本文選題：人參皂苷Rg1 + 人臍血源基質(zhì)細(xì)胞��； 參考：《重慶醫(yī)科大學(xué)》2017年博士論文

【摘要】：造血微環(huán)境(hematopoietic inductive microenvironment,HIM)是支持和調(diào)節(jié)造血干細(xì)胞(haemopoietic stem cells,HSCs)生長發(fā)育的內(nèi)環(huán)境,其結(jié)構(gòu)和功能的完整是維系正常造血功能的重要因素�；|(zhì)細(xì)胞作為HIM的核心組分,不僅通過形成HSCs生長發(fā)育的“龕”、分泌造血因子和細(xì)胞外基質(zhì)等方式支持和調(diào)控HSCs自我更新與分化,還與多種血液系統(tǒng)疾病的發(fā)生、發(fā)展和預(yù)后密切相關(guān)。本課題組長期從事人臍血源基質(zhì)細(xì)胞(human umbilical cord blood-derived stromal cells,h UCBDSCs)及臍血造血微環(huán)境的研究。我們的前期體外研究證實(shí)h UCBDSCs具有造血基質(zhì)細(xì)胞的基本生物學(xué)特征,能有效支持臍血CD34+細(xì)胞數(shù)量擴(kuò)增;動(dòng)物實(shí)驗(yàn)也表明,h UCBDSCs與造血細(xì)胞聯(lián)合移植于輻照后裸鼠體內(nèi)具有促進(jìn)造血重建、修復(fù)受損微環(huán)境和減輕移植物抗宿主病(GVHD,graft-versus-host disease)的多重效應(yīng)。然而隨著研究的深入,我們發(fā)現(xiàn)h UCBDSCs存在移植后存活狀況差和植入效率較低等問題,分析主要原因可能是在體外培養(yǎng)和體內(nèi)輸注過程中會(huì)面臨營養(yǎng)缺乏、炎癥反應(yīng)和氧化應(yīng)激等多種不良因素刺激。這些因素導(dǎo)致胞內(nèi)自由基和活性氧(reactive oxygen species,ROS)含量增加,細(xì)胞結(jié)構(gòu)破壞和功能失調(diào),進(jìn)而造成細(xì)胞衰老、死亡或凋亡。線粒體是細(xì)胞內(nèi)ROS的主要來源,也是最先被攻擊的細(xì)胞器。線粒體ROS(mitochondrial ROS,mt ROS)的穩(wěn)態(tài)調(diào)節(jié)是維系線粒體和細(xì)胞正常功能的關(guān)鍵所在。線粒體氧化應(yīng)激,即mt ROS的產(chǎn)生與抗氧化防御之間的不平衡,將導(dǎo)致線粒體功能障礙及一系列相關(guān)疾病。為確保h UCBDSCs活力和移植療效,研究如何提高細(xì)胞內(nèi)源性抗氧化保護(hù)功能和維持mt ROS穩(wěn)態(tài)調(diào)節(jié)的有效途徑具有重要的理論價(jià)值和應(yīng)用前景意義。人參是中醫(yī)臨床“補(bǔ)氣要藥”,已有數(shù)千年的臨床用藥歷史�，F(xiàn)代醫(yī)學(xué)研究認(rèn)為,人參皂苷是人參中主要的藥理學(xué)活性成分,有多達(dá)幾十種皂苷,人參皂苷Rg1(Ginsenoside Rg1,G-Rg1)是其中最重要的單體皂苷成分之一。本課題組和其他學(xué)者的研究證實(shí),人參皂苷Rg1在抗腫瘤、抗炎癥、抗衰老、抗糖尿病、抗神經(jīng)原退化和促干/祖細(xì)胞增殖等方面有廣泛藥理學(xué)作用。近年來研究還發(fā)現(xiàn),人參皂苷Rg1具有拮抗氧化致衰劑對(duì)多種器官和細(xì)胞的氧化損傷與促細(xì)胞凋亡作用,提示人參皂苷Rg1是人參中重要的抗氧化皂苷。課題組既往研究證明,人參皂苷Rg1不僅能促進(jìn)骨髓基質(zhì)細(xì)胞(bone marrow stromal cells,BMSCs)增殖分化與造血生長因子分泌,還能通過增強(qiáng)其在D-半乳糖誘導(dǎo)的應(yīng)激條件下的抗氧化和抗炎能力以延緩細(xì)胞衰老。然而,人參皂苷Rg1是否對(duì)于氧化應(yīng)激誘導(dǎo)的h UCBDSCs損傷與凋亡發(fā)揮一定的保護(hù)作用以及可能的分子機(jī)制尚未見相關(guān)報(bào)道。目的:在本研究中,我們體外分離、培養(yǎng)和擴(kuò)增h UCBDSCs,并采用叔丁基過氧化氫(tert-butyl hydroperoxide,t-BHP)損傷h UCBDSCs構(gòu)建氧化應(yīng)激損傷細(xì)胞模型,研究人參皂苷Rg1拮抗h UCBDSCs氧化損傷、促進(jìn)細(xì)胞存活、抑制凋亡以及維持線粒體ROS穩(wěn)態(tài)的重要作用,深入探索轉(zhuǎn)錄調(diào)控因子叉頭框蛋白O3a(Fokhead box O3a,Fox O3a)介導(dǎo)的相關(guān)信號(hào)通路在人參皂苷Rg1發(fā)揮這些效應(yīng)中的作用。旨在為闡釋人參皂苷Rg1抗氧化效應(yīng)的現(xiàn)代分子生物學(xué)機(jī)制提供新的理論依據(jù);為提高h(yuǎn) UCBDSCs臨床移植治療效果提供新的輔助手段。方法:1.體外分離、培養(yǎng)和擴(kuò)增h UCBDSCs,采用t-BHP處理細(xì)胞構(gòu)建氧化應(yīng)激損傷體外模型。分別以不同濃度人參皂苷Rg1處理損傷后的h UCBDSCs,CCK-8法檢測人參皂苷Rg1對(duì)h UCBDSCs細(xì)胞活力、細(xì)胞增殖的影響,在倒置相差顯微鏡下觀察成纖維細(xì)胞集落形成單位(colony-forming unit of fibroblast,CFU-F)的形成。以試劑盒分別檢測氧化應(yīng)激相關(guān)指標(biāo),包括丙二醛(malondialdehyde,MDA)含量,乳酸脫氫酶(lactate dehydrogenase,LDH)和超氧化物歧化酶(superoxide dismutase,SOD)的活力。2.采用不同濃度人參皂苷Rg1分別處理t-BHP誘導(dǎo)的h UCBDSCs,流式細(xì)胞術(shù)檢測細(xì)胞凋亡率,闡明人參皂苷Rg1對(duì)細(xì)胞凋亡的影響。Western blot法檢測人參皂苷Rg1對(duì)t-BHP誘導(dǎo)的h UCBDSCs凋亡相關(guān)蛋白(Caspase-3、Bim、Bax和Bcl-2)表達(dá)水平的影響。Western blot法檢測Akt-Fox O3a信號(hào)通路在人參皂苷Rg1下調(diào)Bim蛋白表達(dá)水平中的作用,Western blot法檢測人參皂苷Rg1對(duì)Fox O3a在h UCBDSCs中胞核/質(zhì)轉(zhuǎn)位的調(diào)節(jié)作用。3.采用不同濃度人參皂苷Rg1處理t-BHP誘導(dǎo)的h UCBDSCs,激光掃描共聚焦顯微鏡(laser scanning confocal microscope,LSCM)分別檢測人參皂苷Rg1對(duì)總ROS和mt ROS生成的影響,利用LSCM檢測人參皂苷Rg1對(duì)t-BHP誘導(dǎo)的h UCBDSCs線粒體膜電位(MMP,mitochondrial membrane potential)的影響。以試劑盒檢測人參皂苷Rg1對(duì)錳超氧化物岐化酶(Mn-SOD)和過氧化氫酶(Catalase)活力的影響。采用CCK-8法、LSCM和相應(yīng)試劑盒檢測等方法明確Sirt1在人參皂苷Rg1阻抑線粒體氧化損傷中的作用,Western blot法檢測AMPK-Sirt1信號(hào)通路在人參皂苷Rg1上調(diào)Fox O3a去乙�；街械淖饔�。結(jié)果:1.成功構(gòu)建t-BHP誘導(dǎo)h UCBDSCs的氧化應(yīng)激損傷體外模型研究發(fā)現(xiàn),不同濃度t-BHP損傷h UCBDSCs后,細(xì)胞存活率隨t-BHP濃度的增加而逐漸降低。在t-BHP濃度(80μM)處理細(xì)胞6 h時(shí),細(xì)胞存活率降至(52±3.10)%,即IC50約為80μM。根據(jù)上述實(shí)驗(yàn)結(jié)果,以80μM劑量t-BHP處理細(xì)胞6 h的方法構(gòu)建t-BHP誘導(dǎo)h UCBDSCs的氧化應(yīng)激損傷體外模型。2.人參皂苷Rg1抑制t-BHP誘導(dǎo)的h UCBDSCs損傷和凋亡(1)在本研究涉及的濃度范圍內(nèi),人參皂苷Rg1能濃度依賴的抑制t-BHP誘導(dǎo)h UCBDSCs的活力下降,當(dāng)人參皂苷Rg1濃度為0.1、1、10和50μM時(shí)作用最為顯著。人參皂苷Rg1(50μM)還對(duì)t-BHP誘導(dǎo)的細(xì)胞增殖下降和CFU-F形成減少具有明顯的恢復(fù)作用,且對(duì)于正常h UCBDSCs也具有一定促增殖作用。(2)t-BHP損傷h UCBDSCs后MDA含量和LDH酶活性顯著上升,而SOD酶活性明顯降低。不同濃度(1、10和50μM)人參皂苷Rg1處理均能顯著降低MDA和LDH水平;當(dāng)濃度為10μM和50μM時(shí)人參皂苷Rg1還能明顯提高SOD的酶活性。(3)t-BHP損傷h UCBDSCs后凋亡率較正常組顯著增加,而不同濃度(1、10和50μM)人參皂苷Rg1處理均能夠有效抑制t-BHP誘導(dǎo)的h UCBDSCs凋亡。人參皂苷Rg1(50μM)還能顯著抑制促凋亡蛋白酶Caspase-3的活化、降低促凋亡蛋白Bim和Bax的表達(dá)及提高抑凋亡蛋白Bcl-2的表達(dá)。3.Akt-Fox O3a-Bim信號(hào)通路在人參皂苷Rg1抑制凋亡中的作用人參皂苷Rg1(50μM)能激活A(yù)kt和Fox O3a磷酸化,促進(jìn)Fox O3a由胞核向胞質(zhì)轉(zhuǎn)位,這一改變抑制了Fox O3a對(duì)下游凋亡誘導(dǎo)基因Bim的表達(dá)調(diào)控,進(jìn)而下調(diào)了促凋亡蛋白Bim和Bax的表達(dá),并上調(diào)了抑凋亡蛋白Bcl-2的表達(dá)。而人參皂苷Rg1作用能被PI3K抑制劑LY29004所抑制,提示Akt-Fox O3-Bim信號(hào)通路在人參皂苷Rg1阻抑t-BHP誘導(dǎo)h UCBDSCs的凋亡中發(fā)揮重要作用。4.人參皂苷Rg1減輕t-BHP誘導(dǎo)的線粒體氧化損傷(1)激光掃描共聚焦顯微鏡檢測發(fā)現(xiàn),t-BHP損傷h UCBDSCs后DCFH-DA熒光強(qiáng)度較正常對(duì)照組明顯升高,提示胞內(nèi)總ROS水平上升。不同濃度(1、10和50μM)人參皂苷Rg1處理細(xì)胞后總ROS水平隨著人參皂苷Rg1濃度增加而逐漸下降,表明人參皂苷Rg1能抑制t-BHP誘導(dǎo)的胞內(nèi)活性氧水平提升。(2)激光掃描共聚焦顯微鏡檢測發(fā)現(xiàn),t-BHP損傷h UCBDSCs后Mito SOX Red紅色熒光變強(qiáng),線粒體O2·-水平明顯升高。人參皂苷Rg1濃度依賴的抑制t-BHP誘導(dǎo)的線粒體O2·-增加,當(dāng)其濃度為10μM和50μM時(shí),O2·-水平顯著低于t-BHP單處理組,提示人參皂苷Rg1具有降低線粒體活性氧水平的作用。(3)激光掃描共聚焦顯微鏡觀察JC-1探針標(biāo)記的h UCBDSCs,并采用紅/綠色熒光強(qiáng)度的比值代表線粒體膜電位(Δψm)。結(jié)果表明,與正常對(duì)照組比較,t-BHP處理細(xì)胞后Δψm明顯降低。不同濃度(1、10和50μM)人參皂苷Rg1處理細(xì)胞后Δψm隨人參皂苷Rg1濃度增加而逐漸增加,提示人參皂苷Rg1能抑制t-BHP誘導(dǎo)的h UCBDSCs線粒體膜電位丟失。(4)t-BHP損傷h UCBDSCs后導(dǎo)致Mn-SOD和Catalase的酶活力下降,而人參皂苷Rg1處理能濃度依賴的提高這兩種線粒體抗氧化酶的活力。當(dāng)人參皂苷Rg1濃度為10μM和50μM時(shí),Mn-SOD酶活力水平顯著高于t-BHP單處理組;人參皂苷Rg1濃度為1、10和50μM時(shí),Catalase酶活力較t-BHP單處理組有顯著提高。結(jié)果提示人參皂苷Rg1可通過提高M(jìn)n-SOD和Catalase酶活力發(fā)揮線粒體抗氧化作用。5.AMPK-Sirt1-Fox O3a信號(hào)通路在人參皂苷Rg1調(diào)控線粒體活性氧水平中的作用(1)人參皂苷Rg1(50μM)能顯著提高t-BHP誘導(dǎo)的h UCBDSCs細(xì)胞Sirt1的表達(dá)水平,且對(duì)于正常細(xì)胞Sirt1的表達(dá)也有促進(jìn)作用。使用Sirt1的si RNA干擾后,人參皂苷Rg1對(duì)線粒體O2·-生成的抑制作用被明顯減弱,Mn-SOD和Catalase酶活性降低,細(xì)胞活力也隨之下降,提示Sirt1活化對(duì)于人參皂苷Rg1發(fā)揮線粒體抗氧化作用是必要的。(2)t-BHP損傷h UCBDSCs后磷酸化的AMPK、Sirt1表達(dá)及去乙�；腇ox O3a表達(dá)水平均明顯降低。與t-BHP處理組比較,人參皂苷Rg1(50μM)能顯著促進(jìn)AMPK磷酸化激活和上調(diào)Sirt1表達(dá)水平,進(jìn)而提高Fox O3a去乙�；�。分別利用AMPK si RNA和Sirt1 si RNA干擾處理后,人參皂苷Rg1這一系列效應(yīng)被抑制,表明人參皂苷Rg1能夠通過激活A(yù)MPK-Sirt1信號(hào)通路促進(jìn)Fox O3a去乙�；�,進(jìn)而上調(diào)Fox O3a介導(dǎo)的Mn-SOD和Catalase兩種酶的表達(dá),發(fā)揮線粒體活性氧清除作用。結(jié)論:1.人參皂甙Rg1能夠促進(jìn)t-BHP誘導(dǎo)h UCBDSCs的存活、提高增殖能力,并通過調(diào)控Akt-Fox O3a-Bim信號(hào)通路在抑制細(xì)胞凋亡過程中發(fā)揮重要作用。2.人參皂苷Rg1通過清除過多mt ROS來保護(hù)h UCBDSCs免受t-BHP誘導(dǎo)的線粒體氧化損傷,而這一效應(yīng)主要受到AMPK-Sirt1-Fox O3a信號(hào)通路的調(diào)控。
[Abstract]:Hematopoietic inductive microenvironment (HIM) is an internal environment that supports and regulates the growth and development of haemopoietic stem cells (HSCs). Its structure and function are an important factor in maintaining normal hematopoiesis. As the core component of HIM, the matrix cells not only form a "niche" for HSCs growth and development. "The secretion of hematopoietic factors and extracellular matrix supports and regulates the self renewal and differentiation of HSCs, and is closely related to the occurrence, development and prognosis of a variety of blood system diseases. The research group has long been engaged in the study of human umbilical cord blood stromal cells (human umbilical cord blood-derived stromal cells, h UCBDSCs) and umbilical blood hematopoietic microenvironment. Our previous in vitro studies have confirmed that h UCBDSCs has the basic biological characteristics of hematopoietic stromal cells and can effectively support the number amplification of CD34+ cells in umbilical cord blood. In animal experiments, the combined transplantation of H UCBDSCs with hematopoietic cells in irradiated nude mice can promote hematopoietic reconstitution, repair the damaged microenvironment and alleviate graft-versus-host disease (GVH). The multiple effects of D, graft-versus-host disease). However, with the further study, we found that h UCBDSCs has poor survival and low implantation efficiency. The main reason for the analysis may be that many adverse factors such as nutritional deficiency, inflammatory reaction and oxidative stress may be stimulated in the process of in vitro culture and in vivo infusion. These factors lead to increased intracellular free radicals and reactive oxygen species (reactive oxygen species, ROS), cell structure destruction and dysfunction, resulting in cell senescence, death or apoptosis. Mitochondria are the main source of ROS in cells, and also the first organelles to be attacked first. The steady-state regulation of mitochondrial ROS (mitochondrial ROS, MT ROS) is maintained. Mitochondrial and cell normal function key. Mitochondrial oxidative stress, that is, the imbalance between MT ROS production and antioxidant defense, will lead to mitochondrial dysfunction and a series of related diseases. In order to ensure h UCBDSCs vitality and transplantation effect, how to improve cellular endogenous antioxidant protection function and maintain MT ROS homeostasis regulation The effective way has important theoretical value and application prospect. Ginseng is a clinical medicine for replenishing qi in Chinese medicine. It has a history of thousands of years of clinical medication. Modern medical research holds that ginsenoside is the main pharmacological active component of ginseng, with as many as dozens of saponins and ginsenoside Rg1 (Ginsenoside Rg1, G-Rg1) is the heaviest of them. The research group and other scholars have confirmed that ginsenoside Rg1 has extensive pharmacological effects on anti-tumor, anti-inflammatory, anti aging, antidiabetic, neurogenic degradation and stem / progenitor cell proliferation. In recent years, the research also found that ginsenoside Rg1 has the antagonist to a variety of organs and the antagonist of antioxidation. The effects of oxidative damage and apoptosis promoting cell apoptosis suggest that ginsenoside Rg1 is an important antioxidant saponins in ginseng. Previous studies have shown that ginsenoside Rg1 not only promotes the proliferation and differentiation of bone marrow stromal cells (bone marrow stromal cells, BMSCs) and hematopoiesis, but also enhances its induction in D- galactose. Anti oxidative and anti-inflammatory ability under stress can delay cell senescence. However, there is no report on whether ginsenoside Rg1 has protective effect on H UCBDSCs injury induced by oxidative stress and possible molecular mechanism. Objective: in this study, we isolated, cultured and amplified h UCBDSCs in this study, and used tert. Tert-butyl hydroperoxide (t-BHP) damage h UCBDSCs to construct oxidative stress damage cell model, and to study the important role of ginsenoside Rg1 to antagonize the oxidative damage of H UCBDSCs, promote cell survival, inhibit apoptosis and maintain the mitochondrial ROS homeostasis, and explore the transcriptional regulatory factor fork head frame protein O3a (Fokhead box). A) the mediated signaling pathway plays the role of ginsenoside Rg1 in these effects. The purpose is to provide a new theoretical basis for the interpretation of the modern molecular biological mechanism of the antioxidant effect of ginsenoside Rg1, and to provide a new auxiliary means for improving the therapeutic effect of H UCBDSCs in clinical transplantation. Method: 1. in vitro isolation, culture and amplification of H UCBDSCs, and t -BHP treated cells to construct an in vitro model of oxidative stress injury. H UCBDSCs after injury was treated with different concentrations of ginsenoside Rg1. CCK-8 method was used to detect the activity of ginsenoside Rg1 on H UCBDSCs cells and the effect of cell proliferation. The colony forming unit of fibroblasts was observed under the inverted phase contrast microscope (colony-forming unit of fibroblast). Detection of oxidative stress related indicators, including malondialdehyde (MDA), lactic dehydrogenase (lactate dehydrogenase, LDH), and superoxide dismutase (superoxide dismutase, SOD) activity.2. using different concentrations of ginsenoside Rg1 to treat t-BHP h induction, flow cytometry The effect of ginsenoside Rg1 on the apoptosis, the effect of ginsenoside Rg1 on the expression level of H UCBDSCs apoptosis related protein (Caspase-3, Bim, Bax and Bcl-2) induced by t-BHP was detected by.Western blot method. N blot method was used to detect the effect of ginsenoside Rg1 on the cytoplasmic translocation of Fox O3a in H UCBDSCs.3. using different concentrations of ginsenoside Rg1 processing t-BHP induced H UCBDSCs. The effect of ginsenoside Rg1 on the mitochondrial membrane potential of H UCBDSCs (MMP, mitochondrial membrane potential) induced by t-BHP. The effect of ginsenoside Rg1 on the activity of manganese superoxide dismutase (Mn-SOD) and catalase (Catalase) was detected by a kit. The role of glycoside Rg1 in inhibition of mitochondrial oxidative damage, Western blot method was used to detect the role of AMPK-Sirt1 signaling pathway in the up-regulation of Fox O3a deacetylation level by ginsenoside Rg1. Results: 1. a successful construction of an in vitro model of oxidative stress injury induced by t-BHP to induce h UCBDSCs was studied. The cell survival rate was reduced to (52 + 3.10)% when the concentration of t-BHP concentration (80 u M) was 6 h, that is, the IC50 was about 80 mu M.. According to the experimental results, the method of t-BHP treatment of 6 h with 80 M dose t-BHP was used to construct the t-BHP induced oxidative stress injury model.2. ginseng saponins Injury and apoptosis (1) in the concentration range involved in this study, ginsenoside Rg1 can decrease the activity of H UCBDSCs induced by the concentration dependent inhibitory t-BHP. When the concentration of ginsenoside Rg1 is 0.1,1,10 and 50 mu M, the effect is most significant. The ginsenoside Rg1 (50 u M) also has a significant recovery on the decrease of cell proliferation and the decrease of CFU-F formation induced by t-BHP. Use, and also have a certain proliferation effect on normal h UCBDSCs. (2) after t-BHP damage to h UCBDSCs, MDA content and LDH enzyme activity increased significantly, while SOD enzyme activity decreased significantly. The Rg1 treatment of different concentrations (1,10 and 50 mu M) could significantly reduce MDA and levels. (3) after t-BHP damage h UCBDSCs, the apoptosis rate was significantly higher than that of the normal group, while the different concentrations (1,10 and 50 mu M) could effectively inhibit the UCBDSCs apoptosis induced by t-BHP. Ginsenoside Rg1 (50 mu M) could significantly inhibit the activation of apoptotic protease Caspase-3, and reduce the expression of apoptotic protein and inhibit the expression and inhibition of H. The expression of apoptotic protein Bcl-2.3.Akt-Fox O3a-Bim signaling pathway in ginsenoside Rg1 inhibition of apoptosis, ginsenoside Rg1 (50 mu M) activates Akt and Fox O3a phosphorylation, promotes Fox O3a from the nucleus to the cytoplasm, and this change inhibits the regulation of the expression of the downstream apoptosis induced gene and down regulates the apoptotic protein. The expression of Bax and the expression of anti apoptotic protein Bcl-2, and the effect of ginsenoside Rg1 can be inhibited by PI3K inhibitor LY29004, suggesting that Akt-Fox O3-Bim signaling pathway plays an important role in the inhibition of t-BHP induced H UCBDSCs apoptosis by ginsenoside Rg1, and.4. ginsenoside alleviates the induced mitochondrial oxidative damage (1) laser scanning Confocal microscopy showed that after t-BHP damage h UCBDSCs, the fluorescence intensity of DCFH-DA was significantly higher than that of the normal control group, suggesting that the total ROS level in the cell increased. The total ROS level of the ginsenoside Rg1 treated cells gradually decreased with the increase of the concentration of ginsenoside Rg1, indicating that the ginsenoside Rg1 could inhibit the t-BHP induced cells. (2) the laser scanning confocal microscope showed that the red fluorescence of Mito SOX Red was stronger and the mitochondrial O2 - level increased obviously after t-BHP damage h UCBDSCs. The concentration dependent t-BHP induced mitochondria O2. - increase of ginsenoside Rg1, when the concentration was 10 mu M and 50 mu M, was significantly lower than the single treatment It was suggested that ginsenoside Rg1 had the effect of reducing the level of mitochondrial reactive oxygen species. (3) the JC-1 probe labeled h UCBDSCs was observed by laser scanning confocal microscopy, and the ratio of red / green fluorescence intensity was used to represent the mitochondrial membrane potential (delta m). The results showed that, compared with the normal control group, the m UCBDSCs was significantly reduced after t-BHP treated cells. (1,10 and 50 mu M) after ginsenoside Rg1 treated cells, the delta m gradually increased with the increase of the concentration of ginsenoside Rg1, suggesting that ginsenoside Rg1 could inhibit the loss of t-BHP induced H UCBDSCs mitochondrial membrane potential loss. (4) t-BHP damage h UCBDSCs leads to the decrease of the enzyme activity, and the two species of ginsenoside treatment can increase the concentration of the two species. When the concentration of ginsenoside Rg1 was 10 mu M and 50 mu M, the activity level of Mn-SOD enzyme was significantly higher than that of t-BHP single treatment group. When the concentration of ginsenoside Rg1 was 1,10 and 50 u M, the activity of Catalase enzyme was significantly higher than that of the t-BHP single treatment group. The role of body antioxidant.5.AMPK-Sirt1-Fox O3a signaling pathway in the regulation of mitochondrial reactive oxygen species by ginsenoside Rg1 (1) ginsenoside Rg1 (50 mu M) can significantly increase the expression level of Sirt1 in H UCBDSCs cells induced by t-BHP, and also promote the expression of Sirt1 in normal cells. 1 the inhibition of mitochondrial O2 - formation was obviously weakened, the activity of Mn-SOD and Catalase decreased, and the activity of cell decreased. It was suggested that Sirt1 activation for ginsenoside Rg1 played a necessary role in mitochondrial antioxidant activity. (2) AMPK, Sirt1 expression and Fox O3a expression level of deacetylation of t-BHP after t-BHP damage h UCBDSCs Compared with the t-BHP treatment group, ginsenoside Rg1 (50 mu M) can significantly promote the activation of AMPK phosphorylation and the up-regulation of Sirt1 expression level, and then improve the level of Fox O3a deacetylation. After the AMPK Si RNA and Sirt1 Si, this series of effects are suppressed. The signal pathway promotes the deacetylation of Fox O3a, and then up-regulates the expression of two enzymes of Mn-SOD and Catalase mediated by Fox O3a, and plays the role of mitochondrial reactive oxygen scavenging. Conclusion: 1. ginsenoside Rg1 can promote the survival of H UCBDSCs and increase the proliferation ability of t-BHP, and regulate the apoptosis process by regulating Akt-Fox O3a-Bim signal pathway. The important role of.2. ginsenoside Rg1 is to protect h UCBDSCs from mitochondrial oxidative damage induced by t-BHP by removing excessive MT ROS, and this effect is mainly regulated by the O3a signaling pathway of AMPK-Sirt1-Fox.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R457.7

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王玉堂;李緒文;金海燕;于永;游景艷;張昆;丁蘭;張寒琦;;人參中人參皂苷的直接高壓微波輔助降解[J];高等學(xué)�；瘜W(xué)學(xué)報(bào);2007年12期

2 韓冬;張鐵軍;唐鋮;田成旺;;人參皂苷的藥動(dòng)學(xué)研究進(jìn)展[J];中草藥;2009年02期

3 余瀟苓;苗青;方翠芬;;人參皂苷水溶液熱穩(wěn)定性研究[J];中國現(xiàn)代應(yīng)用藥學(xué);2011年12期

4 付聰;楊艷芳;王業(yè)靜;;藥典法和新方法測定人參皂苷含量比較[J];亞太傳統(tǒng)醫(yī)藥;2013年11期

5 楊雨;鄭斯文;金銀萍;姚春林;王英平;;人參皂苷的提取分離方法研究進(jìn)展[J];江蘇農(nóng)業(yè)科學(xué);2014年05期

6 于超,刁長發(fā),夏文娟,周碧珍;反相高效液相色譜法測定人參及其制劑中人參皂苷Rg1含量[J];藥物分析雜志;2000年04期

7 王啟祥,呂修梅,張晉秀;人參皂苷含量變化研究概況[J];西北藥學(xué)雜志;2002年05期

8 于榮敏,宋永波,張輝,葉文才,張蔭麟,姚新生;西洋參冠癭組織培養(yǎng)及其人參皂苷Re和人參皂苷Rg_1的產(chǎn)生[J];生物工程學(xué)報(bào);2003年03期

9 盧新政,張曉文,黃峻,馬根山,侯麥花;人參皂苷Rg_1對(duì)培養(yǎng)豬骨髓基質(zhì)細(xì)胞增殖的影響[J];中國藥理學(xué)通報(bào);2003年03期

10 董淑華,陳波,馬忠澤,侯秀云,陳英杰;人參皂苷的體內(nèi)代謝反應(yīng)研究[J];人參研究;2003年01期

相關(guān)會(huì)議論文 前10條

1 朱偉;易瓊;王魯;付本懂;賀常亮;呂爽;畢文巖;;人參皂苷Rh2的藥理學(xué)研究進(jìn)展[A];中國畜牧獸醫(yī)學(xué)會(huì)2010年學(xué)術(shù)年會(huì)——第二屆中國獸醫(yī)臨床大會(huì)論文集(下冊(cè))[C];2010年

2 王悅;白玉;劉虎威;;高效液相色譜法測定人參中人參皂苷的方法[A];全國生物醫(yī)藥色譜學(xué)術(shù)交流會(huì)（2010景德鎮(zhèn)）論文集[C];2010年

3 尚文斌;楊穎;姜博仁;金華;周麗斌;劉尚全;陳名道;;人參皂苷Rb1促進(jìn)3T3L1細(xì)胞的脂肪形成和抑制脂肪分解[A];2006年中華醫(yī)學(xué)會(huì)糖尿病分會(huì)第十次全國糖尿病學(xué)術(shù)會(huì)議論文集[C];2006年

4 周紅祖;胡勝全;余惠e，

本文編號(hào)：2052582