Twist2调控小鼠Lewis肺癌干细胞的干性

发布时间：2018-04-13 19:03

本文选题：小鼠Lewis肺癌干细胞 + 悬浮聚球　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:探索从LLC-P中分离能够在体外长期培养的CSCs的方法以及CSCs自我更新的调控机制。方法:在体外贴壁条件下培养LLC-P,通过连续富集悬浮生长的细胞,经过8代后,成功富集出可悬浮成球的LLC-SE。为了验证LLC-SE的干细胞特性,我们使用荧光定量PCR法检测表示干细胞特性的基因Bmi1、Cd133、Aldh1a1、Oct4、Sox2、Nanog和Klf4的表达。6孔板琼脂成球实验检测其增殖和悬浮成球能力。96孔板单细胞克隆形成实验检测单个细胞的克隆形成能力。裸鼠皮下移植这一干细胞特性验证的经典模型检测其干性和C57小鼠左肺原位种植模型验证其体内成瘤能力和转移能力。我们通过基因的差异表达和基因干扰的方法探究可能调控LLC-SE干性和自我更新的机制。通过对LLC-SE进行连续5代单克隆实验可进一步纯化出以对称分裂为主的LLC-SD和以非对称分裂为主的LLC-ASD肿瘤干细胞样细胞,并且通过BrdU标记验证LLC-SD和LLC-ASD确实存在对称分裂和非对称分裂。结果:RT-PCR结果显示,LLC-SE中Bmi1的表达水平显著高于LLC-P。6孔板琼脂成球实验、96孔板单细胞克隆形成实验表明LLC-SE的悬浮成球能力和单细胞克隆能力显著高于LLC-P。动物实验表明LLC-SE的体内致瘤能力、肿瘤转移能力强于LLC-P。RT-PCR显示Twist2在LLC-SE中的表达显著高于LLC-P。随后,我们对LLC-SE中的Twist2进行干扰,结果表明,Twist2被有效干扰后,LLC-SE琼脂成球能力和单细胞克隆形成能力下降,裸鼠皮下成瘤能力也发生变化。结论:首次运用连续8次悬浮聚球法分离获得大量的可稳定传代的具有干细胞特性的LLC-SE,为建立CSCs细胞模型提供了新的方法,为进一步研究CSCs的生物学特性奠定了基础。通过连续5代单克隆方法,纯化出具有与正常干细胞基本特性一致的,未/低分化的、可以进行对称分裂和非对称分裂的细胞模型,并初步探究了EMT转录调控因子Twist2对小鼠Lewis肺癌干细胞的干性的调控。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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