视网膜色素变性患者自体诱导多潜能干细胞系的建立与应用

发布时间：2018-07-28 14:36

【摘要】：背景：视网膜变性疾病是一类以进行性视功能下降为临床特征的疾病,具有遗传性或后天获得性,是临床上多见而又难治的一类致盲性眼病。视网膜色素变性作为最常见的遗传性视网膜变性疾病,其遗传方式多样,致病基因多种,深入的致病机制仍然不明确,目前尚无有效的治疗方法。对于视网膜变性疾病,由于其取材的受限性,难以建立体外细胞研究模型。2007年日本科学家Yamanaka等人发明了人类体细胞诱导的多潜能干细胞(human induced pluripotent stem cell, hiPSC)技术。通过这一技术,理论上可产生大量的与胚胎干细胞同等分化潜能的多潜能干细胞。利用患者自身的体细胞诱导产生的hiPS细胞及其分化产生的视网膜细胞,可为视网膜变性疾病建立体外的细胞研究模型。hiPS细胞以及其分化产生的各类视网膜细胞还可在各类视网膜变性疾病中探索干细胞替代治疗,为攻克此类难治的致盲性眼病带来希望。目的：本课题研究目的在于：(1)将视网膜色素变性患者的皮肤成纤维细胞,通过导入外源基因,诱导其成为多潜能干细胞(hiPSC);(2)在此过程中探索视网膜变性患者体细胞重编程的机制过程,并为进一步向视网膜细胞的定向分化及机制研究奠定基础。材料和方法：本研究取IFT140基因突变的视网膜色素变性的1名患者及1名正常对照受试者的皮肤组织样本进行原代培养,建立人皮肤成纤维细胞系(human dermal fibroblast, HDF)。并取CF-1品系小鼠13.5天胚胎,分离并培养小鼠胚胎成纤维细胞(mouse embryonic fibroblast, MEF),培养至P3代后,经丝裂霉素C处理后制成饲养层细胞(feeder cell)。实验中利用含Oct4、Klf4、Sox2、c-Myc四种重组基因的慢病毒质粒以及慢病毒包装质粒PVSVG和△8.91,在人胚肾细胞(293HEK-T)中包装出四种分别含有Oct4、Klf4、Sox2和c-Myc重组基因的慢病毒颗粒。在P1代人皮肤成纤维细胞中按1：10的比例加入过量的慢病毒颗粒和助转剂polybrene,诱导人成纤维细胞转变成诱导多潜能干细胞。诱导第5天后消化细胞,转至MEF制作成的饲养层细胞上继续培养,直至出现圆形类胚胎干细胞形态的诱导多潜能干细胞克隆。挑出克隆进行扩大培养和维持。对感染前和感染后的人皮肤成纤维细胞进行相关基因的荧光实时定量PCR的检测,鉴定干细胞相关基因和成纤维细胞特异基因的表达变化。结果： (1)成功建立视网膜色素变性患者及正常受试者的人皮肤成纤维细胞系,形态为长梭形,呈编织状、漩涡状密集排列生长,大量表达FSP-1基因。冻存后复苏率达50-75%不等。 (2)成功利用CF-1品系小鼠的MEF细胞,制作成饲养层细胞。冻存后复苏率达75%。 (3)利用含Oct4、Klf4、Sox2和c-Myc四种重组基因的慢病毒质粒以及慢病毒包装质粒PVSVG和△8.91,在人胚肾细胞(293HEK-T)中包装出四种分别含有Oct4、Klf4、Sox2和c-Myc重组基因的慢病毒颗粒。滴度分别为1.4×107TU/ml;1.2×107TU/ml;2.0×107TU/ml和3.5×106TU/ml。 (4)慢病毒感染人皮肤成纤维细胞后,显微镜下观察到细胞逐渐变圆,细胞核增大,在18-20天左右感染后细胞聚集成圆形、类似胚胎干细胞的克隆。 (5)对比感染前、后的人皮肤成纤维细胞的相关基因表达水平,感染后,FSP-1mRNA水平显著下调,endo-Oct4/Sox2/Klf4/c-Myc和Nanog基因的mRNA水平显著上调(p0.05)。结论：本研究首次利用IFT140基因突变的视网膜色素变性患者的皮肤成纤维细胞,用慢病毒诱导的方式,导入外源基因Oct4、Klf4、Sox2和c-Myc,成功诱导体细胞出现类似人胚胎干细胞的形态变化,并表达干细胞特异的内源性基因。
[Abstract]:Background:
Retinal degeneration is a kind of disease characterized by progressive visual function decline, hereditary or acquired acquired. It is a kind of blind eye disease which is frequently seen and difficult to treat in clinical. Retinitis pigmentosa is the most common hereditary retinal degeneration disease. It has many ways of inheritance, many kinds of pathogenic genes and deep pathogenicity. The mechanism is still not clear and there is no effective treatment. For the retinal degeneration disease, because of its limitations, it is difficult to establish an in vitro cell research model.2007 Japanese scientist Yamanaka and others invented the human somatic cell induced pluripotent stem cells (human induced pluripotent stem cell, hiPSC). Technology, in theory, produces a large number of pluripotent stem cells that have the same differentiation potential as embryonic stem cells. The hiPS cells and their differentiated retinal cells, induced by the somatic cells of the patient's own, can be used to establish the cell research model.HiPS cells in vitro for the retina denatured disease and the various types of retina produced by its differentiation. Cells can also explore stem cell replacement therapy in various types of retinal degenerative diseases, hoping to overcome such refractory blinding eye diseases.
Objective:
The purpose of this research is as follows: (1) the skin fibroblasts of patients with retinitis pigmentosa can be introduced into the multipotential stem cells (hiPSC) by introducing foreign genes, and (2) to explore the mechanism of reprogramming of the somatic cells of the retinal degeneration patients in this process, and to lay a foundation for further directional differentiation and mechanism research of retinal cells. Set the foundation.
Materials and methods:
In this study, 1 patients with retinitis pigmentosa with IFT140 gene mutation and 1 normal control subjects were cultured for primary culture, and the human skin fibroblast line (human dermal fibroblast, HDF) was established, and 13.5 day embryos of CF-1 strain mice were taken to isolate and cultivate mouse embryonic fibroblasts (mouse embryonic fibro). Blast (MEF), cultured to P3 generation, was treated with mitomycin C to form feeder layer cells (feeder cell).
In the experiment, four lentivirus plasmids containing Oct4, Klf4, Sox2, c-Myc and lentivirus package plasmids PVSVG and delta 8.91 were used in the experiment to pack four kinds of lentivirus particles containing Oct4, Klf4, Sox2 and c-Myc recombinant genes in human embryonic kidney cells (293HEK-T).
In the P1 generation of human skin fibroblasts, an excessive amount of lentivirus particles and auxiliary agent Polybrene were added at 1:10 proportion to induce human fibroblasts to induce pluripotent stem cells. Fifth days later, the digestive cells were induced to continue to be cultured on the feeder cells made from MEF until the appearance of the morphology of the round embryonic stem cells appeared. Potential stem cell clones. Pick out clones for expansion and maintenance. Detection of fluorescence real-time quantitative PCR for the related genes of human skin fibroblasts before and after infection, and identify the changes in the expression of stem cell related genes and fibroblast specific genes.
Result:
(1) the human skin fibroblast line of the patients with retinitis pigmentosa and normal subjects was successfully established. The morphology of the human skin fibroblast cell line was long spindle shaped, woven and swirling in dense arrangement of the FSP-1 gene. The resuscitation rate was not equal to 50-75% after the cryopreservation.
(2) successful use of MEF cells from CF-1 strain mice was made into feeder cells. After cryopreservation, the recovery rate reached 75%.
(3) using four lentivirus plasmids containing Oct4, Klf4, Sox2 and c-Myc, and lentivirus package plasmids PVSVG and delta 8.91, four lentivirus particles containing Oct4, Klf4, Sox2 and c-Myc recombinant genes were packed in human embryonic kidney cells (293HEK-T). The titers were 1.4 * 107TU/ml, 1.2 * 107TU/ml, 2 * and 3.5.
(4) after infection of the human skin fibroblasts of the lentivirus, the cells gradually became round and the nuclei were enlarged under the microscope. After 18-20 days of infection, the cells were aggregated and round, similar to the cloning of embryonic stem cells.
(5) the level of related gene expression in human skin fibroblasts was compared before and after infection. After infection, the level of FSP-1mRNA was significantly down, and the mRNA level of endo-Oct4/Sox2/Klf4/c-Myc and Nanog genes was significantly up (P0.05).
Conclusion:
In this study, we first used IFT140 gene mutation in the skin fibroblasts of the patients with retinitis pigmentosa. The exogenous gene Oct4, Klf4, Sox2 and c-Myc were introduced in the way of lentivirus induction. The morphological changes of somatic cells similar to human embryonic stem cells were induced, and the specific endogenous genes were expressed in the stem cells.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R774.13

【相似文献】