| ObjectiveTumor necrosis factor -α(TNF-α) is a kind of important cytokine produced by activated macrophage and thymus dependent lymphocyte - T cell, it can be separated to trans - membrane form and excretion form. TNF-α posses wide biological activity including the regulation to the making blood, immune and inflammation, the influence to the blood vessel and blood coagulation and the effect to the multiple organs. Early investigation revealed that TNF — a can induce tumor cells to necrosis and apoptosis. Later confirmed that TNF-α is a main kind of cytokine leading to inflammatory reaction, cellular immunity response and tumor immunity. At present, the diseases related to TNF —a include : AIDS,anemia,autoimmunity disease,tumor,hemorrhagic shock,grafting rejection,tuberculosis,leukemia,diabetes mellitus,rheumatoid arthritis. TNF-α takes part in the occurrence and development course of many diseases, so it can inhibit TNF -α action in different level and may yield therapeutical effect for the diseases related to TNF-α. RNA interference is a kind of post - transcrip-tional gene silencing phenomenon that induced by homology complementary sequence siRNA. It can inhibit the expression of protein and simulate gene knock - out technique. It is a natural and ancient biological pathway that exists in many species resisting virus invasion, inhibiting transposon and regulating gene expression. High efficiency of suppression and strict sequence specificity is the outstanding character of RNA interference. From synthesizing siRNA in vitro to utilizing plasmid for stable expression of siRNA, this technique is widely utilized. RNAi provides a sthenic tool and new method to investigate mammal gene function,viral prevention and gene therapy of tumor. Our investigation selectsfive target sites from TNF - a gene sequence of C57 BL/6 mice and carries out RNAi experiment. Our purpose is to use synthesized siRNA to inhibit target gene expression and to regulate the TNF - a secretion. By interference to five siRNA we can select susceptible target site quickly and provide the most powerful interferential sequence. This lecture will discuss the RNAi of site two.Methods1. Spleen lymphocytes culture: C57 BL/6 mouse , male ( provided by China Medical University animal department) ,8-12 weeks old, body weight is about 20 gram. Kill mice with cervical vertebra dislocation method, get spleen in 0^1 and sterile environment, grind and filter through nylon web to get cell suspend liquid, then separate spleen lymphocytes by lymphocyte demixing liquid, adjust cell density to lXlOVml with DMEM, finally inoculate in 96 hole culture plate.2. UPS stimulate lymphocytes to secret TNF - a: After cell' s normal growth, add 10p:g/ml UPS to stimulate lymphocytes and action time is six hours. Collect the cell culture upper liquid at 24h^36h^48h^60h after stimulation is terminated. Use ELISA method to assay the concentration of TNF - a.3. Cell's dividing and treatment: After cell' s normal growth, divide into four groups: interference group with siRNA of TNF - a > blank - control group with DMEM^negative - control group with siRNA of 1L - 1 and transfection control group with pEGFP-Nj. 48h after transfection, collect the cell culture upper liquid and use ELISA method to assay the concentration of TNF - a and IL- 1 in former three groups; To the transfection control group , we use fluorescence microscope to observe the expression of GFP and monitor transfection efficiency.4. Assay the suppression effect of siRNA: Use the target sequence selection software and principle provided by Promega and select target sites. Next synthesis downstream primer sequence: even- downstream primer includes pair - matching sequence with U6^ target sequence or complementation sequence and U6 terminator sequence. Synthesized downstream primer A and B (transcripted to antisenseN sense RNA) , after PCR reaction XDNA amplification and DNA purifi-cation, mix with SilentGene? transfection reagent, then transfect spleen lymphocytes. After 48h, collect the cell culture upper liquid and use ELISA method to assay the concentration of TNF - a.5. Assay the specificity of RNAi action;To the collected cell culture upper liquid in former three groups, use ELISA method to assay the concentration of IL- 1 and verify the specificity of RNAi effect.6. Assay transfection efficiency of SilentGene transfection reagent; Mix DH -5a and pEGFP -N, , then suspend bacteria with liquid LB, drip to solid LBplate. Cultivate it in 37°C acteriological incubator overnight. Pick colony and inoculate it in 10ml liquid LB, shake it intensively at 31°C overnight. Get plas-mid DNA with the SDS method; use V - gene no - endotoxin super purification reagent kit to purify plasmid DNA; mix SilentGene? transfection reagent (}xl) and DNA(fxg) with the ratio of 3;1 and transfect lymphocytes. After 24h, use fluorescence microscope to observe the expression of GFP.Results1. LPS stimulate lymphocytes to secret TNF - a: ELISA result means that the concentration of TNF - a reached the top point after stimulation is terminated for 48 hours.2. The suppression effect of RNA interference; ELISA result means that the concentration of TNF - a in interference group is obviously lower than in blank- control group( P < 0. 05 ) and negative - control group( P < 0. 05 ) , the rate of inhibition is about 16% ; but there is no obviously difference between blank — control group and negative - control group( P >0. 05 ).3. The specificity of RNAi action.-ELISA result means that the concentration of IL - 1 in negative - control group is obviously lower than in interference group ( P < 0. 05 ) and blank - control group ( P < 0. 05 ) , but there is no obviously difference between interference group and blank -control group( P >0. 05) .
|
PDF Full Text Request