EMILIN3Gene Stimulates Human Mesenchymal Stem Cells Proliferation And Differentiation Into Cartilage Cells

Research background:Repair ability of articular cartilage defect is limited, which leads to irreversibledamage in structure and function. At present, the treatment of articular cartilage defect inthe clinical is the bone marrow stimulating and cartilage transplantation, the former effectgradually decreases with time; the latter material source is limited, and allogeneic tissueshave immunogenicity. With the development of biomedical engineering, autologouschondrocytes with the same function, no immune rejection, are used as seed cells of tissueengineering cartilage. But the amplification ability of chondrocytes is limited in vitro, andmaterial sourcer has great limitations. Because human mesenchymal stem cells (hBMSCs)have multidirectional differentiation proficiency, strong proliferation ability, and noimmune rejection, hBMSCs are widely used as the ideal seed cells in tissue engineering.The differentiation of hBMSCs into cartilage cells need specific inductionenvironment, and especially needs certain growth factors to ensure the migration,proliferation, survival of hBMSCs. At present, there have been many previous studieswhich have identified some stimuli or signal molecules controlling their migration,proliferation (PDGF-B FGF-2), and cartilage differentiation (TGF-beta1, TGF-beta3,IGF-I)[7-11]. EMILIN3is the member of EMILIN gene families (EMILIN1EMILIN2EMILIN3),which is a glycoprotein of the extracellular matrix belonging to a family thatcontains a characteristic N-terminal cysteinerich EMI domain.Mitsuhito Doi[12]found thatEMILIN3was high expression in perichondrium and around the development of thecartilage, but no expression in the mature osteoblast When hBMSCs were induced tocartilage in vitro, with the extension of time EMILIN3expression was increased. Wespeculated that EMILIN3gene may play an important role in the skeleton development.Research purposes:1. The adenovirus with EMILIN3gene was constructed. To explore the effects ofEMILIN3gene on hBMSCs proliferation. 2. To investigate the effects of EMILIN3gene on hBMSCs differentiation intocartilage cells and to provide a new basis for cartilage repair.Research methods:1. HBMSCs were isolated by density gradient centrifugation and detected by flowcytometry.2. EMILIN3gene was amplified by the polymerase chain reaction (PCR), thenrecombinated into adenovirus expressing plasmid. The positive clones was identified byPCR, and then sequenced by sequencer. EMILIN3protein expression was detected byWestern bloting. Adenovirus plasmid and the packing plasmid were amplified in the E.colion virus. Adenovirus was packed in293T cells, and then adenovirus was purified by CsCldensity gradient centrifugation. The drop of adenovirus was determinated after theadenovirus infection into293T cells.3. The hBMSCs were cultured in96-well plates, and the hBMSCs were infectedrespectively with MOI=10,50,100. After48h, choose the proper MOI for the subsequentexperiments.4. The cell proliferation of hBMSCs with EMILIN3protein overexpression determinedby MTT.5. The expression of Collagen II, Aggrecan and SOX9mRNA was detected byreal-time PCR in hBMSCs with EMILIN3protein overexpression and induced tochondrocytes.6. The expression of Collagen II, Aggrecan and SOX9proteins was detected byWestern bloting in hBMSCs with EMILIN3protein overexpression and induced tochondrocytes.Results:1. hBMSCs with high purity could be achieved by density gradient centrifugation.2. EMILIN3gene with2336bp was amplified by the polymerase chain reaction (PCR).EMILIN3gene which was inserted into the adenovirus expression vector was proved tobe correct through PCR and sequencing. EMILIN3expression plasmid has positive band inabout100KD by western bloting. Adenovirus droplets detect kit results showed controladenovirusis droplets was5x1010ifu/ml, and EMILIN3virus droplets was1.4x1011ifu/ml. 3. The fluorescence microscope results showed that it was best when adenovirusinfected hBMSCs in MOI=100.4. MTT experiments showed the proliferation ability was arrested in the hBMSCsinfected by the control adenovirus compared with the pure hBMSCs group, but there wasno difference. the proliferation ability was increased in the hBMSCs infected by theadenovirus with EMILIN3gene than that in the hBMSCs infected by the control adenovirusand the pure hBMSCs group(p<0.05) in4,5and6days.5. The expression of CollagenII, Aggrecan, SOX9mRNA was increased about2.5times、2.7times、2.1times in the hBMSCs infected by the adenovirus with EMILIN3genethan that in the hBMSCs infected by the control adenovirus (p<0.05). Compared with thepure hBMSCs group, there was no difference in the expression of CollagenII, aggrecan,SOX9mRNA in the hBMSCs infected by the control adenovirus.6.The expression of CollagenII, Aggrecan, SOX9proteins was increased about0.75times、0.74times、0.40times in the hMSCs infected by the adenovirus with EMILIN3genethan that in the hBMSCs infected by the control adenovirus (p<0.05). Compared with thepure hBMSCs group, there was no difference in the expression of CollagenII, aggrecan,SOX9proteins in the hBMSCs infected by the control adenovirus.Conclusion:1.Higher purity of hBMSCs could be isolated and cultured by combining densitygradient centrifugation with plastic adherence.2.PCR and Western bloting results showed adenovirus with EMILIN3gene is correct.3.The infection hBMSCs result was best by adenovirus with MOI=100, and follow-upexperiments carried out in accordance with the values of the MOI.4.EMILIN3gene increased hBMSCs proliferation5.EMILIN3gene stimulated hBMSCs differentiation into cartilage cells. It mayprovide new strategy for cartilage repair.