本文選題：衰老 + 間充質(zhì)干細(xì)胞�。� 參考：《昆明醫(yī)科大學(xué)》2017年碩士論文

【摘要】：[目的]篩選并獲得C57BL/6小鼠衰老模型;利用衰老模型評(píng)價(jià)人的臍帶間充質(zhì)干細(xì)胞(hUCMSC)對(duì)衰老小鼠老年性器官功能退變的治療作用;制備hUCMSC來(lái)源外泌體和HUVEC衰老氧化應(yīng)激細(xì)胞模型,利用細(xì)胞模型評(píng)價(jià)hUCMSC來(lái)源外泌體在hUCMSC延緩或逆轉(zhuǎn)衰老中的抗氧化應(yīng)激作用及其機(jī)制,為hUCMSC治療老年退變性疾病提供理論基礎(chǔ)。[方法]1、hUCMSC的制備組織塊直接貼壁法培養(yǎng)hUCMSC,倒置顯微鏡觀察細(xì)胞狀態(tài),流式細(xì)胞儀檢測(cè)hUCMSC細(xì)胞表面標(biāo)志物,hUCMSC成脂、成骨和成軟骨誘導(dǎo)分化實(shí)驗(yàn)鑒定其多向分化能力。體外通過(guò)表達(dá)GFP慢病毒轉(zhuǎn)染標(biāo)記hUCMSC,并通過(guò)熒光計(jì)數(shù)顯微鏡GFP標(biāo)記陽(yáng)性率。2、小鼠衰老模型篩選與評(píng)價(jià)常規(guī)飼料喂養(yǎng)C57BL/6小鼠至72周,共得60只老年鼠,根據(jù)小鼠毛色、身體活動(dòng)力、精神狀態(tài)、大便情況觀察以及體重檢測(cè)的方法進(jìn)行篩選,按照毛色光澤度發(fā)暗,出現(xiàn)白色毛發(fā),精神狀態(tài)欠佳,活動(dòng)減少、久坐,便干或者便稀頻率增加,體重在(20±5)g范圍的標(biāo)準(zhǔn),將符合該標(biāo)準(zhǔn)的老年鼠納入本實(shí)驗(yàn),作為自然衰老小鼠模型,共40只,雌雄各20只;利用觀察年輕小鼠一般情況,毛色發(fā)黑、發(fā)亮、順滑,活動(dòng)較多,精神飽滿、飲食正常、大便正常,體重在(15±2.5)g范圍的標(biāo)準(zhǔn)隨機(jī)選取年輕小鼠20只,為年輕組。隨機(jī)取年輕鼠(8w)與老年鼠(72w)各8只,為年輕組與模型組,對(duì)比評(píng)價(jià)模型組自然衰老小鼠的一般情況;心臟、肝臟、脾臟、肺、腎臟主要器官HE染色后觀察組織結(jié)構(gòu)變化,以及免疫組化檢測(cè)p16、p53、SOD2和Catalase衰老相關(guān)蛋白在模型小鼠重要器官(心、肝、脾、肺、腎)中的表達(dá)水平;qPCR檢測(cè)p16、p53、SOD2和Catalase等衰老相關(guān)蛋白在模型小鼠重要器官(心、肝、脾、肺、腎)中的轉(zhuǎn)錄水平,以及組織勻漿中總SOD活力。3、分組及hUCMSC移植將衰老小鼠隨機(jī)分為實(shí)驗(yàn)組和對(duì)照組,各16只;實(shí)驗(yàn)組按照1×107個(gè)/kg的標(biāo)準(zhǔn),經(jīng)尾靜脈輸入GFP基因標(biāo)記的hUCMSC,每只2×105個(gè)、200ul體積,每周1次,連續(xù)4次;對(duì)照組輸入等體積生理鹽水。常規(guī)飼養(yǎng),移植4w后觀察評(píng)價(jià)。4.hUCMSC移植療效與機(jī)制評(píng)價(jià)hUCMSC移植結(jié)束1個(gè)月后分別比較兩組小鼠的一般情況、脫頸處死后利用組織切片觀察重要器官(心、肝、脾、肺、腎)結(jié)構(gòu)差異以及qPCR、免疫組化分別檢測(cè)衰老相關(guān)基因(p16、p53、SOD2和Catalase)在各重要器官中轉(zhuǎn)錄水平和表達(dá)水平的差異;檢測(cè)兩組小鼠組織勻漿中總SOD活力差異以及qPCR檢測(cè)主要臟器組織(心、肝、脾、肺、腎)中自噬相關(guān)基因(becline1,LC3b,sirt1,sirt5)水平的差異。5.外泌體的制備與鑒定利用超高速離心法獲得純化hUCMSC來(lái)源外泌體,3%磷鎢酸染色電鏡觀察外泌體形態(tài),Western Blot檢測(cè)外泌體陽(yáng)性標(biāo)志物HSP90、HSP70、CD63和陰性標(biāo)志物TAPA。6.機(jī)制研究利用400nmol/L H202處理HUVEC細(xì)胞構(gòu)建HUVEC衰老細(xì)胞氧化應(yīng)激模型,并建立與hUCMSC的transwell共培養(yǎng)體系,Western Blot檢測(cè)LC3a/b、mTOR和p-mTOR蛋白水平,共聚焦顯微鏡觀察細(xì)胞模型自噬流,透射電子顯微鏡檢測(cè)自噬泡形成情況,評(píng)價(jià)hUCMSC抗氧化應(yīng)激效果。建立hUCMSC來(lái)源的外泌體與細(xì)胞衰老模型共培養(yǎng)體系,CCK8法檢測(cè)外泌體對(duì)衰老HUVEC細(xì)胞增殖的影響,流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況,Western Blot檢測(cè)LC3a/b、p62自噬相關(guān)蛋白水平以及外泌體對(duì)衰老HUVEC細(xì)胞重要信號(hào)轉(zhuǎn)導(dǎo)通路的激活情況。[結(jié)果]1、hUCMSC的制備組織塊直接貼壁法培養(yǎng)hUCMSC 5天后可見(jiàn)細(xì)胞爬出,傳至第三代后細(xì)胞呈長(zhǎng)梭形并且具有典型的渦旋式生長(zhǎng)特征;第三代hUCMSC表達(dá)間充質(zhì)干細(xì)胞陽(yáng)性標(biāo)記物CD29和CD44的表達(dá)率為97.1%、99.6%,間充質(zhì)干細(xì)胞陰性標(biāo)志物CD34、CD45和CD105表達(dá)率為3.18%、0.04%、0%;成脂誘導(dǎo)分化10天后飽和油紅O染色為陽(yáng)性,成骨誘導(dǎo)分化3周后,茜素紅染色陽(yáng)性;成軟骨誘導(dǎo)4周后,阿爾新藍(lán)染色陽(yáng)性,免疫組化可觀察到GFP標(biāo)記的細(xì)胞在心臟、肝臟、脾臟、肺、腎臟。2、模型評(píng)價(jià)衰老模型小鼠(72w)與年輕小鼠(8w)相比身體活動(dòng)力減少,毛色光澤度下降,出現(xiàn)白色毛發(fā),精神狀態(tài)欠佳,便干或者便稀頻率增加;HE染色后可見(jiàn)不同程度的炎癥浸潤(rùn),細(xì)胞水腫、壞死,纖維組織增生等退行性變;免疫組化顯示,衰老小鼠重要器官p16、p53表達(dá)水平,差異具有統(tǒng)計(jì)學(xué)意義(p0.05),而SOD2和catalase低水平,差異具有統(tǒng)計(jì)學(xué)意義(p0.05),組織勻漿中總SOD活性衰老組明顯低于年輕組,差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。3、hUCMSC移植療效評(píng)價(jià)hUCMSC移植后,實(shí)驗(yàn)組小鼠毛色光澤度提高,身體活動(dòng)度增加,精神狀態(tài)較好,大便異常率下降;器官組織結(jié)構(gòu)有不同程度改善;免疫組化和qPCR檢測(cè)p16和p53水平下降,差異具有統(tǒng)計(jì)學(xué)意義(p0.05),而SOD2和Catalase水平上升差異具有統(tǒng)計(jì)學(xué)意義(p0.05);小鼠組織勻漿中總SOD活性提高,差異具有統(tǒng)計(jì)學(xué)意義(p0.05);主要臟器組織中自噬相關(guān)基因(becline1,LC3b)轉(zhuǎn)錄水平提高,差異具有統(tǒng)計(jì)學(xué)意義(p0.05)。4、外泌體制備超高速離心分離所得hUCMSC源外泌體(hUCMSC-exo)呈立體的杯狀結(jié)構(gòu),直徑約136nm,外泌體標(biāo)志性蛋白CD63、HSP90和HSP70高表達(dá),而外泌體陰性標(biāo)志物TAPA1不表達(dá)。5、hUCMSC的抗氧化應(yīng)激作用機(jī)制評(píng)價(jià)H2O2處理HUVEC細(xì)胞后,細(xì)胞增殖能力下降,凋亡增加;經(jīng)過(guò)與hUCMSC非接觸共培養(yǎng)后,自噬標(biāo)記物L(fēng)C3a/b表達(dá)增加,電鏡顯示自噬小體及自噬流增加;經(jīng)過(guò)與hUCMSC-exo共培養(yǎng)后,H2O2對(duì)HUVEC細(xì)胞增殖的抑制降低,HUVEC凋亡減少,自噬標(biāo)記物L(fēng)C3a/b表達(dá)增加,p62表達(dá)下降,細(xì)胞自噬泡明顯增加,自噬抑制信號(hào)通路p38、mTOR、hsp27、stat3磷酸化水平下降,信號(hào)通路被抑制。[結(jié)論]1.正常飼養(yǎng)法飼養(yǎng)小鼠72w可獲得具有衰老的一般特征及組織結(jié)構(gòu)退行性改變的衰老小鼠模型。2.靜脈注射hUCMSC,可以改善毛發(fā)、精神、活動(dòng)、飲食以及體重狀態(tài)生理狀態(tài)和緩解組織結(jié)構(gòu)病理狀態(tài);從分子水平hUCMSC能夠抑制衰老基因的表達(dá)而促進(jìn)抗氧化基因的表達(dá),延緩或逆轉(zhuǎn)機(jī)體氧化應(yīng)激狀態(tài)。因此,研究結(jié)果表明hUCMSC具有一定的治療衰老退行性變的作用。3.hUCMSC的抗衰老作用可能是通過(guò)hUCMSC分泌外泌體通過(guò)調(diào)節(jié)多條信號(hào)轉(zhuǎn)導(dǎo)通路促進(jìn)衰老細(xì)胞自噬,清除衰老細(xì)胞中ROS,減少ROS引起的氧化損傷實(shí)現(xiàn)的。
[Abstract]:[Objective] to screen and obtain the aging model of C57BL/6 mice, and to evaluate the therapeutic effect of human umbilical cord mesenchymal stem cells (hUCMSC) on senile organ functional degeneration in aging mice by aging model, to prepare hUCMSC derived Exocyst and HUVEC aging oxidative stress cell model, and to evaluate the delay of hUCMSC source exocrine in hUCMSC by the cell model. The antioxidation stress and its mechanism in reverse senescence provide a theoretical basis for the treatment of senile degenerative disease by hUCMSC. [methods]1, the tissue block of hUCMSC was prepared by the direct adherence of the tissue block to hUCMSC, the inverted microscope was used to observe the cell state, the flow cytometry was used to detect the surface markers of hUCMSC cells, hUCMSC fat, osteogenesis and chondrogenic differentiation. GFP lentivirus was expressed in vitro by expression of GFP lentivirus, and the positive rate of hUCMSC was marked by the expression of lentivirus, and the positive rate was.2 by the fluorescence counting microscope. The mice senescence model was screened and evaluated by conventional feed to feed C57BL/6 mice for 72 weeks. 60 old mice were obtained, and the hair color, physical activity, mental state, and stool condition were observed and observed, as well as in the condition of stool and stool. The method of body weight detection was screened, with white hair dark, white hair, poor mental state, less activity, more sedentary activity, and an increase in the frequency of dry or stool, and the standard of weight in the range of (20 + 5) g. The old mice in accordance with this standard were included in this experiment as a model of natural aging mice, with a total of 40 males and 20 males, using observation. Young mice general condition, hair color black, bright, smooth, more active, full of spirit, normal diet, normal stool, weight at (15 + 2.5) g range of young mice randomly selected 20 young mice, young rats (8W) and the elderly rats (72W) 8 each, for young group and model group, compare the evaluation model group of natural aging mice of the same The expression level of the main organs of the heart, liver, spleen, lung and kidney was observed by HE staining, and the expression level of p16, p53, SOD2 and Catalase senescence related proteins in the important organs of model mice (heart, liver, spleen, lung, kidney) were detected by immunohistochemistry; qPCR detection of p16, p53, SOD2, Catalase and other aging related proteins in p16, p53, SOD2 and Catalase in model mice important organs. The transcriptional level in the official (heart, liver, spleen, lung, kidney), and the total SOD activity in the tissue homogenate,.3, group and hUCMSC transplantation were randomly divided into experimental and control groups, each of 16; the experimental group entered the GFP gene labeled hUCMSC by the tail vein according to the standard of 1 x 107 /kg, 200ul volume, 1 times a week, 4 times a week; the control group was 4 times a week. Input equal volume physiological saline. Routine rearing, observation and evaluation of the effect and mechanism of.4.hUCMSC transplantation after transplantation of 4W; the general situation of the two groups of mice was compared after 1 months of the end of the transplantation. The structural differences between the important organs (heart, liver, spleen, lung, kidney) and qPCR were observed after 1 months of removal of the neck, and the immunohistochemical staining was used to detect the senescence correlation respectively. The difference in the level of transcription and expression of genes (p16, p53, SOD2 and Catalase) in various important organs, the difference of total SOD activity in the homogenate of two groups of mice and the difference of the level of autophagy related genes (becline1, LC3b, SIRT1, sirt5) in the main organs (heart, liver, spleen, lung and kidney) detected by qPCR (becline1, LC3b, SIRT1, sirt5) The exocrine source of hUCMSC was purified by high speed centrifugation, the morphology of Exocyst was observed by 3% phosphotungstic acid staining electron microscopy, and Western Blot was used to detect HSP90, HSP70, CD63 and negative markers in TAPA.6. mechanism of TAPA.6. mechanism, which used 400nmol/L H202 treatment HUVEC cells to construct HUVEC senescence fine cell oxidative stress model, and to establish a hUCMSC Co culture system, Western Blot detected the level of LC3a/b, mTOR and p-mTOR protein. The autophagic flow of cell model was observed by confocal microscope, the formation of autophagic vesicles was detected by transmission electron microscope, and the effect of hUCMSC antioxidant stress was evaluated. The co culture system of exocrine and cell senescence was established by hUCMSC, and CCK8 method was used to detect the senescence HUVEC of exocrine. The effect of cell proliferation, flow cytometry to detect the cell apoptosis, Western Blot detection of LC3a/b, p62 autophagy related protein level and the activation of important signal transduction pathway in senescent HUVEC cells. [results]1, hUCMSC's preparation tissue block directly adhered to hUCMSC 5 days later, the cells climbed out and passed to third generations. The cells showed long spindle shape and typical vortex growth characteristics; the expression rates of third generation hUCMSC expressed MSCs positive markers, CD29 and CD44, were 97.1%, 99.6%, CD34, CD45 and CD105 expression rates of 3.18%, 0.04%, 0%, and 10 days after lipid induced differentiation, saturated oil red O staining was positive, osteogenic induction After 3 weeks, alizarin red staining was positive. After 4 weeks of induction of cartilage, alizarin staining was positive. The GFP labeled cells were observed in the heart, liver, spleen, lung, and kidney.2. The model evaluated aging model mice (72W) to reduce body dynamic power, hair color decline, white hair and mental state. After HE staining, inflammatory infiltration, cell edema, necrosis, fibrous tissue hyperplasia and other degenerative changes were seen in different degrees. Immunohistochemistry showed that the expression level of p16 and p53 in the important organs of aging mice was statistically significant (P0.05), but the difference of SOD2 and catalase was statistically significant (P0.05). The total SOD active senescence group in the weave homogenate was significantly lower than that of the young group, the difference was statistically significant (P0.05).3. The effect of hUCMSC transplantation was evaluated by hUCMSC transplantation, the hair color of the experimental group increased, the body activity increased, the mental state was better, the abnormal rate of the stool decreased, and the organization structure of the organs was improved in different degrees; immunohistochemistry and qPCR were used to detect P1. The difference between 6 and p53 was statistically significant (P0.05), but the difference between SOD2 and Catalase was statistically significant (P0.05), and the total SOD activity in the homogenate of mice was improved (P0.05), and the transcriptional level of autophagy related groups (becline1, LC3b) in the main organs was statistically significant (p0.). The difference was statistically significant (p0.). 05).4, hUCMSC source Exocyst (hUCMSC-exo) derived from exocrine centrifugation was a stereoscopic cup-shaped structure with a diameter of about 136nm, 136nm, CD63, HSP90 and HSP70, but TAPA1 did not express.5, and the antioxidant activation mechanism of hUCMSC was evaluated by H2O2 treatment of HUVEC cells and cell proliferation. Decreasing and increasing apoptosis, the expression of autophagic marker LC3a/b increased after co culture with hUCMSC, and the autophagic body and autophagic flow increased. After co culture with hUCMSC-exo, the inhibition of the proliferation of HUVEC cells decreased, the apoptosis of HUVEC decreased, the expression of autophagic marker LC3a/b was increased, the expression of p62 was decreased, and the autophagic vesicles were significantly increased. Adding, autophagy inhibition signal pathway p38, mTOR, HSP27, STAT3 phosphorylation level decreased, signal pathway was suppressed. [conclusion]1. normal feeding mice 72W can obtain aging mice model with aging general characteristics and tissue structural degenerative changes.2. intravenous hUCMSC, can improve hair, spirit, activity, diet, and body weight status. The physiological state and the pathological state of tissue structure can be alleviated. HUCMSC can inhibit the expression of senescence genes and promote the expression of antioxidant genes and delay or reverse the oxidative stress state of the body. Therefore, the results show that hUCMSC has a certain effect of treating aging degenerative changes, and the antiaging effect of.3.hUCMSC may be through hUCM SC secrete exocrine promotes the autophagy of senescent cells by regulating multiple signal transduction pathways, clearing ROS in senescent cells, and reducing oxidative damage caused by ROS.

【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R457.7

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