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

【摘要】：背景:心血管疾病一直嚴重威脅著人類健康,雖然隨著研究的進展,逐漸了解和掌握了部分相關疾病的流行病學特點、臨床表現及有效的治療方式等,但相關機制研究一直進展緩慢,特別是部分遺傳相關性心血管疾病。原因有很多,其中之一是疾病特異性相關研究模型匱乏或已有模型無法完全模擬人體內疾病過程。而且,由于心肌細胞自身特性,人類心肌細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)揮相應功能,嚴重時可導致心力衰竭,危及患者生命�，F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,多數是控制癥狀,延緩病情進展,無法達到根治疾病的目的。即使是心臟移植,也面臨供體來源不足、免疫反應等多方面不利因素限制。然而,誘導多能干細胞(induced pluripotent stem cells,iPSCs)技術及誘導型心肌細胞(induced cardiomyocytes,iCMs)定向分化技術的出現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體特異性成體細胞,利用重編程技術和定向分化技術先后誘導獲得iPSCs及iCMs,用于疾病機制研究和藥物篩選應用方法與結果:收集心血管疾病患者尿液及外周血液標本,分離出病患特異性的尿液細胞和單個核細胞,進而對兩種成體細胞進行培養(yǎng)和擴增。然后通過電轉方式,將編碼Oct4、Sox2、Klf4、L-Myc、Lin28、mp53DD和EBNA1的質粒導入體細胞,重編程培養(yǎng)得到病人和疾病特異性iPSCs。經鑒定證實獲得的iPSCs顯微鏡下具備和人類胚胎干細胞系同樣顯著的核仁結構、較高的細胞核質比、細胞克隆團致密整體呈鋪路石樣外觀及堿性磷酸酶染色陽性特點,免疫熒光染色OCT4、SOX2、NANOG、SSEA-4及TRA-1-60及熒光定量PCR有多能性標志物結構表達,體外擬胚體法自發(fā)三胚層分化實驗和體內畸胎瘤形成實驗證實有較高的分化發(fā)育潛能。之后,利用小分子Rapamycin、CHIR99021、KY02111和XAV939分階段單層無細胞因子定向誘導iPSCs向心肌細胞分化,在誘導培養(yǎng)的第10天觀察到細胞出現收縮跳動功能,進一步免疫熒光和定量PCR證實有心肌特異性標志物cTnT、cTnI、MHY6、a-Actinin表達。結論:本研究通過重編程技術和定向誘導分化技術,成功建立了心血管疾病特異性尿液和單個核細胞成體細胞庫、誘導多能干細胞和誘導型心肌細胞模型,可廣泛用于疾病機制研究和藥物研發(fā)領域,為個體化醫(yī)療及精準醫(yī)療打下基礎。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R54
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本文編號：2084285