本文選題：誘導(dǎo)性多能干細(xì)胞 + 神經(jīng)干細(xì)胞　； 參考：《南昌大學(xué)》2012年碩士論文

【摘要】：研究背景與目的： 誘導(dǎo)多能干細(xì)胞(induced pluripotent stem cells, iPSCs)是一類與胚胎干細(xì)胞（embryonic stem cells, ESC）具有相似全向分化潛能的干細(xì)胞，體外可分化為神經(jīng)干細(xì)胞（Neural Stem Cells，NSC）、神經(jīng)元等用于中樞神經(jīng)系統(tǒng)疑難疾病的細(xì)胞替代治療，臨床應(yīng)用前景廣闊，有望成為干細(xì)胞移植治療的新的種子細(xì)胞來源。然而，目前人們對iPSCs向NSC分化的調(diào)控機制知之甚少，要獲得數(shù)量豐富且來源穩(wěn)定的iPSCs源性的NSC用于臨床治療還存在相當(dāng)?shù)募夹g(shù)困難。因此，進一步了解iPSCs向NSC定向分化的具體調(diào)控機制，對于iPSCs將來的臨床推廣應(yīng)用意義重大。 由于iPSCs與ESC生物學(xué)特性極其相似,而ESC的自我更新和定向分化機制又一直是生命科學(xué)領(lǐng)域的研究熱點和重點,這就為我們深入探討誘導(dǎo)iPSCs向NSC分化的調(diào)控機制提供了必要的前提和基礎(chǔ)。研究表明ESC的自我更新和定向分化受Notch、Wnt等外部信號通路、轉(zhuǎn)錄因子、和表觀遺傳修飾等多種因素的調(diào)控。Notch通路是生物體極其重要的信號傳導(dǎo)通路之一，其在干細(xì)胞的增殖和分化等方面均起著重要的作用。Notch受體與配體結(jié)合激活時，干細(xì)胞就表現(xiàn)為增殖；當(dāng)Notch信號通路被抑制時，干細(xì)胞就分化為功能細(xì)胞。微小RNA（microRNA，miRNA）在生物體發(fā)生發(fā)育、干細(xì)胞增殖分化和腫瘤發(fā)生發(fā)展中同樣發(fā)揮著極其重要的調(diào)控作用，在人類，microRNA能夠調(diào)控至少30%的基因，一個microRNA可負(fù)調(diào)控數(shù)百個靶基因的蛋白表達(dá)，幾乎可參與調(diào)控所有的生物學(xué)過程。諸多文獻(xiàn)研究提示在ESC的NSC定向分化過程中，microRNA能夠調(diào)控可下調(diào)干細(xì)胞內(nèi)維持其未分化狀態(tài)的基因表達(dá)水平，同時激活干細(xì)胞譜系特異性基因，從而促進ESC定向分化。 然而目前尚未見iPSCs神經(jīng)定向分化相關(guān)調(diào)控機制的研究報道,為探討iPSCs向NSC定向分化的機制以及iPSCs來源的NSC對缺血性腦損傷疾病的修復(fù)效果，本課題擬分為體外實驗及體內(nèi)實驗兩部分：體外實驗首先將iPSCs定向分化為NSC，利用實時定量PCR、Western blot等方法檢測分化過程中各時間點Notch信號分子、miRNAs的表達(dá)變化，初步探討Notch信號分子、miRNAs與iPSCs向NSC定向分化過程的關(guān)系；體內(nèi)實驗擬通過將iPSCs來源的NSC立體定向移植至大鼠中動脈栓塞模型（middle cerebral arterial occlusion，MCAO）模型中，觀察其對大鼠缺血性腦損傷所致神經(jīng)功能缺陷的修復(fù)作用，為利用干細(xì)胞和再生醫(yī)學(xué)技術(shù)治療中樞神經(jīng)系統(tǒng)疑難疾病提供新的理論依據(jù)和技術(shù)準(zhǔn)備。 研究內(nèi)容和方法： 1體外實驗 1.1人iPSCs的培養(yǎng)及向NSC的誘導(dǎo)分化：采用本課題組已建立的培養(yǎng)及分化體系培養(yǎng)人iPSC細(xì)胞并誘導(dǎo)其向NSC分化，應(yīng)用免疫熒光染色對分化后的細(xì)胞進行NSC標(biāo)志物Nestin、Sox2表達(dá)的鑒定。 1.2采用實時熒光定量PCR檢測分化過程中mir-9，mir-34a、mir-200b的表達(dá)，與iPS組比較，觀察mir-9，mir-34a、mir-200b的動態(tài)表達(dá)變化。 1.3采用實時熒光定量PCR檢測分化過程中Notch1、Hes1的基因表達(dá)水平，與iPS組比較，觀察Notch1、Hes1基因的動態(tài)表達(dá)變化。 1.4采用免疫熒光染色和Western blot法檢測誘導(dǎo)過程中Notch1、Hes1的蛋白表達(dá)水平，與iPS組比較，觀察誘導(dǎo)過程中Notch1、Hes1蛋白的表達(dá)變化。 1.5利用Notch信號阻斷劑DAPT進一步驗證Notch信號通路在iPSCs向NSC定向分化過程中的作用。實驗分為RA對照組，DAPT誘導(dǎo)組，，RA+DAPT誘導(dǎo)組，RA+DMSO對照組；倒置顯微鏡下觀察各組細(xì)胞的形態(tài)變化，實時熒光定量PCR誘導(dǎo)第7天的各組細(xì)胞Nestin、β-tubulinШ、GFAP、Notch1和Hes1基因的表達(dá)情況，免疫熒光染色同時檢測Nestin、β-tubulinШ、和GFAP蛋白的表達(dá)。 2體內(nèi)實驗 2.1參考Longa法制作大腦中動脈栓塞（MCAO）模型，CM-DiI標(biāo)記iPS來源的NSC進行大鼠紋狀體立體定向移植，觀察移植的NSC在大鼠腦內(nèi)的存活、遷移狀況，并以免疫熒光染色法檢測神經(jīng)細(xì)胞相關(guān)標(biāo)志物Nestin，β-tublinIII，GFAP的變化，以觀察其分化情況。同時利用平衡木行走實驗、抓握實驗及水迷宮實驗對大鼠進行神經(jīng)功能測評。 結(jié)果： 1體外實驗 1.1成功將iPSCs誘導(dǎo)分化為NSC。 iPSCs經(jīng)RA結(jié)合無血清培養(yǎng)基誘導(dǎo)3天后，可觀察到細(xì)胞球形成神經(jīng)rosette結(jié)構(gòu)，免疫熒光染色顯示細(xì)胞球呈Nestin及Sox2陽性表達(dá)，貼壁培養(yǎng)1個月后，細(xì)胞形成神經(jīng)網(wǎng)絡(luò)狀結(jié)構(gòu)，免疫熒光鑒定提示細(xì)胞高表達(dá)NSC標(biāo)志物Nestin及Sox2。 1.2實時熒光定量PCR結(jié)果顯示，與iPSCs組相比較，mir-9、-34a、-200b的表達(dá)水平在iPSCs的NSC定向分化過程中顯著上調(diào)。 1.3實時熒光定量PCR結(jié)果顯示，在iPSCs形成EB的過程中，與iPSCs組相比較， Notch1和Hes1mRNA的表達(dá)明顯上調(diào)，隨著RA及無血清培養(yǎng)基誘導(dǎo)iPSCs分化的開始，Notch1和Hes1mRNA的表達(dá)下調(diào)。 1.4免疫熒光染色結(jié)果顯示，與自然分化組比較，誘導(dǎo)28d后RA無血清培養(yǎng)基誘導(dǎo)組的Notch1以及Hes1蛋白表達(dá)的陽性細(xì)胞率顯著下降，Western blot結(jié)果也顯示，在iPSCs向NSC的分化過程中, Notch1、Hes1蛋白的表達(dá)隨著RA結(jié)合無血清培養(yǎng)基誘導(dǎo)分化，Notch1、Hes1蛋白的表達(dá)逐漸降低，誘導(dǎo)分化后貼壁培養(yǎng)14d時最低，而后表達(dá)水平逐漸增高。 1.5DAPT加入后，大部分細(xì)胞球容易貼壁，細(xì)胞球周圍可見爬出細(xì)長的神經(jīng)絲樣觸角；第3d左右，貼壁的細(xì)胞球內(nèi)可見大量神經(jīng)rosette結(jié)構(gòu)，培養(yǎng)2W后，細(xì)胞球分化形成神經(jīng)網(wǎng)絡(luò)狀結(jié)構(gòu)。結(jié)合實時熒光定量PCR和免疫熒光鑒定各誘導(dǎo)組神經(jīng)標(biāo)志物Nestin、β-tubullinIII及GFAP的表達(dá)結(jié)果表明， DAPT能夠促進iPSC向NSC的定向分化；與RA對照組及RA+DMSO對照組比較，加入DAPT后，iPSC向NSC的分化速度加快，同時神經(jīng)元細(xì)胞及少突膠質(zhì)細(xì)胞所占分化后細(xì)胞的比例也增加，說明加入DAPT后，Notch信號被抑制，使得iPSC能夠快速的向NSC定向分化，而由于DAPT對Notch信號的抑制作用是持續(xù)的，使得部分NSC繼續(xù)分化為神經(jīng)元及少突膠質(zhì)細(xì)胞。 2體內(nèi)實驗 2.1移植后iPSCs來源的NSC在大鼠腦組織內(nèi)能夠長期存活，移植1周和2周后免疫熒光染色提示移植區(qū)和腦缺血區(qū)分別可見移植細(xì)胞，且細(xì)胞分別表達(dá)神經(jīng)細(xì)胞標(biāo)志物β-tubulinШ、GFAP，表明移植后的細(xì)胞在腦組織內(nèi)能夠向缺血區(qū)遷移并進一步分化為神經(jīng)細(xì)胞；分別利用抓握實驗、平衡木行走實驗及水迷宮實驗在模型制備后0，1，2，3W對各組SD大鼠進行評分，與正常組比較，大鼠腦缺血損傷后各項神經(jīng)功能檢測指標(biāo)均明顯降低；與對照組比較，NSC細(xì)胞移植組在移植2周后大鼠的抓握力、平衡行走能力和記憶功能均有一定程度的恢復(fù)。 結(jié)論： 1. RA結(jié)合無血清培養(yǎng)基誘導(dǎo)法能夠有效將人iPSCs定向分化為NSC； 2. Notch信號通路參與了人iPSCs向NSC定向分化過程的調(diào)控； 3. mir-9、-34a及mir-200b有可能通過調(diào)控Notch信號參與了人iPSCs向NSC定向分化的調(diào)控； 4. iPSCs來源的NSC定向移植至MCAO大鼠腦組織后能夠長期存活，定向遷移至腦缺血區(qū)并分化為神經(jīng)細(xì)胞，一定程度促進大鼠腦缺血損傷后的神經(jīng)功能恢復(fù)。
[Abstract]:Research background and purpose:
Induced pluripotent stem cells (iPSCs) is a kind of stem cells with similar omnidirectional differentiation potential with embryonic stem cells (embryonic stem cells, ESC). In vitro it can be differentiated into neural stem cells (Neural Stem Cells), neurons and other cell replacement therapy for the central nervous system, before clinical application. It is promising to be a new source of seed cells for stem cell transplantation. However, little is known about the regulatory mechanism of iPSCs to NSC differentiation. There are considerable technical difficulties in obtaining a rich and stable source of iPSCs derived NSC for clinical treatment. Therefore, further understanding of the specific differentiation of iPSCs to NSC is specific. The regulation mechanism is of great significance for the clinical popularization and application of iPSCs in the future.
As the biological characteristics of iPSCs and ESC are very similar, the mechanism of self renewal and orientation differentiation of ESC has been the focus and focus in the field of life science. This provides the necessary premise and basis for us to explore the regulation mechanism of inducing iPSCs to NSC differentiation. The self-renewal and directional differentiation of ESC is determined by Notch, Wnt. The regulatory.Notch pathway, such as external signaling pathways, transcription factors, and epigenetic modification, is one of the most important signaling pathways in organisms. It plays an important role in the proliferation and differentiation of stem cells. When the.Notch receptor is activated by the ligand binding to the ligand, the stem cells are proliferated, and when the Notch signaling pathway is used. When suppressed, stem cells differentiate into functional cells. Small RNA (microRNA, miRNA) also plays an extremely important regulatory role in the development of organisms, proliferation and differentiation of stem cells and the development of tumor. In humans, microRNA can regulate at least 30% of the gene, and a microRNA can negatively regulate the expression of hundreds of target genes. It is possible to participate in the regulation of all biological processes. Many literature studies suggest that microRNA can regulate the level of gene expression that can maintain its undifferentiated state in stem cells during the NSC directional differentiation of ESC, and activates the specific genes of stem cell lineage, thus promoting the ESC differentiation.
However, there is no research report on the regulation mechanism of iPSCs neurodirectional differentiation. In order to explore the mechanism of iPSCs directed differentiation to NSC and the effect of iPSCs source NSC on the repair of ischemic brain damage, this subject is divided into two parts: in vitro experiment and in vivo experiment: in vitro, iPSCs is first differentiated into NSC, and the real time is used in real time. Quantitative PCR, Western blot and other methods were used to detect the expression of Notch signal molecules and miRNAs in the process of differentiation. The relationship between Notch signal molecules, miRNAs and iPSCs to NSC directional differentiation was preliminarily investigated. In vivo experiments were designed to transplant NSC stereotaxis of iPSCs sources to the rat middle artery embolism model. In the occlusion, MCAO) model, the repair of neural functional defects caused by ischemic brain injury in rats was observed and a new theoretical basis and technical preparation were provided for the use of stem cells and regenerative medicine to treat the difficult diseases of the central nervous system.
Research contents and methods:
1 in vitro experiment
The culture of 1.1 iPSCs and induction of differentiation into NSC: the culture and differentiation system established by this group were used to cultivate human iPSC cells and induce it to differentiate into NSC. Immunofluorescence staining was used to identify the NSC marker Nestin and Sox2 expression of the differentiated cells.
1.2 real-time fluorescence quantitative PCR was used to detect the expression of miR-9, miR-34a and mir-200b in the differentiation process. Compared with iPS group, the dynamic expression of miR-9, miR-34a and mir-200b was observed.
1.3 real-time fluorescence quantitative PCR was used to detect the expression level of Notch1 and Hes1 in differentiation. Compared with group iPS, the dynamic expression of Notch1 and Hes1 genes was observed.
1.4 the protein expression level of Notch1 and Hes1 during induction was detected by immunofluorescence staining and Western blot, and the expression of Notch1 and Hes1 protein in the induction process was observed.
1.5 Notch signal blocking agent DAPT was used to further verify the role of Notch signaling pathway in the directional differentiation of iPSCs to NSC. The experiments were divided into RA control group, DAPT induction group, RA+DAPT induction group and RA+DMSO control group, and the morphological changes of each cell were observed under the inverted microscope, and the real-time fluorescence quantitative PCR induced seventh days of Nestin, beta -tubu. The expressions of Lin, GFAP, Notch1 and Hes1 genes were detected, and the expression of Nestin, beta -tubulin and GFAP proteins were detected by immunofluorescence staining.
2 in vivo experiment
2.1 Longa method was used to make the middle cerebral artery embolism (MCAO) model, and the CM-DiI labeled iPS derived NSC for the stereotactic transplantation of the rat striatum. The survival and migration of the transplanted NSC in the rat brain were observed and the changes of the neurons related markers Nestin, beta -tublinIII, and GFAP were detected by immunofluorescence staining, in order to observe the differentiation. At the same time, we used the balance beam walking test, grasping experiment and water maze test to evaluate the neurological function of rats.
Result:
1 in vitro experiment
1.1 the iPSCs induced differentiation into NSC. iPSCs was induced by RA and serum-free medium for 3 days. The rosette structure of the cell ball was observed. The immunofluorescence staining showed that the cells showed positive expression of Nestin and Sox2. After 1 months of adherent culture, the cells formed a neural network structure, and the immunofluorescence identification suggested that the cell expressed the NSC marker. Nestin and Sox2.
1.2 the results of real-time quantitative PCR showed that the expression levels of miR-9, -34a and -200b in iPSCs were significantly higher than those in iPSCs group.
1.3 real time fluorescence quantitative PCR results showed that in the process of iPSCs formation, the expression of Notch1 and Hes1mRNA was obviously up-regulated compared with the iPSCs group. The expression of Notch1 and Hes1mRNA decreased with the initiation of iPSCs differentiation induced by RA and serum-free medium.
1.4 the results of immunofluorescence staining showed that, compared with the natural differentiation group, the Notch1 and the positive cell rate of Hes1 protein expression in the RA serum-free medium induction group decreased significantly after the induction of 28d, and the Western blot results also showed that the expression of Notch1, Hes1 protein was induced by RA combined with serum-free medium during the differentiation of iPSCs to NSC. The expression of H1 and Hes1 protein decreased gradually, and the lowest expression level was observed after adherent culture, and the expression level of 14d increased gradually.
After the addition of 1.5DAPT, most of the cell spheres were easily adhered to the wall, and the long nerve filament like tentacles were found around the cell spheres. A large number of nerve rosette structures were visible in the cell spheres on the wall of the cell. After the culture of 2W, the cell spheres were differentiated into neural network structure. The neural markers of the induced groups were identified by real-time fluorescent quantitative PCR and immunofluorescence. The expression of Nestin, beta -tubullinIII and GFAP showed that DAPT could promote the directional differentiation of iPSC to NSC. Compared with the RA control group and RA+DMSO control group, the differentiation rate of iPSC to NSC increased after the addition of DAPT, while the proportion of neuron cells and oligodendrocytes also increased. Inhibited, iPSC can rapidly differentiate into NSC, and the inhibition of DAPT to Notch signals is continuous, making some NSC continue to differentiate into neurons and oligodendrocytes.
2 in vivo experiment
After 2.1 transplantation, the NSC derived from iPSCs could survive in the rat brain for a long time. After 1 and 2 weeks, immunofluorescence staining showed the transplanted cells in the transplanted area and the cerebral ischemia area, respectively, and the cells expressed the nerve cell marker, beta -tubulin, respectively, GFAP, indicating that the transplanted cells could migrate into the ischemic region in the brain tissue and further further migrate to the ischemic region. The rats were divided into nerve cells, and the SD rats were scored by 0,1,2,3W after the model was prepared by grasping the grip experiment, the balance Wood Walking experiment and the water maze test. Compared with the normal group, the nerve function indexes of the rats were obviously decreased after the cerebral ischemia injury. Compared with the control group, the NSC cell transplantation group was transplanted 2 weeks after the transplantation. Grip strength, balance walking ability and memory function were restored to some extent.
Conclusion:
1. RA combined with serum-free medium induction method can effectively differentiate human iPSCs into NSC.
2. the Notch signaling pathway is involved in the regulation of the directional differentiation of human iPSCs into NSC.
3., miR-9, -34a and mir-200b may participate in the regulation of NSC induced differentiation by regulating Notch signaling.
4. iPSCs derived NSC can be transplanted to the brain tissue of MCAO rats for a long time and migrate to the cerebral ischemia area and differentiate into nerve cells, to a certain extent, to promote the recovery of nerve function after cerebral ischemia injury in rats.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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相關(guān)期刊論文 前1條

1 馮年花;謝安;婁遠(yuǎn)蕾;阮瓊芳;郭菲;楊陽;潘長福;鄧志鋒;汪泱;;人誘導(dǎo)性多能干細(xì)胞向神經(jīng)干細(xì)胞分化的方法探討[J];中國病理生理雜志;2010年08期

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