【摘要】：骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)是一類存在于骨髓中的非造血干細(xì)胞,具有自我更新、增殖及多向分化的潛能,在體外可誘導(dǎo)分化為骨、軟骨、肌肉、脂肪、神經(jīng)細(xì)胞和心肌細(xì)胞等。腫瘤干細(xì)胞(cancer stem cell, CSC)是指存在于腫瘤組織中的一小部分具有干細(xì)胞性質(zhì)的細(xì)胞群體,具有無限增殖和自我更新的能力,是腫瘤發(fā)生的根源。目前已有研究者在白血病、乳腺癌及腦瘤中發(fā)現(xiàn)CSC,但它的起源仍存在諸多爭議沒有定論。隨著對CSC研究的深入,BMSCs與CSC起源的關(guān)系成為了研究的關(guān)鍵點。 本文旨在從細(xì)胞增殖分化的角度研究CSC與干細(xì)胞的相關(guān)性。實驗選用大鼠BMSCs為研究對象,建立大鼠BMSCs的神經(jīng)細(xì)胞分化模型,以此為基礎(chǔ)觀察不同時間點阻斷正常分化過程后細(xì)胞發(fā)生的變化,探討干細(xì)胞與腫瘤發(fā)生的相關(guān)性。具體方法如下： 1、采用全骨髓培養(yǎng)法分離大鼠BMSCs,實現(xiàn)體外培養(yǎng)和擴(kuò)增； 2、建立誘導(dǎo)分化模型：用β-巰基乙醇誘導(dǎo)BMSCs向神經(jīng)細(xì)胞分化,通過免疫組化檢測神經(jīng)元特異性烯醇化酶(NSE),神經(jīng)絲蛋白(NF),膠質(zhì)纖維酸性蛋白(GFAP)的表達(dá),鑒定誘導(dǎo)后的細(xì)胞； 3、建立分化阻斷模型,探討干細(xì)胞與腫瘤發(fā)生的相關(guān)性：BMSCs分為分化組和阻斷組,分化組細(xì)胞正常誘導(dǎo)與此同時阻斷組選擇不同的時間點阻斷正常的誘導(dǎo)過程,利用形態(tài)學(xué)觀察,免疫組化,核型分析等手段檢測阻斷誘導(dǎo)后的細(xì)胞。通過對正常BMSCs、正常分化的神經(jīng)細(xì)胞及阻斷組中各時間點細(xì)胞的比對研究,探討干細(xì)胞分化阻斷與腫瘤發(fā)生的相關(guān)性。 研究結(jié)果如下： 1、成功分離了大鼠BMSCs,并實現(xiàn)體外擴(kuò)增培養(yǎng)。傳代培養(yǎng)的BMSCs生長狀態(tài)良好,克隆形成能力強(qiáng)。 2、成功誘導(dǎo)大鼠BMSCs分化為神經(jīng)細(xì)胞。誘導(dǎo)后的細(xì)胞顯示典型的神經(jīng)細(xì)胞形態(tài)特征,經(jīng)免疫組化檢測后NSE、NF陽性表達(dá),GFAP陰性表達(dá)。 3、進(jìn)行了大鼠BMSCs分化阻斷與腫瘤相關(guān)性的探索研究。分化受阻的BMSCs發(fā)生了惡性轉(zhuǎn)變,阻斷分化的時間點不同,細(xì)胞的神經(jīng)細(xì)胞特定蛋白表達(dá)量及惡性程度均不同。受阻的干細(xì)胞分化程度越低,相應(yīng)產(chǎn)生的細(xì)胞分化程度越低、增殖速度越快,惡性程度越高,反之亦然。 綜上所述,干細(xì)胞分化過程受到阻斷,細(xì)胞停止在分化的中間階段,大量增殖可能發(fā)生了干細(xì)胞向CSC的轉(zhuǎn)化。通過對正常干細(xì)胞分化阻斷的研究,本實驗為CSC起源于正常干細(xì)胞提供了新的參考實驗數(shù)據(jù)。
[Abstract]:Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) are non-hematopoietic stem cells that exist in bone marrow. They have the potential of self-renewal, proliferation and multidirectional differentiation. They can be induced to differentiate into bone, cartilage, muscle and fat in vitro. Nerve cells and cardiomyocytes, etc. Tumor stem cell (cancer stem cell, CSC) is a small group of stem cells that exist in tumor tissue. It has the ability of unlimited proliferation and self-renewal. It is the origin of tumorigenesis. At present, researchers have found CSC, in leukemia, breast cancer and brain tumors, but there are still many controversies about its origin. With the deepening of the study of CSC, the relationship between BMSCs and the origin of CSC has become the key point of the study. The aim of this study is to study the relationship between CSC and stem cells from the perspective of cell proliferation and differentiation. The neural cell differentiation model of rat BMSCs was established by using rat BMSCs as the object of study. Based on this model, the changes of cell formation after blocking the normal differentiation process at different time points were observed, and the correlation between stem cells and tumorigenesis was discussed. The specific methods were as follows: (1) isolation of rat BMSCs, by whole bone marrow culture to achieve in vitro culture and amplification; 2. To establish differentiation model: BMSCs was induced to differentiate into nerve cells by 尾-mercaptoethanol. The expression of neuron-specific enolase (NSE), neurofilament protein (NF), glial fibrillary acidic protein (GFAP) was detected by immunohistochemistry. The induced cells were identified. (3) to establish a differentiation-blocking model to explore the correlation between stem cells and tumorigenesis: BMSCs was divided into two groups: differentiation group and blocking group. At the same time, the differentiation group selected different time points to block the normal induction process, and the differentiation group was divided into differentiation group and blocking group. Morphological observation, immunohistochemistry and karyotype analysis were used to detect the cells induced by blocking. The relationship between stem cell differentiation blocking and tumorigenesis was studied by comparing the normal BMSCs, differentiated nerve cells and the cells in the blocking group at different time points in order to explore the relationship between stem cell differentiation blocking and tumorigenesis. The results were as follows: 1. Rat BMSCs, was successfully isolated and cultured in vitro. The BMSCs cultured in subculture had good growth condition and strong clone forming ability. 2. The rat BMSCs was successfully induced to differentiate into nerve cells. The induced cells showed typical morphological features of nerve cells, and the expression of NSE,NF was positive and GFAP negative was detected by immunohistochemistry. 3. The correlation between BMSCs differentiation blocking and tumor in rats was studied. The differentiation-blocked BMSCs had a malignant transformation. The expression of neuron-specific protein and the degree of malignancy of the cells were different at different time points of blocking differentiation. The lower the differentiation degree of the blocked stem cells, the lower the differentiation degree of the corresponding cells, the faster the proliferation rate, the higher the malignant degree, and vice versa. In conclusion, stem cell differentiation process is blocked, cells stop in the intermediate stage of differentiation, a large number of proliferation may occur stem cells to CSC transformation. Through the study of normal stem cell differentiation blocking, this experiment provides new experimental data for the origin of CSC from normal stem cells.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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本文編號：2451589