VEGF Induces Mesenchymal Stem Cells To Differentiate Into Vascular Endothelial Cells Combined With BFGF And AFGF Respectively In Vitro

BACKGROUNDSelection of suitable seed cells is basic to tissue engineering research. Due to the ethical issues, immunological rejection and teratoma result from transplantation, embryonic stem cells has been limited in the application of clinical practice and scientific research. The mesenchymal stem cells are adult stem cells which originated in the interstitial mesoderm, they have self-renew and multiple differentiation potential, and have the capacity to develop into many cell types in suitable enviroment both in vitro and in vivo, especially cells from the mesoderm and neural ectoderm, such as osteoblasts, cartilage cells, fat cells, cardiac cells, vascular endothelial cells and so on. Furthermore, they maintain the characteristic of stem cells after reproduction and expansion. The mesenchymal stem cells were found in various tissues, researchers have isolated MSCs from a variety of tissues of fetal and adult, such as bone marrow, periosteum, fat, milk teeth, umbilical cord and so on. The isolation of mesenchymal stem cells does not distruct blastocyst, and thus will not cause ethical problem.The mesenchymal stem cells also have immunomodulatory functions, they can inhibit the proliferation and immune response of T cells by the interaction of the cells and producing cytokines, which play the function of the immune reconstitution. What is more, many studies show that the transplantation of MSCs results in no immune rejection, and will not lead to teratoma. All these characteristics make MSCs be a suitable candidate for use in regenerative medicine and cell-based therapy.Bone marrow is the best source of MSCs, but they constitute a mere0.001%to0.01%percent of the bone marrow cells with nucleus. The common separation methods of MSCs are the whole bone marrow adherence method, density gradient centrifugation, cell sorting by flow cytometry, immunomagnetic separation. The whole bone marrow adherence method and density gradient centrifugation are easier to inplement, but it is difficult to achieve the purification of the cell by applying one method, thus will cause an adverse effect on the experimental results. Cell sorting by flow cytometry and immunomagnetic separation can isolate highly purfied MSCs, but the operating processes are complex, the cost expense is high, so both of the metheds are not widely promoted. Currently, density gradient centrifugation and adherent cultivation method are combined to isolate and purify the MSCs, whether the purity of the saparated cells meets the requriment of the experimental research and clinical treatment, the cell viability whether changes after passages, there is no systematic and comprehensive assessment up to now.Many studies have shown that mesenchymal stem cells can differentiate into vascular endothelial cells (VECs) in vivo and in vitro. Vascular endothelial growth factor (VEGF) plays a major role in the differentiation process, and basic fibroblast growth factor (bFGF) can create a microenvironment which is appropriate for the differentiation of MSCs, which means that bFGF has a synergistic effect. VEGF combined with bFGF are widely used in inducing MSCs to differentiate into VECs in vitro. Acidic fibroblast growth factor (aFGF) has a similar biological activity with bFGF, its isoelectric point is5.16, and studies show that bioactivities of aFGF play better than those of bFGF in the acid environment. We all know that the PH of the medium decreases due to metabolic activities of the cells, this make it possible that aFGF may be more suitable for assisting VEGF inducing MSCs to differentiate into VECs.The first chapter:Isolation, culture, identification and cell viability testing of rabbit bone marrow mesenchymal stem cellsOBJECTIVETo isolate mesenchymal stem cells (MSCs) from rabbit bone marrow, and identify MSCs from mophological characteristics, detecting cell phenotypes, osteogenic and adipogenic induction. Test the viabilities and compare the differences of the passage cells.METHODS1、Isolate and purify the MSCsThe rabbit bone marrow was extracted from femur and tibia in sterile condition, and the cells with different proportions were saparated by gradient centrifugation method, we select the cells with single nucleus as the subjects, and culture them in the37℃CO2incubator。The non-adherent cells were removed by replacing the medium. Mononuclear cells and fiber cells have stronger adherent capacity, so MSCs are easier to be digested by trypsin, we strictly controlled the amount of trypsin and digestion time in the course of passage, collected the digested MSCs and abandoned the undigested cells. In this way, MSCs were purified.2、Identification of MSCs(1) Mophological characteristics:We observed the mophological characteristics of cells at different passages under inverted microscope, and the cells were stained by hematoxylin and eosin.(2) Expession of cell phenotype:We detected the expression efficiency of CD14、 CD29、CD34、CD44、CD45and CD90by flow cytometry(3) Multi-lineage differentiation potential:MSCs were subjected to a osteogenic differentiation protocol, alizarin red S staining was performed to identify the calcium nodules; adipogenic induction was carried out as well, Oil Red O staining was performed to identify adipocyte cells.3. Test the cell viabilities(1) Drawing growth curve:We choosed cells in good state at passage1,3and5as the subjects, cultured the same number of cells in the petridish with a diameter of3.5cm. From the second day, we took out three dishes at random, digested cells by trypsin, counted the cell numbers and struck an average. We used excel software to draw the growth curve, time was set as the abscissa, and cell number as the longitudinal axis.(2) Seeding efficiency:We choosed cells which came into logarithm growth period at passage1,3and5as the subjects, cultured the same number of cells in the petridish with a diameter of3.5cm. From seeding the cells then on, we took out three dishes at random every two hours, removed the non-adherent cells and the medium, digested cells by trypsin, ounted the cell numbers in every dish and struck an average. The corresponding seeding efficiency was caculated by using the formula. Eighteen hours later, the trend curve was drawn by setting time as the abscissa, and cell number as the longitudinal axis.(3) Colony-forming efficiency:We choosed cells which came into logarithm growth period at passage1,3and5as the subjects, digested them by trysin, seeded100cells on each well of the six-mesh plate. The cells were constantly cultured for sixteen days, and stained by trypan blue. The standard of cell colony is that there are at least50cells in each unit.RESULTS1、Mophological characteristics:MSCs are adherent cells. Most of the cells attached to the dish after seeding for48hours. The primary cells were spindle-shaped, circular and triangular. Seeding for five or six days, cell colonies formed. After8or10days, cells reached80%-90%confluency and the subculture cycle was3to5days. After several passages, homogeneity and uniformity of the cells inproved well.2、Immunophenotype:Flow cytometry demonstrated that cells were negative for CD14, CD34and CD45, the expression efficiencies were5.36%,0.36%and2.03%respectively. But they were positive for CD29, CD44and CD90, the expression efficiencies were97.21%,98.87%and96.20%respectively.3%Multi-lineage differentiation potential:Osteoinduction for21days, calcium nodules were formed in the experimental group, and they could be stained crimson by alizarin red, while there was no significant change in the control group. Inducing adipocyte formation for14days, the cells in the experimental group became round or polygonal, there were visible lipid droplets within the cytoplasm, the lipid droplets became red stained by oil red O, no significant change was foud in the control group.4、The growth curve:The growth curves of cells at passage1and3were similar, both of them showed typical "S" shape. The proliferation of cells at passage5was slower than cells at passage1and3.5、Seeding efficiency:The adherent rates of cells at passage1and3were consistent. Seeding for14hours, more than96%of the cells had attached to the dishes, there was not significant difference between the two passages (P>0.05).The rate of cells at passage5was lower, only87%of the cells attached to the dishes14hours later from seeding. The differences were significant between cells at passage5and cells at passage1or3(P<0.01)6、Colony-forming efficiency:Under the microscope, cell clones were formed from cells at passage1,3and5. Cells in the clones packed tightly, and they were similar with primary cells in terms of mophology, we could see some aging or mutated cells on the edge of the clones. The differecnces of cloning efficiency between two of the three passages were significant (P<0.01), colony forming efficiency decreased as the cell passage.CONCLUSION:1、MSCs maintain the multi-lineage differentiation potential, and they could be purified in the course of passage.2、Cells at passage1and3are keeping a high level of viabilities, but the viabilities will decrease as the cell passage.3、Bone marrow derived MSCs within3th generation are suitable for experimental study of tissuse engineering, as they can attach to the stents tihgtly. The second chapter:Inducing bone marrow mesenchymal stem cells to differentiate into vascular endothelial cells:A comparison of biological activity between aFGF and bFGFOBJECTIVETo induce differentiation into vascular endothelial cells (VECs) from mesenchymal stem cells by VEGF combined with aFGF and bFGF respectively. VECs were identified from mophological characteristics, detecting uptaken functions, the expression of CD31and the No secretion.comparing the differences of CD31expression and No secretion between the two inducing methods.METHOD1、Isolate and purify the rabbit bone marrow MSCs:Be the same as the first part.2、Inducing differentiation from MSCs to VECs:Cells at passage3which came into logarithm growth were selected as subjects, they were seeded in the six-mesh plate. Inducing medium of the experimental group consisted of VEGF(20ng/ml) and aFGF (10ng/ml), and the compositions of inducing medium in the control group were VEGF(20ng/ml) and bFGF (10ng/ml)3、Identify the VECs(1)Observe the mophological changes under the inverted mcriscope before and after the introduction.(2)Uptaken function:Double staining method was applied to identify VECs. VECs can phagocytize Dil-ac-LDL and show red fluorescence, they aslo can bind FITC-UEA-I and distribut green fluorescence. Double positive cells show yellow. fluoresence.(3)Detecting CD31expression:On the15th and24th days, the expression of CD31was detected by flow cytometry.(4)NO secretion detection:The inductive supernatant was collected on the15th and24th days, and stored in the-80℃refrigerator。Our operation was instructed by user manual of the NO assay kit.RESULT1、Mophological characteristics of MSCs:Be the same as first part.2、Identification for VECs(1)Cell mophology:The cells had been induced for24days, they were smaller, and changed round, oval or short spindle shape from long spindle shape. The endothelioid cells showed slabstone performance which was considered as the typical characteristic of vascular endothelial cells of adult.(2) Uptaken function:We could saw that more than50%of the cells were double positive for Dil-Ac-LDL and FTIC-UEA-I under the fluorescence microscope.(3) Expression of CD31:On the15th day of induction, expression efficiencies of CD31in the experimental group and the control group were (34.20±2.90)%and (46.23±2.87)%respectively, the difference was significant(P>0.05). On the24th day, the efficiencies of the two groups were (53.35±2.12)%and (56.14±3.93)%, there was no significant difference between the two groups(P <0.01).(4) NO secretion detection:On the15th day of induction,the concentration of NO in the supernatant of the experimental group and the control group was (70.27±3.45)μmol/L and (79.19±5.34)μmol/L respectively, the difference was significant(P>0.05). On the24th day, the results were (105.16±5.89)μmol/Land (109.97±5.99)μmol/L, there was no significant difference between the two groups(P<0.01).CONCLUSION1、VEGF can successfully induce MSCs to differentiate into VECs combined with bFGF and aFGFrespectively. On the24th day of induction, there was no significant difference of induce efficiency between the two methods.2、In the process of induced differentiation, although the biological activity of bFGF is better than that of aFGF, aFGF is more stable and less affected by cell metabolism compared with bFGF.