IGF-â…  And PDGF-AA To Promote Proliferation Of The Rat Nucleus Pulposus Cell In Vitro And The Comparison Between The Two

Background:Neck shoulder pain since ancient times which beset the lives of people, but can not find the real reason for the delay. Until 1934, Mixter and Barr that "disc era (Dynasty of the disc)", that people began to slowly recognize this disease, which began to carry out its occurrence and development in further studies. In recent years, with the tissue engineering studies have also extended to the field of spinal surgery, foreign scholars began to carry out a tissue-engineered intervertebral disc-related basic research work. Although the number of published studies was small, yet reflects the encouraging prospects of this study, but on the intervertebral disc cells cultured in vitro research and awareness are relatively small. However, as cell and tissue culture technology has good repeatability, low cost, result analysis is relatively simple, non-ethical issues and other characteristics so that in disc degeneration research attention and continue to be applied.Objective:With the development of molecular biology techniques which have begun to involve intervertebral disc degeneration in this area. Insulin-like growth factor and platelet-derived growth factor can exist with binding protein in soluble form,which play a key role in cartilage formation and cartilage homeostasis regulation and has to change the disc extracellular matrix changes and to prevent apoptosis of disc cells. From cultured rat intervertebral disc nucleus pulposus cells in vitro,we could find the effects of different concentrations of insulin-like growth factor and platelet-derived growth factor on the nucleus pulposus cells, apoptosis, proliferation and extracellular matrix metabolism and the difference between the two.And provides a theoretical basis on human intervertebral disc study in further.Method:1st, Isolated and digested wistar rats's intervertebral disc, received a single nucleus pulposus cells and completed culturtion of primary cells; 2st, After the cell fusion of original generation, established the passage control group, experimental group I (1-4 groups, imposed with 1ng/ml, 10ng/ml, 100ng/ml, 1000ng/ml different concentrations of insulin-like growth factor in DMEM culture medium) and experimental groupⅡ(5-8 groups, to impose containing 1ng/ml, 10ng/ml, 100ng/ml, 1000ng/ml different concentrations of platelet-derived growth factor in DMEM culture medium);3rd, Used inverted microscope to observe the cell morphology,proliferation changes of the control group and experimental group in the growth process;4th,Using HE, toluidine blue to observe the proliferation of three generations nucleus pulposus cells,use immune staining to detected the expression of collagen typeⅡin each group; 5th, Using MTT (MTT colorimetric) to determine the absorbance values of mass third-generation nucleus pulposus cells in control group and experimental groupⅠ,Ⅱ, which be stimulated with different concentrations of growth factorion after 3,5,7days and drafting the mapping of Cell growth; Using flow cytometry to determine the cell content during the S phase (DNA synthesis phase) in each group;6th, Make a conclusion with statistical software analysis.Results:1st,Observed the cells under the inverted phase contrast microscope,more than 95%cells adherent after 10 days, primary cells are mostly spindle-shaped, with protruding pseudopodia; by trypsin digestion after generation, the cells adherent growth rate accelerated, while the Join insulin-like growth factor and platelet-derived growth factor to stimulate the growth of nucleus pulposus cells the growth and reproduction of which were markedly increased; 2st, nucleus pulposus cells which are stain normal in HE are spindle, cytoplasm-rich, there are two nuclei, in the control group without the stimulation of IGF-1 or PDGF-AA nucleus pulposus cells grow slowly, the number of cells are little, cross-connect with each other less.The shape of cell tends to irregular cell morphology, Cellular Tunisia was prolonged, which was slender cord-like, cytoplasmic content was decreased, while the group of the IGF-1 or PDGF-AA action cells, compared with the control group, growth factor-group cell morphology in the training of early (<72h) changed little, but in the latter phase of training, the number of cells are much more than the control group,The cells morphology are mostly as a short spindle shape, polygon, abundant cytoplasm and cross-cutting reticular. The nucleus are stained purple and cytoplasm was stained dark blue by toluidine blue. TypeⅡcollagen immunohistochemical staining showed positive results.3st, MTT assay showed absorbance values, when the third day the 4 groups and 7 groups are different with the rest of group (p<0.05),4and7 group of non-discriminatory; when the five days control group,1and 5 groups were different with each other groups (p<0.05), control group, land 5 group are non-discriminatory, the 7groups and 8 groups are different with other groups (p<0.05),the 7and 8 group are non-discriminatory; when the 7 days the control group,1 group and 5 group were different with the remaining groups (p<0.05), control group,1 dna 5 group are non-discrimination,2,4,6 group are different with 3 group (p<0.05),2,4,6 group are non-discrimination,7,8group are ifferent with other groups (p<0.05),7 group are different with 8 (p<0.05).4,The concentration of growth factor are showed positive correlation with the number of cells in S phase by flow cytometry.Conclusion:This experiment, which provides a simple and effective method to culture nucleus pulposus cells.During the process of culture nucleus pulposus cells in vitro, the insulin growth factor (IGF-1) and platelet-derived growth factor (PDGF-AA) can promote effectively the nucleus pulposus cell proliferation, and with the increasing concentration significantly increased proliferation, promote cells metabolism, linear relationship with concentration.With comparison of these two cytokines,the facilitation of PDGF-AA is no difference with IGF-1 in low concentration, and when the initial training the facilitation of PDGF-AA is no difference with IGF-1 in high concentration, but by the time the late train, the effect of PDGF-AA on cell role in promoting is stronger than IGF-1。...