The Study Of Inhibiting Process Of Rat's Hepatic Febrosis By Matrix Metalloproteinal Inhibitor-1 SiRNA With The Microbubble Of Ultrasound Contrast Agent

Matrix metalloproteinases (MMPs) is one of the most major proteinases to degradate all compositions of extracellular matrix (ECM), 26 kinds, such as collagen enzyme,gelatine enzyme etc. have been discovered. However, 4 tissue inhibitor of Matrix metalloproteinase (TIMPs) are the natural inhibitors to MMPs, TIMP-1,TIMP-2,TIMP-3 and TIMP-4. And they play a very impotant effect on ECM rebuilt and the pathological process of hepatic febrosis by inhibiting the function of MMPs to regulate the balance of ECM, hold-up the process of new production of blood vessels and prohibit the endothelium cells shifting.As the result of disequilibrium between fibre synthetic and degradeation, the hepatic febrocise is a "coalescence reaction"to chronic damage of liver, with the increase of collagen sedimentation and decrease of degradation as its main points, and in such courses, the balance between fibre synthetic and degradation is kept by the disequilibrium between MMPs and TIMPs. As we all known, all negative factors of liver can stimulate hepatic stellate cells (HSC)to become myroblast. So as to the increase of synyhetic and secretion of matrix metalloproteinase inhibitors.TIMPs inhibit the expression of MMPs in two aspects: Firstly, TIMP combine with MMPs steadily to impede zymogen of MMPs activating itself in zymogen activating period; while in the zymogen activated period, it works through TIMP directly combining with activated MMPs in tha rate of 1:1. In liver, TIMP-1, which mainly comes from HSC, can decrease activity of all MMPs except MMP-14 and MMP-19. Especially, its combination with MMP-1 decomposes collagen-I and collagen-â…¢,lead the mesenchymal collagen and FN to substitute for collagenâ…£,â…¤,â…¥,â…©â…£in the febrosis of Disse cavity.The study of treatment to hepatic febrosis is to inhibit collagen sedimentating in one hand; and to increase the degradation of deposited collagen in hepatic sinus in another hand.Inhibiting the expression of TIMP-1 can set free the function of MMPs and make MMPs decompose sedimentated collagen, eventually, contributing to mend the structure-of hepatic fibrosis and provide an effective method to inhibit or reverse the process of hepatic fibrosis.Compared with the technology of antisense oligonucleotides(ODNs), ribozymes, DNAzymes and RNA interference(RNAi) in gene express inhibition, RNA interference brought about a striking effect. And small interference RNA(siRNA) being constructed on the basis of the theory of RNA interference had much more significant effect on inhibiting expression of gene, superior to the sigle strain antisense RNA and antisense DNA, which shows a high activity without toxicity in low content; and the dose-reaction curve shows the IC50 of siRNA to inhibit gene expression by 100 times than that of antisense oligonucleotides in the same target-sequence; RNA interference is of speciality, for the change of sigle nucleotide at middle sit of siRNA will lead to the lose of the efficacy of inhibition, and much similar with all isogenesis gene as the siRNA with same sequence to isogenesis gene; Besides, siRNA keeps its function steadily, the half-life period of it keeps 24 hours in serum,and antisense oligonucleotides will be decomposed in several minutes.RNA interference is post-transcriptional gene silence(PTGS) when the double strand siRNA entered with the same sequence to aim gene cells.The technic of RNA interference makes the double strand RNA(dsRNA) owing 21-23bp decomposed the message RNA with isogenesis sequence to siRNA, which inhibits the process of translation from aim gene, and makes the actived protein lose their function rather than interferes the transcription of gene. In the thesis, we designed and constructed the eukaryotic express lentiverus vectors of TIMP-1 to decompose the message RNA of TIMP-1 to inhibit the process of TIMP-1 translation in order to set free the function of MMPs.Then,the key problem is how to transfer the TIMP-1 siRNA eukaryotic express lentivirus vector to liver in high efficiency with safety so as to realize the inhabitation to aim gene expression steadily.Cytomenbrance is the barrier which inhibits gene molecule to enter into the cell, and is of the passability of prerequisite of gene transferring. Ways making gene enter into cells:l)direct injection: direct injection of gene to muscle can realize the expression of gene effectively since the muscle cell canswallow exoticgene to express relative protein, but it comparatively shows low efficiency in other tissues 2) vein injection:owing to all kinds of enzyme in blood, exoticgene always been degradated, still there are so many difficulties utilizing gene in clinic. To find high efficient method of gene transfecting is also the hotspot of research.The study of ultrasound contrast agent is to improve the diagnosis rate in clinic originally. In a new discovery, the negative electricity on microbubble surface of ultrasound contrast agent is combined with the gene moleculars to produce the microbubble-gene-complexes,and released the gene moleculars when the microbubble broke under the ultrosound irradiated to them.Shock wave produced from microbubble breaking makes blood capillary increase passability and produce sound-aperture (cytomenbrance of endothelium cell appears aperture observed with the electron microcope when soundpower as 0.8W/cm～1.0W/cm), in order to use drugs and realize the transfer and expression of aim gene in the target tissue and cells.Lawrie proved that ultrasound improved the transfer rate of nake plasmid by 100 times, and 300 times when combine with ultra soundcontrast agent microbuble. In addition, the moving of microbubble is observed in realtime through ultrasound image by putting the irradiation to target tissue and broke micobubble in the special period to improve the efficiency of gene treatment.The eukaryotic express lentiverus of TIMP-1 pRNAT-U6.2/Lenti siRNA (TIMP-1 siRNA) is constructed acting on the theory of RNA interference and transference with microbubble of ultrasound contrast agent to hepatic fibrosis rats, the effect of rats in hepatic struction and blood serum element is observed aiming at providing a high efficiency and special target gene and a safety method to transfer and treat hepatic fibrosis.This experiment is divided into four parts.Part 1: Construction and identification of matrix metalloproteinal inhibitor-1 siRNA eukaryotic express vectorMethods1. According the principle of small interference design, two specific sequences were choosen (447- 465nt, 552-540nt) in TIMP-1 mRNA of rat and nonspecific sequence in contrast.2. Synthetise hairpin DNA pieces ,cloned them to vector of pGEM-T, cut pGEM-T and express vector ofpRNAT-U6.2 with BamHI and XhoI enzyme,retrieved these specific piece with sticky terminal then subcloned to pRNAT-U6.2 to construct the eukaryotic express plasmid of TIMP-1 pRNAT-U6.2, identified by double enzyme digesting analysis and DNA sequencing.3. Package three TIMP-1 siRNA plasmids with ViraPower^TM Packaging Mix to construct TIMP-1 pRNAT-U6.2/Lenti lentivirus express vector.4. Plasmids with LipofectAmine^TM 2000 and lentivirue vectors were transfected into T6. Fluorescent expressions of TIMP-1 siRNA plasmid and lentivirus were observed through fluorescent microcope, expression of TIMP-1 mRNA in T6 were evaluated by RT-PCR and full quantitative-PCR.5. Statistical analysis: All the dates were analyzed by SPSS 10.0 statistical package, the count information calculated the positive rate, enumeration date are expressed by standard deviation [(?)±s] The comparison of positive rates uses the Chi-square and Fisher, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variable are analyzed by the correlation analysis.The level of significant difference isα=0.05.Results 1.Specific sequences were inserted into TIMP-1 pRNAT-U6.2 siRNA fit with designed pieces identified by the double enzyme and DNA sequencing, with successfully constructed TMP-1 pRNAT-U6.2/Lenti siRNA eukaryotic expression plasmid and lentivirus vector.2.Content of TIMP-1 pRNAT-U6.2/Lenti siRNA express vector were 1.2～2.8×10^6-7 IU/ml.3.The efficiency to transfect T6 of TIMP-1 pRNAT-U6.2/Lenti siRNA lentivirus vector are higher than that of TIMP-1 pRNAT-U6.2 siRNA plasmid with LipofectAmine^TM 2000((P＜0.05).4.The expression of TIMP-1 mRNA in these groups of TIMP-1 siRNA vector decrease significantly than in nonspecific sequence group and contrast group(P＜0.01);Rates of TIMP-1 gene silence of TIMP-1 (447) siRNA and TIMP-1(552) siRNA plasmid are (78.6±8.72)% and (54.3±5.51)% separately; Rates of TIMP-1 gene silence of TIMP-1(447) siRNA and TIMP-1(552)siRNA lentivirus express vector are (81.2±7.61)% and (66.3±8.37)% separately.Part 2: Construct the modle of hepatic fibrosis of ratsMethods1.Male Wistar rats in SPF ,weight (150±10)g,1% dimethylnitrosa-mine(DMN),abdominal injection,3 times series in first week,2 times series in the second,third,fouth,fifth and sixth week; same dose saline injection at same time.2.After the first injection,random execute 5 rats differently at the first,second,fouth,sixth and eighth week. Sample blood and liver to detect the content ofTIMP-1,MMP-1,HA,LN,CP-â… ,CP-â…¢by ELISA and observe the hepatic structure(classfy criterion of hepatic fibrosis in 1995), and detect collagen in liver by Masson, TIMP-1 protein by immunohistochemistry,TIMP-1 mRNA by in situ hybridization.3.Statistical analysis: Figer analisis use analyse the All the dates were analyzed by SPSS 10.0 statistical package, the count information calculated the positive rate, entimeration date are expressed by standard deviation [(?)±s] The comparison of positive rates uses the Chi-square and Fisher, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variable are analyzed by the correlation analysis.The level of significant difference isα=0.05.Results1.16 days later, ruine of models changed deep, rare drink and food;after 32 days, rats showed bellicose and drowsiness; 4 rats appeared abdominal dropsy after 45 days, and at the 8th week,all appeared abdominal dropsy,2 rats died. Content of TIMP-1,MMP-1,HA,LN,CP-â… ,CP-â…¢trend inserum:2.Content of TIMP-1 raised from the first week after the first DMN injection, and continue to the top at the 4th week;content of MMP-1 locateded peak at second week, gradually decrease to the lowest,but still higher than contrast group ;Content of HA raised by double times at first week,continue to peak, LN gradually increase to peak at the 6th week; CP-â… raise from first week,then continue to the top at the 8th week; CP-â…¢,peak also at the 8th week.3.Expression of TIMP-1 mRNA ( by in situ hybridization) and TIMP-1 protein (by immunohistochemistry)raised from first week, showed great differences to thecontrast group, P＜0.001,continued to peak at the 8th week;Collageb raised gradiuly to 46.3±5.44 at the 4th week ,and showed marked difference to contrast group,to peak at 8th week.4.One week after injection DMN,hepatic cell showed denaturation and drops of necrosis, the inflammation was appeared with circumference and middle of sinus hepaticus little fibre sedimentation;2 weeks later, the fibro interval is formed and part of trabecula appeared drop and piece of necrosis increased fibre sedimentation at circumference and middle of sinus hepaticus;4 weeks later,great part of liver showed denaturation of cells and harmonized necrosis, the fibro interval and confusion of trabecula were structured with S3 of hepatic fibrisis being classified,6 weeks later, classified to S2～3,S4 at the 8th week.Part 3: Effects of TIMP-1 siRNA virus expression vector on blood and liver of Hepatic Fibrosis Rat Methods1. Rats were divided into several groups: TIMP-1 siRNA group ((24 model rats, TIMP-1 siRNA lentivirus vector 20μl+200μl saline),model contrast group (24 model rats,220μl saline),normal group (24 Wister rats, 220μl saline),injection of medicine to penic vein.After 2 days,1 week and 2 weeks, execut 8 rats differently,take blood and tissue of liver.2.Content Of TIMP-1,MMP-1,HA,LN,CP-I,CP-â…¢by ELISA(ng/ml); Observe structure of liver by HE; TIMP-1 mRNA by in situ hybridization: TIMP-1 protein by immunohistochemistry:fluorescent microcope observe the expression of transfection rate of TIMP-1 pRNAT-U6.2/Lenti lentivirus vector.3.Statistical analysis: The SPSS 10.0 statistical package was used for all analyses.Using the Picture analyses system, take 5 regions of positive reaction in every piece, calculated the positive rate. the count information, enumeration date are expressed by standard deviation [(?)±s]. The comparison of positive rates uses the Chi-square and Fisher, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variation are analyzed by the correlation analysis. The level of significant difference isα=0.05.Results1. 507 nm fluorescent imagin under 488 nm fluorescent microcope.2 days later, fluorescent expression of TIMP-1 pRNAT-U6.2/Lenti siRNA lentivirus vector shows a little drops in pieces of liver, heart and kidney in group of TIMP-1 siRNA.2.Executed at the 2th day, expression of collagen,TIMP-1 mRNA and TIMP-1 protein in liver had nondifferency with contrast group differently, P＞0.05;1 week, expression of TIMP-1 mRNA,TIMP-1 protein and collagen in liver decreased much more significantly than that of contrast group, P＜0.05;2 week, expression of TIMP-1 mRNA and TIMP-1 protein in liver reduced markedly than contrast group, P＜0.05;expression of collagen in 2 week showed marked difference with collagen in 1 week, P＜0.05,more difference with the contrast group, P＜0.001.3.Detected by Elisa:2 day,content of TIMP-1, MMP-1 and LN showed much more differences than the contrast group in serum,P＜0.05, expression of HA,CP-â… ,CP-â…¢show nondifferency than contrast group, P＞0.05; 2 weeks, content of TIMP-1,HA,LN,CP-â… ,CP-â…¢in serum deduced significiently with that of contrast group, P＜0.001;content of MMP-1 increased a lot compared with that of contrast group, P＜0.001.Part 4: Effect of TIMP-1 siRNA expression vector on Blood and liver structure under the interference by ultrasound microbubble and ultrasound irradiation on hepatic fibrosis ratsMethods1.Compounded ultrasound contrast agent with TIMP-1pRNAT-U6.2/Lenti lentivirous vector: Sono Vue hexahedron microbubble, diameter 2.5μm, potency (1～5)×10⁸/ml,Rate of TIMP-1 pRNAT-U6.2/Lenti siRNA vector and microbubble is 1:10,put the complexes in refrigerator at 4℃for 30 miniters.2.Rats are divided into following groups:TIMP-1 siRNA (group V): TIMP-1 siRNA 20μl＋200μl saline; TIMP-1 siRNA compound microbubble (group V＋B): TIMP-1siRNA 20μl＋200μl microbubble; TIMP-1 siRNA compound with microbubble＋irradiation on liver (group V+B+U): TIMP-1siRNA 20μl＋200μlmicrobubble,simultaneously irradiate liver in MI 1.0 for 5 mins;model contrast(group C): 220μl saline,all medicine injected to penic vein of rats.Differently executed 8 rats in random at 2 day, 1 week and 2 week,take blood and liver,heart and kidney.3.Detect content of TIMP-1,MMP-1,HA,LN,CP-I,CP-â…¢by Elisa; and observe structure of liver by HE and Masson; TIMP-1 mRNA by in situ hybridization; TIMP-1 protein by immunohisto chemistry; flu orescent microcope is to observe the expression of transfection rate of TIMP-1 pRNAT-U6.2/Lenti lentivirus vector; full quantitative-PCR detect TIMP-1 mRNA of liver.4. Statistical analysis: The SPSS 10.0 statistical package was used for all analyses.Using the Picture analyses system, take 5 regions of positive reaction in every piece, calculated the positive rate. the count information, enumeration date are expressed by standard deviation [(?)±s]. The comparison of positive rates uses the Chi-square and Fisher, the mean of two groups uses the t-test. The mean of more groups use the ANVOA. The relation of two variation are analyzed by the correlation analysis. The level of significant difference isα=0.05.Results1.Transfected 2 days later, the expression of TIMP-1 siRNA fluorescent in group V+B+U exceeds to other groups significantly, P＜0.001;there are scattered green fluorescent expression in group V and group V＋B, and with no nondifferences, P＞0.05;there are expression of branch fluorescent in group V＋B＋U.2.Collagen in liver:2 days later, after injection epression of TIMP-1 mRNA,TIMP-1 protein and collagen in group V＋B＋U deduced much more than the group contrast, P＜0.05;expression of them in group V and group V＋B had nondifference to the group contrast, , P＞0.05;1 week and 2 weeks later, expression of TIMP-1 mRNA, protein and collagen in group V＋B＋U deduced significantly than group contrast, group V and group V＋B, P＜0.001;between group V and group V＋B there aren't differences. P＞0.05.3.ELISA detect:2 days later,expression of TIMP-1 in group V and group V＋B deduced compared with the group contrast, P＜0.05;other data hadn't different to group contrast, P＞0.05; rxpression of MMP-1 in group V＋B＋U increased to the contrast, other data deduced much markedly than the contrast, P＜0.05; 1 week and 2 weeks, content of data in serum was markedly different with group contrast,V and V＋B, P＜0.001; between group V and group V＋B there wasn't markedly differences, P均＞0.05.4.Content of TIMP-1 mRNA in liver by full quantitative-PCR:1 week,interference rate of TIMP-1 mRNA in group V＋B＋U is 73.1%, differently 47.1% and 40.3% in group V and group V＋B; 2 week, inter fereence rate of group V＋B＋U is 85.5%,differently 68.4% in group V＋B and 66.4 %in group V.Conelusions 1. The eukaryotic express plasmid of TIMP-1 pRNAT-U6.2 siRNA and the lentivirus express vector of TIMP-1 pRNAT-U6.2/Lenti siRNA are successfully chosen and constructed.2. The TIMP-1 pRNAT-U6.2 siRNA plasmid and TIMP-1 pRNAT-U6.2/Lenti siRNA lentivirus vector to hepatic stellate cell(T6) are primarily transfected and works had been done to identify the efficiency of silence to TIMP-1. TIMP-1 siRNA eukaryotic express vector inserted 447-470 specific sequence brought about a striking effect to silence the expression of TIMP-1.3. The hepatic fibrosis modle of rats are successfully established by abdominal injection of dimethylnitrosamine (DMN), systematicly observed the change of hepatic structure and dynamic parameters of serums in various period, which provided the experimental models in order to research the hepatic pathological mechanism and hepatic fibrosis treatment.4.The injected TIMP-1 siRNA express vector to penic vein of rats, resulted thatTIMP-1 siRNA silenced the expression of TIMP-1 effectily, increase the content of MMPs, and decrease the content of TIMP-1,HA,collagen-â… .The experiment showed that the febrosis in hepatic tissue decreased and hepatic structure improved.5.That TIMP-1 siRNA lentivirus vector combined with ultrasound contrast agend microbubble to hepatic fibrosis rats in the meantime irradiated the liver in high mechanical index (MI) provided an effective and safety method to improve the expression of TIMP-1 siRNA lentivirus vector to liver.Inhibiting on expression of TIMP-1 could ameliorate the structure of hepatic fibrosis in rats.Creative points(1) The base of hepatic fibrosis is the collagen sedimentation ofextracellular matrix(ECM),but the core is the disequilibrium between MMPS and TIMPs.Previous research of hepatic fibrosis treatment just concentrated the process of initiation of hepatic stellate cell(HSC) but inhibited all initiation factors. In this experiment, specific sequence of TIMP-1 is chosen according to the advanced technology of RNA interference and the new gene express system of pRNAT-U6.2/Lenti is utilized to constructe TIMP-1 siRNA express vector aiming to inhibit the core gene TIMP-1, and try to find a specific target gene to treat hepatic fibrosis.(2) TIMP-1 siRNA letivirus vector is primarily transfected by combining with ultrasound contrast agend microbubble to hepatic fibrosis rats in the meantime irradiated the liver in high mechanical index (MI),which improved the expression of TIMP-1 siRNA lentivirus vector in liver and target aim gene to aim organ, and provided an effective method to treat hepatic fibrosis in safity.