| Objective: Recombined human interleukin-10 gene expression vector (pcDNA4/hIL-10) was constructed, and the recombined vector was transfected into the cultured rabbit corneal cells in vivo and injected local tisse of the eye in vitro , so we want to know if the human recombined interleukin-10 vector expressed in cultured rabbit cells and its founction on suppress of high risk corneal rejection. The recombined interleakin-10 vector may be used in gene therapy for corneal immune rejection in future. Materials and methods: The full genomic DNA was gained from the high immune patient blood and PCR techniqae was used for the human interleukin-10 gene cloning, then the human interleukin-10 gene fragment was cloned into the pcDNA4/HisMaxB to be a combined exppression vector pcDNA4/hIL-10 .Report GFP gene system was used for transfected rate and then pcDNA4/hIL-10 plasmid were transfected into the cultured rabbit corneal cells in vitro. The GFP cells could be observed with general fluorescent microscope, The human IL-10 protein was detected with ELISA method and immunohistochemistry and immunofluorescence technique. Results : Firstly, New Zealand white rabbit (NZW) coneal cells were cultured with modified sliced tisse culture technique , pure cell layers of epithelium, stromal fibral cells and endotheliam were gained successfully, in culturing procedure , trypsin and EDTA were used for mixed cells removing. Secondly, recombined pcDNA4/hIL-10 plasmid could be transfected into the three types of cornea cells in vitro with effectene transfection reagent , a unique non-liposomal lipid formulation. Green fluorescent protein (GFP) reporter gene system was a good visually gene expression for cultured corneal cells in situ . Thirdly, pcDNA4/hIL-10 expression was detected very high with the technique of ELISA, immunohistochemistry and immunofluorescence technique manifested hIL-10 protein in the cell . Fourthly, pcDNA4/hIL-10 recombined vecor injected into the anterior chamber and subconjunctiva of the high risk immune rejection model of keratoplasty could not stop the rejection after alkali burning. The human inlerleukin-10 gene was cloned and combined with pcDNA4/HisMaxB vector successfully obseved with PCR and gene seqence analysis. Corneal cell types of epithelium, stromal fiberal cells and endothelium could be cultured to from a pure local single layer by modified sliced tisse culture, sometimes trypsin and EDTA could be used for cells puring, The DNA and the effetene transfection reagent were no influence to the cells . Effectene? were effectived for pcDNA4/hIL-10 transfection in rabbit corneal cell types. In the cultured corneal cells, hIL-10 could be detected in the three types of the cells. With the ELISA technigue, hIL-10 protein could be detected very higher, and the highest level were two times of the standard control at 36h after transfecting. hIL-10 also could be detected with the immunohistochemistry and immunofluorescence technique in the epithelium, stromal fileral cells and endothelium. The hIL-10 excreted expression of protein was very low at 72h. In the high risk immune rejection model of the gene therapy,all the rabbit cornea were muddy in about 2 weeks, rejection could not be stopped for gene therapy, conclusion: Primary corneal cells can be used as a successful model for gene transfection, combined pcDNA4/hIL-10 can be expressed in the corneal epethelium,stromal cells and endothelium. Effectene transfection reagent is effectively for transfecting hIL-10 gene into the three types of corneal primary cells. Though the hIL-10 gene therapy could not prevent the occurrence of immunorejection on high risk rejection model, but this study provide a first study of hIL-10 gene transfection to corneal cells, the influence factors of transfection in vitro are complicated, this study may be helpful for future investigation of the gene therapy for corneal immunorejection, because the IL-10 can be delay the cell living time for cardiac and liver transplantation.
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