血管紧张素转化酶抑制剂对表皮干细胞增殖、分化，迁移的影响及机制研究：ACE在皮肤的新功能

发布时间：2018-04-20 01:39

  本文选题：血管紧张素转化酶 + 皮肤　； 参考：《暨南大学》2014年硕士论文

【摘要】：背景: 肾素-血管紧张素-醛固酮系统（RAS）在肾病的进展过程中起着举足轻重的作用，其中血管紧张素转化酶（ACE）则是其关键限速酶。故血管紧张素转化酶抑制剂已被作为一线抗高血压药物，广泛应用于心血管、肾疾病的治疗。然而各种血管紧张素转化酶抑制剂（ACEI）药物使用后近50％的副反应发生在皮肤，如皮疹、荨麻疹、脱发，甚至严重的皮肤反应等，，其具体的作用机制目前尚不完全清楚。糖尿病人的治疗中长期服用ACE抑制剂者发生足溃疡和下肢截肢的风险增加。而表皮干细胞(ESC)是皮肤组织自我更新、修复与再生的重要靶器官，因此弄清两者间的关系有着重大的意义。 目的： 观察探讨血管紧张素转化酶（ACE）与表皮干细胞(ESC)两者的关联性，并研究血管紧张素转化酶抑制剂卡托普利（Captoprol）对表皮干细胞增殖、迁移，分化等生物学行为的影响，及探讨其作用的信号机制。 方法：利用差速贴壁法获得人表皮干细胞进行离体培养。利用表皮干细胞公认的表面标记物对其鉴定；实验检测ESC表面ACE的表达。分实验组和对照组：实验组为含有不同浓度卡托普利的表皮干细胞培养基（K-SFM），对照组为单纯的K-FSM培养基。 (1).根据细胞酶联免疫XTT法检测ESC的增殖活力并确立卡托普利最佳浓度； (2).体外细胞创伤模型观察卡托普利实验组与对照组中表皮干细胞的迁移能力； (3).流式细胞仪检测K10的表达观察卡托普利对ESC分化的影响； (4).细胞克隆形成率分析和细胞粘附能力分析观察卡托普利对ESC的影响； (5).流式细胞仪检测卡托普利对ESC凋亡的影响； (6). Elisa法检测细胞上清液中ANG2和ANG1-7的变化，探讨ACEI作用ESC的途径。 (7).检测ANGⅡ下游底物AT1和AT2在ESCs的表达，分组观察ANGⅡ，AT1阻断剂（Losartan），AT2阻断剂(PD123,319)，ACEI+Losartan和ACEI+PD123，319在表皮干细胞增殖，迁移中的作用，从而分析ACEI对ESC的作用点。 (8).蛋白质印迹法（Western blot）观察加入ACE抑制剂处理前后ESC细胞外信号调节激酶（ERK）、磷酸化细胞外调节激酶（phospho-ERK）,(AKT)、(phospho-AKT)、(STAT1)、(phospho-STAT1)、(STAT3)、(phospho-STAT3)的变化，通过分析其信号通路探讨ACE在维持ESCs生物学功能中所起的作用。 结果： (1).在体外培养的细胞经β1-整合素和K19免疫荧光双标记染色，双标阳性者约83.55%，即ESCs。免疫荧光染色和RT-PCR结果显示ESC表达ACE。流式细胞仪定量检测培养的细胞ACE阳性率为74.2%。 (2).卡托普利明显抑制了ESCs的增殖（P0.05），且10-6mol/l的浓度最为明显，同时于第5天达到峰值。10-6mol/l的卡托普利明显抑制了细胞的迁移能力（P0.05）、粘附能力（P＜0.05）和克隆能力（P0.05）。 (3).流式结果表明：10-6mol/l的卡托普利并没有影响K10的表达（P0.05)及凋亡（P0.05）。 (4). Elisa法检测卡托普利处理前后表皮干细胞上清液中的变化，实验组与对照组间的ANG2表达差异具有统计学意义（P0.05），而ANG1-7表达无明显统计学差异（P0.05）。 (5). RT-PCR结果示ESC表达AT1和AT2，细胞酶联免疫XTT法检测ANGⅡ，Losartan， PD123,319，ACEI+Losartan和ACEI+PD123，319对ESCs增殖的结果差异具有统计学意义（P0.05）；体外创伤模型观察ANGⅡ， Losartan，PD123,319，ACEI+Losartan和ACEI+PD123，319对ESCs迁移结果差异具有统计学差异（P0.05）。 (6).蛋白质印迹法（Western blot）观察加入ACE抑制剂处理前后ESCphospho-ERK, phospho-STAT1、phospho-STAT3的蛋白表达差异具有统计学意义（P0.05）。 结论： ACE通过调控ANG2的浓度及调节AT1与AT2之间的平衡起作用，可能为激活磷酸化细胞外信号调节激酶（P-ERK）、phospho-STAT1、phospho-STAT3信号通路影响ESC的增殖，迁移和粘附从而影响皮肤的损伤修复和自我更新。
[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锛

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