Study On The Construction Of Sirna Eukaryotic Expression Vector For Tgf-β1 And Its Transfection Into A549

BackgroundPulmonary interstitial fibrosis is a diffused disease of pulmonary caused by many factors, it is a heterogeneity disease as well as the common end of the pulmonary disease. its pathogenesy involved the interaction of multiple factors, such as proteolytic ferment,clllagen metabolism,immunologic cell,cell factors,free radical damage and apoptosis.Transforming growth factor -β1 received as the key cell factor shaped the pulmonary fibrosis. It is play an important part in the development of pulmonary fibrosis as a multifunction and superactive cell factor, it can refulation the hyperplasy,differentiate and the extemalization of extracellular matrix, it is the most immediate cell factor in pulmonary fibrosis. So the specificity treatment on account of the TGF-β1 has a wide development prospect in cure of pulmonary fibrosis. we can get important information for therapy strategy by the study of TGF-β1. Maybe one day it can become the important target in the cure of Pulmonary interstitial fibrosis.RNA interference is a new molecular biology technology, it can import the corresponding plus sense RNA and antisense RNA which is called double strand RNA to induce specificity target mRNA degradation in the recipient cell had the homologization sequence, and then to prevent the translation process of protein and make its homologous genes silencing. RNA interference widespread reside in living nature, from prokaryotic organism to plants,true fungus,invertebrate as well as mammal and its mechanism is more complex.Recent study found that, small interfering RNA is the important intermedial effector molecule in RNAi, it is a effective therapeutic tool to silent the gene expression by the use of small molecular substance. Many studies now create original therapeutic drug in use of the molecular substance, small interfering RNA maybe become the new member of these medicines. The study and utilize of small interfering RNA and RNAi have vital important theory and practical meanings.ObjectiveTo construct the specific small interfereing RNA(siRNA) eukaryotic expression vector that can block the rat TGF-β1 gene according to the mRNA of rat TGF-β1,then transfect to eukaryotic cell (A549) and observe the expression of it for the therapy of pulmonary interstitial fibrosis by gene silencing technique in post-transcriptional level.Materials and methods1. The siRNA target sequences of 339 and 1965 sites were designed by using the siRNA software according to the mRNA of rat TGF-β1, then two pieces of DNA sequences with short hairpin RNA (shRNA) structure were synthesized and annealed to clone into the pSilencer^TM 2.0-U6 Expression Vector. The recombinants were evaluated by using enzyme cutting and sequencing analysis.2. After the base sequence was confirmed by sequence determination, extracted the plasmid of recombinate expression vector TGF-β1siRNA-pSilencer^TM 2.0-U6 and transfected it into A549 by liposome, then observed its expression in eukarya cell by flurescence.3. compared with the blank control group and the empty vectors control group (pSilencer^TM 2.0-U6 vector without siRNA),extract the mRNA in eukarya cell transfected with recombinate expression vector TGF-β1 siRNA -pSilencer^TM 2.0-U6,The mRNA expression of TGF-β1 in A549 were detected by reverse transcription-PCR. Result1. Two exons in 339 and 1965 situs of TGF-β1mRNA were ensured, which were 5'-GTACTTCACCAACTGCAAG-3' and 5'-AAACAATGTGGTGAGT GTC-3', two pieces of DNA sequences with short hairpin RNA (shRNA) structure were synthesized according to its target sequence.2. After evaluated by using enzyme cutting, we can get linearity pSilencer^TM 2.0-U6 gene fragment with BamHⅠand HindⅢsitus located in about 2000bp ,and the hairpin siRNA purpose gene in about 100bp.Theory and experimental result was conformity.3. Recombinant plasmid was send to biological engineering company of Shanghai to one-way sequencing, the result showed that TGF-β1 siRNA sequence cloned into pSilencer^TM 2.0-U6 expression vector was completely consistent with designed.4. The experimental group can obviously down- regulation the mRNA expression of TGF-β1(P<0.01) compared with the control group has little change (P>0.05) after them respectively transfected into the A549,especially the 339 siRNA has the higher silencing effect(P<0.05).Conclusion1. The siRNA oligonucleotide liked hairpin target TGF-β1 gene was successfully designed.2. The eukaryotic express vector of TGF-β1siRNA-pSilencer^TM 2.0-U6 was constructed successfully.3. The 339 siRNA which can highly inhibit the expression of TGF-β1 gene was screened successfully, which make the foundation of inhibit pulmonary interstitial fibrosis rat by using the RNA interference technique and will become the new clinic therapy target in the future.