| Objective: To investigate the effect of B-cell lymphoma / leukemia-xl gene(Bcl-xL)on directional differentiation of human umbilical cord mesenchymal stem cells(HUCMSCs),and to observe the efficacy of Bcl-xL gene modified human umbilical cord stem cells in repairing rabbit articular cartilage injury.The main contents were as follows:(1)acquisition,isolation and culture of human umbilical cord mesenchymal stem cells and induction of differentiation into cartilage cells;(2)Human umbilical Cord Blood Stem cells infected with apoptosis inhibitor Gene Bcl-xL carried by lentivirus;(3)detection of the effect of overexpression of Bcl-xL gene on apoptosis of stem cells.(4)to detect the effect of overexpression of Bcl-xL gene on the secretion of type II collagen,Caspase-3 and MMP3 by stem cells;(5)to establish rabbit knee cartilage injury model;(6)to implantation and repair rabbit cartilage injury with sodium alginate scaffold coated with Bcl-xL gene modified stem cells;(7)to detect whether the implanted stem cells can survive in heterogeneic animals;(8)the repair of cartilage defect was observed by HE,MASSON and modified red O staining,and(9)the effect of Bcl-xL gene expression on the secretion of type II collagen,Caspase-3 and MMP3 by cartilage cells was detected by immunohistochemistry and real-time PCR,WB.Materials and Methods:(1)obtaining human umbilical cord mesenchymal stem cells in a normal delivery process,The effects of induction and differentiation were identified by toluidine blue staining and immunohistochemical staining,and compared with the control group.(2)modifying the human umbilical cord mesenchymal stem cells with a lentivirus encoding Bcl-xL,inoculating the third generation cord blood stem cell to a 6-well plate,and transfecting human umbilical cord mesenchymal stem cells with a lentiviral vector encoding a Bcl-xL gene when the cell fusion reaches 50~70 percent;The cells were divided into four groups: the control group,the induction and differentiation group,the induction group,the Bcl-xL slow virus transfection group,the induction group and the slow virus no-load.In the control group,only the cultured stem cells were isolated and no inducers were added;the induction group was added with the inducing factors such as TGF-?1,and the induction group and the Bcl-xL lentiviral transfection group were added with the slow disease coding the Bcl-xL gene on the basis of the induction group.The no-load group is a slow disease which does not encode the Bcl-xL gene only on the basis of the induction group.Poison.The above four groups continue to culture 48 hours,the morphological characteristics of the cells were continuously observed and recorded under an inverted microscope;the staining of the toluidine blue confirmed that the stem cells transfected with the Bcl-xL gene still had the characteristics of the chondrocytes;and the surface resistance characteristic of the chondrocyte was identified by flow cytometry.For example: CD90 and CD105 Expression of Bcl-xL gene on stem cells by flow cytometry to detect the apoptosis of each group of cells.The expression of Bcl-xL gene in the level of protein was detected by fluorescent PCR,and the expression of Bcl-xL gene was detected by Western blotting(WB).The expression of Bcl-xL gene and its expression in type II collagen,Caspase-3 and MMP3 were detected by real-time PCR and WB.Effect of SPSS 17.0 software on the expression of mRNA and protein in each group(3)preparing sodium alginate support and HUCMSCs-stent composite;dropping 1.2% sodium alginate solution into a 102 m M calcium chloride solution to form a cell-free sodium alginate support in a grinding tool;adding 1.2% sodium alginate solution into the HUCMSCs cell to prepare 1-106/ ml of monofine Cell suspension,dropping of 102 m M calcium chloride solution in a grinding tool to form a sodium alginate scaffold containing hUCMSCs cells;(4)manufacturing a rabbit cartilage damage model,and 30 Japanese large-ear rabbits of 3-month-old,Five groups were divided into 5 groups.The internal incision of the back leg of the two-sided rear leg was introduced into the knee joint.The joint surface of the femoral condyle had a diameter of 4 mm and a depth of about 3 mm.The subchondral bone was penetrated through the subchondral bone,resulting in a total layer of articular cartilage injury.Each group of 12 knee joints was repaired in 5 different ways.Cartilage defect: sham operation group(control group): only the sham operation and no damage to the cartilage were performed;the model group: the model group was not used for treatment and direct suture;the empty vector treatment group: the lentiviral empty vector was transfected into the stem cells,and the sodium alginate was coated to treat the area of the cartilage injury defect;and the dry thin The treatment group: the stem cells transfected with the lentivirus and the sodium alginate are coated to treat the cartilage damage Defect area.Bcl-xL gene modified treatment group: Bcl-xL modified lentivirus transfect stem cell,sodium alginate coated to treat cartilage injury defect area;all animals were sacrificed after 8 weeks of feeding,and the knee was obtained.To determine whether the stem cells implanted in the cartilage defect can survive in a rabbit with normal immune function by using the anti-human nuclear specific antibody,and observe the effect of the Bcl-xL gene-modified HUCSCs transplantation in the treatment of articular cartilage damage.The effects of the repair of articular cartilage injury were observed by HE,MASSON,modified red O-solid green cartilage staining and etc.The expression of Bcl-xL gene was detected by immunohistochemistry,real-time PCR and WB and other protein tables such as type II collagen,Caspase-3 and MMP3.The effect of data on the level of mRNA and protein in each group was compared by SPSS 17.0 software.Results: 1.Mesenchyma stem cells could be successfully isolated from human umbilical cord and cultured in vitro and induced to differentiate into cartilage cells.2.Human umbilical cord mesenchymal stem cells were modified with lentivirus encoding Bcl-xL,and the differentiation of stem cells still had the characteristics of cartilage cells.Bcl-xL gene increased at mRNA and protein level.Bcl-xL gene could inhibit the apoptosis of stem cells.Bcl-xL gene could promote the expression of type II collagen in cartilage cells at mRNA and protein level,and significantly inhibit the expression of Caspase-3,MMP3.There was significant difference between groups(P<0.05).3.The stem cells survived when the sodium alginate scaffold-stem cell complex was implanted into the animal body,and the pathological staining showed that the cartilage injury in each group was repaired,and the three staining methods showed that the cartilage structure of the stem cell group modified by Bcl-xL gene was the most satisfactory,while that of the model group was the least satisfactory.The results of PCR and WB were basically the same: cartilage injury promoted the expression of type II collagen fibers at mRNA and protein levels,while Bcl-xL gene modification further increased the expression of type II collagen fibers.The results of Caspase-3 and MMP3 showed that the expression of caspase-3 and MMP-3 in the model group was significantly up-regulated compared with the pseudo-operation group,and the expression of caspase-3 and MMP-3 in the Bcl-xL gene modified HUCSCs group was significantly lower than that in the model group.There was significant difference between the two groups(P<0.05).In addition,compared with normal HUCSCs,Bcl-xL modification could further decrease the expression of caspase-3 and MMP-3 induced by cartilage injury and increase the expression of type II collagen fibers.Conclusion: 1.Human umbilical cord mesenchymal stem cells can be induced to differentiate into cartilage cells,and Bcl-xL gene modification can reduce the apoptosis of stem cells.2.Transplantation of HUCSCs can repair cartilage injury,while Bcl-xL modification can improve the curative effect of HUCSCs transplantation. |
PDF Full Text Request