本文選題：Fli-1　切入點(diǎn)：RNA干擾　出處：《吉林大學(xué)》2012年碩士論文

【摘要】：目的：Fli-1（Friend leukemia integration-1）是屬于Ets(E26transformation-specific)家族的轉(zhuǎn)錄因子, Fli-1的異常表達(dá)是某些類型細(xì)胞惡性轉(zhuǎn)化起始階段的重要因素。研究發(fā)現(xiàn)Fli-1不僅在血細(xì)胞和血管生成中起著至關(guān)重要的作用[2]，而且還具有促進(jìn)腫瘤發(fā)生發(fā)展的作用[3]。基于此，本實(shí)驗(yàn)利用RNA干涉（RNA interference,RNAi）技術(shù)，以小鼠Fli-1基因?yàn)榘谢颍O(shè)計(jì)Fli-1基因特異性的小干擾RNA（small interferenceRNA,siRNA）,構(gòu)建制備目的基因Fli-1的siRNA慢病毒載體，以最適合的感染復(fù)數(shù)(optimal multiplicity of infection,MOI)感染CB3細(xì)胞，通過檢測各實(shí)驗(yàn)組CB3細(xì)胞中Fli-1mRNA及蛋白質(zhì)的表達(dá)，驗(yàn)證其對CB3細(xì)胞中Fli-1基因的沉默效果，并觀察沉默F(xiàn)li-1基因后對CB3細(xì)胞生物學(xué)特性的影響，為今后探討其作用機(jī)制奠定了基礎(chǔ)。 方法： 1．RNAi慢病毒載體的制備：針對小鼠Fli-1基因mRNA序列設(shè)計(jì)三條針對目的基因Fli-1的干擾序列，并設(shè)計(jì)陰性對照序列。合成干擾序列的單鏈DNA寡核苷酸，然后退火配對產(chǎn)生雙鏈DNA寡核苷酸，再通過其兩端所含酶切位點(diǎn)直接連入Age I、EcoR I酶切后的RNA干擾慢病毒載體上,將連接產(chǎn)物轉(zhuǎn)入制備好的細(xì)菌感受態(tài)細(xì)胞DH5α，對長出的單克隆菌落進(jìn)行PCR鑒定,PCR鑒定陽性菌落進(jìn)行測序鑒定。 2．RNAi慢病毒的包裝與滴度檢測：按Lipofectamine2000使用說明，將慢病毒載體與輔助載體共感染293T細(xì)胞，，收集富含慢病毒顆粒的細(xì)胞上清液，離心后收集得到高滴度的慢病毒濃縮液。在293T細(xì)胞中，利用逐孔稀釋法測定病毒的滴度。 3．慢病毒感染CB3細(xì)胞：離心收集生長狀態(tài)良好的CB3細(xì)胞進(jìn)行病毒感染，為摸索CB3細(xì)胞最佳感染復(fù)數(shù)（multiplicity of infection,MOI）,根據(jù)MOI值將實(shí)驗(yàn)分為100、10和1三組，感染3天后，通過熒光顯微鏡觀察綠色熒光表達(dá)情況，篩選感染效率最高組。 4．慢病毒感染CB3細(xì)胞后基因沉默效果的鑒定：慢病毒感染CB3細(xì)胞96h后，分別用RT-PCR及Western blot方法測定Fli-1mRNA及蛋白質(zhì)表達(dá)量。 5．MTT法檢測各實(shí)驗(yàn)組CB3細(xì)胞生長。流式細(xì)胞儀檢測慢病毒感染72h后的CB3細(xì)胞的凋亡情況。 結(jié)果： 1．成功構(gòu)建攜帶有Fli-1干涉序列的慢病毒載體（分別命名為Fli-1-RNAi-LV1、Fli-1-RNAi-LV2和Fli-1-RNAi-LV3），并篩選出陽性克隆，經(jīng)PCR擴(kuò)增及DNA測序，證實(shí)重組慢病毒表達(dá)載體構(gòu)建成功。 2．慢病毒載體包裝后收集得到高滴度的慢病毒濃縮液，在293T細(xì)胞中利用逐孔稀釋法測定病毒滴度，3個靶序列的慢病毒滴度均為:1.5E+9TU/mL。 3．將病毒以不同的濃度感染CB3細(xì)胞，測得當(dāng)MOI值為100時CB3細(xì)胞感染效率最高為80%。 4．與陰性對照組(Negative control,NC)和未感染組(Non-infected)相比，各感染組在mRNA水平及蛋白質(zhì)水平表達(dá)量明顯減少(p＜0.001)，其中以Fli-1-RNAi-LV3組抑制效果最好。 5．MTT法檢測并分析各實(shí)驗(yàn)組CB3細(xì)胞生長曲線，發(fā)現(xiàn)各感染組較對照組及未感染組生長增殖減慢（p0.05），各感染組間無明顯差異。流式細(xì)胞儀檢測慢病毒感染CB3細(xì)胞72h后細(xì)胞的凋亡情況，同對照組相比，感染組CB3細(xì)胞發(fā)生了顯著的凋亡。 結(jié)論： 1．成功構(gòu)建了Fli-1基因特異性siRNA慢病毒載體。 2．利用構(gòu)建的重組慢病毒包裝生產(chǎn)出慢病毒顆粒，成功測定重組慢病毒顆粒的滴度，證實(shí)重組慢病毒可高效感染CB3細(xì)胞。 3．以最適MOI值感染CB3細(xì)胞，經(jīng)RT-PCR及Western blot檢測，證實(shí)達(dá)到了特異性沉默CB3細(xì)胞Fli-1基因表達(dá)的目的，并可使干涉組CB3細(xì)胞生長增殖減慢，為進(jìn)一步研究Fli-1基因?qū)B3細(xì)胞生物學(xué)行為的影響，探討其作用機(jī)制奠定了基礎(chǔ)。
[Abstract]:Objective : Fli - 1 ( Friend leukemia integration - 1 ) is a transcription factor belonging to Ets ( E26transformation - specific ) family . The abnormal expression of Fli - 1 is an important factor in the initiation of malignant transformation of some types of cells . Based on this , the Fli - 1 gene was used as the target gene and Fli - 1 gene was designed to infect CB3 cells . The effect of Fli - 1 mRNA and protein expression in CB3 cells was determined by detecting Fli - 1mRNA and protein expression in CB3 cells . The effect of silencing Fli - 1 gene on the biological characteristics of CB3 cells was observed .

Method :

1 . Preparation of RNAi lentivirus vector : Three interfering sequences for the target gene Fli - 1 were designed for the mRNA sequence of the Fli - 1 gene of mouse , and the negative control sequence was designed . Then the double - stranded DNA oligonucleotide was synthesized by annealing and pairing . Then , the digested RNA of EcoR I was directly linked into the vector of Age I and EcoR I , then the ligated product was transferred to the prepared bacterial competent cell DH5.alpha . , PCR was carried out on the long - output monoclonal colony , and the positive bacteria were identified by PCR .

2 . packaging and titer detection of RNAi slow virus : the slow virus carrier and the auxiliary carrier are co - infected by Lipofectamine 2000 , the supernatant is collected from the cells rich in the slow virus particles , and the slow virus concentrated solution with high titer is collected after centrifugation .

3 . CB3 cells were infected by slow virus infection : CB3 cells with good growth status were collected by centrifugation to infect the CB3 cells . In order to find out the best infection complex infection at CB3 cells , the experiment was divided into 100 , 10 and 1 groups . After 3 days of infection , the expression of green fluorescence was observed by fluorescence microscope , and the highest infection efficiency group was screened .

4 . Identification of gene silencing effect after slow virus infection of CB3 cells : After 96 h of slow virus infection , Fli - 1mRNA and protein expression were measured by RT - PCR and Western blot respectively .

5 . MTT assay was used to detect CB3 cell growth in each experimental group . Flow cytometry was used to detect the apoptosis of CB3 cells after 72 hours .

Results :

1 . A lentivirus vector ( Fli - 1 - RNAi - LV1 , Fli - 1 - RNAi - LV2 and Fli - 1 - RNAi - LV3 ) carrying the Fli - 1 interference sequence was constructed successfully , and positive clones were screened out . After PCR amplification and DNA sequencing , the recombinant lentivirus expression vector was confirmed to be successful .

2 . The slow virus concentrated solution with high titer was collected after the slow virus carrier was packaged , and the virus titer was measured by a hole - by - hole dilution method , and the slow virus titer of the three target sequences was 1.5E + 9TU / mL .

3 . The virus was infected with CB3 cells at different concentrations . The efficiency of CB3 cell infection was 80 % at 100 .

4 . Compared with the negative control group ( NC ) and non - infected group ( Non - infected ) , the level of mRNA and protein level of each infection group were significantly decreased ( p < 0 . 001 ) , and the Fli - 1 - RNAi - LV3 group had the best inhibition effect .

5 . The growth curve of CB3 cells was detected and analyzed by MTT assay . It was found that the growth and proliferation of CB3 cells in the infected group were slower than those in the control group and the non - infected group ( p < 0.05 ) .

Conclusion :

1 . Fli - 1 gene specific siRNA lentivirus vector was successfully constructed .

2 . By using the constructed recombinant lentivirus package to produce the slow virus particles , the titer of the recombinant lentivirus particles was successfully determined , and it was confirmed that the recombinant lentivirus could infect CB3 cells efficiently .

3 . The expression of Fli - 1 gene was confirmed by RT - PCR and Western blot in CB3 cells infected with CB3 cells by RT - PCR and Western blot . The effect of Fli - 1 gene on the biological behavior of CB3 cells was further investigated .

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R733.73;R-332

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1 李巖;慢病毒載體介導(dǎo)的小鼠紅白血病CB3細(xì)胞Fli-1基因沉默的研究[D];吉林大學(xué);2012年

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