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系統識別號 U0026-0812200910421301
論文名稱(中文) 一氧化氮對懷孕大白鼠子宮重塑的角色探討
論文名稱(英文) The Role of Nitric Oxide on Uterine Remodeling in Pregnant Rats
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 91
學期 2
出版年 92
研究生(中文) 王中林
研究生(英文) Jong-Lin Wang
電子信箱 jonglin59@yahoo.com.tw
學號 s3687103
學位類別 碩士
語文別 英文
論文頁數 79頁
口試委員 口試委員-李文森
指導教授-蔡美玲
口試委員-劉哲育
口試委員-江美治
中文關鍵字 子宮重塑  一氧化氮  平滑肌肌凝蛋白重鏈 
英文關鍵字 uterine remodeling  nitric oxide  smooth muscle myosin heavy chain 
學科別分類
中文摘要 當胚胎著床後,為了適應日漸生長的胚胎,懷孕子宮會進行一連串時間的與空間的重塑,包括細胞外基質重新排列以及平滑肌的改變。隨著懷孕的進行,懷孕早期子宮肌層的平滑肌細胞是呈現些微肥大而且組織間也輕微水腫,而在懷孕後期,平滑肌細胞肥大現象則非常明顯。平滑肌肌凝蛋白重鏈 (smooth muscle myosin heavy chain)在平滑肌細胞肥大時表現量增加。有研究指出,在大白鼠主動脈血管平滑肌細胞培養的實驗中,平滑肌肌凝蛋白重鏈的表現是與一氧化氮 (nitric oxide, NO)-環化鳥糞嘌呤核苷酸依賴性蛋白激酶 (cyclic GMP-dependent protein kinase, PKG) 訊息路徑有關。一氧化氮會藉由抑制平滑肌增生而減弱血管重塑 (vascular remodeling),即使已經知道處於長期慢性高血壓的情況,血管平滑肌細胞的肥大與增生會造成血管壁的增加,是否因為一氧化氮的增加透過降低平滑肌肌凝蛋白重鏈的表現而抑制平滑肌肥大,目前並不清楚。因此,我的研究是要探討在懷孕子宮,一氧化氮是否透過一氧化氮-環化鳥糞嘌呤核苷酸依賴性蛋白激酶訊息路徑降低平滑肌肌凝蛋白重鏈的表現而抑制平滑肌細胞肥大,進而導致子宮的重塑 (uterine remodeling)。我們的實驗以懷孕階段的大白鼠子宮作為動物模式,探討:1) 懷孕對子宮重塑的影響;2) 一氧化氮供應者 (NO donor) 對平滑肌肌凝蛋白重鏈以及一氧化氮-環化鳥糞嘌呤核苷酸依賴性蛋白激酶訊息路徑的影響;3) 懷孕對與一氧化氮有關的蛋白產量的影響。
本實驗以西方點墨轉漬法 (western blot analysis) 來測量平滑肌肌凝蛋白重鏈、平滑肌肌動蛋白 (SM-actin)、內皮型一氧化氮合成酶 (endothelial nitric oxide synthase, eNOS) 和環化鳥糞嘌呤核苷酸依賴性蛋白激酶的蛋白質表現量,以免疫組織染色法 (immunohistochemistry) 觀察內皮型一氧化氮合成酶和平滑肌肌動蛋白存在的位置,以Sievers 280一氧化氮分析儀偵測一氧化氮產生量,並利用免疫沉澱法 (immunoprecipitation) 測量環化鳥糞嘌呤核苷酸依賴性蛋白激酶蛋白磷酸化的程度。
根據組織染色形態觀察的結果顯示,懷孕第七天的子宮在著床部位內膜層 (endometrium) 厚度比非著床部位厚,相對地,懷孕第十八天子宮著床部位內膜層與肌肉層 (myometrium) 厚度比非著床部位薄。
在使用一氧化氮供應者實驗結果顯示:1) 在高濃度SNP (sodium nitroprusside) 10-4M作用下,平滑肌肌凝蛋白重鏈表現量下降;2) 平滑肌肌動蛋白 (SM-actin)與非平滑肌肌動蛋白 (non SM-actin) 表現量不受SNP濃度改變的影響;3) 溶解型鳥糞嘌呤核苷酸環化酶 (soluble gunylate cyclase) 蛋白質表現量不受SNP濃度改變的影響;4) SNP在10-7 和10-4M濃度時會降低環化鳥糞嘌呤核苷酸依賴性蛋白激酶蛋白質表現量;5) 在高濃度SNP 10-4M作用一小時及二十四小時後會增加環化鳥糞嘌呤核苷酸依賴性蛋白激酶蛋白磷酸化。
在偵測一氧化氮產生量的結果顯示:1) 整個子宮組織,懷孕第七天非著床部位一氧化氮產量比著床部位高;然而,懷孕第十八天著床部位與非著床部位一氧化氮產量是相當的;2) 子宮內膜所產生的一氧化氮,在懷孕第七天非著床部位是最高的;3) 投以SNP體外培養二十四小時後,培養液所偵測到的一氧化氮產量是隨著SNP濃度增加而增加。
在懷孕的影響的西方點墨轉漬法實驗結果顯示:1) 當比較懷孕時間因素時,平滑肌肌凝蛋白重鏈蛋白表現量懷孕第十八天比第七天高;平滑肌肌動蛋白與非平滑肌肌動蛋白表現量沒有差異;內皮型一氧化氮合成酶表現量懷孕第七天比第十八天高;環化鳥糞嘌呤核苷酸依賴性蛋白激酶表現量並沒有差異;2) 當比較懷孕部位因素時,平滑肌肌凝蛋白重鏈蛋白、非平滑肌肌動蛋白以及環化鳥糞嘌呤核苷酸依賴性蛋白激酶,三者蛋白質的表現量並不會因為著床位置不同而有差異;平滑肌肌動蛋白表現量是非著床部位比著床部位高;內皮型一氧化氮合成酶表現量是著床部位比非著床部位高。
在免疫組織染色的結果指出:相較於懷孕第七天非著床部位,較多量的內皮型一氧化氮合成酶分布在著床部位的子宮內膜層,而較少量分布在子宮肌肉層;而相較於懷孕第十八天非著床部位,較多量的內皮型一氧化氮合成酶分布在著床部位的子宮肌肉層。
由本研究得到三個結論:1) 在高濃度的一氧化氮情況,一氧化氮降低平滑肌肌凝蛋白重鏈的表現可能不是透過環化鳥糞嘌呤核苷酸依賴性蛋白激酶活化所導致;2) 著床可以促進局部內皮型一氧化氮合成酶表現量的增加;3) 隨著懷孕時間的進行,內皮型一氧化氮合成酶在著床部位產生不同的分布,可能是與一氧化氮調節平滑肌肌凝蛋白重鏈表現有關。
英文摘要 After implantation, pregnant uteri undergo a series of spatial and temporal remodeling including extracellular matrix rearrangement and smooth muscle adaptation to accommodate growing embryos. With the processing of pregnancy, smooth muscle cells of the myometrium are slightly hypertrophic with mild edema in the interstitial compartment in early- gestation uteri. In late-gestation, smooth muscle cells are prominently hypertrophic. Expression of smooth muscle myosin heavy chain (SMMHC), one of the most specific smooth muscle differentiation markers, increased in the hypertrophied state. In rat aortic vascular smooth muscle cells (VSMC) culture system, the expression of SMMHC in cultured vascular smooth muscle cells is related to NO-PKG signaling pathway. Nitric oxide (NO) attenuates vascular remodeling by inhibiting smooth muscle proliferation. Even though it is known that hypertrophy and hyperplasia of vascular smooth muscles contribute to the increase of vascular wall in response to chronic pulmonary hypertension, it is not clear whether the increased NO suppresses muscle hypertrophy by lowering the expression of SMMHC. Since hypertrophy also contributes to vascular remodeling, we further explored whether the elevation of NO lowers smooth muscle hypertrophy by reducing the expression of contractile proteins. Therefore, we hypothesize nitric oxide decreased the expression of SMMHC mediate through NO-PKG dependent pathway.
In this regards, pregnant uteri were used as the experimental model. Three objectives were designed 1) to characterize the influence of pregnancy on uterine remodeling, 2) to determine the effect of a NO donor on the expression of SMMHC related to NO-cGMP dependent pathway, and 3) to examine the influence of pregnancy on the protein abundance of the molecules related to the NO. Western blot analysis was used to measure the protein abundance of SM-MHC, SM α-actin, eNOS and PKG. Immunohistochemistry was used to localize SM α-actin and eNOS. The level of NO production was measured by Sievers 280 nitric oxide analyzer. The immunoprecipitation was used to detect the level of PKG phosphorylation.
The morphological data showed that, the implanted regions in G7 uteri have thicker wall with increased endometrial thickness than non-implanted regions. In contrast, the implanted regions of G18 had much thinner wall with dramatic decrease of both endometrial and myometrial thickness than the non-implanted regions of G18 uteri.
In NO donor modulating effects, our data shown: 1) SMMHC was decreased in response to the highest concentration of SNP (10-4M). 2) SM α-actin and non SM-actin was not affected by increasing concentrations of SNP (10-7M-10-4M). 3) sGC was not affected by increasing concentrations of SNP (10-7M-10-4M).4) SNP at 10-7 and 10-4M significantly lowered PKG abundance. 5) PKG phosphorylation was increased in response to SNP (10-4M) after incubated 1 hr and 24 hr.
In NO production data indicated: 1) In whole tissue extracts, the production of NO in non-implantation regions of G7 uteri was greater than that in implantation regions of G7. However, non-implanted regions of G18 uteri generated a similar amount of NO as implanted regions of G18 uteri did. 2) Endometrial NO from non-implanted regions of G7 uteri had much greater production of NO than other regions tested but there was no difference in implanted regions between G7 and G18 uteri. 3) After incubated with SNP for 24-hr, medial NO was increased with increasing concentrations of SNP (10-7 – 10-4 M).
In the influence of pregnancy, Western blot analysis indicated: 1) When compare the temporal effect: SMMHC expressed higher in late-gestation (G18) than early-gestation (G7); SM α-actin and non SM-actin was not significantly different between G7 and G18; eNOS expressed higher in G7 than G18; PKG was not significantly different between G7 and G18. 2) When compare the spatial effect: SMMHC, non SM-actin and PKG were not significantly different between non-implanted and implanted regions; SM α-actin in implanted regions was slightly lower than in non-implanted regions; eNOS expressed higher in implanted regions than in non-implanted regions.
Immunostaining results shown: When compared with non-implanted regions of G7 uteri, implanted regions of G7 uteri had relatively intense staining for the immunoreactive eNOS in the inner decidual reaction zone of endometrium but had similar intensity of the staining in myometrium layers. When compared with non-implanted regions of G18 uteri, implanted regions of G18 uteri had relatively intense staining for the immunoreactive eNOS in myometrial layers.
Our data suggested, at high concentration, NO-decreased SMMHC expression may not be mediated by PKG activation. Implantation enhances local expression of eNOS. With the progression of pregnancy, the differential distribution of eNOS in the implantation region may be related to regulate the NO level mediated SMMHC expression in pregnant uteri.
論文目次 口試證明………………………………………………………… 2
Content……………………………………………………………3
中文摘要………………………………………………………… 4
Abstract………………………………………………………… 7
Introduction…………………………………………………… 11
Materials and Methods…………………………………………16
Results……………………………………………………………23
Discussion……………………………………………………… 35
References……………………………………………………… 43
Figures……………………………………………………………52
Appendix
Curriculum vitae
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