慢病毒载体介导的小鼠红白血病CB3细胞Fli-1基因沉默的研究

发布时间：2018-04-01 08:31

本文选题：Fli-1　切入点：RNA干扰　出处：《吉林大学》2012年硕士论文

【摘要】：目的：Fli-1（Friend leukemia integration-1）是属于Ets(E26transformation-specific)家族的转录因子, Fli-1的异常表达是某些类型细胞恶性转化起始阶段的重要因素。研究发现Fli-1不仅在血细胞和血管生成中起着至关重要的作用[2]，而且还具有促进肿瘤发生发展的作用[3]。基于此，本实验利用RNA干涉（RNA interference,RNAi）技术，以小鼠Fli-1基因为靶基因，设计Fli-1基因特异性的小干扰RNA（small interferenceRNA,siRNA）,构建制备目的基因Fli-1的siRNA慢病毒载体，以最适合的感染复数(optimal multiplicity of infection,MOI)感染CB3细胞，通过检测各实验组CB3细胞中Fli-1mRNA及蛋白质的表达，验证其对CB3细胞中Fli-1基因的沉默效果，并观察沉默Fli-1基因后对CB3细胞生物学特性的影响，为今后探讨其作用机制奠定了基础。方法： 1．RNAi慢病毒载体的制备：针对小鼠Fli-1基因mRNA序列设计三条针对目的基因Fli-1的干扰序列，并设计阴性对照序列。合成干扰序列的单链DNA寡核苷酸，然后退火配对产生双链DNA寡核苷酸，再通过其两端所含酶切位点直接连入Age I、EcoR I酶切后的RNA干扰慢病毒载体上,将连接产物转入制备好的细菌感受态细胞DH5α，对长出的单克隆菌落进行PCR鉴定,PCR鉴定阳性菌落进行测序鉴定。 2．RNAi慢病毒的包装与滴度检测：按Lipofectamine2000使用说明，将慢病毒载体与辅助载体共感染293T细胞，，收集富含慢病毒颗粒的细胞上清液，离心后收集得到高滴度的慢病毒浓缩液。在293T细胞中，利用逐孔稀释法测定病毒的滴度。 3．慢病毒感染CB3细胞：离心收集生长状态良好的CB3细胞进行病毒感染，为摸索CB3细胞最佳感染复数（multiplicity of infection,MOI）,根据MOI值将实验分为100、10和1三组，感染3天后，通过荧光显微镜观察绿色荧光表达情况，筛选感染效率最高组。 4．慢病毒感染CB3细胞后基因沉默效果的鉴定：慢病毒感染CB3细胞96h后，分别用RT-PCR及Western blot方法测定Fli-1mRNA及蛋白质表达量。 5．MTT法检测各实验组CB3细胞生长。流式细胞仪检测慢病毒感染72h后的CB3细胞的凋亡情况。结果： 1．成功构建携带有Fli-1干涉序列的慢病毒载体（分别命名为Fli-1-RNAi-LV1、Fli-1-RNAi-LV2和Fli-1-RNAi-LV3），并筛选出阳性克隆，经PCR扩增及DNA测序，证实重组慢病毒表达载体构建成功。 2．慢病毒载体包装后收集得到高滴度的慢病毒浓缩液，在293T细胞中利用逐孔稀释法测定病毒滴度，3个靶序列的慢病毒滴度均为:1.5E+9TU/mL。 3．将病毒以不同的浓度感染CB3细胞，测得当MOI值为100时CB3细胞感染效率最高为80%。 4．与阴性对照组(Negative control,NC)和未感染组(Non-infected)相比，各感染组在mRNA水平及蛋白质水平表达量明显减少(p＜0.001)，其中以Fli-1-RNAi-LV3组抑制效果最好。 5．MTT法检测并分析各实验组CB3细胞生长曲线，发现各感染组较对照组及未感染组生长增殖减慢（p0.05），各感染组间无明显差异。流式细胞仪检测慢病毒感染CB3细胞72h后细胞的凋亡情况，同对照组相比，感染组CB3细胞发生了显著的凋亡。结论： 1．成功构建了Fli-1基因特异性siRNA慢病毒载体。 2．利用构建的重组慢病毒包装生产出慢病毒颗粒，成功测定重组慢病毒颗粒的滴度，证实重组慢病毒可高效感染CB3细胞。 3．以最适MOI值感染CB3细胞，经RT-PCR及Western blot检测，证实达到了特异性沉默CB3细胞Fli-1基因表达的目的，并可使干涉组CB3细胞生长增殖减慢，为进一步研究Fli-1基因对CB3细胞生物学行为的影响，探讨其作用机制奠定了基础。
[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 .

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R733.73;R-332

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