Functional Identification Of RPS20Gene Based On Growth Of Intestinal Cells And Effects Of Chinese Medicine That Invigorates Spleen

Background and ObjectiveSpleen deficiency is a common syndrome of Chinese medicine. In modern medicine, the clinical symptoms of spleen deficiency are throught to be relevant to the abnormal functions of digestion, absorption and metabolism. To elucidate the pathophysiology of spleen deficiency, scholars have investigated the syndrome of spleen deficiency from the perspectives of digestive system, immune system, material and energy metabolism, et al. But the recent studies can't fully reflect the essence of spleen deficiency due to the systemic and complex nature of Chinese medicine syndromes. In recent years, reseaches on the differentially expressed gene profiles of Chinese medicine syndromes develops based on the genomic technology, which can reflect the biological basis of Chinese medicine syndromes from the overall level. Exploration of differentially expressed genes in Chinese medicine syndromes based on gene microarray provides new thoughts and methods to interpret the essence of Chinese medicine syndromes and have made some progress.In the previous research project on the differentially expressed gene profile of chronic superficial gastritis patients with spleen deficiency syndrome, investigators of our lab found that genes of ribosomal proteins such as ribosomal protein S20(RPS20) showed down-regulated tendency, which was validated by fluorescence quantitative PCR. Subsequently we carried out researches in vitro to identify the gene functions of RPS20on rats'small intestinal epithelial cells (IEC-6) and the research results preliminarily showed that genes of ribosomal protein may play an important role in the process of the development of spleen deficiency syndrome. After RNA interference (RNAi) for RPS20gene, the morphology and structure of IEC-6cells were changed and the migration and proliferation function of IEC-6cells were both suppressed, which indicated that down-regulated expression of RPS20gene may influence the digestion and absorption function and mucosal repair function of intestinal epithelial cells. Besides, in order to identify the gene functions of RPS20in vivo in the future, investigator selected the effective sequence of RNAi for RPS20gene on mice's colonic tumor cells (CT26), recombinated and constructed the RNAi lentiviral vectors for RPS20. Based on the previous studies, firstly we aim to carry out further investigations on the gene function of RPS20in this thesis. We will investigate the effects of Chinese medicines that nourish Qi invigorate spleen on the impaired morphology and function of IEC-6after RNAi for RPS20gene, to prove the previous study results from the viewpoint of the medicine effect and to support the findings in the previous researches. Secondly we aim to verify the validity of RNAi sequence for RPS20previously selected on CT26cell and to provide reference for further functional identification of RPS20by RNAi in mice, by producing lentivirus of RNAi for RPS20based on the recombinant lentiviral vector, transfecting CT26cells with it and investigating its effect on the growth of CT26cell.Methods1. Transfect IEC-6cells with siRNA and treat them with the extract from Chinese medicines that nourish Qi to invigorate spleen. And then determine proliferation, migration, morphology and structure, RPS20expression and polyamine content of IEC-6cells.(1) Investigation of the growth conditions of IEC-6cells:Investigate the growth curves of IEC-6cells by the methods of cell counting and xCELLingence real-time cell analyzer (RTCA), to provide reference for choosing the appropriate cell planting density and the right time for interventiont in the following experiments.(2) Selection of the transfection condition of RNA interference:Add a sequence expressing fluorescence into the RNAi sequence previously selected. Monitor the growth status of IEC-6cells transfected by different transfection mixtures with xCELLingence RTCA, which can reflect the effect of RNA interference. Select the suitable transfection condition according to the growth status and the fluorescence expression of IEC-6cells.(3) Effect of Chinese medicine on proliferation of IEC-6cells:Use xCELLingence RTCA to study the effects of extracts from Radix Astragali, Radix Glycytthizae and Radix Codonopsis on proliferation of normal IEC-6cells and that of IEC-6cells after RNA interference.(4) Effect of Chinese medicine on migration of IEC-6cells:Use the migration model of scarification to study the effects of extract from Radix Astragali on migration of normal IEC-6cells and that of IEC-6cells after RNA interference.(5) Effect of Chinese medicine on morphology and structure of IEC-6cells:Observe the morphology and structure of IEC-6cells after RNA interference and after treatment with extract from Radix Astragali by phase contrast microscope and transmission electron microscope respectively to investigate the restoring effect of Chinese medicine on the impaired morphology and structure of IEC-6cells after RNA interference.(6) Effect of Chinese medicine on RPS20expression of IEC-6cells:Determine the RPS20mRNA and protein expression of IEC-6cells after RNA interference and after treatment with extract from Radix Astragali by quantitative RT-PCR and Westerblot respectively. (7) Effect of Chinese medicine on polyamine content of IEC-6cells:Prepare the samples for HPLC determination with IEC-6cells after RNA interference and after treatment with extract from Radix Astragali. And determine the content of spermidine in the samples with the method of HPLC pre-column derivatization.2. Effect of lentivirus of RNAi for RPS20on the growth of CT26cells:Co-transfect293FT cells with the recombinant lentiviral vectors against RPS20in CT26cells and packing reagents to produce lentivirus of RNAi for RPS20. Determine the titer of lentiviral stocks and transfect CT26with them. Monitor the growth of CT26post-transfection by xCELLigence RTCA to investigate the effect of lentivirus of RNAi on the growth of CT26cells.Results1. RNAi for RPS20of IEC-6cells and the effects of Chinese medicines that nourish Qi to invigorate spleen:(1) The growth conditions of IEC-6cells:The growth curve of IEC-6cells planted with the density of1×104cells/well in the24-well plate was drawn by cell counting, which showed the cells grew into the logarithmic phase from the3rd day after planted and reached the plateau from the10th day. By xCELLigence RTCA, the growth curves of IEC-6cells of different passages (23rd and26th) planted in different densities were obtained. Cells of23rd and26th passages showed similar growth curves in the same planting density. As the planting density increased, the time of which cells'growth reached the plateau shortened.(2) The transfection condition of RNA interference:The results from RTCA showed the transfection agent (Lipofectamine2000) of high and medium dosage was significantly harmful to the cells. Transfection mixtures of different dose-combinations showed similar inhibitory effects on the growth of IEC-6cells at the beginning of transfection, but the inhibitory effects of mixtures with higher dosage of siRNA lasted longer. Considering the fluorescence expression with the growth status of the cells, we selected the transfection condition as follow:0.3μL Lipofectamine2000and16pmol siRNA per well in the E-plate, the concentrations of which were1.5mg/L and80nmol/L respectively.(3) Effect of Chinese medicine on proliferation of IEC-6cells:Extracts from Radix Astragali and Radix Codonopsis promoted proliferation of normal IEC-6cells, but the effect of Radix Glycytthizae on normal IEC-6cells was not significant. Proliferation of IEC-6cells after RNA interference was suppressed. Extracts from Radix Astragali, Radix Glycytthizae and Radix Codonopsis significantly promoted proliferation of IEC-6cells after RNA interference. The effect of Radix Astragali was better. The results showed that Chinese medicines that invigorates spleen can restore the pathology of depressed proliferation of IEC-6cells after RNA interference.(4) Effect of Chinese medicine on migration of IEC-6cells:High and medium dosage of extract from Radix Astragali could effectively promote migration of normal IEC-6cells which were wounded of scarification. Migration of IEC-6cells after RNA interference was suppressed, which was significantly enhanced after treatment with extract from Radix Astragali, compared with the transfected group. But it wasn't restored to the normal level. The results showed that the impaired function of migration of IEC-6cells after RNA interference can be restored to some extent by Chinese medicines that invigorates spleen.(5) Effect of Chinese medicine on morphology and structure of IEC-6cells:It was observed by optical microscope that ceH numbers of IEC-6cells were decreased, cell morphology was changed and intercellular spaces were enlarged after RNA interference, which was improved after treatment with extract from Radix Astragali. The increasing of endochylema vacuolization and lysosome numbers and the reduction of mitochondria was observed by transmission electron microscope. The impaired ultrastructure of cells was restored to some extent after treatment.(6) Effect of Chinese medicine on RPS20expression of IEC-6cells:The expressions of RPS20mRNA of IEC-6cells were significantly inhibited after RNA interference, which could be enhanced after treatment with extract from Radix Astragali. The group of medium dosage showed significant difference compared with the group of transfection. The expressions of RPS20protein were also inhibited markedly after RNA interference, but they weren't elevated after treatment.(7) Effect of Chinese medicine on polyamine content of IEC-6cells:The regression equation of spermidine was Y=25.69X-1.337and its linear correlation coefficient R=0.9998, which showed spermidine was of good linearity in the range from0.7600to7.600nmol. The contents of spermidine of IEC-6cells after RNA interference were increased but without significant difference compared with control group. The contents of spermidine of IEC-6cells in groups treated with extract from Radix Astragali were markedly higher than that of control groups, but without significant difference compared with the transfection group.2. Effect of lentivirus of RNAi for RPS20on the growth of CT26cells:The titer of lentiviral stocks was1.2X105TU/mL. The increasing of cell index of CT26cells was significantly inhibited after transfected by the lentivirus of RNAi and the inhibitory rate of cell index reached82.24%30hours after transfection. The results showed that the RNAi sequence selected previously can be constructed into lentivirus successfully and inhibit the growth of CT26cells after transfection effectively.ConelusionsTransfecting IEC-6cells by the RNA interference condition selected based on the results of xCELLigence RTCA determination and cell fluorescence expression can effectively inhibit RPS20expression of IEC-6cell and also inhibit its ability of proliferation and migration and change its morphology and structure. After treatment with extract from Radix Astragali, the expression of RPS20mRNA of IEC-6cell can be enhanced, the ability of proliferation and migration of IEC-6cell can be promoted and the impaired morphology and structure of IEC-6cell can be restored. The result that the abnormal morphology and function of IEC-6cell initiated by deficient expression of RPS20can be improved after treatment with Chinese medicine that nourishes Qi to invigorate spleen, suggests that RPS20may be relevant to the development of spleen deficiency syndrome and further verified the findings of our previous research on the differentially expressed genes of spleen deficiency syndrome and the results of RPS20functional identification of our research team. Our research can provide a reference for functional identification on differentially expressed genes of Chinese medicine syndromes.Results in this thesis also confirmed that the recombinant lentiviral vectors for RPS20on CT26cells can be used to produce lentiviral stock of RNAi successfully and transfecting CT26cells with it can inhibit cell growth effectively, which suggests that lentivirus of RNAi may be used to identify RPS20function in the further study in mice.