Homing Efficiency Of Mesenchymal Stem Cells Transfected TIMP-1-shRNA Gene Were Enhanced In Rat Infarcted Myocardium

Background: Animal experiments and early clinical studies have shown that intravenous administration of adult bone marrow-derived Mesenchymal stem cells (MSCs) could home to the ischemic myocardial tissue, and improve cardiac function through a variety of mechanisms, thus intravenously administered MSCs may be a new therapeutic strategy for the treatment of myocardial infarction. However, a loss of a large number of stem cells in the process of homing affected stem cell homing therapeutic effect. The molecular mechanisms, however, that control MSC homing which require invasion through extracellular matrix (ECM) barriers are almost unknown. Matrix metalloproteinases (MMPs) were believed to play a pivotal role in invasion behavior of stem cells. Some studies by RNA interference revealed that gene knock-down of MMP-2, MT1-MMP, or TIMP-2 substantially impaired MSC invasion, whereas silencing of TIMP-1 enhanced cell migration. Lentiviral vectors provide an efficient means for TIMP-1-shRNA delivery into MSCs. Accordingly, we investigated whether intravenously transplanted TIMP-1-silencing MSCs can improve the invasion and homing capacity in vivo and in vitro, increase the number of homing MSCs, and further explore stem cell therapy for ischemic cardiomyopathy mechanism.PartⅠConstruction of lentiviral expression vector of TIMP-1-shRNA geneObjective: Designed short hairpin RNA (shRNA), targeted to silence TIMP-1 gene of bone marrow-derived mesenchymal stem cells, constructed lentiviral expression vector carrying TIMP-1-shRNA gene, then dentified and purified packaged plasmid.Methods: Designed and synthesized three pairs for the TIMP-1 gene-specific oligonucleotide, annealed and connected with Pll3.7 vector that was digested by Hpa I and Xho1 enzyme. Plasmids were amplified, extracted, identified by double enzyme digestion and then sequenced. Vectors that have the correct sequence and the lentiviral packaging plasmid were co-transfected 293T cells. At last, collect, purify and concentrate the virus particles which are rich in cell supernatant.Results: Double enzyme digestion and sequencing results showed that three groups of recombinant lentiviral vector of shRNA exactly have a fragment and were consistent with the target chain that were designed and synthesized by our.Conclusion: Successfully synthesized oligonucleotide sequences to silence the rat TIMP-1 gene, and constructed carrying shRNA-TIMP-1 gene lentiviral expressing vector, then packaged by the 293T cells to produce high titer lentivirus particles.PartⅡUsing an HIV-based lentiviral vector to transfect Mesenchymal stem cells to knock-out TIMP-1 gene stablyObjective: Using an HIV-based lentiviral vector to transfect Mesenchymal stem cells to knock-out TIMP-1 gene stably in vitro,then selecting out cell which have the highest efficiency of gene silencing in three cell linesMethods: Three lentiviral expression vector of carrying different shRNA-TIMP-1 gene was transfected into MSCs (SH1, SH2, SH3 group), and set up an empty vector transfection group and negative control group. The morphology of cells was observed in each group before and after cell transfection. After five day culturing, GFP (green) was detected in the fluorescence microscope. Protein was extracted, Western blot was carried out to detect TIMP-1 protein expression levels in each group cells and RT-PCR was carried out to detect TIMP-1 mRNA levels in each group cells,and GAPDH was used as a housekeeping gene.Results: On the lentivirus-transfected MSCs 5th days, strong fluorescence can be detected in each groups, cell morphology did not change before and after transfection. Western blot analysis showed that SH2 group expressed the lowest TIMP-1 Protein levels; RT-PCR results showed that the largest decline in the levels of TIMP-1 mRNA expression could be detected distinctly in SH2 group.Conclusion: Lentiviral vector carrying shRNA-TIMP-1 gene could make MSCs stably to knockout TIMP-1 gene.Cell lines of SH2 group has the highest efficiency of gene silencing in three cell lines.PartⅢSection A Culture of Mesenchymal Stem Cells from Rat Bone MarrowObjective: To establish a method for isolation and cultivation of mesenchymal stem cells (MSCs) from rat bone marrow and to study their phenotypical properties to offerg an experimental foundation for their further differentiation and actual application.Methods: Marrow was isolated from the long bonesof rat by centrifugation and plated in tissue-culture dishes. The proliferation and growth characteristics were observed in primary and passage culture. Cell cycle was analyzed by measuring DNA content with flowcytometer. Epitope analyses were detected by flow cytometry.Results: The adherent, fibroblast-like cells were confluent in single layer after plating for 10～14 days. The cell cycle analysis showed that 89.43% of MSCs was in G0/G1 phase. More than 90% of rat bone marrow-derived mesenchymal stem cells expressed CD29, CD90 and CD44, but not CD34 which was hematopoietic stem cell surface marker. Conclusion: Application of MSCs adherent properties in vitro conditions, rat bone marrow MSCs which were successful isolated, cultured and amplificated can be used to study stem cell homing. It is suggesting that the method can provide cell material for cell homing experiment.Section B A Model of Myocardial Infarction in RatObjective: Myocardial infarction (MI) was induced by left anterior descending artery (LAD) ligation.Methods: MI was induced by ligation of the LAD in 24 rats. Doppler echocardiography was performed to compare myocardial function in two groups of rats. Electrocardiogram (ECG) changes were observed in the operation and postoperative. Pathology was also performed 4 weeks later. Results: Coronary artery ligation resulted in anterior and lateral infarction of the left ventricular wall. The immediate changes visible post ligation were immediately pallor of the myocardium. LVEDd (the LV end-diastolic dimension) in MI increased highly compare to control group (control versus MI: 5.68±0.41 mm vs 7.35±0.67 mm, p<0.01), and the ejection fraction (EF) decreased (control versus MI: 88.36±3.83% vs 49.36±6.25%, p<0.001) in the MI group. Histological tests confirmed that coronary artery ligation resulted in anterior and lateral infarction of the left ventricular wall.Conclusion: This rat model of MI is reliable, reproducible, and similar to the human condition.PartⅣContrast MSCs transfected TIMP-1-shRNA and MSCs in vitro invasive ability and investigate its mechanism in vitro.Objective: Contrast MSCs transfected lentiviral empty vector, MSCs and MSCs transfected shRNA-TIMP-1 gene chemotactic invasive ability in vitro, while detected the gelatin activity in cell culture supernatant, as well as TIMP-1 and MMP-9 expression, and investigated its mechanism affecting the cells invasive ability in vitro.Methods: Using Costar trans-membrane chamber system to study MSCs transfected lentiviral empty vector, P2 generation MSCs and MSCs transfected shRNA-TIMP-1 gene the chemotactic invasion ability in vitro.After culturing in serum-free medium in 5% CO2 at 37℃for 24 h,harvested culture supernatants, detected gelatinase activity and TIMP-1, MMP-9 levels in culture supernatants of different cell lines.Results: Compared with three groups, the invasive ability of MSCs line transfected shRNA-TIMP-1 gene was the highest (P<0.05), gelatin activity of cell culture supernatant also increased, TIMP-1 content of cell culture supernatant decreased, while MMP-9 expression levels increased(P<0.05).Conclusion: MSCs line transfected shRNA-TIMP-1 gene can increase the MMP-9 activity and expression in cell culture supernatant, and increase invasion ability of MSCs. The improvement of MSCs invasion ability in vitro was associated with knockout TIMP-1 gene. PartⅤEffect of Intravenously Injected Marrow Mesenchymal Cells in Rat Infarcted MyocardiumObjective: To investigate whether intravenously injected MSCs improved cardiac function in rat myocardial infarction model, and explore whether the combined application of MSCs and shRNA-TIMP-1 gene in the treatment of MI is feasible, as well as investigate stem cell homing mechanism for the treatment of MI.Methods: A commercially available self-inactivating lentiviral vector has been modified for the delivery of TIMP-1-siRNA into MSCs. MSCs from healthy rat were isolated, cultured and transduced with a lentiviral vector containing either TIMP-1-siRNA and green fluorescent protein (GFP; TIMP-1-siRNA /GFP vector) or GFP alone (control vector).MI model was induced by occluding the LAD in 71 SD rats (230-385g). After the establishment of MI model, survival 56 rats were randomly divided into 4 groups: control group (A group, n = 14), MSCs group (B group, n = 14), GFP-MSCs group (C group, n = 14), and TIMP-1-KD-MSCs group (D group, n = 14). One week later, intravenously injected MSCs or suspensions were performed.The echocardiographic studies were performed to measure cardiac function in the preoperative and postoperative 4 weeks. Intravenously injected MSCs three days later, four rats were killed randomly. TUNEL technology was performed to detect myocardial cell apoptosis. Four weeks later, triphenyltetrazolium chloride (TTC) staining was performed to compare infarct sizes (IS) in each group and assess the number of blood perfusion capillary in the infarct zone and infarct border zone. In addition, ELISA assay were performed to measure serum TGF-β1 content before MI and stem cell transplantation preoperative and postoperative for treatment of myocardial infarction. Finally, the number of MSCs homing to myocardial tissue was measured by flow cytometry.Results: Myocardial infarction was defined by echocardiography as any segmental wall motion abnormality. Left ventricular end-diastolic diameter (LVEDd) increase and contractility decreased significantly after MI (P<0.05). After intravenously injected MSCs, the cardiac function in B, C, D group improved compared with control group (P<0.05), the cardiac function improvement in D group was better than the other three groups (P<0.05). MSCs transplantation can prevent post-infarction ventricular dilatation, increased cardiac contractility.TUNEL test showed: Apoptotic myocytes of B group and C group were no significantly difference (P>0.05), but lower than the control group, higher than D group (P<0.05). Myocardial infarct size of D group, B group and C group were reduced significantly compared with the control group (P<0.05), Myocardial infarct size in D group was smaller than B group and C groups (P<0.05). Compared with the control group, B, C, D group infarct zone capillary density was significantly increased (P <0.05), the number of blood perfusion capillary of D group in the infarct zone was the most highest in B, C, D group (P <0.05). The serum TGF-β1 levels began to increase in post-infarction and gradually decreased after cells transplantation, the level of TGF-β1 of D group decreased significantly Compared with A,B, C group (P<0.05). The number of MSCs homing to myocardial tissue was measured by flow cytometry confirmed D group was significantly higher than the other three groups (P<0.05).Conclusion: MSCs may both migrate and differentiate extensively, might improve the cardiac function by attenuating contractile dysfunction and pathologic thinning in this model of left ventricular wall infarction. This improvement might result from myocardial regeneration and angiogenesis in injured hearts by engrafted cells. Some cytokines maybe play an important role in repairing the ischemic myocardium.TIMP-1 gene knockout increased the MMP-9 activity and promoted the invasive ability of MSCs, and increased the number of MSCs homing to ischemic myocardium, so that the quantitations of MSCs that participating in myocardial repair increased, and the therapeutic benefit was more powerful.Use of lentiviral vectors for delivery of TIMP-1-shRNA enhanced invasion and homing efficiency of intravenously injected MSCs in rat infarcted myocardium, so the strategies will be an effective approach for the treatment of MI.