壳聚糖纳米细胞介导基因转染的研究

Study on Gene Transfection Mediated by Chitosan Nanocell

目的构建核壳型壳聚糖（chitosan，CTS）纳米细胞（nanocell，NC），探讨其作为基因转染载体的可行性。方法以壳聚糖为载体材料，pEGFP-N1为模型质粒，制备质粒复合物pEGFP-N1／CTS—NP。以pEGFP-N1／CTS-NP为核心，用复乳法外包含十八胺（stearylamine，SA）的脂质体膜制备纳米细胞pEDFP-N1／CTS—SANC，用CTS对其进行包覆制成CTS-（pEGFP-N1／CTS-SANC），测定其形态、粒径与电位。用MTF法测定NC的细胞毒性，用荧光显微镜及流式细胞仪定性、定量地评价其在HeLa细胞中的转染率。结果所制备的样品多呈类球形，粒径与电位分别分布在120—220nm与40-65mV之间。pEGFP-N1／CTS-SANC与CTS-（pEGFP-N1／CTS—SANC）均可将质粒转染到HeLa细胞并表达绿色荧光蛋白。CTS-（pEGFP-N1／CTS—SANC）可降低pEGFP-N1／CTS-SANC的毒性，在相同质粒用量下，使HeLa细胞存活率从81．8％增至100．9％。转染率从3．90％增至8．13％。达市售脂质体转染试剂的76％。结论壳聚糖纳米细胞有望作为基因转染的载体。

Objective To develop a core-shell type chitosan nanocell （CTS-NC） and to investigate its feasibility as a vector of gene transfection. Methods The plasmid complex, pEGFP- N1/CTS-NP was prepared by using chitosan as the carrier material and pEGFP-N1 as the model plasmid. The pEGFP-N1/CTS-NP core was coated with a liposome membrane containing stearylamine （SA） to obtain nanocell pEGFP-N1/CTS-SANC by double emulsifying method. The pEGFP- N1/CTS-SANC was additionally coated with chitosan outside to prepare CTS- （pEGFP-N1/CTS- SANC ） . The morphology, size and zeta potential of all these nano-size particles were measured. The cytotoxicity was measured in HeLa cells by the MTY method. The gene transfection efficiency was evaluated in Hela cells by fluorescence inverted microscopy and flow cytometry qualita- tively and quantitatively. Results Most of the prepared nano-size particles were spherical. The size and zeta potential was within 120-220 nm and 40-65 mV, respectively. Both pEGFP-N1/CTS-SANC and CTS- （pEGFP-N1/CTS-SANC） could be transfected into Hela cells, which afterwards expressed green fluorescence protein. CTS- （pEGFP-N1/CTS-SANC） reduced the cytotoxicity of pEGFP-N1/CTS-SANC by additional chitosan coating. With the same amount of plasmid used, the viability of Hela cells was increased from 81.8% to 100. 9% and the transfection efficiency enhanced from 3.90% to 8. 13% , which was about 76 % of that of commercial DNAFect transfection reagent. Conclusion The chitosan nanocell is expected to become a candidate vector for gene transfection.