本文選題：人誘導(dǎo)多能干細(xì)胞 + 重編程��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：背景:心血管疾病一直嚴(yán)重威脅著人類健康,雖然隨著研究的進(jìn)展,逐漸了解和掌握了部分相關(guān)疾病的流行病學(xué)特點(diǎn)、臨床表現(xiàn)及有效的治療方式等,但相關(guān)機(jī)制研究一直進(jìn)展緩慢,特別是部分遺傳相關(guān)性心血管疾病。原因有很多,其中之一是疾病特異性相關(guān)研究模型匱乏或已有模型無法完全模擬人體內(nèi)疾病過程。而且,由于心肌細(xì)胞自身特性,人類心肌細(xì)胞出生后幾乎不再具備生長增殖能力,使得心肌一旦受損傷后無法發(fā)揮相應(yīng)功能,嚴(yán)重時(shí)可導(dǎo)致心力衰竭,危及患者生命�，F(xiàn)有高血壓、冠心病、心律失常、心肌病及先天性心臟病等治療措施,除心臟移植外,多數(shù)是控制癥狀,延緩病情進(jìn)展,無法達(dá)到根治疾病的目的。即使是心臟移植,也面臨供體來源不足、免疫反應(yīng)等多方面不利因素限制。然而,誘導(dǎo)多能干細(xì)胞(induced pluripotent stem cells,iPSCs)技術(shù)及誘導(dǎo)型心肌細(xì)胞(induced cardiomyocytes,iCMs)定向分化技術(shù)的出現(xiàn)及日趨成熟有望改變這種局面。目的:通過收集和分離臨床心血管疾病患者個(gè)體特異性成體細(xì)胞,利用重編程技術(shù)和定向分化技術(shù)先后誘導(dǎo)獲得iPSCs及iCMs,用于疾病機(jī)制研究和藥物篩選應(yīng)用方法與結(jié)果:收集心血管疾病患者尿液及外周血液標(biāo)本,分離出病患特異性的尿液細(xì)胞和單個(gè)核細(xì)胞,進(jìn)而對(duì)兩種成體細(xì)胞進(jìn)行培養(yǎng)和擴(kuò)增。然后通過電轉(zhuǎn)方式,將編碼Oct4、Sox2、Klf4、L-Myc、Lin28、mp53DD和EBNA1的質(zhì)粒導(dǎo)入體細(xì)胞,重編程培養(yǎng)得到病人和疾病特異性iPSCs。經(jīng)鑒定證實(shí)獲得的iPSCs顯微鏡下具備和人類胚胎干細(xì)胞系同樣顯著的核仁結(jié)構(gòu)、較高的細(xì)胞核質(zhì)比、細(xì)胞克隆團(tuán)致密整體呈鋪路石樣外觀及堿性磷酸酶染色陽性特點(diǎn),免疫熒光染色OCT4、SOX2、NANOG、SSEA-4及TRA-1-60及熒光定量PCR有多能性標(biāo)志物結(jié)構(gòu)表達(dá),體外擬胚體法自發(fā)三胚層分化實(shí)驗(yàn)和體內(nèi)畸胎瘤形成實(shí)驗(yàn)證實(shí)有較高的分化發(fā)育潛能。之后,利用小分子Rapamycin、CHIR99021、KY02111和XAV939分階段單層無細(xì)胞因子定向誘導(dǎo)iPSCs向心肌細(xì)胞分化,在誘導(dǎo)培養(yǎng)的第10天觀察到細(xì)胞出現(xiàn)收縮跳動(dòng)功能,進(jìn)一步免疫熒光和定量PCR證實(shí)有心肌特異性標(biāo)志物cTnT、cTnI、MHY6、a-Actinin表達(dá)。結(jié)論:本研究通過重編程技術(shù)和定向誘導(dǎo)分化技術(shù),成功建立了心血管疾病特異性尿液和單個(gè)核細(xì)胞成體細(xì)胞庫、誘導(dǎo)多能干細(xì)胞和誘導(dǎo)型心肌細(xì)胞模型,可廣泛用于疾病機(jī)制研究和藥物研發(fā)領(lǐng)域,為個(gè)體化醫(yī)療及精準(zhǔn)醫(yī)療打下基礎(chǔ)。
[Abstract]:Background: cardiovascular disease has been a serious threat to human health. Although with the progress of the research, the epidemiological characteristics, clinical manifestations and effective treatment methods of some related diseases have been gradually understood and mastered, but the related mechanisms have been progressing slowly, especially some genetic related cardiovascular diseases. There are many reasons. One is that the disease specific related research model is deficient or the existing model can not fully simulate the disease process in the human body. Moreover, because of the characteristics of cardiac myocytes, human cardiomyocytes are almost no longer capable of growing and proliferating after birth, making the myocardium unable to perform the function of the phase once the myocardium is damaged, which can cause heart failure and endanger the patient. The existing treatment measures such as hypertension, coronary heart disease, arrhythmia, cardiomyopathy and congenital heart disease, except for heart transplantation, mostly control the symptoms, delay the progress of the disease, can not achieve the purpose of curing the disease. Even the heart transplant, it is also faced with many adverse factors such as insufficiency of donor source and immune response. Induced pluripotent stem cells (iPSCs) technology and the emergence and maturity of directed differentiation of induced cardiomyocytes (induced cardiomyocytes, iCMs) are expected to change this situation. Objective: by collecting and separating individual specific adult cells of patients with clinical cardiovascular disease, reprogramming techniques and directional differentiation techniques are used. IPSCs and iCMs were successfully induced and used for disease mechanism research and drug screening. The urine and peripheral blood samples of patients with cardiovascular disease were collected, urine cells and mononuclear cells were isolated from the patients, and then two adult cells were cultured and amplified. Then Oct4, Sox would be encoded by electric transfer. 2, Klf4, L-Myc, Lin28, mp53DD and EBNA1 plasmid transfected somatic cells. Reprogramming culture obtained patients and disease specific iPSCs. confirmed that the iPSCs microscope has the same significant nucleolar structure as human embryonic stem cell lines, higher cell nuclear ratio, compact whole cell clones as a paving stone like appearance and alkaline phosphoric acid. OCT4, SOX2, NANOG, SSEA-4, TRA-1-60 and fluorescent quantitative PCR have multiple markers of structural expression. In vitro, the embryogenic spontaneous three germ layer differentiation experiment and the formation of teratoma in vivo proved to have high differentiation potential. After that, small molecules Rapamycin, CHIR99021, KY02111 and XAV939 points are used. The single cell monolayer was directed to induce iPSCs to differentiate into cardiomyocytes. In the tenth day of induction and culture, the cell appeared contraction and beating function. Further immunofluorescence and quantitative PCR confirmed the expression of myocardial specific markers, cTnT, cTnI, MHY6, and a-Actinin. Conclusion: This study was carried out with heavy programming and directed differentiation. An adult cell library of specific urine and mononuclear cells of cardiovascular disease has been established to induce the model of pluripotent stem cells and inducible cardiomyocytes, which can be widely used in the field of disease mechanism research and drug research and development, which lays the foundation for individualized medical treatment and precision medical treatment.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R54
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本文編號(hào)：2084285