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目的构建含小分子肽P1-GFP融合基因慢病毒载体,用携带P1-GFP融合基因的慢病毒感染MSC,使MSC具有靶向性,将靶向MSC注入小鼠体内后观察MSC在小鼠脾脏的定位及与淋巴细胞的关系。方法用组织片贴壁法培养健康人脐带间充质干细胞,用基因工程技术构建含小分子肽P1-GFP融合基因慢病毒载体并感染人脐带间充质干细胞,通过尾静脉将转入P1-GFP融合基因的MSC注入小鼠体内,18 h后免疫组化染色观察GFP在小鼠脾脏的定位。结果培养的健康人脐带间充质干细胞生长良好,MSC感染含P1-GFP融合基因的慢病毒18 h后MSC开始出现绿色荧光,随着培养时间的延长,荧光强度逐渐增强,72 h达高峰。靶向MSC表达髓系干细胞的表面标记CD105(90.0%)/CD44(98%),CD73(85.0%)/CD90(98.5%)。将靶向MSC经尾静脉注入小鼠体内,18 h后小鼠脾脏出现大量GFP阳性细胞,并与脾脏淋巴细胞密切接触。结论本研究成功构建了含P1-GFP融合基因的靶向MSC,靶向MSC成功定向脾脏,并与脾脏淋巴细胞密切接触,可用于后续的实验研究。
Objective To construct a lentiviral vector containing small molecule peptide P1-GFP fusion gene and infect MSCs with lentivirus carrying P1-GFP fusion gene, so that MSC could be targeted. After targeting MSC were injected into mice, MSCs were observed in mouse spleen The location and the relationship with lymphocytes. Methods Human umbilical cord mesenchymal stem cells (MSCs) were cultured with tissue adherent method. The lentiviral vector containing small molecule peptide P1-GFP fusion gene was constructed and infected with human umbilical cord mesenchymal stem cells by genetic engineering. The cells were transfected into P1- GFP fusion gene injected into the mouse MSC, 18 h after immunohistochemical staining GFP in mouse spleen localization. Results The cultured human umbilical cord mesenchymal stem cells grew well. MSC infected with lentivirus containing P1-GFP fusion gene showed green fluorescence after 18 h, and reached the peak at 72 h with the prolongation of culture time. Surface markers CD105 (90.0%) / CD44 (98%), CD73 (85.0%) / CD90 (98.5%) targeting MSC expressing myeloid stem cells. The targeted MSCs were injected into mice via the tail vein. After 18 h, a large number of GFP positive cells appeared in the spleen of mice and were in close contact with spleen lymphocytes. Conclusion The target MSCs containing P1-GFP fusion gene were constructed successfully. The targeted MSCs were successfully targeted to the spleen and closely contacted with spleen lymphocytes, which could be used in subsequent experimental studies.