發(fā)布時(shí)間：2018-02-20 07:19

  本文關(guān)鍵詞： EPCs miR-22 衰老 增殖 遷移 血管形成 Akt3　出處：《上海交通大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：背景及目的內(nèi)皮祖細(xì)胞(Endothelial progenitor cells,EPCs)是血管內(nèi)皮細(xì)胞的前體細(xì)胞,廣泛存在于臍帶血、外周血及骨髓中,參與缺血性疾病血管新生和損傷后的血管內(nèi)皮的愈合修復(fù)。當(dāng)血管受到損傷后,EPCs從骨髓中釋放進(jìn)入外周血中,遷移至損傷部位,然后增殖分化為成熟內(nèi)皮細(xì)胞,促進(jìn)損傷血管再內(nèi)皮化。已有的大量研究表明,隨著年齡的增長,EPCs數(shù)量減少,其粘附、增殖、遷移和血管形成等功能也隨之衰退。這一結(jié)論表明年齡的增長使EPCs呈現(xiàn)細(xì)胞衰老表型,但其確切的作用機(jī)制仍不清楚。Micro RNAs(mi RNAs)是一類長度約為22個(gè)核苷酸左右的單鏈內(nèi)源性非編碼小RNA,廣泛存在于動(dòng)物及植物中。mi RNAs能夠通過與其靶基因的3′端非編碼區(qū)(3′untranslated region,UTR)進(jìn)行完全或不完全互補(bǔ)配對的方式直接降解其靶基因或者對其靶基因進(jìn)行轉(zhuǎn)錄后調(diào)控,從而參與細(xì)胞存活、衰老、增殖、分化、遷移等重要的細(xì)胞生理過程。mi RNAs的異常表達(dá)與多種疾病如心血管疾病等密切有關(guān),而心血管疾病與EPCs又有著密切的關(guān)系。近年來,EPCs的衰老與mi RNAs的關(guān)系受到人們的重點(diǎn)關(guān)注,如Zhao及其同事報(bào)道m(xù)i R-34a通過抑制沉默信息調(diào)控子1(Sirt1)的表達(dá),加速EPCs的衰老,并降低細(xì)胞血管形成能力。Zhu課題組研究發(fā)現(xiàn)mi R-10a和mi R-21通過靶向Hmga2來調(diào)節(jié)小鼠EPCs的衰老、遷移、增殖、自我更新和體內(nèi)外血管形成能力;該研究的芯片結(jié)果也顯示mi R-22在小鼠老年組中表達(dá)量要高于小鼠年輕組。那么,mi R-22在人老年組中是否也是高表達(dá)?mi R-22是否在EPCs衰老過程中發(fā)揮生物學(xué)作用呢?為了解決這些問題,我們首先設(shè)置了年輕組(21歲左右)和老年組(66歲左右)兩組樣本,分離出EPCs,然后采用多種研究方法來研究mi R-22對EPCs的細(xì)胞衰老、增殖、遷移和血管形成的影響。通過預(yù)測軟件的預(yù)測和生物信息學(xué)分析,推測Akt3可能是mi R-22的潛在靶基因,最后采用各種實(shí)驗(yàn)方法探討Akt3與mi R-22之間的關(guān)系,從而來闡明mi R-22對EPCs衰老的作用機(jī)制。方法(1)本研究設(shè)置兩組樣本,一組是年輕組(21歲左右),一組是(66歲左右),每組各3個(gè)樣本,采取其外周血,然后采用密度梯度離心法(淋巴分離液Ficoll)來分離外周血,獲得單個(gè)核細(xì)胞,最后運(yùn)用貼壁分離法獲得較純的EPCs。(2)采用Di I-AC-LDL和FITC-UEA-I雙熒光法和表面抗原CD133、CD31和CD34檢測,對分離的EPCs進(jìn)行鑒定。采用熒光定量PCR(q RT-PCR)實(shí)驗(yàn)方法檢測mi R-22在年輕組和老年組中的表達(dá)量。(3)采用分子生物學(xué)方法構(gòu)建慢病毒載體p LVTHM-mi R-22和p LVTHM-anti-mi R-22,然后與病毒包裝質(zhì)粒共同感染293T細(xì)胞,收集上清,檢測病毒滴度。(4)慢病毒Lv-mi R-22和Lv-anti-mi R-22分別g感染青年組和老年組EPCs,采用q RT-PCR實(shí)驗(yàn)方法檢測轉(zhuǎn)染慢病毒的青年組和老年組EPCs中mi R-22的表達(dá)量,采用β-半乳糖苷酶檢測、MTT增殖法、transwell細(xì)胞遷移法、體外血管形成檢測來檢測和觀察轉(zhuǎn)染后青年組和老年組EPCs的衰老、增殖、遷移和血管形成等生物行為的變化。(5)利用Target Scan,mi Randa和Pic Tar靶基因預(yù)測軟件,預(yù)測出Akt3可能是mi R-22的一個(gè)潛在的靶基因。利用雙熒光素酶報(bào)告基因試驗(yàn)驗(yàn)證mi R-22對Akt3的負(fù)向調(diào)控作用。Mi R-22在年輕組EPCs中過表達(dá)或老年組EPCs中抑制表達(dá)后利用q RT-PCR和免疫印跡實(shí)驗(yàn)檢測Akt3 m RNA和蛋白水平的變化。(6)構(gòu)建Akt3和Akt3-3′UTR慢病毒載體,感染年輕組EPCs-mi R-22穩(wěn)轉(zhuǎn)株后,檢測其衰老、增殖、遷移和血管形成等生物行為的變化。結(jié)果(1)剛分離的單個(gè)核細(xì)胞形狀呈圓形,且體積很小,1天后可見貼壁細(xì)胞形狀呈較粗的圓形、橢圓形或不規(guī)則形。培養(yǎng)7天后,長梭形的內(nèi)皮樣細(xì)胞數(shù)增多且體積也增大,部分視野中可見由數(shù)個(gè)細(xì)胞形成的細(xì)胞集落。(2)在激光共聚焦顯微鏡下觀察到分離得到的單個(gè)核細(xì)胞分化到第7天出現(xiàn)橙黃色雙染陽性細(xì)胞,表明細(xì)胞攝取了Di I-AC-LDL和FITC-UEA-I并證實(shí)了橙黃色雙染陽性細(xì)胞為正在分化的EPCs。(3)流式細(xì)胞儀檢測表面抗原CD133、CD31、CD34和VEGFR-2,陽性率分別為33.3%、51.7%、45.4%和47.2%。(4)老年組mi R-22的表達(dá)量明顯高于年輕組。(5)年輕組和老年組EPCs分別感染mi R-22和anti-mi R-22慢病毒后,與對照組相比,感染mi R-22的年輕組中mi R-22表達(dá)量顯著上調(diào),衰老細(xì)胞數(shù)明顯增加,細(xì)胞增殖、遷移和血管形成能力明顯下降;另一方面,感染anti-mi R-22的老年組中mi R-22表達(dá)量明顯下降,衰老細(xì)胞數(shù)也明顯下降,其增殖、遷移和血管形成等生物學(xué)功能卻上調(diào)。(6)熒光素酶報(bào)告基因?qū)嶒?yàn)證實(shí),mi R-22可以抑制野生型Akt3-3′UTR-WT的熒光報(bào)告基因的活性,而對突變型Akt3-3′UTR-MUT沒有影響。此外,mi R-22過表達(dá)引起Y-EPCs中Akt3的基因和蛋白質(zhì)水平的表達(dá)明顯低于對照組,而anti-mi R-22導(dǎo)致A-EPCs中Akt3的表達(dá)水平明顯上調(diào)。(7)Akt3過表達(dá)能夠逆轉(zhuǎn)mi R-22對Y-EPCs的增殖、遷移和血管形成的抑制作用和對細(xì)胞衰老的促進(jìn)作用。結(jié)論(1)mi R-22在A-EPCs中表達(dá)量明顯升高。(2)mi R-22促進(jìn)EPCs衰老,抑制其增殖、遷移和血管形成能力。(3)mi R-22通過抑制其靶基因Akt3的表達(dá)來促進(jìn)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.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R363

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本文編號：1519073