The Effect Of RNA Interference To The Excretion Of TNF-α In Stimulated Splenic Lymphocytes

ObjectiveTumor necrosis factor - α (TNF - α) is a kind of important cytokine produced by activated macrophage and dependent lymphocyte T cell. TNF - a posses wide biological activity. At first , the function of TNF - a we know is only that it can kill tumor cells particularly. Then it is found that TNF - α also can regulate immunity function, participate in some inflammation. Appropriate TNF -α can regulate immunity. It plays an important part in maintaining organism stabilization and resisting nosogenesis.However, exceptional exudation of TNF - a is the important mudium in inflammation , damnification and shock. At present ,the diseases related to TNF -α induce AIDS, anemia,, tumor, hemorrhagic shock , grafting regection, tuberculosis, leukemia, diabetes mellitus, rheumatoid arthritis. TNF - α takes part in the occurrence and depentment course of many disease, so it can inhibit TNF - a action in different level and may yield therapeutical effect for the diseases related to TNF - a.RNA interference is a kind of post - transcriptional gene silencing phenomenon that induced by homology complementary sequence siRNA . It can inhibit the expression of protein and simulate gene knock - out technique. RNAi can be found in plant, epiphyte and animal cells. It has a broad applied foreground. RNAi is high efficiency and sequence specificity. Our investigation uses Silent-Gene^tm U6 RNAi system to compound siRNA expression carrier which is taken into cells and induce RNAi. We select 2 target sites from TNF - α gene sequence to use synthesized siRNA to inhibit target gene expression and to regulatethe TNF - a secretion. By interference to 2 siRNA we can select susceptible target site quickly and provide the most powerful interferential sequence. The lecture will discuss the RNAi of site 1.Methods1. Spleen lymphocytes culture : C57 BL/6 mouse , male (provided by ChinaMedical University animal department). Kill mice with cervical vertebra dislocation method ,get spleen in 0^ 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 1 x 105/ml with DEME , finally inoculate in 96 hole culture plate.2. Ability of lymphocytes to secret TNF — a: After cell 's normal growth , add 10|xg/ml LPS 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 : four groups;Interference group;add siR-NA expression carrier aiming at target site 1 from TNF - agene sequence. Positive — control group: add siRNA expression carrier aiming at IL - 1 gene sequence. Negative —control group:add normal DEME culture liquid . Transfection control group :add pEGFP — Nl former 3 groups;collect the cell culture upper liquid to assay the concentration of TNF — a and IL - 1 with ELISA. To the transfection control group , we use fluorescence microscope to observe the expression of GFP and monitor transfection efficiency.4. Prepare siRNA expression carrier of RNA interference group and positive - control group: Find TNF - a and IL - 1 gene sequence of C57BL/6 mice fromGeneBank. Use the target sequence selection software and principle provided by promega and select target sites. Next synthesis PCR reaction synthesize siRNA expression carrier. After purification, 2% agarose gel electrophoresis detect DNA.5. Assay the specificity of RNAi action: IL - 1 is an important cytokine lymphocytes excrete. Use IL - 1 as positive - control which is inhibited. This canprove specificity of RNAi.6. Prepare pEGFP - Nl plasmid and transfection: The pEGFP - Nl plasmid was amplified in classical way and was parified by the Endotoxin - free Ultrapure Plasmid DNA Parification Kit,then transport pEGFP - Nl plasmid into original cultured splenic T lymphcytes.7. Interference: After lymphcytes grow normally, add corresponding transfection reagent, observe transfection - control group under Fluorescence Microscope. Other groups detect concentration of TNF - a and IL - 1.8. Detect cytokine: Use ELISA to detect concentration of TNF - a and IL -1.Results1. LPS stimulate lymphocytes to secret TNF - a: The concentration of TNF - a reached the top point after stimulation is terminated for 48 hours.2. Prepare siRNA expression carrier of RNAi group and positive - control group:downstream primer of RNAi group :sense chain5' CAAAAACTGTAAAAA - GAGCACAGAAAGCATGATC -GGTGTTTCGTCCTTTCCACAAGA3'antisense chain5' CAAAAACTGTAAAAA - GATCATGCTTTCTGTGCTC -GGTGTTTCGTCCTTTCCACAAGA3'downstream primer of positive - control group;sense chain5' CAAAAACTGTAAAAA - GCAAGCTATGGCTCACTTC -GGTGTTTCGTCCTTTCCACAAGA3'antisense chain5' CAAAAACTGTAAAAA - GAAGTGAGCCATAGCTTGC -GGTGTTTCGTCCTTTCCACAAGA3'3. RNAi effect the concentration of TNF - a: ELISA result means that theconcentration of TNF - a in interference group is obviously lower than in negative - control group(P < 0.05) and in positive - control group(P < 0. 05).4. The specificity of RNAi action: ELISA result means that the concentration of IL - 1 in positive - control group is obviously lower than in interference group ( P < 0. 05 ) and negative - control group ( P < 0.05 ) .5. The transfection effect of pEGFP - Nl plasmid : Observe cells under fluorescence microscope.Conclusion1. RNAi technique can interfere the mice spleen lymphocytes from primary culture and inhibit the secretion of TNF - a.2. RNAi has strict sequence specificity.3. The siRNA of site 1 can specifically inhibit the secretion of TNF - a, so we can select this site as sensitive target site for the later experiment.In a word, RNAi specifically inhibit the expression of cytokine TNF - a. It promotes the application of RNAi technique in mammal cells and opens up a new way to the gene therapy of TNF - a.