The Expression Of YAP In Different Segment Nucleus Pulposus Cells Of Rat

Objective:Examine the expression of YAP in different segment nucleus pulpous cells of rat; observe the activation of YAP pathway in different cell density; test the lever of YAP in the serum-deprived nucleus pulpous cells.Methods:1. Culture normal lumbar nucleus pulposus cell and coccygeal vertebra nucleus pulposus cell of SD rats with 10%FBS/DMEM-F12 in vitro, observe the proliferation of different subgroups in lumbar NP cells.2. Culture all the cells to the third generation, observe the morphology of different cells.3. Detect the expression of the YAP in third generation of the lumbar NP cells by immunofluorescence.4. The expression YAP mRNA was examined in lumbar NP cells with 50% fusion state(group A)?lumbar NP cells with 90% fusion state(group B)?coccygeal vertebra NP cells with 50% fusion state(group C) and coccygeal vertebra NP cells with 90%fusion state(group D) by RT-PCR in step 2.5. Detect the expression and the localization of YAP protein in lumbar and coccygeal vertebra NP cells by Western blot in step 4, respectively named group A nucleus?group A cytoplasm?group B nucleus?group B cytoplasm?group C nucleus?group C cytoplasm?group D nucleus and group D cytoplasm.6. Culture the third generation of coccygeal vertebra NP cells with DMEM-F12 in vitro to set up serum-deprived induced model, examine the expression of YAP protein in normal group and serum-deprived induced model by Western Blot after ten days.Results:1. The NPCs were isolated successfully. The adherence time of the primary cells(the cell fusion reached 90%) in lumbar NPCs was longer than caudal NPCs. The most of lumbar NPCs were short fusiform or polygonal in primary cells, the caudal NPCs were round or oval. The cells with vacuoles were found in caudal NPCs.2. Immunofluorescence staining showed strongly positive expression of YAP in the nucleus of lumbar NPCs when cells were in low density. Otherwise, the YAP was highly expressed in the cytoplasm.3. According to the results of RT-PCR, the expression of YAP mRNA in the group A was statistically significant compared with group B(P<0.05), but no significant compared with C. And the expression of YAP in group D was decreased significantly compared with group C(P<0.05).4. Western blot revealed that the YAP expressed highly in nucleus when the lumbar or caudal NPCs fusion reached 50%. And when the cells fusion reached 90%, the expression of nucleus become decreased.5. Western blot showed that the expression of YAP was decreased in serum-deprived group than normal group.Conclusion:1. YAP is examined in the NPCs.2. The proliferation of caudal NPCs is faster than lumbar NPCs, but their levers of YAP is no significance.3. Cell contact and serum-deprived restrain the expression of YAP in NPCs and could arouse the localization between nucleus and cytoplasm.