| Objective:Articular cartilage injury,one of the most common age-related degenerative diseases,has a high incidence worldwide.The autologous regenerative capacity of articular cartilage is relatively weak,and cartilage injury often leads to osteoarthritis.Due to internal reasons such as difficulty in obtaining progenitor cells,lack of blood supply in surrounding tissues after cartilage injury,and weak ability of articular cartilage cells to migrate and produce extracellular matrix,cartilage tissue after injury is different from other tissues in human body and lacks sufficient regenerative capacity.To solve this dilemma,cell transplantation technology can provide new methods and ideas for cartilage regeneration.The ideal seed cells to be transplanted and repaired should not only have a wide range of sources and a large number of cells,but also be able to reproduce vigorously and expand their size in a short time.In addition,the seed cells can secrete enough extracellular matrix to complete the corresponding biological functions.So far,there are two kinds of seed cells that are widely used: one is the fibroblast,but the source of the cell is less abundant,small in size,and more likely to decay as they grow.The second is the bone marrow mesenchymal cell,which is also small in size and weak in differentiation,unable to meet the needs of bioremediation.Adipose Stem Cells,abbreviated as ADSCs,have a wide range of sources,high survival rate and strong differentiation ability.Under certain conditions,it can differentiate into chondrocyte,tendon cells and so on according to people's needs.In addition,adipose stem cells will be an important source of seed cells for repairing cartilage injury due to their high productivity and good anti-aging performance.Until now,the application of adipose stem cells in cartilage injury repair has not achieved breakthrough research results,because the cartilage injury site cannot produce the substance that makes adipose stem cells differentiate.Currently,special cellular material is needed to make adipose stem cells differentiate into chondrocytes and repair damaged cartilage tissue.Bone morphogenetic protein-7,abbreviated as BMP-7,is a cellular substance belonging to the TGF-?series.Although it does not directly stimulate the formation of cartilage tissue,it can cause bone marrow mesenchymal cells to differentiate into cartilage tissue.There are experts and scholars pointing out: BMP can improve the biological properties of?type collagen and elastin,and plays an important role in improving the stress tolerance of cartilage tissue.In this study,the in vitro gene transfection of ADSCs with bone morphogenetic protein-7(BMP-7)mediated by liposome vector is used as the treatment strategy.The BMP-7 gene is introduced into rat adipose stem cells(ADSCs)and implanted into animal models of cartilage injury in rats to observe the effects of cartilage repair.This provides reliable guidance for the innovative treatment of cartilage injury and tissue repair.Method:The first part,the primary culture and identification of the rat adipose stem cells:collection and culture of autologous adipose stem cells:After local skin preparation and sterilization,the fat around the kidney of rats is cut aseptically for about 3cm3.After treatment,type I collagenase with a concentration of 0.1% at 5 times the volume of adipose tissue is added for digestion for one hour.After centrifugation,the floating fat is removed from the upper layer and is filtered by 200 mesh filter.The precipitation obtained after centrifugation is suspended in complete medium.Finally,the cells are moved to the cell culture bottle and placed in an environment where the temperature is 37 degrees Celsius,the humidity is saturated and the carbon dioxide concentration is 5 %.Replace the cell culture solution after two days and then every two days.Identification of adipose stem cells:Morphological characteristics of cells(observation by light and electron microscope);Cell immunophenotype(CD106,CD44,and CD49d);Multiple differentiation ability(induction of differentiation into osteoblast).The second part,Eukaryotic expression plasmid pc DN3.1+BMP-7 is restructured and sequence verification analysis is conducted on it.A pc DNA3.1+BMP-7-ADSCs cell line with stable expression of BMP-7 gene is synthesized.The expression of BMP-7 is detected by immunofluorescence and Western-Blot.RT-PCR detects the expression of ? type collagen of transgenic adipose stem cells,at the same time under phase contrast microscope immunofluorescence method is used to detect the expression of ? type collagen.The third part,animal model of rat cartilage injury:after SD rats take monosodium iodoacetate(MIA)orally and osteoarthritis is induced,the knee joint tissue is embedded with fixed paraffin and sectioned.The Mankin osteoarthritis score is identified and modelled after HE and toluidine blue staining.After 8 weeks,the spinal dislocation is performed to kill the rats and the cartilage of the knee joint is exposed.Observe the expression of BMP-7 and repair of cartilage injury after implantation of transfected adipose stem cells:grouping:Bmp-7 gene transfected adipose stem cells group,pure transplanted adipose stem cells group,osteoarthritis model group,normal control group.Curative effect evaluation: 8 weeks after cell transplantation,the comparison between groups,measurement indicators as follows,observation on repair of articular cartilage injury: Immunohistochemistry method is used to detect the expression of ? type collagen in each group,and Western blot method is used to detect the expression of ? type collagen in each group.Results:The first part: the primary culture and identification of the rat adipose stem cells:The cultured adipose stem cells were in good growth and reproduction state both for the primary cells and the passage cells.The two generations of cells are basically the same,some are long spindle,some are polygonal.The results of CD44 and CD49 d of cell membrane tested by cellular immunophenotype are all positive,but the results of CD106 are negative.In addition,the test results of dexamethasone,alizarin red staining,and alizarin red staining in the ?-glycerophosphate inducer group are positive,indicating that the cultured adipose stem cells have multiple differentiation abilities.It can be seen from the in-depth analysis of the proliferation curve of ADSCs that the adipose stem cells have strong growth and reproduction capacity after the passage treatment.The second part:reconstruction of recombinant bone morphogenetic protein gene plasmid and verification results of adipose stem cells differentiation into chondrocyte: A complete and reasonable eukaryotic expression plasmid pc DNA3.1+BMP-7 cell strain is constructed,and the transfection of eukaryotic expression plasmid pc DNA3.1+BMP-7 in rat adipose stem cells is successfully conducted.After bone morphogenetic protein transfection,adipocyte stem cells show good ability of differentiation and reproduction.Compared with untreated BMP adipose stem cells,it effectively demonstrates a high expression of biological performance of ? type collagen.The third part:the establishment of animal model of rat cartilage injury:HE and toluidine blue staining indicate the changes of cartilage injury in the cartilage injury group compared with the control group,and OARSI score indicates the presence of cartilage injury in the experimental group,with statistically significant differences.Observe the expression of BMP-7 and repair of cartilage injury after implantation of transfected adipose stem cells.8 weeks after cell transplantation,immunohistochemical staining detects COL?is closer in transgenic cell transplantation group than the normal group with obvious advantages.Using western blot method for validation and analysis,in the adipose stem cell group of gene transfection with the transplantation of BMP-7,? type collagen content is most close to that of normal cartilage,much higher than the transplanted stem cell group and model group,and the difference is statistically significant.Conclusion:1.Cartilage injury model was prepared by injection of articular cavity with iodoacetic acid,the cartilage surface of the experimental group was rough,a pathological condition in which a part or whole layer is absent,OARSI scores indicated that the osteoarthritis model was successfully established.2.Bmp-7 transfected adipose stem cell in vivo transplantation to repair articular cartilage injury,Compared with the control group,the morphology of cartilage matrix in the experimental group was restored to normal shape,the number of chondrocytes increased significantly,and the chondrocytes were arranged like normal ones,the arrangement of cartilage matrix is normal,and it may have some repair effect.3.Western-Blot detection transplantation to BMP-7 gene adipose stem cells group model COL ? expression is higher than the control group is the most close to the normal control group,the difference has statistical significance. |
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