Pax6基因转录本对HLE-B3细胞生物学功能调控的实验研究

发布时间：2018-03-13 22:29

  本文选题：后发障　切入点：Pax6基因　出处：《天津医科大学》2012年博士论文　论文类型：学位论文

【摘要】：目的:1.建立大鼠后囊膜浑浊模型并测定Pax6转录本的表达变化。2.在HLE-B3细胞系通过敲减及回复表达Pax6转录本,观察其对B3细胞的影响。3.利用基因芯片技术进行全基因表达谱检测观察随着Pax6转录本表达变化对下游基因的影响。方法:1.32只大鼠随机分组右眼行ECLE,分别取手术后不同时间点的大鼠后囊膜组织,通过Real-time PCR测定后囊膜上Pax6基因转录本的表达水平。2.利用半定量PCR测定了人HLEB3细胞中Pax6的水平,构建靶向Pax6的shRNA慢病毒载体,应用shRNA干扰慢病毒感染B3细胞,观察Pax6基因敲减后对B3细胞活力增殖的影响。3.构建分别表达Pax6基因两个转录本的慢病毒载体,在Pax6敲减后的细胞中进行回复表达,研究两个转录本对B3细胞活力增殖的影响。4.利用上述shRNA干扰后及分别回复转录本1或转录本2,分别抽提RNA,再用Agilent 4X44K人类全基因表达谱芯片进行检测,分析Pax6基因变化对下游基因的影响及2个转录本的功能差异。结果:1.成功复制了大鼠后发障模型,构建了大鼠Pax6基因的质粒并进行测序,获得大鼠Pax6两个转录本的序列。检测Pax6两个转录本的表达,在手术后7天,Pax6两种转录本的表达均有所增加,在术后14天时又有一定程度的回复,至28天时降至最低。两种转录本的表达趋势比较相似。2.成功构建了 Pax6 shRNA重组慢病毒载体,抑制Pax6基因表达后,B3细胞凋亡的数目成倍增加。3.敲减Pax6基因的表达水平后,单独回复表达其中一个Pax6转录本,不但不能逆转敲减Pax6造成的细胞活力下降、增殖减缓,反而加重了这种变化。特别是回复表达转录本2,导致了更明显的细胞活力下降和增殖减缓,并且凋亡细胞数量也显著的增加。4.基因芯片的结果显示Pax6基因被敲减后,有224个基因表达被上调,144个基因表达被下调;RNAi的同时回复转录本1的表达,引起758个基因表达上调和211个基因表达下调;RNAi的同时回复转录本2的表达,引起2033个基因表达上调和617个基因表达下调。回复表达所引起的基因表达的变化要更加明显,尤其是2号转录本。结论:1.大鼠后发障模型Pax6基因的表达存在变化,而且在术后即刻、7d、14d、28d表达量有规律性改变,两种转录本的表达趋势比较一致。2.Pax6基因表达下调、在Pax6基因下调后单独上调转录本1或转录本2的表达,都表现出一致的抑制细胞增殖,凋亡增加,但程度上是有差异的。3.基因芯片结果显示:Pax6两个转录本的回复表达,比Pax6的基因敲减,引起了更多与细胞周期和凋亡相关的基因表达的变化,包括WNT、p53信号通路的一些基因和Caspase相关的基因。
[Abstract]:Objective 1. To establish a rat model of posterior capsule opacification and to determine the expression of Pax6 transcripts. 2. To express Pax6 transcripts by knockdown and response in HLE-B3 cell lines. To observe its effect on B3 cells. 3. To observe the effect of Pax6 transcripts on downstream genes with the change of Pax6 transcripts. Methods one hundred and thirty-two rats were randomly divided into right eyes and treated with ECLE.After the operation, the whole gene expression profile was detected. The posterior capsule tissue of rats at different time points, The expression level of Pax6 gene transcripts on the posterior capsule was determined by Real-time PCR. 2. The Pax6 level in human HLEB3 cells was determined by semi-quantitative PCR. ShRNA lentivirus vector targeting Pax6 was constructed, and Lentivirus was infected by shRNA interfering lentivirus in B3 cells. To observe the effect of Pax6 knockout on the proliferation of B3 cells. 3. To construct the lentivirus vector expressing two transcripts of Pax6 gene, and to express them in the cells after Pax6 knockout. To study the effect of two transcripts on the proliferation of B3 cells. (4) by using the shRNA interference and reverting transcripts 1 or 2 respectively, RNAs were extracted separately, and then detected by Agilent 4X44K whole gene expression microarray. The effects of Pax6 gene changes on downstream genes and the functional differences of two transcripts were analyzed. Results: 1. The rat model of posterior barrier was successfully established, and the plasmid of rat Pax6 gene was constructed and sequenced. We obtained the sequence of two transcripts of rat Pax6. The expression of two transcripts of Pax6 increased 7 days after operation and returned to a certain extent at 14 days after operation. The expression trend of the two transcripts was similar. 2. The recombinant lentivirus vector of Pax6 shRNA was successfully constructed, and the number of apoptosis of B3 cells increased exponentially after inhibiting the expression of Pax6 gene. After knocking down the expression level of Pax6 gene, the expression level of Pax6 gene was reduced. The expression of one of the Pax6 transcripts alone could not reverse the decrease in cell viability caused by Pax6 knockout, and the proliferation of cells was slowed down. In particular, the response to transcription 2 resulted in a more significant decrease in cell viability and slower proliferation, and a significant increase in the number of apoptotic cells. The results of the microarray showed that the Pax6 gene was knocked out. The expression of 224 genes was up-regulated, 144 genes were down-regulated and the expression of transcription 1 was restored simultaneously, which resulted in 758 gene expression upregulation and 211 gene expression down-regulation. 2 033 genes were up-regulated and 617 genes were down-regulated. The changes of gene expression induced by reverse-expression were more obvious, especially in transcription number 2. Conclusion: 1. The expression of Pax6 gene in rat model of posterior impairment is changed. The expression of Pax6 gene was down-regulated at 14d ~ 28d after Pax6 down-regulation. After down-regulation of Pax6 gene, the expression of transcription 1 or transcription 2 was upregulated. Both showed consistent inhibition of cell proliferation and increased apoptosis, but the degree of apoptosis was different. The results of gene chip showed that the two transcripts of the two transcripts of Pax6 were less expressed than those of Pax6 gene knockout. It has caused more changes in cell cycle and apoptosis related gene expression, including some genes in the p53 signaling pathway and Caspase related genes.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R776.1

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