| Background and purpose:Empirical study showed that adipose cell proliferation and hypertrophia in marrow of the head of femur at the alcoholic head of femur necrosis induced adipose cell accumulation and induced osteocyte degenerated and expired for lipidoses. The gene of PPARγwas an adipogenic transcription factor, which could not express in preadipocyte, but it could express in preadipocyte-to-adipocyte differentiation, and it could express preceding other adipocyte genes. Alcohol could induce adipogenic regulator PPARγmRNA high expression in fibroblast of MSCs NIH3T3, induce cells differentiate into lipoids in a great quantity and restrain cells differentiate into bone. This was an original pathogeny of alcoholic osteonecrosis. PPARγplayed a critical regulate effect in the process of differentiation MSCs to adipocyte, and it was an important target gene in alcoholic osteonecrosis. So we used the technology of RNAi, the gene which interfered the expression of PPARγwas brought into stem cells in vitro or body or the targeted cells transfected gene which interfered the expression of PPARγwere implanted into alcoholic osteonecrosis, it would persistently interfere the expression of the gene of PPARγin the alcoholic osteonecrosis tissue and promote prevention and treatment of alcoholic osteonecrosis. There were two retroviral vectors expressing the PPARγshRNA and established substantial foundations for implementing the gene therapy to alcohol osteonecrosis.Methods:1. Select the destination fragments The destination fragments selected were located at 497-515 codon and 677-695 codon (GenBank Accession AF013266), we designed siRNA sequences with http://www.ambion.com/techlib/misc/siRNA_finder.html soft program, and then designed double strands hairpin structure with siRNA Hairpin Oligonucleotide Sequence Designer software. There was Bam H I at the 5'end, Cla I at the 3'end in the double strands, There were adhesive ends that the two enzyme incid sites. The 19 nucleotides were used to form hairpin loop structure. There was a TTTTTT terminal signal near the 3'end, which was of the U6 promotor.2. The two retroviral vectors pSINsi-hU6-PPARγcontaining PPARγgene had been constructed.Quadri-oligonucleotide bands composited were annealed into two double strands. The two annealed double strands were connected with pGEM-T vector. We runned PCR amplification with T7/SP6 universal primer, extracted plasmid and sequenced bar DNA. We connected the 497 DNA and 677 DNA which had the BamH I and Cla I sticking terminals with plasmid pSINsi-hU6 which had the BamH I and Cla I sticking terminals too; We identified the colonies with PCR amplification with the primer PPA1/PPA2;We incised and identified the product with BamH I HindIII, and Cla I HindIII either, definited the size of insertion element.3. The two retroviral vectors suppressed the expression of the gene of PPARγ.The two retrovirus vector expressing siRNA was transfected into the EC9706 cells, according to the description of transfection agent; 48 hours later, we collected EC9706 transfected cells. Total the transfected cellular RNA was isolated, and RT-PCR was applied to detect cellular PPARγmRNA.Consequence:1. Identification of retrovirus vector pSINsi-hU6-PPARγwith PCR. The size of part was 510bp or so.2. The retrovirus vector pSINsi-hU6-PPARγwere identified by inciding the sites with BamH I, HindIII and Cla I, HindIII either, there were twelve electrophoresis strips with 350bp, 1100bp, 5000bp and 430bp, 1100bp, 5000bp.3. Sequencing of cloning vectors pGEM-497 and pGEM-677 were at equal pace with sequences which we designed completely.4. After the EC9706 was transfected pSINsi-hU6-PPARγ, 48 hours later, the EC9706 transfected cells did not express PPARγmRNA, verified by RT-PCR. They could explain that the expression of PPARγof the EC9706 transfected cells was suppressed completely, and we had achieved the purpose of the experiment.Brief summary:1. PPARγcDNA was correctly cloned;2. The retrovirus expression vector pSINsi-hU6-PPARγwas constructed successfully.
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