一. Lipid Rafts And The Forward Signal Of Two Types Of TNF-α 二. Construction Of Topic Correlative TNFR1 Gene, Mutants And Mcs Of PTriEx4.0 Vector And Trimerization Mutant

Lipid rafts as special mirodomains reside in biomembrane, rich of lipid, such as cholesterol, sphingolipid and load associated protein, as GPI anchoring protein, caveolin. Lipid rafts are dynamic, floating in disorder cellular membrane lipid bilayer. Due to the distinct biochemical composition and characteristics, they were known as the most active domains for the biological action. Lipid rafts also can provide recruitment platform for protein interaction, playing an important effect in signal transduction and cystoskeleton construction.TNF-αmolecule expresses on the cell surface by means of transmembrane formation (tmTNF-α) and can be catalyzed by TNF-converting enzyme (TACE) to release secretory TNF-α(sTNF-α). The role of amphitypy TNF-αis not identical. Our previous work has confirmed that tmTNF-α-47 site is palmitoylated and a part of tmTNF-αlocalize in lipid rafts. But the relationship between tmTNF-αmediated cytotoxicity and lipid rafts is not clear now. In addition, a part of TNFR1 also localize in lipid rafts. The issue that sTNF-αbind to TNFR1 inside or outside of lipid rafts transmitting the uniform or different signal is also not clear. To clarify the questions, we extract lipid rafts constitution using sucrose density gradient centrifugation and establish lipid rafts destruction model by MCD, studying the contribution of lipid rafts in amphitypy TNF-αsignaling.1. Destruction the lipid rafts of target cells enhanced the sTNF-αmediated killing ability, but inhibited the cytotoxicity of tmTNF-α: Prior experiment showed that destroyed lipid rafts by MCD resulting in sTNF-αcytotoxicity increased, but tmTNF-αcytotoxicity decreased signaificantly. To furher illustrate the changes of two types of TNF-αcytotoxicity were caused by lipid rafts, rather than the impact of MCD itself, we employ another lipid rafts destruction reagent named cholesterol oxidase to treat target cell. The effect was destruction of lipid rafts resulted in the sTNF-αcytotoxicity increasing about 44% (p<0.01), but tmTNF-αdecreasing about 30% (p<0.05), which was same to MCD. It suggests that lipid rafts played a different effect in sTNF-αand tmTNF-αinduced cytotoxicity. Lipid rafts promoted the cytotoxicity of tmTNF-α, but inhibited the lethal effect of sTNF-α. 2. Destruction of lipid rafts inhibits adhesion ability, thereby block tmTNF-αcytotoxicity: We treated HeLa target cells with MCD for 45min and then found the T24 cells (stably transfected tmTNF-α-pIRES2-EGFP) adhering on HeLa cells were decreased obviously, from about one hundred cells per field to about thirty cells. It demonstrates that MCD blocked the cytotoxicity of tmTNF-αbecause of adhesion decline. One of the mechanism lipid rafts mediated the cytotoxicity of tmTNF-αwas the adhesive attraction by the effector-target cells.3. Destruction of lipid rafts made sTNF-αmediated survival signal pathway─NF-κB activity decrease: In order to investigate the changes in sTNF-αcytotoxic mechanism, we first observed the lipid rafts on NF-κB pathway. HeLa cells were treated with MCD and prestimulated with sTNF-α(400ng/ml) for 30min, the level ofΙκB-αwas augmented but P65 in nucleus was decreased which indicated the inhibition of NF-κB activity. These data showed that destruction of lipid rafts weakened the NF-κB mediated survival signal and enhanced the apoptosis signal. One of the mechanisms that lipid rafts inhibited the lethal effect of sTNF-αwas the activity of NF-κB by means of promoting survival signal.4. Disruption of lipid rafts made sTNF-αmediated internalization of TNFR1 increase: sTNF-α-TNFR1 interaction in addition to activatied NF-κB pathway, but also mediated apoptotic signaling. The TNFR1 internalization was principally responsible for the sTNF-αmediated cytotoxicity. HeLa cells transfected with TNFR1 increased killing rate of sTNF-α, about one fold (p<0.01), but transfected with Y236A-TNFR1 which failed to endocytosis had no significant change as compared with control, which was only 19.3% (p<0.01). These results indicated that cytotoxicity of sTNF-αwas concerned with TNFR1 internalization. THP1 cells were pretreated with MCD and then stimulated by sTNF-α. FCS detected the TNFR1 expression of THP1 suggesting that disruption of lipid rafts resulted in the decrease of TNFR1 expression on THP1 cells. Moreover, Western Blot showed the MCD had no effect to the total expression of TNFR1. It was shown that the augmentation of internalization is responsible for the reduction of TNFR1 expression. Fluorescence microscope detection confirmed this result that sTNF-αstimulated the 293T cells transfected with TNFR1-pEGFP-N1 and pretreated with MCD inducing the increased internalization of TNFR1. It intensively indicated the main reason for disruption of lipid rafts resulting in the increased cytotoxicity of sTNF-αwas the increased internalization of TNFR1 and thereby strengthening the apoptosis signal of sTNF-α. The second mechanism that lipid rafts inhibited the lethal effect of sTNF-αwas the decrease TNFR1 internalization by means of weaken apoptosis signal.5. Two types of TNF-αrecruitment different molecues to lipid rafts: Using sTNF-αand tmTNF-αstimulated HeLa cells and then we extracted the structure of lipid rafts and identified that the sTNF-αcan induce the aggregation of TRADD,RIP and TRAF2 to lipid rafts. But tmTNF-αcan only recruit TRADD,FADD and Caspase-8 to lipid rafts. These molecules distributed outside of the lipid rafts in non-stimulation. sTNF-αused lipid rafts as a platform to recruitment anti-apoptosis compound TRADD-RIP-TRAF2 transmiting anti-apoptosis signaling. And tmTNF-αused lipid rafts as a platform to recruitment apoptosis compound TRADD-FADD-Caspase-8 transmiting apoptosis signaling. The third mechanism that disruption of lipid raft resulting the augmented cytotoxicity of sTNF-αwas that the anti-apoptosis signal compound failed to form in lipid rafts.But tmTNF-αmediated apoptosis signal in lipid rafts. It can explain disruption of lipid raft induced the decreased cytotoxicity of tmTNF-α.Conclusion: Lipid rafts reacting as a signal transduction platform played a different role in two types of TNF-αctyotoxicity. Lipid rafts enhancemented the cytotoxicity action of tmTNF-αto target cell. The mechanisms were: lipid rafts participated in adhesion of effector-target, thereby promoted the tmTNF-αlethal effect; simultaneously, tmTNF-αcan recruitment apoptosis signal compound TRADD-FADD-Caspase-8 to lipid rafts to mediated apoptosis and killing signal. Lipid rafts inhibited sTNF-αlethal effect, the mechanisms were: on the one hand, lipid rafts induced sTNF-αmediated survival signal─NF-κB activity up-regulate; on the other hand, lipid rafts made sTNF-αmediated apoptosis signal─TNFR1 internalization decrease. And sTNF-αrecruitment anti-apoptosis signal compound TRADD-RIP- TRAF2 to lipid rafts to mediate anti-apoptosis signal, thereby inhibited the cytotoxicity of sTNF-α. 一. Construction of TNFR1 and mutants recombinant plasmidTumor necrosis factor receptor includes TNFR1 and TNFR2, whose relative molecular mass is 55kD and 75kD respectively. Only when TNF-αcombine with amphitypy TNFR, can play biological effect. sTNF-αmediates cell survival or apoptosis through TNFR1 cytoplasmic domain recruitmenting different signal complex. tmTNF-αcan also induce cytotoxicity by TNFR1, but tmTNF-αis type II protein anchoring in membrane, once cell and cell come into contact, killing is then mediated. So tmTNF-α/TNFR1 interactions induce apoptosis signaling pathways may be different from sTNF-α.The project is planned to study TNFR1-mediated tmTNF-αapoptosis pathway through the TNFR1 cytoplasmic area consisting of internalization district (TRID), neutral sphingomyelinase district (NSD) and the death domain (DD). We obtain the whole length TNFR1 gene and TNR1 mutants by RT-PCR and overlapping PCR respectively. The mutant Y236A-TNFR1 is inhibition of internalization,ΔNSD-TNFR1 is lack of neutral sphingomyelinase andΔDD-TNFR1 is missing death domain. Then clone the full-length TNFR1 and mutants into pEGFP-N1 vector and transfect into 293T cells, doing the cell biology experiments subsequently.二. Reconstruction of pTriEx 4.0 vector multiple cloning sitesIn our scientific research practice, we need transfer the same gene fragment to vectors of different function and characteristics frequently. Using the traditional methods of molecular biology, every time when we need to replace the vector for another, we should experience the whole cloning process, including PCR, endonucleases digestion, connectivity, transformation, identification and DNA sequencing analysis. In this process, PCR primer synthesis and DNA sequencing is time consuming, and PCR amplification of purpose gene, may appear base mutation and deletion, which will easily lead to the failure of molecular cloning. It is undoubt that this will increase the workload and influence follow-up experiments carrying out smoothly.In order to solve the problem transferring gene fragments into different vectors conveniently, we design a set of programme for vector reconstruction. The basic strategy is: replacing the multiple cloning sites (MCS) of pTriEx 4.0 with pEGFP-N1 MCS. Because the MCS of pEGFP-N1 is too short to recycle, we insert a 238bp fragment into it at the EcoRI site. After digesting the 238bp fragment, the vector was successfully constructed. This topic is tmTNF-αand TNFR1 apoptotic signaling upon bingding to pass in-depth study provides clues to the tmTNF-αand clinical application of basic research has important theoretical and practical value.三. Construction ofΔ115-118-tmTNF-α-pIRES2-EGFP-XhoI recombinant plasmidTumor necrosis factor alpha (TNF-α), a pleiotropic cytokine with wide biological activitiy, is mainly produced by monocytes/macrophages cells. TNF-αis one of the tumor necrosis factor superfamily (TNF-SF) members, which have different biological funcionns. But the amino acid sequences 46-58,119-130,150-157 of the members in protein molecule primary structure are homologous (highly conserved domain). Moreover, they must be in the form of homologous trimer to play biological functions through interacting with TNF receptors. Preliminary work has confirmed that trimeric formation is relevant to highly conserved amino acids sequences. So we further conjecture neighbouring anmio acids of conserved sequences whether influence TNF-αto shape trimer. In this study, mutant TNF-αwas amplified through recombinant ploymerase chain reaction (PCR) from tmTNF-α-pcDNA3.0 by site-directed deletion of 115-118 position. Then the recombinant plasmidΔ115-118 tmTNF-α-pIRES2-GFP-XhoI was constructed and transfected into 293T cell lines, which expressed mutant TNF-αon the membrane surface. We investigated the changes of the cytotoxicity of mutant TNF-αcompared with wide TNF-α, which will give supports for further study of the relationship between the structure and fuction of tmTNF-αand its clinical application in the future.The main results are as follow:1. Construction and verification of TNFR1 and mutants recombinant plasmid1.1 Construction of recombinant plasmid: Using the cDNA of THP1 as template, the TNFR1 gene was amplified by ploymerase chain reaction (PCR). After digestion with XhoI and BamHI endonucleases, the TNFR1 fragment was inserted into pEGFP-N1 plasmid by T4 DNA ligase. Then the recombinant TNFR1-pEGFP-N1 was transformed into E.coli DH5αand the positive clones were screened by kanamycin resistance. Using TNFR1-pEGFP-N1 plasmid as template, we obtained the Y236A-TNFR1-pEGFP-N1,ΔNSD-TNFR1-pEGFP-N1 andΔDD-TNFR1-pEGFP-N1 mutants by overlapping PCR.1.2 Verification of positive clones: The positive clones were confirmed by colony PCR and restriction enzyme digestion, showing the cleaved fragment from the recombinant plasmid with the expected molecule magnitude. DNA sequence analysis proved that there were no other point mutations, frame-shift or deletion mutations but expected ones.2. Reconstruction of pTriEx 4.0 vector2.1 Construction and identification of pEGFP-N1/238 recombinant: Using the plasmid tmTNF-α-pcDNA3.0 as template, fragment of 238bp was amplified by PCR and EcoRI restriction enzyme site was introduced at the same time. After digestion with endonucleases, the 238bp fragment was inserted into MCS of pEGFP-N1 plasmid by T4 DNA ligase. The recombinant was named pEGFP-N1/238 and transformed into E.coli DH5αand the positive clones were screened by kanamycin resistance. The positive clones were confirmed by colony PCR and EcoRI digestion, showing the cleaved fragment from the recombinant with the molecule magnitude of 238bp, suggesting plasmid pEGFP-N1/238 was successfully constructed.2.2 Construction and identification of pTriEx 4.0/MCS-238 recombinant: Using the pEGFP-N1/238 plasmid as template, MCS-238 was amplified by PCR. NcoI and EcoR81I restriction sites and His tag in downstream primer only were introduced. After digestion with NcoI and EcoR81I, MCS-238 was inserted into MCS of pTriEx 4.0 plasmid by T4 DNA ligase. The recombinant was named pTriEx 4.0/MCS-238 and transformed into E. coli DH5αand the positive clones were screened by ampicillin resistance. The positive clones were confirmed by colony PCR were confirmed by colony PCR and EcoRI digestion, showing the cleaved fragment from the recombinant with the molecule magnitude of 350bp, suggesting pTriEx 4.0His/MCS-238 plasmid was successfully constructed.2.3 Digestion of 238bp with EcoRI and construction of pTriEx4.0His/N1: pTriEx 4.0His/MCS-238 was digested by EcoRI, than connected by T4 DNA ligase. The recombinant was transformed into E. coli DH5αand the positive clones were screened by ampicillin resistance. The positive clones were confirmed by colony PCR. DNA sequence analysis showed that MCS of pEGFP-N1 successfully replaced the MCS of pTriEx 4.0.3. Construction ofΔ115-118 tmTNF-α-pcDNA3.0 /pIRES2-EGFP-XhoI recombinant plasmid3.1 Construction ofΔ115-118 tmTNF-α-pcDNA3.0 recombinant plasmid: Using the plasmid tmTNF-α-pcDNA3.0 as template, the mutant TNF-αwas amplified with overlapping PCR. After digestion with endonucleases, the mutant TNF-αfragment was inserted into pcDNA3.0 plasmid at BamHI and XhoI by T4 DNA ligase. Then the tmTNF-α-pcDNA3.0 recombinant plasmid was transformed into E. coli DH5αand the positive clones were screened by ampicillin resistance.3.2 Identification of positive clones: The positive clones were confirmed by colony PCR, and endonucleases digestion, showing the cleaved fragment from the recombinant plasmid with the molecule weights of 690bp.DNA sequence analysis also proved that there were no other point mutations, frame-shift or deletion mutations but expected ones.3.3 Construction ofΔ115-118 tmTNF-α-pIRES2-EGFP-XhoI recombinant plasmid: DigestΔ115-118 tmTNF-α-pcDNA3.0recombinant plasmid with endonucleases BamHI and XhoI simultaneously and digest pIRES2-EGFP-XhoI plasmid with Bgl II and XhoI sequencely. Then theΔ115-118 tmTNF-αfragment was inserted into pIRES-EGFP-XhoI plasmid at Bgl II and XhoI by T4 DNA ligase. Then theΔ115-118 tmTNF-α-pIRES2-EGFP -XhoI recombinant plasmid was transformed into E. coli DH5αand the positive clones were screened by kanamycin resistance.3.4 Identification of positive clones: The positive clones were confirmed by colony PCR, and endonucleases digestion with NheI and XhoI, showing the cleaved fragment from the recombinant plasmid with the molecule weights of 690bp.In this study, we obtained TNFR1 and its mutants by PCR and successfully replaced the multiple cloning sites of pTriEx 4.0 with pEGFP-N1 MCS, providing important experimental tools and instruments for the following biological function research.