miR-22在内皮祖细胞衰老中的作用及机制研究

发布时间：2018-02-20 07:19

本文关键词： EPCs miR-22 衰老增殖迁移血管形成 Akt3　出处：《上海交通大学》2015年博士论文　论文类型：学位论文

【摘要】：背景及目的内皮祖细胞(Endothelial progenitor cells,EPCs)是血管内皮细胞的前体细胞,广泛存在于脐带血、外周血及骨髓中,参与缺血性疾病血管新生和损伤后的血管内皮的愈合修复。当血管受到损伤后,EPCs从骨髓中释放进入外周血中,迁移至损伤部位,然后增殖分化为成熟内皮细胞,促进损伤血管再内皮化。已有的大量研究表明,随着年龄的增长,EPCs数量减少,其粘附、增殖、迁移和血管形成等功能也随之衰退。这一结论表明年龄的增长使EPCs呈现细胞衰老表型,但其确切的作用机制仍不清楚。Micro RNAs(mi RNAs)是一类长度约为22个核苷酸左右的单链内源性非编码小RNA,广泛存在于动物及植物中。mi RNAs能够通过与其靶基因的3′端非编码区(3′untranslated region,UTR)进行完全或不完全互补配对的方式直接降解其靶基因或者对其靶基因进行转录后调控,从而参与细胞存活、衰老、增殖、分化、迁移等重要的细胞生理过程。mi RNAs的异常表达与多种疾病如心血管疾病等密切有关,而心血管疾病与EPCs又有着密切的关系。近年来,EPCs的衰老与mi RNAs的关系受到人们的重点关注,如Zhao及其同事报道mi R-34a通过抑制沉默信息调控子1(Sirt1)的表达,加速EPCs的衰老,并降低细胞血管形成能力。Zhu课题组研究发现mi R-10a和mi R-21通过靶向Hmga2来调节小鼠EPCs的衰老、迁移、增殖、自我更新和体内外血管形成能力;该研究的芯片结果也显示mi R-22在小鼠老年组中表达量要高于小鼠年轻组。那么,mi R-22在人老年组中是否也是高表达?mi R-22是否在EPCs衰老过程中发挥生物学作用呢?为了解决这些问题,我们首先设置了年轻组(21岁左右)和老年组(66岁左右)两组样本,分离出EPCs,然后采用多种研究方法来研究mi R-22对EPCs的细胞衰老、增殖、迁移和血管形成的影响。通过预测软件的预测和生物信息学分析,推测Akt3可能是mi R-22的潜在靶基因,最后采用各种实验方法探讨Akt3与mi R-22之间的关系,从而来阐明mi R-22对EPCs衰老的作用机制。方法(1)本研究设置两组样本,一组是年轻组(21岁左右),一组是(66岁左右),每组各3个样本,采取其外周血,然后采用密度梯度离心法(淋巴分离液Ficoll)来分离外周血,获得单个核细胞,最后运用贴壁分离法获得较纯的EPCs。(2)采用Di I-AC-LDL和FITC-UEA-I双荧光法和表面抗原CD133、CD31和CD34检测,对分离的EPCs进行鉴定。采用荧光定量PCR(q RT-PCR)实验方法检测mi R-22在年轻组和老年组中的表达量。(3)采用分子生物学方法构建慢病毒载体p LVTHM-mi R-22和p LVTHM-anti-mi R-22,然后与病毒包装质粒共同感染293T细胞,收集上清,检测病毒滴度。(4)慢病毒Lv-mi R-22和Lv-anti-mi R-22分别g感染青年组和老年组EPCs,采用q RT-PCR实验方法检测转染慢病毒的青年组和老年组EPCs中mi R-22的表达量,采用β-半乳糖苷酶检测、MTT增殖法、transwell细胞迁移法、体外血管形成检测来检测和观察转染后青年组和老年组EPCs的衰老、增殖、迁移和血管形成等生物行为的变化。(5)利用Target Scan,mi Randa和Pic Tar靶基因预测软件,预测出Akt3可能是mi R-22的一个潜在的靶基因。利用双荧光素酶报告基因试验验证mi R-22对Akt3的负向调控作用。Mi R-22在年轻组EPCs中过表达或老年组EPCs中抑制表达后利用q RT-PCR和免疫印迹实验检测Akt3 m RNA和蛋白水平的变化。(6)构建Akt3和Akt3-3′UTR慢病毒载体,感染年轻组EPCs-mi R-22稳转株后,检测其衰老、增殖、迁移和血管形成等生物行为的变化。结果(1)刚分离的单个核细胞形状呈圆形,且体积很小,1天后可见贴壁细胞形状呈较粗的圆形、椭圆形或不规则形。培养7天后,长梭形的内皮样细胞数增多且体积也增大,部分视野中可见由数个细胞形成的细胞集落。(2)在激光共聚焦显微镜下观察到分离得到的单个核细胞分化到第7天出现橙黄色双染阳性细胞,表明细胞摄取了Di I-AC-LDL和FITC-UEA-I并证实了橙黄色双染阳性细胞为正在分化的EPCs。(3)流式细胞仪检测表面抗原CD133、CD31、CD34和VEGFR-2,阳性率分别为33.3%、51.7%、45.4%和47.2%。(4)老年组mi R-22的表达量明显高于年轻组。(5)年轻组和老年组EPCs分别感染mi R-22和anti-mi R-22慢病毒后,与对照组相比,感染mi R-22的年轻组中mi R-22表达量显著上调,衰老细胞数明显增加,细胞增殖、迁移和血管形成能力明显下降;另一方面,感染anti-mi R-22的老年组中mi R-22表达量明显下降,衰老细胞数也明显下降,其增殖、迁移和血管形成等生物学功能却上调。(6)荧光素酶报告基因实验证实,mi R-22可以抑制野生型Akt3-3′UTR-WT的荧光报告基因的活性,而对突变型Akt3-3′UTR-MUT没有影响。此外,mi R-22过表达引起Y-EPCs中Akt3的基因和蛋白质水平的表达明显低于对照组,而anti-mi R-22导致A-EPCs中Akt3的表达水平明显上调。(7)Akt3过表达能够逆转mi R-22对Y-EPCs的增殖、迁移和血管形成的抑制作用和对细胞衰老的促进作用。结论(1)mi R-22在A-EPCs中表达量明显升高。(2)mi R-22促进EPCs衰老,抑制其增殖、迁移和血管形成能力。(3)mi R-22通过抑制其靶基因Akt3的表达来促进EPCs的衰老。
[Abstract]:Endothelial progenitor cells background and objective (Endothelial progenitor, cells, EPCs) are the precursor cells of vascular endothelial cells, widely exists in umbilical cord blood, peripheral blood and bone marrow, healing in endothelial angiogenesis and ischemic disease after injury. When blood vessels are damaged after the release of EPCs in the peripheral blood from the bone marrow, migrate to the injury site, then proliferate and differentiate into mature endothelial cells, promote reendothelialization. A large number of studies show that with the increase of age, the amount of EPCs decreased, the adhesion, proliferation, migration and formation of blood vessels and other functions will also decline. This conclusion shows that the growth of the age to EPCs showing the cell senescence phenotype, but the exact mechanism is still not clear whether the.Micro RNAs (MI RNAs) is a kind of endogenous single strand length is approximately about 22 nucleotides encoding non small RNA, widely exists in animal and plant in.Mi Through the RNAs and its target gene 3 'non encoding region (3' untranslated region, UTR) to complete or incomplete complementary way direct degradation of its target genes or transcription of its target gene regulation, which is involved in cell survival, proliferation, differentiation, senescence, closely related to the abnormal expression of cells with a variety of diseases.Mi RNAs and other important physiological processes such as migration of cardiovascular disease, and cardiovascular disease and EPCs have a close relationship. In recent years, the relationship between aging and MI RNAs EPCs's attention, such as Zhao and colleagues reported mi R-34a through the inhibition of silent information regulator 1 (Sirt1) expression. EPCs accelerated aging, and reduce cell angiogenesis research group.Zhu mi R-10a found that the subject ability and MI R-21 by targeting Hmga2 to EPCs mice aging, migration, proliferation, self-renewal and in vivo angiogenesis can The study of the chip; results also show that the expression of MI R-22 in mice in the elderly group was higher than that of young mice group. Then, whether mi R-22 is highly expressed in the elderly group? Mi R-22 might play a biological role in the aging process of EPCs? In order to solve these problems, we first set up the young group (21 years old) and the elderly group (66 years old) two samples, isolated EPCs, and then to study the MI R-22 of EPCs cell aging, using a variety of methods influence the proliferation, migration and angiogenesis. The prediction software of prediction and bioinformatics analysis, we speculated that Akt3 may be a potential target gene of MI R-22 finally, to investigate the relationship between Akt3 and MI R-22 by using various experimental methods, so as to elucidate the mechanism of MI R-22 on EPCs aging. Methods (1) this study set two group samples, a group of young group (21 years old), one group (66 years old), each Each group of 3 samples taken in peripheral blood, and then by density gradient centrifugation (lymph fluid separation Ficoll) to separate peripheral blood mononuclear cells, finally using adherent separation method to obtain relatively pure EPCs. (2) by Di I-AC-LDL and FITC-UEA-I double fluorescence and surface antigen CD133, CD31 and CD34 detection on the separation of EPCs were identified by fluorescence quantitative PCR (Q RT-PCR) expression experiment method for detection of MI R-22 in the young and old groups. (3) the construction of lentiviral vector of P LVTHM-mi R-22 and P LVTHM-anti-mi R-22 by molecular biological method and virus packaging plasmid co infected 293T cells collected the supernatant, detect virus titer. (4) Lv-mi R-22 and Lv-anti-mi R-22 lentivirus were G infection in the young group and the old group EPCs expression by Q RT-PCR assay transfection of lentivirus EPCs in the young group and the old group mi R-22, using beta Galactosidase assays, MTT proliferation assay, Transwell cell migration detection method, to detect and observe the transfection of the young group and the old group EPCs aging, the formation of in vitro vascular proliferation, migration and angiogenesis changes and other biological behavior. (5) by Target Scan, MI Randa and Pic Tar target gene prediction software, forecast Akt3 may be a potential target gene of MI R-22. The Akt3 negative regulation of.Mi R-22 in the young group EPCs overexpression or EPCs inhibits the expression of elderly group by changes of Q RT-PCR and Western blotting assay of Akt3 m RNA and protein levels by dual luciferase R-22 (MI test. 6) construction of Akt3 and Akt3-3 'UTR lentiviral vector infection of young group of EPCs-mi detected in R-22 after the detection of aging, proliferation, migration and angiogenesis changes and other biological behavior. Results (1) the newly isolated mononuclear cells were round in shape, And the volume is small, 1 days after the adherent cell shape of a thick round, oval or irregular in shape. After 7 days of culture, the number of endothelial cells increased and spindle volume increased in the visible part of the field of vision is formed by a number of cell colonies. (2) in a laser confocal microscope platform. Under the microscope to observe isolated mononuclear cells to seventh days of orange double staining cells showed that the cellular uptake of Di, I-AC-LDL and FITC-UEA-I and confirmed the orange double staining cells are differentiated EPCs. (3) surface antigen detection CD133, flow cytometry, CD31, CD34 and VEGFR-2 positive. Rates were 33.3%, 51.7%, 45.4% and 47.2%. (4) expression of MI R-22 was significantly higher than that of the old group of young group. (5) the young and old groups were infected with EPCs mi R-22 and anti-mi R-22 lentivirus, compared with the control group, the amount of MI R-22 expression in R-22 infected Mi young group With the increased number of senescent cells significantly increased, cell proliferation, migration and angiogenesis significantly decreased; on the other hand, anti-mi R-22 infection in elderly group mi R-22 expression was significantly decreased, the number of senescent cells also decreased significantly, the proliferation, migration and angiogenesis and other biological functions are raised. (6) luciferase reporter genetic experiments confirmed that the fluorescent reporter gene mi R-22 can inhibit the wild-type Akt3-3 'UTR-WT activity, but had no effect on the mutant Akt3-3' UTR-MUT. In addition, the MI R-22 Y-EPCs over expression induced Akt3 gene and protein expression level was significantly lower than the control group, while anti-mi R-22 leads to the expression level of A-EPCs is significantly increased in Akt3. (7) the overexpression of Akt3 can reverse mi R-22 on Y-EPCs proliferation, inhibition of migration and angiogenesis and promotes cellular senescence. Conclusion (1) the expression of MI R-22 in A-EPCs was significantly higher (2) mi R-22 promotes EPCs senescence and inhibits its proliferation, migration and angiogenesis. (3) mi R-22 promotes the senescence of EPCs by inhibiting the expression of its target gene Akt3.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R363

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