Therapeutic Effects Of Mesenchymal Stem Cells Infected By The RNAi Targeting CTGF Gene Mediated By A Recombinant Lentivirus On Liver Fibrosis In Rats

Part OneEstablishment of a Standardized Liver Fibrosis Model with Different Pathological Stages in RatsObjective:To establish a standardized and stable liver fibrosis model with different pathological stages in rats. On one hand, it was necessary to lay foundation for later experimental procedures of this research, on the other hand, to explore a method to establish a standardized and stable liver fibrosis model creatively would contribute to evaluate other liver fibrosis model in rats.Material and Methods:The Sprague-Dawley (SD) rats were chose as experimental animals and endured intraperitoneal and subcutaneous injections of10%CC14liver oil solution at a dose of1□mL/kg administered twice a week for15weeks. The5%edible ethanol solution was used as the only drink for experimental rats. Five rats in the control group were sacrificed each week from the2nd to the1.5th week, and the blood-related indices(such as hyaluronic acid (HA), laminin (LN), procollagen Ⅲ N-terminal peptide (PIIINP), collagen type IV (CIV), conective tissue growth factor (CTGF) etc.) and pathological sections were detected for hepatic pathological grading analysis. The pathological grading was based on the criteria of Histological Grading and Staging of Chronic Hepatitis for Fibrosis.Results:Based on the mothods above, the regularity that the blood-related indices and the pathological grading changed along with the weeks after experiment can be observed.Conclusion:By taking the method above, the standardized and stable liver fibrosis model with different pathological stages (S0-S4) in rats could be established. Attributing to the chronic lesion induced by taking Low toxicity medicine long-termly and associatedly, the modeling success rate was89.33%, and the death rate was17.3%. Part TwoAmplification And Identification in Vitro of Bone Marrow Mesenchymal Stem CellsObjective:To isolate, culture and purify establish the bone marrow mesenchymal stem cells(BMSCs) in vitro successfully.Material and Methods: The simple adherent method was adopted in isolating and culturing experiments of bone marrow mesenchymal stem cells(BMSCs). The inverted microscope was used to observe cells. The flow cytometry was used in checking cell mark antigens.Results:The morphological characteristics of the major of the BMSCs were unified into polygonal or fusiform, germinated into a spiral shape. Both high expression of CD29、CD71、CD90and low expression of CD11b and CD45were detected by the flow cytometry.Conclusion:The simple adherent method was a feasible way to isolate, culture and purify BMSCs successfully. It layed foundation for later experimental procedures of this research. Part ThreeConstruction And Functional Identification of A Recombinant Lentiviral Vector Mediated RNAi Targeting CTGF GeneObjective:Afer confirming that it was feasible to modify the gene of rats by RNA interference, a recombinant lentiviral vector mediated RNAi targeting CTGF gene was constructed and was used to infect the BMSCs gotten from "Part Two". Then, the cell line infected and labled was cultured and become so stable that it could be indentified and named.Material and Methods:Firstly, whether it was high expression that the CTGF gene of the BMSCs gotten from " Part Two" should be tested by using qPCR method in order to confirm the later experimental plan. Secondly, the most calculated Multiplicity of infection (MOI) was confirmed after infected experiment by means of different MOI tested. Thirdly, everyone of the four interfering plasmids(pcDNATM6.2-X147-1, pcDNATM6.2-X147-2, pcDNATM6.2-X147-3, pcDNATM6.2-X147-4) and the vector with the hign expression of CTGF gene(pCDNA3.1(+)-CTGF) were chosen to infected the HEK293cell line. Fourthly, the most effective plasmid was used to construct the lentiviral vector by means of BPrecombination,LR recombination and gateway technique. Fifthly, the lentiviral vector and packaging plasmid (mix) were also used to infected293T cell line. NEXT, the lentiviral with viral titer determined was harvested after the viral fluid was concentrated by ultrafiltration. At last, to get the stable BMSCs cell line infected by the recombinant lentiviral which contains CTGF gene, GFP gene and Ampicillin resistance gene and to remove the other by means of resistance selection.Results:There were high expressions of CTGF gene in rats. The most calculated MOI value was200. The results of gene sequencing demonstrated that the four interferential plasmids(pcDNATM6.2-X147-1, pcDNATM6.2-X147-2, pcDNATM6.2-X147-3, pcDNATM6.2-X147-4), the vector with the high expression of CTGF gene(pCDNA3.1(+)-CTGF) and the lentiviral vector (pLenti6.3-X147-2) were constructed successfully. The effect of RNA interference confirmed that pcDNATM6.2-X147-2was the most effective plasmid and the interferential efficience was81%. The stable BMSCs cell line infected by the recombinant lentiviral which contains CTGF gene, GFP gene and Ampicillin resistance gene was harvested after resistance selection. Conclusion:The recombinant lentiviral vector mediated RNAi Targeting CTGF Gene (pLenti6.3-X147-2) were constructed successfully. A stable BMSCs cell line infected by the recombinant lentiviral was harvested. Part FourTherapeutic Effect of Bone Marrow Mesenchymal Stem Cells Infected by the RNAi Targeting CTGF Gene Mediatedby A Recombinant Lentivirus on Liver Fibrosis in RatsObjective:To observe the therapeutical impact of bone marrow mesenchymal stem cells infected by the RNAi targeting CTGF gene mediated by a recombinant lentivirus on liver fibrosis in rats and the difference between different pathological stages.Material and Methods:At first, A total of95male Sprague-Dawley (SD) were provided and established into a standardized and stable liver fibrosis model with different pathological stages in rats by means of "Part One". Next, the rats were divided into different parts with different pathological stages. The4groups were draw from different parts at random and designed as following:Group A:received opening abdominal wall, hepatic caudate lobectomy, closing abdominal wall surgery and appendix intravenous injection of lml PBS solution. Group B:received opening abdominal wall, hepatic caudate lobectomy, closing abdominal wall surgery and appendix intravenous injection of1ml BMSCs(without infected by virus) solution(1X107cells/ml).Group C:received opening abdominal wall, hepatic caudate lobectomy, closing abdominal wall surgery and appendix intravenous injection of1ml BMSCs(infected the by lentivirus only with the high expression of EGFP gene sequence and without the RNAi targeting CTGF gene (pLenti6.3-X147-2) sequence) solution(1X107cells/ml).Group D:received opening abdominal wall, hepatic caudate lobectomy, closing abdominal wall surgery and appendix intravenous injection of1ml BMSCs(infected the by lentivirus not only with the high expression of EGFP gene sequence and but also with the RNAi targeting CTGF gene (pLenti6.3-X147-2) sequence) solution(1X107cells/ml).At the end, the animals were sacrificed at7day after operation, the liver and serum specimen labeled with numeral were harvested and analysed as following:(1) To test with fluorescent identification of liver tissue harvested after BMSCs transplantation;(2) To test the serum level of liver fibrosis markers (such as hyaluronic acid (HA), laminin (LN), procollagen Ⅲ N-terminal peptide (PⅢNP), collagen type Ⅳ (CⅣ), conective tissue growth factor (CTGF) etc.) ith enzyme-linked immuno sorbent assay (Elisa);(3) To examine the pathologic change with pathological section of liver tissue and HE staining(4) To examine the alteration of CTGF protein level in histotomy with Immunohistochemistry(IHC);(5) To test the mRNA expression level of CTGF gene with Realtime PCR;(6) To test the protein expression level of CTGF with Western blot.Results:(1) The photo of fluorescent identification of liver tissue harvested after BMSCs transplantation via the portal vein confirmed that the BMSCs could grow in diseased region and repair the damaged tissue.(2) The serum level of liver fibrosis markers (such as hyaluronic acid (HA), laminin (LN), procollagen III N-terminal peptide (PⅢNP), collagen type Ⅳ (CIV), conective tissue growth factor (CTGF) etc.) of the3groups treated with BMSCs decreased obviously. And Group D held the biggest magnitude of the drop than Group B and C.(3) The pathological section of liver tissue and HE staining confirmed that the degeneration cell number in hepatic lobule, the area of focal necrosis, expanding degree between portal regions and the quantity of fiber rope decreased obviously among the3groups:Group D> Group B≈Group C (the magnitude of the alteration).(4) The results of Immunohistochemistry(IHC) demonstrated that the average optical density(AOD) value of the positive area of CTGF protein expression changed from49.53±4.03to31.98±3.53. The variation Reached the significant level(p=0.0083).(5) The test of Realtime PCR confirmed that mRNA expression level of CTGF gene decreased distinctly in Group B and Group C, but no more obviously than Group D.(6) Afer the protein expression level of CTGF was tested with Western blot, it was indicated that the CTGF protein increased with the severity of hepatic fibrosis, decreased obviously after BMSCs transplantation among the3groups:Group D> Group B≈Group C (the magnitude of the alteration).Conclusion:The BMSCs infected by the RNAi targeting CTGF gene mediated by a recombinant lentivirus could grow in diseased region and repair the damaged tissue. Moreover, the method of RNA interference down-regulated the over-expression of CTGF gene dramaticlly evoked by fibrosis likely. These were probable mechanism that the BMSCs infected by the RNAi targeting CTGF gene mediated by a recombinant lentivirus could alleviate liver fibrosis in rats in the test of fluorescent identification, the serum level of liver fibrosis markers (HA, LN, PⅢNP, CⅣ, CTGF, et al), pathologic change, IHC, Realtime PCR, Western blot, et al.