發(fā)布時間：2018-04-20 01:39

  本文選題：血管緊張素轉(zhuǎn)化酶 + 皮膚��； 參考：《暨南大學(xué)》2014年碩士論文

【摘要】：背景: 腎素-血管緊張素-醛固酮系統(tǒng)（RAS）在腎病的進展過程中起著舉足輕重的作用，其中血管緊張素轉(zhuǎn)化酶（ACE）則是其關(guān)鍵限速酶。故血管緊張素轉(zhuǎn)化酶抑制劑已被作為一線抗高血壓藥物，廣泛應(yīng)用于心血管、腎疾病的治療。然而各種血管緊張素轉(zhuǎn)化酶抑制劑（ACEI）藥物使用后近50％的副反應(yīng)發(fā)生在皮膚，如皮疹、蕁麻疹、脫發(fā)，甚至嚴(yán)重的皮膚反應(yīng)等，，其具體的作用機制目前尚不完全清楚。糖尿病人的治療中長期服用ACE抑制劑者發(fā)生足潰瘍和下肢截肢的風(fēng)險增加。而表皮干細(xì)胞(ESC)是皮膚組織自我更新、修復(fù)與再生的重要靶器官，因此弄清兩者間的關(guān)系有著重大的意義。 目的： 觀察探討血管緊張素轉(zhuǎn)化酶（ACE）與表皮干細(xì)胞(ESC)兩者的關(guān)聯(lián)性，并研究血管緊張素轉(zhuǎn)化酶抑制劑卡托普利（Captoprol）對表皮干細(xì)胞增殖、遷移，分化等生物學(xué)行為的影響，及探討其作用的信號機制。 方法：利用差速貼壁法獲得人表皮干細(xì)胞進行離體培養(yǎng)。利用表皮干細(xì)胞公認(rèn)的表面標(biāo)記物對其鑒定；實驗檢測ESC表面ACE的表達(dá)。分實驗組和對照組：實驗組為含有不同濃度卡托普利的表皮干細(xì)胞培養(yǎng)基（K-SFM），對照組為單純的K-FSM培養(yǎng)基。 (1).根據(jù)細(xì)胞酶聯(lián)免疫XTT法檢測ESC的增殖活力并確立卡托普利最佳濃度； (2).體外細(xì)胞創(chuàng)傷模型觀察卡托普利實驗組與對照組中表皮干細(xì)胞的遷移能力； (3).流式細(xì)胞儀檢測K10的表達(dá)觀察卡托普利對ESC分化的影響； (4).細(xì)胞克隆形成率分析和細(xì)胞粘附能力分析觀察卡托普利對ESC的影響； (5).流式細(xì)胞儀檢測卡托普利對ESC凋亡的影響； (6). Elisa法檢測細(xì)胞上清液中ANG2和ANG1-7的變化，探討ACEI作用ESC的途徑。 (7).檢測ANGⅡ下游底物AT1和AT2在ESCs的表達(dá)，分組觀察ANGⅡ，AT1阻斷劑（Losartan），AT2阻斷劑(PD123,319)，ACEI+Losartan和ACEI+PD123，319在表皮干細(xì)胞增殖，遷移中的作用，從而分析ACEI對ESC的作用點。 (8).蛋白質(zhì)印跡法（Western blot）觀察加入ACE抑制劑處理前后ESC細(xì)胞外信號調(diào)節(jié)激酶（ERK）、磷酸化細(xì)胞外調(diào)節(jié)激酶（phospho-ERK）,(AKT)、(phospho-AKT)、(STAT1)、(phospho-STAT1)、(STAT3)、(phospho-STAT3)的變化，通過分析其信號通路探討ACE在維持ESCs生物學(xué)功能中所起的作用。 結(jié)果： (1).在體外培養(yǎng)的細(xì)胞經(jīng)β1-整合素和K19免疫熒光雙標(biāo)記染色，雙標(biāo)陽性者約83.55%，即ESCs。免疫熒光染色和RT-PCR結(jié)果顯示ESC表達(dá)ACE。流式細(xì)胞儀定量檢測培養(yǎng)的細(xì)胞ACE陽性率為74.2%。 (2).卡托普利明顯抑制了ESCs的增殖（P0.05），且10-6mol/l的濃度最為明顯，同時于第5天達(dá)到峰值。10-6mol/l的卡托普利明顯抑制了細(xì)胞的遷移能力（P0.05）、粘附能力（P＜0.05）和克隆能力（P0.05）。 (3).流式結(jié)果表明：10-6mol/l的卡托普利并沒有影響K10的表達(dá)（P0.05)及凋亡（P0.05）。 (4). Elisa法檢測卡托普利處理前后表皮干細(xì)胞上清液中的變化，實驗組與對照組間的ANG2表達(dá)差異具有統(tǒng)計學(xué)意義（P0.05），而ANG1-7表達(dá)無明顯統(tǒng)計學(xué)差異（P0.05）。 (5). RT-PCR結(jié)果示ESC表達(dá)AT1和AT2，細(xì)胞酶聯(lián)免疫XTT法檢測ANGⅡ，Losartan， PD123,319，ACEI+Losartan和ACEI+PD123，319對ESCs增殖的結(jié)果差異具有統(tǒng)計學(xué)意義（P0.05）；體外創(chuàng)傷模型觀察ANGⅡ， Losartan，PD123,319，ACEI+Losartan和ACEI+PD123，319對ESCs遷移結(jié)果差異具有統(tǒng)計學(xué)差異（P0.05）。 (6).蛋白質(zhì)印跡法（Western blot）觀察加入ACE抑制劑處理前后ESCphospho-ERK, phospho-STAT1、phospho-STAT3的蛋白表達(dá)差異具有統(tǒng)計學(xué)意義（P0.05）。 結(jié)論： ACE通過調(diào)控ANG2的濃度及調(diào)節(jié)AT1與AT2之間的平衡起作用，可能為激活磷酸化細(xì)胞外信號調(diào)節(jié)激酶（P-ERK）、phospho-STAT1、phospho-STAT3信號通路影響ESC的增殖，遷移和粘附從而影響皮膚的損傷修復(fù)和自我更新。
[Abstract]:Background:
The renin angiotensin aldosterone system (RAS) plays an important role in the progression of kidney disease, in which angiotensin converting enzyme (ACE) is the key rate limiting enzyme. Therefore, angiotensin converting enzyme inhibitor has been used as a first-line antihypertensive drug, widely used in the treatment of cardiovascular and renal diseases. However, various vascular tension is used. Nearly 50% of the side effects of the hormone converting enzyme inhibitor (ACEI) drug use occur in skin, such as rash, urticaria, hair loss, and even severe skin response. The specific mechanisms of action are still not fully understood. The risk of foot ulcers and lower limb amputation in patients with diabetes mellitus for long-term use of ACE inhibitors is increased. Cell (ESC) is an important target organ for skin tissue self-renewal, repair and regeneration, so it is of great significance to understand the relationship between them.
Objective:
The relationship between angiotensin converting enzyme (ACE) and epidermal stem cell (ESC) was observed and the effects of angiotensin converting enzyme inhibitor Kato Pury (Captoprol) on the biological behavior of epidermal stem cell proliferation, migration and differentiation were investigated, and the signal mechanism of the effect of angiotensin converting enzyme inhibitor (Captoprol) was explored.
Methods: human epidermal stem cells were cultured in vitro by differential adhesion method. The surface markers recognized by epidermal stem cells were used to identify the epidermal stem cells. The expression of ACE on the surface of ESC was tested. The experimental group and control group were divided into the experimental group and the control group with different concentrations of Kato Pury's epidermal stem cell culture medium (K-SFM), and the control group was a simple K-FSM. Medium.
(1) to detect the proliferation activity of ESC and establish the best concentration of Kato Pury according to the enzyme linked immunosorbent assay (XTT).
(2) in vitro cell trauma model was used to observe the migration ability of epidermal stem cells in Kato Pury experimental group and control group.
(3) the expression of K10 was detected by flow cytometry, and the effect of Kato Pury on ESC differentiation was observed.
(4) cell clone formation rate analysis and cell adhesion ability analysis to observe Kato Pury's influence on ESC.
(5) flow cytometry was used to detect the effect of Kato Pury on apoptosis of ESC.
(6) Elisa method was used to detect the changes of ANG2 and ANG1-7 in the supernatant, and to explore the way of ACEI acting on ESC.
(7) to detect the expression of AT1 and AT2 in the downstream substrate of ANG II, and to observe the role of ANG II, AT1 blocker (Losartan), AT2 blocker (PD123319), ACEI+Losartan and ACEI+PD123319 in the proliferation and migration of epidermal stem cells.
(8). Protein imprinting (Western blot) observation of ESC extracellular signal regulated kinase (ERK), phosphorylated extracellular regulated kinase (phospho-ERK), (phospho-AKT), (STAT1), (phospho-STAT1), (STAT3), (phospho-STAT3), before and after the treatment of ACE inhibitors. Play a role.
Result錛

本文編號：1775696