The Effect Of FGL2Gene Silence On Rat Myocardial Microvascular Endothelial Cells Apoptosis And Related Molecular Mechanism Involved

ObjectiveThe expression of Fibrinogen Like Protein2gene in myocardial microvascularendothelial cells is silent specially. Then, we will observe changes of cell morphologyfunction, as well as proliferation and apoptosis ability to explore the mechanism offgl2silence in MMVECs.Methods1. Isolate and foster the MMVECs then identified the cell. MMVECs wereisolated, fostered and identified. We used health and newborn6⁸male wistar ratwhich the weights were between8¹0g. MMVECs were cultured by the method oftissue-sticking. MMVECs had grown in primary monolayer, then third-generationMMVECs were identified by factor VIII and CD31related antigen, which wereimportant markers of MMVECs.2. The nucleotide sequences of hfgl2(NM₀06682) were found in the GeneBank. The four target sequences of FGL2gene could be effectively silent accordingto RNA interference principle. The cDNA containing both sense and antisense OligoDNA fragments of target sequences were designed and cloned into the pGCSIL-GFPvector which was digested by AgeI/EcoRI. The obtained lentiviral vector containingFGL2shRNA was confirmed by digestion and sequencing. Using lentiviral vectors totransfect293T cells, then the lentivirus were collected. The titer of virus was testedaccording to the expression level of GFP. We used Western blot to select the effectivenucleotide sequences. The DNA segments were synthesized by Shanghai GenechemCompany.3. Groups of the experiment: Third-generation MMVECs were divided to threegroups. A: MMVECs without any treatment (control group); B: transfected theMMVECs with empty lentiviral vector; C: FGL2RNAi lentiviral vector weretransfected into MMVECs. When transfection was stable after four days, the mRNAof Bax, Bcl-2, Cox-2was detected by quantitative real-time RT-PCR (qRT-PCR). TheWesternblot was used to detect the FGL2expression of C group. MTT was used to find the difference of cell proliferation among three groups. And cell apoptosis wasdetected by Flow Cytometry (FCM).Results1. Primary MMVECs were cultured successfully, the structure of MMVECspresented typical paving pebble-like, and the purity was about95%.2. Immunofluorescence test showed the positive expression of CD31and FVIIIin upper90%cells. CD31was mainly in the membrane, showing red fluorescence.However FVIII was found in the cytoplasm, showing green fluorescence.3. DNA sequencing demonstrated that lentivirus vector FGL2RNAi wasconstructed successfully. The lentivirus vector tilter was1×109TU/ml tested by seriesdilution method. Transfecting lentivirus vector FGL2RNAi after96hours,70-80%MMVECs expressed green fluorescence. MTT and FCM test showed group C had thegreatest proliferation among the three groups, and cell apoptosis reduced significantly.qRT-PCR revealed the expression of FGL2, Bax and Cox-2in group C were sharpdown, and Bcl-2increased among the three groups. The Westernblot demonstratedFGL2protein was down-regulated obviously in group C(P<0.05). But there was nostatistical differences between A and B group.Conclusion:Primary MMVECs were cultured successfully, the expression of FGL2mRNAand protein reduced significantly after transfected lentivirus vector FGL2RNAi, theexpression of Cox-2and Bax mRNA were down-regulated; however, the Bcl-2mRNA expression increased. The cell proliferation and anti-cell apoptosis wereaugmented after lentivirus vector FGL2RNAi transfection in MMVECs. The possiblemechanism was that Bcl-2upregulation promoted apoptosis factor Bax expressionafter FGL2gene silence, also inhibited inflammation factor Cox-2expression, andtherefore inhibited cell apoptosis.So interfering FGL2gene by RNAi might provide anew strategy for anti-cell apoptosis therapy.