Effects of Inhibition of the Expression of MMP9in Mice Bone Marrow on Hemangioma Formation
|School||Fourth Military Medical University|
|Keywords||hemangioma vascular endothelial growth factor RNA interference endothelial progenitor cells matrix metalloproteinase-9|
BackgroundHemangioma is the most common benign tumor of infants. Most of hemangiomaoccurs in the relatively superficial places of head and neck, but a few occur in themucosa, muscle, bone tissue and internal organs, which not only affect the appearance,disturb physiological functions but the caused deformity and facial defects bringtremendous mental stress or even psychological barriers to patients as well. Due to ulcers,infection, bleeding, high-flow diverted heart failure or tumors at special body parts, somelesions may threaten life. But at present, its exact pathogenesis still remains unclear andclinical treatment lacks ideal prevention method. Therefore, it is of great significance tostudy the pathogenesis of hemangiomas as well as its efficient and specific preventionand treatment methods.ObjectiveThe expression of MMP9in bone marrow of C57mice was inhibited by RNAitechnology, then the model of hemangioma was constructed via VEGF gene transplant toinvestigate whether hemangioma was formed, and to confirm whether VEGF played arole in the formation of the hemangioma by activating MMP9to mobilize EPCs, whichprovided the basis for efficient specific treatment of hemangioma.Methods1. The siRNA sequences was designed for matrix metalloproteinases9(MMP9) target genes, and the DNA of its short hairpin RNA structure was synthesized. The DNAsequence was cloned into the pSIL-RFP vector, which was cut by Age Ι and EcoR Ιenzyme, and transformed competent escherichia coli cells. It was namedMMP9-shRNA lentiviral vector (pSIL-RFP/MMP9-shRNA). The resultinglentiviral vector contained MMP9-shRNA, and it was confirmed by PCR andsequencing. By western blot, the best siRNA lentiviral vector was certificated and itwas pSIL-RFP/MMP9-shRNA. With best MMP9-shRNA lentiviral vector,pHelper1.0and pHelper2.0, MMP9-shRNA lentivirus was produced, and the titer ofvirus was tested.2. Ten female C57mice,6weeks old, were randomly divided into two groups. For theexperimental group, MMP9siRNA lentivirus was injected into the bilateral femoralmarrow cavity; and the equivalent dose of empty viral vector injected into thebilateral femoral marrow cavity in control group. The expression of MMP9wasdetected by Western blot.3. The lenti-VEGF165-EGFP lentivirus expression vector in mouse was transfected tofibroblasts. The expression of VEGF165were checked out from mRNA level andprotein level which were detected by RT-PCR and Western Blot respectively. Cellswith successful transfection were selected by flow cytometry. The NIH/3T3cellswith successful VEGF165transfection were injected by1x107/ml intogastrocnemius of experimental and control groups respectively. Thus the model ofhemangioma was constructed in mice.4. To observe whether hemangioma occurred. Whether CD31cells participated in theformation of the hemangioma was checked by immunohistochemical method.Results1. It was confirmed that the Oligonucleotide chain of MMP9-shRNA lentiviral vectorwas inserted pSIL-RFP carrier correctly by PCR and DNA sequencing, andMMP9-shRNA lentivirus was successful produced, and its titer was1x1010pfu/l.2. The transfection of MMP9siRNA lentiviral virus was observed in mouse bone marrow, and the result was obtained by Western blot. After gray analysis, it wasfound that the MMP9-siRNA lentiviral virus has inhibition effect on femoral bonemarrow.3. Transfected by Lentivirus VEGF165-EGFP, NIH/3T3cell line with humanVEGF165were sorted out by flow cytometry. It was observed that NIH/3T3cellstransfected by lentivirus emited bright green fluorescence by fluorescencemicroscopy.4. A stable model of hemangioma was made in mice by VEGF165gene transplant.43days after that, formation of new vascular tissue was seen cells implanted area by HEstaining. In the regular or irregular tubular structure, high expressions of CD31cellswere indicated by immunohistochemical result. In the experimental group, noobvious fresh vascular tissue was seen in cells implanted area, and no high expressionof CD31cells was detected according to immunohistochemical result.Conclusions1. The interference lentivirus vector targeting at MMP9RNA was successfullyconstructed. And it inhibited the expression of MMP9in C57mice.2. The NIH/3T3cell line with stable expression of human VEGF165was successfullymade.3. Stable angiogenesis model was established in mice by VEGF165genetransplantation.4. The MMP9was confirmed to play an important role in hemangioma formation.