心血管病患者成体细胞源性诱导型心肌细胞的建立

发布时间：2018-06-30 01:38

  本文选题：人诱导多能干细胞 + 重编程　； 参考：《第二军医大学》2017年硕士论文

【摘要】：背景:心血管疾病一直严重威胁着人类健康,虽然随着研究的进展,逐渐了解和掌握了部分相关疾病的流行病学特点、临床表现及有效的治疗方式等,但相关机制研究一直进展缓慢,特别是部分遗传相关性心血管疾病。原因有很多,其中之一是疾病特异性相关研究模型匮乏或已有模型无法完全模拟人体内疾病过程。而且,由于心肌细胞自身特性,人类心肌细胞出生后几乎不再具备生长增殖能力,使得心肌一旦受损伤后无法发挥相应功能,严重时可导致心力衰竭,危及患者生命。现有高血压、冠心病、心律失常、心肌病及先天性心脏病等治疗措施,除心脏移植外,多数是控制症状,延缓病情进展,无法达到根治疾病的目的。即使是心脏移植,也面临供体来源不足、免疫反应等多方面不利因素限制。然而,诱导多能干细胞(induced pluripotent stem cells,iPSCs)技术及诱导型心肌细胞(induced cardiomyocytes,iCMs)定向分化技术的出现及日趋成熟有望改变这种局面。目的:通过收集和分离临床心血管疾病患者个体特异性成体细胞,利用重编程技术和定向分化技术先后诱导获得iPSCs及iCMs,用于疾病机制研究和药物筛选应用方法与结果:收集心血管疾病患者尿液及外周血液标本,分离出病患特异性的尿液细胞和单个核细胞,进而对两种成体细胞进行培养和扩增。然后通过电转方式,将编码Oct4、Sox2、Klf4、L-Myc、Lin28、mp53DD和EBNA1的质粒导入体细胞,重编程培养得到病人和疾病特异性iPSCs。经鉴定证实获得的iPSCs显微镜下具备和人类胚胎干细胞系同样显著的核仁结构、较高的细胞核质比、细胞克隆团致密整体呈铺路石样外观及碱性磷酸酶染色阳性特点,免疫荧光染色OCT4、SOX2、NANOG、SSEA-4及TRA-1-60及荧光定量PCR有多能性标志物结构表达,体外拟胚体法自发三胚层分化实验和体内畸胎瘤形成实验证实有较高的分化发育潜能。之后,利用小分子Rapamycin、CHIR99021、KY02111和XAV939分阶段单层无细胞因子定向诱导iPSCs向心肌细胞分化,在诱导培养的第10天观察到细胞出现收缩跳动功能,进一步免疫荧光和定量PCR证实有心肌特异性标志物cTnT、cTnI、MHY6、a-Actinin表达。结论:本研究通过重编程技术和定向诱导分化技术,成功建立了心血管疾病特异性尿液和单个核细胞成体细胞库、诱导多能干细胞和诱导型心肌细胞模型,可广泛用于疾病机制研究和药物研发领域,为个体化医疗及精准医疗打下基础。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R54
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本文编号：2084285