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背景:单份脐血难以满足成人造血干细胞移植的要求,需对其进行体外扩增,这不仅需要较长的时间和较高的培养条件,而且容易导致干细胞自身分化,从而影响移植效果。目的:克隆人白细胞介素3基因cDNA,构建其真核表达载体并转导脐血CD34+细胞,观察白细胞介素3的表达情况。设计、时间及地点:细胞-基因组学体外实验,于2008年在承德医学院完成。材料:健康成人外周血由承德市中心血站提供,脐血由承德市妇幼保健院提供,提供者对实验均知情同意。方法:采集健康成人外周血,应用Ficoll密度梯度法分离单个核细胞,免疫磁珠法分离脐血CD34+细胞。提取白细胞介素3mRNA,应用RT-PCT扩增白细胞介素3cDNA,构建真核表达载体pcDNA3/IL-3。设立2组:实验组利用基因枪技术将pcDNA3/IL-3导入脐血CD34+细胞中,对照组未行转染。主要观察指标:采用人白细胞介素3ELISA试剂盒检测脐血CD34+细胞上清液中白细胞介素3水平。结果:扩增的白细胞介素3基因cDNA理论上应为616bp,实际PCR产物经琼脂糖凝胶电泳后,紫外线下可见预期大小的条带,反转录合成的cDNA完整。BamHⅠ和XbaⅠ双酶切后,电泳可见616bp的插入片段,与白细胞介素3基因序列相同。转染第1～7天,实验组脐血CD34+细胞上清液中白细胞介素3水平明显高于未转染对照组(t=3.46,P<0.05)。结论:白细胞介素3基因cDNA克隆成功,并成功构建了真核表达质粒pcDNA3/IL-3,pcDNA3/IL-3能在脐血CD34+细胞中短期有效表达。
Background: Single cord blood is difficult to meet the requirements of adult hematopoietic stem cell transplantation, which needs to be expanded in vitro. This not only requires a long time and high culture conditions, but also easily leads to the differentiation of stem cells and affects the transplantation effect. OBJECTIVE: To clone human interleukin-3 gene cDNA and construct its eukaryotic expression vector and transduce cord blood CD34 + cells to observe the expression of interleukin-3. DESIGN, TIME AND SETTING: Cell-genomic in vitro experiments were performed at Chengde Medical College in 2008. MATERIALS: Healthy adult peripheral blood was provided by Blood Center of Chengde City Center. Cord blood was provided by Chengde Maternal and Child Health Hospital, and the providers provided informed consent to the experiment. Methods: Peripheral blood of healthy adults was collected. Mononuclear cells were isolated by Ficoll density gradient method and CD34 + cells were isolated by immunomagnetic beads method. Interleukin-3 mRNA was extracted, and interleukin-3 cDNA was amplified by RT-PCR to construct eukaryotic expression vector pcDNA3 / IL-3. Two groups were set up: experimental group pcDNA3 / IL-3 was introduced into cord blood CD34 + cells by gene gun technology, while the control group was not transfected. MAIN OUTCOME MEASURES: Interleukin-3 levels in cord blood CD34 + cell supernatants were measured using human interleukin-3 ELISA kit. Results: The amplified cDNA of interleukin - 3 gene should be 616bp in theory. After the actual PCR product was electrophoresed on agarose gel, the expected size band could be seen under ultraviolet light and the cDNA synthesized by reverse transcription was intact. After double digestion with BamH I and Xba I, a 616 bp insert was found by electrophoresis, which was identical to the sequence of interleukin 3 gene. Interleukin-3 levels in cord blood CD34 + cell supernatants in the experimental group were significantly higher than those in the untransfected control group (t = 3.46, P <0.05). CONCLUSION: The cDNA of interleukin 3 gene was cloned successfully and the eukaryotic expression plasmid pcDNA3 / IL-3 was successfully constructed. PcDNA3 / IL-3 could be expressed in short term in cord blood CD34 + cells.