本文選題：小鼠Lewis肺癌干細胞 + 懸浮聚球　； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:探索從LLC-P中分離能夠在體外長期培養(yǎng)的CSCs的方法以及CSCs自我更新的調(diào)控機制。方法:在體外貼壁條件下培養(yǎng)LLC-P,通過連續(xù)富集懸浮生長的細胞,經(jīng)過8代后,成功富集出可懸浮成球的LLC-SE。為了驗證LLC-SE的干細胞特性,我們使用熒光定量PCR法檢測表示干細胞特性的基因Bmi1、Cd133、Aldh1a1、Oct4、Sox2、Nanog和Klf4的表達。6孔板瓊脂成球?qū)嶒灆z測其增殖和懸浮成球能力。96孔板單細胞克隆形成實驗檢測單個細胞的克隆形成能力。裸鼠皮下移植這一干細胞特性驗證的經(jīng)典模型檢測其干性和C57小鼠左肺原位種植模型驗證其體內(nèi)成瘤能力和轉(zhuǎn)移能力。我們通過基因的差異表達和基因干擾的方法探究可能調(diào)控LLC-SE干性和自我更新的機制。通過對LLC-SE進行連續(xù)5代單克隆實驗可進一步純化出以對稱分裂為主的LLC-SD和以非對稱分裂為主的LLC-ASD腫瘤干細胞樣細胞,并且通過BrdU標(biāo)記驗證LLC-SD和LLC-ASD確實存在對稱分裂和非對稱分裂。結(jié)果:RT-PCR結(jié)果顯示,LLC-SE中Bmi1的表達水平顯著高于LLC-P。6孔板瓊脂成球?qū)嶒灐?6孔板單細胞克隆形成實驗表明LLC-SE的懸浮成球能力和單細胞克隆能力顯著高于LLC-P。動物實驗表明LLC-SE的體內(nèi)致瘤能力、腫瘤轉(zhuǎn)移能力強于LLC-P。RT-PCR顯示Twist2在LLC-SE中的表達顯著高于LLC-P。隨后,我們對LLC-SE中的Twist2進行干擾,結(jié)果表明,Twist2被有效干擾后,LLC-SE瓊脂成球能力和單細胞克隆形成能力下降,裸鼠皮下成瘤能力也發(fā)生變化。結(jié)論:首次運用連續(xù)8次懸浮聚球法分離獲得大量的可穩(wěn)定傳代的具有干細胞特性的LLC-SE,為建立CSCs細胞模型提供了新的方法,為進一步研究CSCs的生物學(xué)特性奠定了基礎(chǔ)。通過連續(xù)5代單克隆方法,純化出具有與正常干細胞基本特性一致的,未/低分化的、可以進行對稱分裂和非對稱分裂的細胞模型,并初步探究了EMT轉(zhuǎn)錄調(diào)控因子Twist2對小鼠Lewis肺癌干細胞的干性的調(diào)控。
[Abstract]:Aim: to explore the method of isolation of CSCs from LLC-P and the regulatory mechanism of CSCs self-renewal.Methods: LLC-P was cultured in vitro and cultured in vitro. After 8 passages, LLC-SE was successfully enriched by continuous enrichment of suspension-growing cells.To verify the stem cell properties of LLC-SE,Fluorescence quantitative PCR assay was used to detect the expression of the gene Bmi1cCD133H, Oct4, Sox2, Nanog, and Klf4. The proliferation and suspension ability of single cell clone forming were detected by Agar. 96-well plate single cell clone forming assay was used to detect the clone forming ability of single cell.Subcutaneous transplantation of stem cells into nude mice was a classical model to test their dryness and in situ implantation model of left lung of C57 mice to test their tumorigenesis and metastasis ability in vivo.We explored the mechanisms of LLC-SE dryness and self-renewal through gene differential expression and gene interference.The LLC-SD with symmetrical division and LLC-ASD tumor-like cells with asymmetric division can be further purified by 5 consecutive generations of monoclonal experiments on LLC-SE.And the BrdU markers show that LLC-SD and LLC-ASD do exist symmetric splitting and asymmetric splitting.Results the expression level of Bmi1 in LLC-SE was significantly higher than that in LLC-P.6 plate Agar and 96 well plate single cell clone formation test. The results showed that the suspension and single cell clone ability of LLC-SE was significantly higher than that of LLC-P.Animal experiments showed that the ability of LLC-SE to cause tumor in vivo and the ability of tumor metastasis were higher than that of LLC-P.RT-PCR in LLC-SE and the expression of Twist2 in LLC-SE was significantly higher than that of LLC-P.Subsequently, we interfered with the Twist2 in LLC-SE. The results showed that the ability of LLC-SE agglomerating and single cell clone formation decreased after Twist2 was effectively interfered, and the ability of subcutaneous tumorigenesis of nude mice also changed.Conclusion: for the first time, a large number of stable passaged LLC-SEs with stem cell characteristics were obtained by the method of continuous suspension polymerization, which provided a new method for the establishment of CSCs cell model and laid a foundation for further study on the biological characteristics of CSCs.After 5 consecutive generations of monoclonal method, the undifferentiated and undifferentiated cell models with the same basic characteristics as normal stem cells were purified, which could be divided symmetrically and asymmetrically.The effects of EMT transcription regulator Twist2 on the dryness of mouse Lewis lung cancer stem cells were investigated.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R734.2

【參考文獻】

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1 王珊珊;殷勤偉;張洪杰;譚琰;華茜;;非貼壁微球體培養(yǎng)法分離T-淋巴細胞瘤干細胞及鑒定[J];中國細胞生物學(xué)學(xué)報;2014年08期

2 Anfei Liu;Xiya Yu;Shanrong Liu;;Pluripotency transcription factors and cancer stem cells:small genes make a big difference[J];Chinese Journal of Cancer;2013年09期

3 Qi Chen;Guolan Gao;Shiwen Luo;;Hedgehog signaling pathway and ovarian cancer[J];Chinese Journal of Cancer Research;2013年03期

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本文編號：1745795