Effects Of Growth Factors And MAPK Pathway On The Growth Plate Chondrocyte Proliferation And Differentiation

Chapter 1 Studies on isolation and culture of rat costalchondral growth plate chondrocytes and their biological characteristicAim: To establish the in vitro model rat costalchondral growth plate chondrocytes, and investigate their biological characteristic in vitro.Methods: Isolated rat costalchondral growth plate by microdissection, dispersed RGC (rat costochondral growth plate chondrocyte) were obtained by digestion (0.05% hyaluronidase and 0.2% collagenase type Ⅱ) , inoculated and cultured in monolayer. From primary to the fifth passage, growth dynamic analysis of RGC was carried out; the expression and location of collagen type Ⅱ and α -SMA of RGC were detected by ICC(Immuno-cytochemistry), alcine blue staining and western blot.Results: In the primary cultured RGCs, more than 95% cells were positively stained by collagen type Ⅱ, positive staining area was around cell nucleus and membrane, the nucleus were enclosed by positive stained fiber, round cells were strong positive stained. As passage number increasing, the percentage of collagen type II stained cells decreasing, in the sixth passage RGCs, only cells retaining round shape were still positively stained. Primary cultured RGCs expressed abundant GAG (glycosaminoglycan), and were alcine blue stained positively. From passage 2 to passage 3 alcine blue staining of RGCs decreased, from passage 4, RGCs were alcine blue stained negatively. The ICC staining of α -SMA was weak in the primary cultured RGC. As passage number increasing, α -SMA staining augmented gradually . Western blot demonstrated the same expression pattern of collagen typeⅡ and α -SMA.Conclusion: Established in vitro model of rat costalchondral growth plate chondrocyte successfully. The monolayer cultured RGCs lost their phenotype of differentiation after passage 4, and α -SMA could be taken as a new marker of RGC dedifferentiation.Chapter 2 Effects of growth factors and growth factor combinations on proliferation and differentiation of serial passaged RGCAim: To investigate the effects of growth factors and growth factor combinations on proliferation and differentiation of serial passaged RGCs, reveal the influences of dedifferentiation caused by serial passage on the effects of growth factors and growth factor combinations, thus settle the foundation for establishing serum free culturing system for RGC.Methods: Serial passaged RGC was taken as experimental model, the effects of different concentrations L-ascorbic acid (L-AA), bFGF, IGF-1 ,TGF- β1,TGF-β 3, EGF and their combinations on proliferation , collagen type II and GAG synthesis of serum free cultured primary to forth passage RGC were detected by 3H-TdR, 3H -Proline and 35S-Sulfate incorporations.Results: For the primary cultured RGC, 25μg/mL, 50μg/mL L-AA stimulated 10%FBS cultured RGC proliferation and collagen synthesis, but inhibited serum free cultured RGC proliferation and collagen synthesis intensively, thus there was no addition of L-AA to the medium of the following experiments. All the five growth factors detected in this research had vary extent of proliferation and collagen synthesis stimulating effects on primary RGC. Among them, bFGF had the highest proliferation stimulating effect, about 6 times higher than serum free control, and was also higher than 10%FBS control; the collagen synthesis stimulating effect of TGF- β 1 was strongest, about 1.5 times stronger than serum free control ,but it was inferior to 10%FBS control. The promotion effects of growth factor combination (l0μg/L bFGF , 10μg/L IGF- I and 1μg/L TGF- β 1) on proliferation , collagen synthesis andGAG synthesis corresponded to 10% FBS. As passage number increasing, the promotion effects of growth factors and growth factor combinations on proliferation and collagen synthesis of passaged RGC took on a downtrend.Conclusion: bFGF, IGF-Ⅰ, TGF- β 1, TGF- β 3, EGF were able to promote primary RGC proliferation and collagen synthesis in vary extent. The promotion effects of growth factor combination (10μg/L bFGF , 10μg/L IGF-Ⅰ and 1μg/L TGF-β 1) on proliferation ,collagen synthesis and GAG synthesis corresponded to 10% FBS. As passage number increasing, the promotion effects of growth factors and growth factor combinations on proliferation and collagen synthesis of passaged RGC took on a downtrend.Chapter 3 RGC proliferation and differentiation and MAPK pathwayAim: To investigate the effects of three major pathway of MAPK, ERK1/2, JNK and p38 on RGC proliferation and differentiation, and their roles in promotion effect of growth factors on RGC proliferation and differentiation.Method: Examined the impact of PD98059, SP600125 and SB203580, special inhibitor of ERK1/2, JNK and p38, and genistein, non-special inhibitor of tyrosine kinase, on proliferation and collagen synthesis of primary RGC by 3H-TdR and 3H -Proline incorporation. Western blot verified the influences of the aforementioned inhibitor and growth factors and growth factor combinations on ERK1/2, JNK and p38 expressing in primary RGC. In the end of this experiment, the effects f PD98059 on collagen and aggrecan mRNA transcription enhanced by growth factor were observed by RT-PCR.Results: Among the three examined special inhibitor, PD98059 and SP600125 suppressed primary RGC proliferation and collagen synthesis significantly, but SB203580 didn't have remarkable effect on primary RGC, western blot testified that these three inhibitor suppressed phosphated ERK1/2, JNK and p38 expression. Genistein suppressed RGC proliferation and collagen synthesis more deeply than PD 98059 and SP600125, in addition genistein treatment altered RGC morphology.Western blot demonstrated genistein remarkably inhibited phosphated ERK expression in primary RGC. bFGF enhanced phosphated ERK expression in primary RGC, IGF-1 didn't alter phosphated ERK expression, but the phosphated ERK expression enhanced by their combinations was stronger than bFGF alone. Growth factors and growth factor combinations treatment on RGC had no obvious effect on phosphated JNK expression. RT-PCR demonstrated PD98059 could inhibit collagen and aggrecan mRNA transcription enhanced by growth factor。Conclusion: To the primary cultured RGC, ERK and JNK pathway participate in RGC proliferation and differentiation; p38 pathway has no remarkable effect. The promotion effects of bFGF on RGC proliferation and collagen synthesis were transduced by ERK pathway, in addition, ERK and JNK pathway participated in sox9 expression.Chapter 4 Construction of adenovirus vector expressing ERK-2, and studies on the effects of constructed Ad-ERK2 on proliferation and collagen synthesis of primary RGCAim: To construct replication deficient adenovirus vector expressing ERK-2, and study the effects of Ad-ERK2 infection on proliferation and collagen synthesis of primary RGC。Methods: Recombinant adenoviral vector Ad-ERK2 and Ad-CMV were generated by homologous recombinant in bacteria. Constructed Ad-ERK2 and Ad-CMV were identified by fluorescent microscopy and western blot, viral titers were determined by FACS. Detected the infective efficiencies of Ad-ERK2 and Ad-CMV on primary RGC with fluorescent microscopy and FACS. Detected the effects of Ad-ERK2 infection on proliferation collagen synthesis and cell cycle with isotope incorporation and FACS.Results: Constructed Recombinant adenoviral vector Ad-ERK2 and Ad-CMV successfully. The infective efficiencies of primary RGC achieved 98.11% with MOI 100 Ad-CMV. Compared with Ad-CMV, Ad-ERK2 promoted proliferation andcollagen synthesis of infected primary RGC, isotope incorporation of treatment is 1.52 and 1.16 times higher than control. Cell cycle analysis demonstrated that the G2M and S phase ratio of infected RGC increased.Conclusion: Constructed Recombinant adenoviral vector Ad-ERK2 and Ad-CMV successfully. By infecting primary RGC with adenovirus vector expressing ERK-2, this experiment proved that ERK pathway stimulated RGC proliferation and collagen synthesis.