【摘要】：背景： 視網(wǎng)膜變性疾病是一類以進(jìn)行性視功能下降為臨床特征的疾病,具有遺傳性或后天獲得性,是臨床上多見而又難治的一類致盲性眼病。視網(wǎng)膜色素變性作為最常見的遺傳性視網(wǎng)膜變性疾病,其遺傳方式多樣,致病基因多種,深入的致病機(jī)制仍然不明確,目前尚無有效的治療方法。對于視網(wǎng)膜變性疾病,由于其取材的受限性,難以建立體外細(xì)胞研究模型。2007年日本科學(xué)家Yamanaka等人發(fā)明了人類體細(xì)胞誘導(dǎo)的多潛能干細(xì)胞(human induced pluripotent stem cell, hiPSC)技術(shù)。通過這一技術(shù),理論上可產(chǎn)生大量的與胚胎干細(xì)胞同等分化潛能的多潛能干細(xì)胞。利用患者自身的體細(xì)胞誘導(dǎo)產(chǎn)生的hiPS細(xì)胞及其分化產(chǎn)生的視網(wǎng)膜細(xì)胞,可為視網(wǎng)膜變性疾病建立體外的細(xì)胞研究模型。hiPS細(xì)胞以及其分化產(chǎn)生的各類視網(wǎng)膜細(xì)胞還可在各類視網(wǎng)膜變性疾病中探索干細(xì)胞替代治療,為攻克此類難治的致盲性眼病帶來希望。 目的： 本課題研究目的在于：(1)將視網(wǎng)膜色素變性患者的皮膚成纖維細(xì)胞,通過導(dǎo)入外源基因,誘導(dǎo)其成為多潛能干細(xì)胞(hiPSC);(2)在此過程中探索視網(wǎng)膜變性患者體細(xì)胞重編程的機(jī)制過程,并為進(jìn)一步向視網(wǎng)膜細(xì)胞的定向分化及機(jī)制研究奠定基礎(chǔ)。 材料和方法： 本研究取IFT140基因突變的視網(wǎng)膜色素變性的1名患者及1名正常對照受試者的皮膚組織樣本進(jìn)行原代培養(yǎng),建立人皮膚成纖維細(xì)胞系(human dermal fibroblast, HDF)。并取CF-1品系小鼠13.5天胚胎,分離并培養(yǎng)小鼠胚胎成纖維細(xì)胞(mouse embryonic fibroblast, MEF),培養(yǎng)至P3代后,經(jīng)絲裂霉素C處理后制成飼養(yǎng)層細(xì)胞(feeder cell)。 實驗中利用含Oct4、Klf4、Sox2、c-Myc四種重組基因的慢病毒質(zhì)粒以及慢病毒包裝質(zhì)粒PVSVG和△8.91,在人胚腎細(xì)胞(293HEK-T)中包裝出四種分別含有Oct4、Klf4、Sox2和c-Myc重組基因的慢病毒顆粒。 在P1代人皮膚成纖維細(xì)胞中按1：10的比例加入過量的慢病毒顆粒和助轉(zhuǎn)劑polybrene,誘導(dǎo)人成纖維細(xì)胞轉(zhuǎn)變成誘導(dǎo)多潛能干細(xì)胞。誘導(dǎo)第5天后消化細(xì)胞,轉(zhuǎn)至MEF制作成的飼養(yǎng)層細(xì)胞上繼續(xù)培養(yǎng),直至出現(xiàn)圓形類胚胎干細(xì)胞形態(tài)的誘導(dǎo)多潛能干細(xì)胞克隆。挑出克隆進(jìn)行擴(kuò)大培養(yǎng)和維持。對感染前和感染后的人皮膚成纖維細(xì)胞進(jìn)行相關(guān)基因的熒光實時定量PCR的檢測,鑒定干細(xì)胞相關(guān)基因和成纖維細(xì)胞特異基因的表達(dá)變化。 結(jié)果： (1)成功建立視網(wǎng)膜色素變性患者及正常受試者的人皮膚成纖維細(xì)胞系,形態(tài)為長梭形,呈編織狀、漩渦狀密集排列生長,大量表達(dá)FSP-1基因。凍存后復(fù)蘇率達(dá)50-75%不等。 (2)成功利用CF-1品系小鼠的MEF細(xì)胞,制作成飼養(yǎng)層細(xì)胞。凍存后復(fù)蘇率達(dá)75%。 (3)利用含Oct4、Klf4、Sox2和c-Myc四種重組基因的慢病毒質(zhì)粒以及慢病毒包裝質(zhì)粒PVSVG和△8.91,在人胚腎細(xì)胞(293HEK-T)中包裝出四種分別含有Oct4、Klf4、Sox2和c-Myc重組基因的慢病毒顆粒。滴度分別為1.4×107TU/ml;1.2×107TU/ml;2.0×107TU/ml和3.5×106TU/ml。 (4)慢病毒感染人皮膚成纖維細(xì)胞后,顯微鏡下觀察到細(xì)胞逐漸變圓,細(xì)胞核增大,在18-20天左右感染后細(xì)胞聚集成圓形、類似胚胎干細(xì)胞的克隆。 (5)對比感染前、后的人皮膚成纖維細(xì)胞的相關(guān)基因表達(dá)水平,感染后,FSP-1mRNA水平顯著下調(diào),endo-Oct4/Sox2/Klf4/c-Myc和Nanog基因的mRNA水平顯著上調(diào)(p0.05)。 結(jié)論： 本研究首次利用IFT140基因突變的視網(wǎng)膜色素變性患者的皮膚成纖維細(xì)胞,用慢病毒誘導(dǎo)的方式,導(dǎo)入外源基因Oct4、Klf4、Sox2和c-Myc,成功誘導(dǎo)體細(xì)胞出現(xiàn)類似人胚胎干細(xì)胞的形態(tài)變化,并表達(dá)干細(xì)胞特異的內(nèi)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R774.13

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