| Articular cartilage and even cartilage defect damage are common orthopedic disease. The most common reason is trauma, and other factors such as infection, autoimmunity, tumor.These reasons can cause a variety of joint surface cartilage damage. So joint cartilage disease is one of the most common disease in the clinical disease.And at the same time it is very difficult subject. Articular cartilage is composed of cartilage cells, collagen and proteoglycan matrix, no blood vessels and lymphatic vessels and nerve. Lack of blood vessels, articular cartilage chondrocytes not migrate, mature chondrocytes can’t migrate limits the curing reaction. Damage of articular cartilage will affect the normal sliding joints, pain, instability and joint dysfunction, accelerate the degeneration of the joints, lead to the occurrence of osteoarthritis. Serious cartilage injury can lead to joint pain and deformity.For the weight-bearing joints, such as knee arthritis in late usually only lead to artificial joint replacement. Whether do repair cartilage damage in the early days of the articular cartilage damage? To reverse the cartilage injury, even reverse joint degeneration.Therefore cartilage repair after injury has been a hot spot in the traumatic orthopedics and joint surgery. Traditional micro drilling fracture, planing treatment under arthroscopy can solve part of the problem, but the effect is still not satisfied. Autologous cartilage transplantation is a method of obtained satisfactory curative effect, but the patients must be beard a surgical trauma and more sacrifice part of autologous normal cartilage. In the1980s tissue engineerin was been put forward for the first time, offering new hope for cartilage repair. Tissue engineering cartilage repair in the application is to use a small amount of seed cells by in vitro amplification, after amplification of the active cell attached to specific biodegradable stent materials, then transplanted them to the cartilage damage area and the formation of a new, healthy cartilage. Tissue engineering is considered to be most likely to repair cartilage damage. Using autologous chondrocyte as seed cells to repair cartilage damage method of clinical effect is good method, but its shortcoming is also very obvious. Therefore the key to the problem is to find more appropriate seed cells. Mesenchymal stem cells (mesenchymal stem cells, MSCs) was first found in the bone marrow. With the deepening of research, there are succession extracting MSCs from umbilical cord blood, placenta, muscles, blood vessels, fat, and the skin tissue.Mesenchymal stem cells are a kind of pluripotent stem cells, early in the development of mesoderm cells, has the potential of multi-directional differentiation. Under certain induction conditions they can differentiate into osteoblasts, chondrocytes, fat cells, cardiomyocytes, neural cells, etc. They are the ideal seed cells in tissue engineering.Bone marrow mesenchymal stem cells (BMSCs) is present in the bone marrow to have high ability to self-renewal and multi-directional differentiation potential of stem cells. And under the condition of appropriate they can differentiate into bone cells, chondrocytes, fat cells. They are the ideal seed cells in tissue engineering. But due to problems such as materials, safety, ethical, rarely separation reasons, marrow mesenchymal stem cells limits its application. Umbilical cord blood mesenchymal stem cells (hUCB-MSCs) is a kind of multi-directional differentiation potential of mesenchymal stem cells.They have a strong ability of directional differentiation and proliferation. Umbilical cord blood mesenchymal stem cells have the following advantages:smaller invasive; more primitive stem cells, stronger ability of proliferation differentiation; weak immunogenicity; higher security; easy to separate.The umbilical cord blood mesenchymal stem cells have become a hot spot of research at home and abroad. This study combined use of density gradient centrifugation cells and cell adherent method for separation and purification of batches, confirm the cultured cells for BMSCs and UCB-MSCs. BMP was found in1965and has always been a research hot spot. BMP-2is a subtype of BMP. In favor of cartilage cell growth environment, mesenchymal cells can be induced to the cartilage cell proliferation, differentiation, and can maintain the chondrocyte phenotype by BMP-2. Due to BMP diffusion ability, BMP implantation is easy to rinse out by tissue fluid or by enzyme degradation. It makes BMP cannot play full the role. This requires a sustained release of BMP protein, maintain protein concentration of technology to solve this problem. BMP-2gene load is a new research direction. In this study using liposome mediated recombinant plasmid pIRES2EGFP-hBMP-2gene transfect BMSCs and hUCB-MSCs. And it is to explore effective transfection and promote BMSCs and hUCB-MSCs differentiation into cartilage cells in vitro.ObjectiveTo identify whether mesenchymal stem cells can be isolated in umbilical cord blood and marrow and whether recombinat plasmid pIRES2-EGFP-hBMP-2can be transfected into human umbilical cord blood derived-mesenchymal stem cells(hUCB-MSCs) and bone marrow derived stroma cells(BMSCs)with liposome.Methods1.Umbilical cord blood mesenchymal stem cells and bone marrow mesenchymal stem cell collection, isolation, culture and identificationChoose three under the age of30healthy, and acute or chronic diseases, infectious diseases, blood test negative cesarean delivery woman cord blood. Immediately after neonatal output from the umbilical cord ligation of clamps and cut off the placenta, umbilical cord venous puncture the umbilical cord blood lead directly into the one-time blood bag containing sodium citrate for about30ml.Volunteers are5people were men, ages24to36. Because of lower limb fracture internal fixation should be inline fixation admitted use of Qingdao university medical college affiliated hospital orthopaedic trauma. Internal fixation are taken out before the operation after anesthesia, on the needle with bones in front of the iliac crest draw5ml bone marrow puncture.Using density gradient centrifugation and adherent cells combined training method, separation mesenchymal stem cells, develop, extend and purification. Mesenchymal stem cells are detected by flow cytometry instrument detection through surface antigen after purification. There are higher express CD90, CD105, CD29, not express hematopoietic stem cell surface marker CD34, CD45.2. The recombinant plasmid pIRES2EGFP-hBMP-2transfection between umbilical cord blood mesenchymal stem cells and bone marrow mesenchymal stem cellsSelect P3as transfection cells. Add0.25%of pancreatic digestive enzyme digestion,1000r/min, the centrifugal5min, add do not contain fetal bovine serum and green, streptomycin resistance of nutrient solution for single cell suspension, in proportion to the number of cells1×106/ml vaccination within the six orifice to sterilization, each hole with1ml of suspension, plus containing10%FBS DMEM/F12culture to3ml, in cultivation in the box. With have been builded recombinant plasmid pIRES2EGFP-hBMP-2and X-ray treme GENE transfection reagent, after thawing, the recombinant plasmid of1000r/min centrifugal5min. With serum-free culture medium dilution of1micrograms of plasmid DNA/100edged up culture, blending. Take1micrograms of DNA of diluent200ul100<mu>1tips, added to the sterile EP1,2,3,4a tube.2were taken,4,6,8edged X- ray treme GENE added to the above four tube before. Blending incubation for20min. P3cells in proportion to the number of cells30x104vaccination to6orifice, overnight train. Not for transfection cell alignment of more than80%. In DMEM/F12cultures without FBS1ml.2h after DNA complexes of the blending in accordance with the volume ratio of3:1ratio respectively in hole, with serum-free culture medium to2ml, continue to develop. Take out the cell after8h, in DMEM/F12broth containing10%FBS. Every24h to observe a Fluorescent microscope ectomesenchymal stem cells develop Enhanced Green Fluorescent Protein (Enhanced Green Fluorescent Protein; EGFP) expression. Fluorescence with488nm wavelength ultraviolet excitation. When calculating48h cells.Collect mesenchymal cells,3days after transfection cells extracted total RNA. Using PrimeScript rt-pcr Kit (TaKaRa) Kit RNA to cDNA.Through PCR amplification. After the PCR reaction, product line of agarose gel electrophoresis, imaging analyzer imaging with a gel after electrophoresis.UCB-MSCs and BMSCs were isolated using density gradient centrifugation followed by adherent cultures. The plasmid (pIRES2-EGFP-hBMP-2) was transfected into MSCs by using X-treme GENE transfection kit. Successful transfection is determined by both the protein expression of EGFP observed by fluorescence microscopy and the mRNA expression of human bone morphogenetic protein-2(hBMP-2) detected using RT-PCR. Cell surface marker and morphological changes were examined by immunohistochemistry (IHC) after two weeks of transfection.The transfection cells are carried out in accordance with the number15×104cells per hole cells to climb, fixed, add0.5%Triton X-100, incubate. To add the closed serum and incubate cells. Then successively add1:50diluted concentration of rabbit anti people Ⅱ type collagen a resistance and rat rabbit two resistance, resistance to incubate. Add the DAB chromogenic. Wood grain is to be done after dyeing. It is microscopic examination afer rubber sealing piece.Results1. Both UCB-MSCs and BM-MSCs were isolated and expanded successfully for multiple passages and demonstrated slight growth and morphological differences. Differentiation under the certain condition, make its have to cartilage cell differentiation potential of stem cells.2. The recombinant plasmid (pIRES2-EGFP-hBMP-2) is successfully transfected into both UCB-MSCs and BM-MSCs with an efficiency of27.7±7.6%and18.4±5.9%, respectively. The mRNA expression of hBMP-2correlated with increased staining for collagen type II in the transfected cells.Conclusions1. This study successfully separate the umbilical cord blood and bone marrow mesenchymal stem cells. These cells are proved to be mesenchymal stem cells by training, wedding and purification. And mesenchymal stem cells are induced into cartilage cells.2. Recombinant plasmid pIRES2EGFP-hBMP-2gene can effective transfect into human umbilical cord blood and bone marrow mesenchymal stem cells. And gene can promote transformation of cells into cartilage cells.Innovationsl.At present in both human BMSCs and hUCB-MSCs, in terms of BMP-2gene transfection study is less, especially for hUCB-MSCs. This study successful apply recombinant plasmid pIRES2EGFP-hBMP-2to effectively transfect into human umbilical cord blood and bone marrow mesenchymal stem cells. And through the tests confirm it.2.This study confirmed that the recombinant plasmid pIRES2EGFP-hBMP-2can promote BMSCs and hUCB-MSCs into cartilage cells. This result can provide seed cells for tissue engineering, and repair cartilage damage. |
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