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本研究旨在通过慢病毒介导的基因转移方法,使NB4细胞稳定过表达hβc基因,观察过表达hβc基因的NB4细胞在IL-3或GM-CSF作用下分化行为的改变,探讨hβc基因与NB4细胞分化之间的关系。以本实验室前期构建的携带hβc基因ORF的克隆质粒为模板,用带PmeI和BstBI酶切位点的引物PCR获得目的基因,酶切PCR产物,定向克隆至慢病毒载体pRRLSIN.cPPT.PGK/IRES/GFP.WPRE,构建含hβc基因的慢病毒载体,经酶切及测序鉴定其正确性,将构建好的重组质粒与包装质粒一起共转染293T细胞,收集培养液上清,感染NB4细胞,用Western blot鉴定目的基因在NB4细胞中的表达情况。以转染空载体慢病毒的NB4细胞(简称NB4-blank细胞)为对照,观察过表达hβc基因的NB4细胞(简称NB4-hβc细胞)在IL-3、GM-CSF、全反式维甲酸(ATRA)作用下分化行为的改变。结果表明:含有hβc基因的重组慢病毒载体能高效转染NB4细胞,并使NB4细胞稳定过表达hβc基因。NB4-hβc细胞,在IL-3或GM-CSF作用下CD11b表达水平上调,但上调的幅度均低于ATRA作用下的上调幅度,且未观察到形态学改变。NB4-Blank细胞在IL-3或GM-CSF作用下CD11b表达水平无明显变化。结论 :通过慢病毒介导的基因转移方法,成功地使NB4细胞稳定过表达hβc基因,IL-3或GM-CSF在一定程度上能诱导过表达hβc基因的NB4细胞向中性粒细胞分化,但不能使其完全分化成熟。
This study aimed to stably overexpress hβc gene in NB4 cells by lentivirus-mediated gene transfer and to observe the differentiation behavior of NB4 cells overexpressing hβc gene under the action of IL-3 or GM-CSF. NB4 cell differentiation between the relationship. The cloned plasmids harboring ORF of hβc gene in our laboratory were used as templates and the target genes were obtained by PCR with PmeI and BstBI restriction sites. The PCR products were digested and cloned into the lentiviral vector pRRLSIN.cPPT.PGK / IRES / GFP.WPRE to construct a lentiviral vector containing hβc gene. The correctness of the vector was confirmed by restriction enzyme digestion and sequencing. 293T cells were co-transfected with the recombinant plasmids and packaging plasmids, and the supernatant of the culture supernatant was collected to infect NB4 cells The expression of the target gene in NB4 cells was identified by Western blot. The NB4 cells overexpressing hβc gene (referred to as NB4-hβc cells) were transfected with empty vector lentivirus NB4 cells (NB4-blank cells) for IL-3, GM-CSF and all- ATRA) under the action of changes in differentiation. The results showed that the recombinant lentiviral vector containing hβc gene could efficiently transfect NB4 cells and stably overexpress hβc gene in NB4 cells. NB4-hβc cells under the action of IL-3 or GM-CSF, the expression of CD11b was up-regulated but the amplitude of up-regulation was lower than that of ATRA. No morphological changes were observed. NB4-Blank cells in the IL-3 or GM-CSF CD11b expression levels did not change significantly. CONCLUSION: The NB4 cells stably overexpressing hβc gene, IL-3 or GM-CSF can be successfully induced to differentiate into neutrophils by NB4 cells overexpressing hβc gene by lentivirus-mediated gene transfer. But can not completely differentiate and mature it.