發(fā)布時間：2018-03-13 22:29

  本文選題：后發(fā)障　切入點：Pax6基因　出處：《天津醫(yī)科大學》2012年博士論文　論文類型：學位論文

【摘要】：目的:1.建立大鼠后囊膜渾濁模型并測定Pax6轉(zhuǎn)錄本的表達變化。2.在HLE-B3細胞系通過敲減及回復表達Pax6轉(zhuǎn)錄本,觀察其對B3細胞的影響。3.利用基因芯片技術(shù)進行全基因表達譜檢測觀察隨著Pax6轉(zhuǎn)錄本表達變化對下游基因的影響。方法:1.32只大鼠隨機分組右眼行ECLE,分別取手術(shù)后不同時間點的大鼠后囊膜組織,通過Real-time PCR測定后囊膜上Pax6基因轉(zhuǎn)錄本的表達水平。2.利用半定量PCR測定了人HLEB3細胞中Pax6的水平,構(gòu)建靶向Pax6的shRNA慢病毒載體,應(yīng)用shRNA干擾慢病毒感染B3細胞,觀察Pax6基因敲減后對B3細胞活力增殖的影響。3.構(gòu)建分別表達Pax6基因兩個轉(zhuǎn)錄本的慢病毒載體,在Pax6敲減后的細胞中進行回復表達,研究兩個轉(zhuǎn)錄本對B3細胞活力增殖的影響。4.利用上述shRNA干擾后及分別回復轉(zhuǎn)錄本1或轉(zhuǎn)錄本2,分別抽提RNA,再用Agilent 4X44K人類全基因表達譜芯片進行檢測,分析Pax6基因變化對下游基因的影響及2個轉(zhuǎn)錄本的功能差異。結(jié)果:1.成功復制了大鼠后發(fā)障模型,構(gòu)建了大鼠Pax6基因的質(zhì)粒并進行測序,獲得大鼠Pax6兩個轉(zhuǎn)錄本的序列。檢測Pax6兩個轉(zhuǎn)錄本的表達,在手術(shù)后7天,Pax6兩種轉(zhuǎn)錄本的表達均有所增加,在術(shù)后14天時又有一定程度的回復,至28天時降至最低。兩種轉(zhuǎn)錄本的表達趨勢比較相似。2.成功構(gòu)建了 Pax6 shRNA重組慢病毒載體,抑制Pax6基因表達后,B3細胞凋亡的數(shù)目成倍增加。3.敲減Pax6基因的表達水平后,單獨回復表達其中一個Pax6轉(zhuǎn)錄本,不但不能逆轉(zhuǎn)敲減Pax6造成的細胞活力下降、增殖減緩,反而加重了這種變化。特別是回復表達轉(zhuǎn)錄本2,導致了更明顯的細胞活力下降和增殖減緩,并且凋亡細胞數(shù)量也顯著的增加。4.基因芯片的結(jié)果顯示Pax6基因被敲減后,有224個基因表達被上調(diào),144個基因表達被下調(diào);RNAi的同時回復轉(zhuǎn)錄本1的表達,引起758個基因表達上調(diào)和211個基因表達下調(diào);RNAi的同時回復轉(zhuǎn)錄本2的表達,引起2033個基因表達上調(diào)和617個基因表達下調(diào)�；貜捅磉_所引起的基因表達的變化要更加明顯,尤其是2號轉(zhuǎn)錄本。結(jié)論:1.大鼠后發(fā)障模型Pax6基因的表達存在變化,而且在術(shù)后即刻、7d、14d、28d表達量有規(guī)律性改變,兩種轉(zhuǎn)錄本的表達趨勢比較一致。2.Pax6基因表達下調(diào)、在Pax6基因下調(diào)后單獨上調(diào)轉(zhuǎn)錄本1或轉(zhuǎn)錄本2的表達,都表現(xiàn)出一致的抑制細胞增殖,凋亡增加,但程度上是有差異的。3.基因芯片結(jié)果顯示:Pax6兩個轉(zhuǎn)錄本的回復表達,比Pax6的基因敲減,引起了更多與細胞周期和凋亡相關(guān)的基因表達的變化,包括WNT、p53信號通路的一些基因和Caspase相關(guān)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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R776.1

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