人脐带间充质干细胞对小鼠老年性器官功能退变的治疗作用及其机制研究

发布时间：2018-04-20 06:08

本文选题：衰老 + 间充质干细胞　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]筛选并获得C57BL/6小鼠衰老模型;利用衰老模型评价人的脐带间充质干细胞(hUCMSC)对衰老小鼠老年性器官功能退变的治疗作用;制备hUCMSC来源外泌体和HUVEC衰老氧化应激细胞模型,利用细胞模型评价hUCMSC来源外泌体在hUCMSC延缓或逆转衰老中的抗氧化应激作用及其机制,为hUCMSC治疗老年退变性疾病提供理论基础。[方法]1、hUCMSC的制备组织块直接贴壁法培养hUCMSC,倒置显微镜观察细胞状态,流式细胞仪检测hUCMSC细胞表面标志物,hUCMSC成脂、成骨和成软骨诱导分化实验鉴定其多向分化能力。体外通过表达GFP慢病毒转染标记hUCMSC,并通过荧光计数显微镜GFP标记阳性率。2、小鼠衰老模型筛选与评价常规饲料喂养C57BL/6小鼠至72周,共得60只老年鼠,根据小鼠毛色、身体活动力、精神状态、大便情况观察以及体重检测的方法进行筛选,按照毛色光泽度发暗,出现白色毛发,精神状态欠佳,活动减少、久坐,便干或者便稀频率增加,体重在(20±5)g范围的标准,将符合该标准的老年鼠纳入本实验,作为自然衰老小鼠模型,共40只,雌雄各20只;利用观察年轻小鼠一般情况,毛色发黑、发亮、顺滑,活动较多,精神饱满、饮食正常、大便正常,体重在(15±2.5)g范围的标准随机选取年轻小鼠20只,为年轻组。随机取年轻鼠(8w)与老年鼠(72w)各8只,为年轻组与模型组,对比评价模型组自然衰老小鼠的一般情况;心脏、肝脏、脾脏、肺、肾脏主要器官HE染色后观察组织结构变化,以及免疫组化检测p16、p53、SOD2和Catalase衰老相关蛋白在模型小鼠重要器官(心、肝、脾、肺、肾)中的表达水平;qPCR检测p16、p53、SOD2和Catalase等衰老相关蛋白在模型小鼠重要器官(心、肝、脾、肺、肾)中的转录水平,以及组织匀浆中总SOD活力。3、分组及hUCMSC移植将衰老小鼠随机分为实验组和对照组,各16只;实验组按照1×107个/kg的标准,经尾静脉输入GFP基因标记的hUCMSC,每只2×105个、200ul体积,每周1次,连续4次;对照组输入等体积生理盐水。常规饲养,移植4w后观察评价。4.hUCMSC移植疗效与机制评价hUCMSC移植结束1个月后分别比较两组小鼠的一般情况、脱颈处死后利用组织切片观察重要器官(心、肝、脾、肺、肾)结构差异以及qPCR、免疫组化分别检测衰老相关基因(p16、p53、SOD2和Catalase)在各重要器官中转录水平和表达水平的差异;检测两组小鼠组织匀浆中总SOD活力差异以及qPCR检测主要脏器组织(心、肝、脾、肺、肾)中自噬相关基因(becline1,LC3b,sirt1,sirt5)水平的差异。5.外泌体的制备与鉴定利用超高速离心法获得纯化hUCMSC来源外泌体,3%磷钨酸染色电镜观察外泌体形态,Western Blot检测外泌体阳性标志物HSP90、HSP70、CD63和阴性标志物TAPA。6.机制研究利用400nmol/L H202处理HUVEC细胞构建HUVEC衰老细胞氧化应激模型,并建立与hUCMSC的transwell共培养体系,Western Blot检测LC3a/b、mTOR和p-mTOR蛋白水平,共聚焦显微镜观察细胞模型自噬流,透射电子显微镜检测自噬泡形成情况,评价hUCMSC抗氧化应激效果。建立hUCMSC来源的外泌体与细胞衰老模型共培养体系,CCK8法检测外泌体对衰老HUVEC细胞增殖的影响,流式细胞仪检测细胞凋亡情况,Western Blot检测LC3a/b、p62自噬相关蛋白水平以及外泌体对衰老HUVEC细胞重要信号转导通路的激活情况。[结果]1、hUCMSC的制备组织块直接贴壁法培养hUCMSC 5天后可见细胞爬出,传至第三代后细胞呈长梭形并且具有典型的涡旋式生长特征;第三代hUCMSC表达间充质干细胞阳性标记物CD29和CD44的表达率为97.1%、99.6%,间充质干细胞阴性标志物CD34、CD45和CD105表达率为3.18%、0.04%、0%;成脂诱导分化10天后饱和油红O染色为阳性,成骨诱导分化3周后,茜素红染色阳性;成软骨诱导4周后,阿尔新蓝染色阳性,免疫组化可观察到GFP标记的细胞在心脏、肝脏、脾脏、肺、肾脏。2、模型评价衰老模型小鼠(72w)与年轻小鼠(8w)相比身体活动力减少,毛色光泽度下降,出现白色毛发,精神状态欠佳,便干或者便稀频率增加;HE染色后可见不同程度的炎症浸润,细胞水肿、坏死,纤维组织增生等退行性变;免疫组化显示,衰老小鼠重要器官p16、p53表达水平,差异具有统计学意义(p0.05),而SOD2和catalase低水平,差异具有统计学意义(p0.05),组织匀浆中总SOD活性衰老组明显低于年轻组,差异具有统计学意义(p0.05)。3、hUCMSC移植疗效评价hUCMSC移植后,实验组小鼠毛色光泽度提高,身体活动度增加,精神状态较好,大便异常率下降;器官组织结构有不同程度改善;免疫组化和qPCR检测p16和p53水平下降,差异具有统计学意义(p0.05),而SOD2和Catalase水平上升差异具有统计学意义(p0.05);小鼠组织匀浆中总SOD活性提高,差异具有统计学意义(p0.05);主要脏器组织中自噬相关基因(becline1,LC3b)转录水平提高,差异具有统计学意义(p0.05)。4、外泌体制备超高速离心分离所得hUCMSC源外泌体(hUCMSC-exo)呈立体的杯状结构,直径约136nm,外泌体标志性蛋白CD63、HSP90和HSP70高表达,而外泌体阴性标志物TAPA1不表达。5、hUCMSC的抗氧化应激作用机制评价H2O2处理HUVEC细胞后,细胞增殖能力下降,凋亡增加;经过与hUCMSC非接触共培养后,自噬标记物LC3a/b表达增加,电镜显示自噬小体及自噬流增加;经过与hUCMSC-exo共培养后,H2O2对HUVEC细胞增殖的抑制降低,HUVEC凋亡减少,自噬标记物LC3a/b表达增加,p62表达下降,细胞自噬泡明显增加,自噬抑制信号通路p38、mTOR、hsp27、stat3磷酸化水平下降,信号通路被抑制。[结论]1.正常饲养法饲养小鼠72w可获得具有衰老的一般特征及组织结构退行性改变的衰老小鼠模型。2.静脉注射hUCMSC,可以改善毛发、精神、活动、饮食以及体重状态生理状态和缓解组织结构病理状态;从分子水平hUCMSC能够抑制衰老基因的表达而促进抗氧化基因的表达,延缓或逆转机体氧化应激状态。因此,研究结果表明hUCMSC具有一定的治疗衰老退行性变的作用。3.hUCMSC的抗衰老作用可能是通过hUCMSC分泌外泌体通过调节多条信号转导通路促进衰老细胞自噬,清除衰老细胞中ROS,减少ROS引起的氧化损伤实现的。
[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.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R457.7

【参考文献】