荧光标记的米糠多糖及其细胞摄入研究

Study on the Preparation and Cell Uptake of Fluorescently-Labeled Rice Bran Polysaccharide

多糖是一类具有良好生物活性的天然大分子物质,但其结构中缺少易于检测的发光基团,给其研究带来诸多不便。为解决这一问题,试验尝试制备荧光探针标记的米糠多糖,并进一步探究其细胞摄入和安全性。利用米糠多糖还原性末端与罗丹明B活泼的氨基反应制备米糠多糖-罗丹明B化合物(Rice Bran Polysaccharide-Rhodamine B,RBP-Rh B);利用米糠多糖还原性末端与酪胺上的氨基共价偶联,通过氨基与异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)发生亲核反应,得到米糠多糖-FITC化合物(Rice Bran Polysaccharide-Fluorescein isothiocyanate,RBP-FITC)。对两种样品分别进行红外光谱、紫外光谱和荧光光谱表征,结果表明米糠多糖成功标记上荧光探针;对RBP-Rh B和RBP-FITC的荧光取代度的测定中,紫外分光光度法测定的荧光取代度分别为0.7%和0.2%,荧光分光光度法测定的荧光取代度分别为1.0%和0.3%;同时试验通过荧光显微镜和MTT法分别研究了荧光标记多糖的细胞摄入和细胞毒性,结果表明标记后的多糖可被细胞有效摄入且细胞毒性低。

Rice bran polysaccharide（RBP）is a kind of biologically active nature product in rice bran. But the further research of its biologically activity was limited to the lacking of bioluminescent groups in its structure.We tried to introduce fluorophore-containing groups into polysaccharides by covalent binding the rhodamine B（Rh B） and fluorescein isothiocyanate（FITC） to RBP. The infrared spectrum,ultraviolet spectrum and fluorescence spectrums showed the Rh B and the FITC were labeled on the RBP successfully. The uptake of labeled RBP in Hela cells were detected by fluorescence microscope. In the experiment of measuring the fluorescence substitute ratio of RBP-Rh B and RBP-FITC,the ratio measured by ultraviolet spectrophotometry was 0.7 % and 0.2 % respectively,and by fluorescence spectrophotometry was 1.0 % and 0.3 % respectively.The cell uptake was evaluated by fluorescence microscope and cell viability evaluated by MTT assay. The results revealed the labeled RBP were successfully uptaked by the hela cells and the labeled-RBP showed the nearly cytotoxicity as the RBP to Hela cells.