三種RhoGEF其mRNA的表現情形 55
圖3. 血管平滑肌細胞受刺激之後其LARG 蛋白的表現情形 57
圖4. 血管平滑肌細胞受刺激之後，Rho A蛋白活化的情形 60
圖5. 將LARG的小干擾核糖核酸(small interfering RNA, siRNA)遞送至血管平滑肌細胞內以造成LARG基因減弱(gene knockdown)的效果 61
圖6. 血管平滑肌細胞LARG基因減弱(gene knockdown)模式下，Rho A蛋白的活化程度 63
圖7. 血管平滑肌細胞在Ang II第一型受體(AT1)活化之後，MYPT1磷酸化的程度 65
圖8. 血管平滑肌細胞LARG基因減弱(gene knockdown)模式下，MYPT1磷酸化的程度 66
圖9. 以主動脈環(aortic rings)進行等長收縮張力(isometric contractile tension)測量之前，LARG基因減弱效果之確認 68
圖10. 各種處理對於Ang II所引起的主動脈環等長收縮張力的影響 69
圖11. LARG siRNA減弱Ang II所引起的主動脈環等長收縮力 71
圖12. 主動脈環等長收縮張力測量之後，LARG基因減弱之再確認 72
圖13. 螢光染色-1 73
圖14. 螢光染色-2 74
圖15. 免疫染色－DAB呈色 75
圖16. p115-RhoGEF mRNA的表現 76
圖17. PDZ-RhoGEF mRNA的表現 77
圖18. LARG mRNA的表現。 78
圖19. LARG蛋白表現 80
圖20. p115-RhoGEF蛋白表現 83
圖21. PDZ-RhoGEF蛋白表現 85
圖22. 以主動脈環(aortic rings)進行等長收縮張力(isometric contractile tension)測量之前，LARG基因減弱效果之確認 86
圖23. LARG基因減弱對於Ang II所引起的WKY及SHR主動脈環等長收縮張力的影響(代表性曲線圖) 87
圖24. LARG基因減弱對於Ang II所引起的WKY及SHR主動脈環等長收縮張力的影響 88
圖25. AT1和AT2在不同品系及不同週齡的大鼠血管平滑肌細胞的基因表現情形 90
圖26. 組織切片免疫染色--AT1 91
圖27. 組織切片免疫染色—AT2 92
參考文獻 93
附錄：發表之相關論文 100

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