Modulation Of TNFα Expression By RNA Interference In Stimulated Splenic Lymphocytes

ObjectiveTumor necrosis factor - α ( TNF_α) is a kind of cytokine of wide biology activity , which can be produced by stimulated macrophages and T lymphcytes . It plays a very important role during inflammatory reaction ,cellular immunologic response , tumor immunity and apoptosis . Regulating the expression of TNF_α can provide a powerful method to treat the diseases which involved TNF_α. RNA interference ( RNAi) is the process by which double strained RNA (dsRNA ) directs sequence - specific degradation of messenger RNA in animal and plant cells . These mRNAs take the homologous genes with the double RNA ( dsRNA ) . RNAi has been used to study gene function in multiple model organisms ,including plants , Caenorhabditis elegans , Drosophila and etc . However , in most mammalian cells , dsRNAs longer than 30nt activate an interferon response , leading to nonspecific degradation of RNA transcripts and a general shutdown of host cell protein translation . This nonspecific effect can be circumvented by the use of synthetic siRNAs which are 21 nt long with short 3'overhangs . The synthesized siRNA have been shown to induce homology - dependent degradation of cognate mRNA and used to knock down expression of endogenous and heterolo-gous genes in mammalian cell lines.In this experiment ,depending the target sequence selection principle provided by Cold Spring Horbor Laboratory and the selective software of Promega Corporation , we chosed a segment sequence of mice TNF_α gene as silencing target . We utilized the silentgene U6 RNA interference systerm , recombinated U6 promotor and the double strains sequence and transported the compounds to our original cultured splenic T lymphcyles . This design can make the transcrip-tion of the target sequence more stabilized . At the same time ,we co - transport pEGFP - Nl plasmid to monitor the transfection efficiency . Our purpose is to observe the effection of the synthesized siRNA on the expression of TNFa and provide a possible experiment - based method to treat the diseases involved TNFa.Methods1. Dynamic observing the capable of lymphcytes to produce TNFaThe original cultured splenic T lymphcytes were stimulated by LPS ,we collect the supernatant after stimulation 24 hours ,36 hours ,48 hours ,60 hours respectively ,and then use mice TNFa ELISA reagent kit to detect the concentration of TNFO during the different period after the stimulation .2. Constructing the template double - strained DNA2. 1 We used Silentgene? U6 Cassette System to recombinate the U6 pro-motor and the target double strains sequence by PCR . We synthesized a sequence target IL -1 in th same way as a comparison .2. 2 The production was purified after amplification by Silentgene? PCR DNA Purification System and was detected by 2% agarose gel electrophoresis .2. 3 We utilized Silentgene? Transfection Reagent to transport the compounds to our original cultured splenic T lymphcytes .3. Observing the result of transportion3.1 The amplification and purification of pEGFP - Nl plasmidThe pEGFP - Nl plasmid was amplified in classics way , and was purified by the Endotoxin - free Ultrapure Plasmid DNA Purification Kit , then the output was detected wih 1 % agarose gel electrophoresis . we transported EGFP plasmid into original cultured splenic T lymphcytes with Silentgene? Transfection Reagent to monitor the effect of transfection.3.2 The cells transported with EGFP plasmid were observed under the Fluorescence Microscope .4. Detecting the effect of RNAi on the expression of TNFaThe cells were divided into different groups and the cytokines were detectedby mice TNFa ELISA reagent kit : There were 3 groups lymphcytes : negative comparison with no interference ; interfered with siRNA of TNFa; interfered with siRNA of IL - 1 as positive comparison.5. Detecting the specificity of RNAiThe two groups would be detected by IL - 1 ELISA reagent kit: negative comparison with no interference ; interfered with siRNA of TNFa.Results1. The capable of lymphcytes to produce TNFaAfter stimulated by LPS for 6 hours , the splenic lymphcytes were cultured in the common condition . We collected the supernatant at 24h ,36h ,48h and 60h and compared the results ; the secretive ability of the lymphcytes began to raise at 24 h after stopping stimulation and reached the high point at 48 h . From then on ,the secretive ability began to drop down . Here were the TNFa concentration of different periods :24h : 24.58 ±2.06pg/ml ;36h :25.45 ± 1.42pg/ ml ;48h :28.33±1.50pg/ml;60h :25.02 ±1.54pg/ml .2. Obtaining the target template double - strained DNA The sequences were here;sense: GGACAGCAAGGGACTAGCC ; antisense: GGCTAGTCCCTTGCTGTCC.The production of PCR was detected wih 2% agarose gel electrophoresis , which about 3OObp .3. The transprotion was successfulThe pEGFP - Nl plasmid was detected wih 1% agarose gel electrophoresis , which about 4. 7kb . We successfully actualized transprotion : there were lymphcytes express EGFP and emit green fluorescence .4. The effect of RNAi detected by mice TNFO ELISA reagent kit: negative comparison with no interference: 27. 57 ± 1.75pg/ml ; interfered with siRNA of TNFa: 19.46 ± 1. 34pg/ml; interfered with siRNA of IL -1 as positive comparison; 27.88±1.30pg/ml.5. The specificity of RNAi was detected by IL - 1 ELISA reagent kit: nega-...