靶向脐带间充质干细胞的构建及其在小鼠脾脏内的定位

Construction of targeted umbilical cord derived mesenchymal stem cells and their distribution in the mouse spleen

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 lentiviral vectors containing peptide P1-GFP fusion genes. Umbilical cord derived mesenchymal stem cells were infected with lentivirus carrying peptide P1 and GFP fusion genes. To inject the targeted umbilical cord derived mesenchymal stem cells into mice and to detect GFP expression in the spleen. Methods Umbilical cord derived mesenchymal stem cells were cultured with adhered tissues of umbilical cord smaller than 1 mm3.Lentiviral vector containing P1-GFP fusion genes with engineering technology was constructed and infected the umbilical cord derive mesenchymal stem cells. Targeted umbilical cord derived mesenchymal stem cells were intravenously injected in the mouse tail vein and after 18 hours GFP expression was detected with immunohistochamical staining of the spleen tissues. Results Harvested umbilical cord derived mesenchymal stem cells grew well in culture medium. Green fluorescence on umbilical cord derived mesenchymal stem cells were observed under fluorescence microscope at 18 hours after infected with lentivirus. Green fluorescence intensity of umbilical cord derived mesenchymal stem cells was increasing over time and reached a peak at 72 hours. Umbilical cord derived mesenchymal stem cells highly expressed CD105( 90. 0%) /CD44( 98%) and CD73( 85. 0%) / CD90( 98. 5%) molecules. GFP expression was detected in the spleen after intravenous injection of targeted umbilical cord derived mesenchymal stem cells in the mice 18 hours later. GFP expressing cells intimately contacted with lymphocytes. Conclusions Targeted umbilical cord derived mesenchymal stem cells contain P1-GFP fusion genes are constructed. Targeted umbilical cord derived mesenchymal stem cells can be targeted to mouse spleen and intimately contact with lymphocytes after intravenous injection. Our results lay the groundwork for further studies.