组织工程与重建外科杂志 ›› 2024, Vol. 20 ›› Issue (3): 275-.

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不同转染方式用于modRNA转染人脂肪干细胞的初步研究

  

  • 出版日期:2024-06-01 发布日期:2024-07-05

Preliminary study on modRNA transfection of human adipose-derived stem cells with different transfection methods

  • Online:2024-06-01 Published:2024-07-05

摘要:

目的 筛选更优的转染方式,探讨其用于 modRNA 转染人脂肪干细胞(Human adipose-derived stem cell,hADSC)并表达特定蛋白的可行性。

方法 分别采用 2种电转染方式 Optimization4及 Optimization6和脂质体转染试剂RNAiMAX,将modGFP转染到hADSC中去。应用流式细胞分析仪和荧光显微镜分析,比较不同转染方式的转染效率和蛋白表达情况;通过细胞死活染色,评估转染后24 h细胞的存活情况;将更优的电转方式与RNAiMAX用于modVEGFA转染 hADSC,收集其培养 24 h 的上清用于人脐静脉内皮细胞(Human umbilical vein endothelial cells,hUVECs)成管和transwell迁移实验,并与基质胶混合后注射于裸鼠皮下,通过大体观、HE染色及免疫荧光染色,分析其在体内促进血管再生的情况。结果 Optimization6 电转转染 hADSC 的效率为 87.75%±1.52%,高于 Optimization4 转染组(80.85%±1.48%)和 RNAiMAX 转染组(81.80%±1.08%),且荧光显微镜观察及流式分析均表明电转染方式的蛋白表达优于RNAiMAX脂质体转染。3种转染方式对细胞的毒性均较小,无统计学差异。与RNAiMAX相比,Optimization6电转能更高效地介导 modVEGFA 转染 hADSC,能更好地促进体外 hUVECs成管和迁移,皮下血管再生的效果也更佳。

结论相比于 RNAiMAX 和 Optimization4电转染,Optimization6电转方式转染 hADSC的效率更高,蛋白表达高效,且能在体内外快速高效促进血管再生。

关键词:

Abstract:

Objective To select a transfection method with better efficiency and to determine its feasibility in modRNA
transfection of human adipose-derived stem cell (hADSC) as well as specific protein expression pattern. Methods ModGFP
was transfected into hADSC using two kinds of electrotransfection methods (Optimization4 and Optimization6) and liposome
transfection reagent (RNAiMAX).The transfection efficiency and protein expression of different transfection methods were
analyzed by flow cytometry and fluorescence microscope. Cell survival rate at 24 h after transfection was evaluated by AM/PI
staining. Electrotransformation mode with better effect and RNAiMAX were used to transfect modVEGFA into hADSC.
Culture supernatant of hADSC after being transfected for 24 h was collected to incubate human umbilical vein endothelial
cells (hUVECs) followed by tube formation and transwell migration experiments. hADSCmodVEGFA mixed with matrigel was
injected subcutaneously in nude mice. The ability to stimulate angiogenesis in vivo was analyzed by gross observation, HE
staining, and immunofluorescence staining. Results The transfection efficiency was higher in Optimization6 group (87.75%
±1.52%), compared with Optimization4 group (80.85% ±1.48%) and RNAiMAX group (81.8%±1.08%).Fluorescence
microscopy and flow analysis showed that the protein expression pattern was better in electrotransfection groups than that of
RNAiMAX group. The toxicity of these three transfection methods in cells was minimal, no statistical difference was found.
Compared with RNAiMAX, Optimization6 can transfect modVEGFA more efficiently into hADSC and possess better ability of
promoting angiogenesis both in vitro and in vivo. Conclusion Compared with RNAiMAX and Optimization4, Optimization6
has higher transfection efficiency, better protein expression pattern, and stronger angiogenesis promoting effect both in vitro
and in vivo.

Key words:

adipose-derived stem cell