| 1.BackgroundCyclin-dependent kinase 5 (Cdk5) is a member of the family of Cdks, it belongs to serine/threonine kinase family. It was first extracted and purified from mammalian brain cell. Cdk5 named for its sequence homology with Ddk family. Then it's biological features was ascertained. The single unit of Cdk5 can't be activated without binding with the regulator protein p35 and p39. The protein p35 and p39 are the essential activators for Cdk5. The protein p35 and p39 showed different developmental characteristics in the rat nervous system. On the one hand the expression time are different, expression of protein p35 is 1-2 weeks earlier than p39. On the other hand, the distribution of them in the rat nervous system is different. The protein p39 mainly expressed in cerebellum, while the protein mostly distribute in hippocampus and primary olfactory cortex. In neuronal sub-cellular structure, p39 expressed in neuron cell body and axons, while the p35 only expressed in neuron cell body. Such differences of p35 and p39 in the nervous system suggest that they might have a different mechanism of function.The developmental characteristics of p35 and p39 in the rat nervous system have different influences on the regulations of Cdk5/p35 and Cdk5/p39 in the neurons normal growth and differentiation, migration; on the integration of synapse's morphology and function too.When the neurons were ready to polarize, the subcellular localization of Cdk5 vary from the cell body to the axon. Cdk5/p35 played an important role in neurons growth and differentiation since P35 only expresses in neuron cell body.The expression of p39 in neuronal axons indicates that Cdk5/p39 had an important influence on growth and differentiation of axons in the neurons of later developmental stages.Dendritic spines are an important element of synapse. In p35-/- and p39-/- or Cdk5-/- hippocampal neurons, the density of dendritic spines significantly reduced and its morphology didn't develop well. And in p35-/- hippocampal neurons, the development of morphology of dendritic spines are normal, but the activity of dendritic spines significantly decrease. The results above indicate that the synaptic activity caused by dendritic spines is mainly dependent on activity of the Cdk5/p35.For the regulation of synaptic vesicle secretion, Cdk5/p35 conbined with presynaptic membrane protein -18 (Munc-18), leading to phosphorylation of Munc-18. Finally the combination of Munc-18 and Synaptic fusion protein 1A (syntaxin 1A) failed and promoted the secretion of vesicles. On the contrary, the Cdk5/p39 primarily phosphorylated Munc-18; result in external Ca2+ flowing in vesicle, thereby promoted vesicle secretion. The differences between Cdk5/p35 and Cdk5/p39 in the role of regulation of synaptic vesicle secretion indicate that they have different effect in the regulation of growth and development of the nervous system. In addition, compared to Cdk5/p35, the stability of Cdk5/p39 are weaker, purified Cdk5/p39 would be inactivated by Triton X-100. So wasn't the Cdk5/p35. The regulation of Cdk5/p39 activity is mainly dependent on its combination or dissociation, while the regulation of Cdk5/p35 activity is mainly dependent on the degradation and synthesis of p35.Trigeminal neuralgia (TN) characterized by chronic, paroxysmal facial pain, its pathogenesis is still unclear. Spinal trigeminal nucleus caudalis subnucleus (Vc) is a portal that nociceptive information transmits from peripheral to the central. Three is a variety of terminal of neuropeptide-positive fibers which conduct pain in Vc. Most of them derived from the trigeminal ganglion neurons, and formed of synaptic connections with a large number of cell bodies and dendrites that distributed in the superficial layers of Vc neurons.In recent years, many researches have been done in the regulation role of nerve pain signal transduction. In 2006, some scholars researched on the p35 gene knockout model p35-/- mice and p35 over-expression mouse models Tgp35 mice, using the wild WT mice as a control. The experiment found that the pain response of thermal stimulation of p35-/- mice was significantly delayed for its Cdk5 activity was significantly decreased. However, heat stimulation of the pain of Tgp35 mice was allergic reaction, since its Cdk5 levels were significantly upward. These results indicated that the Cdk5/p35 involved in pain transmission in vitro. Inhibiting Cdk5 activity has become a new target of treatment in acute and chronic neurological pain. In our research, we tested the expression of p35 and Cdk5 in various developmental stages in vat Vc tissue and activity of Cdk5/p39. The results would be compared with others2. ObjectiveTo Test expression of p39 and Cdk5 and the activity of Cdk5 in various developmental stages in Vc tissue, analyze the activity of Cdk5 by in different developmental stages in rat Vc, Analyze and Compare the expression characteristic of p35 and p39.3. Methods72 male SD rats were randomly divided into 6 groups according to developmental stages as newborn group, one week group, two weeks group, three weeks group, four weeks group and adult group. The Vc tissue of each group was excised after they were anaesthetic, following by extracted the antigen protein in the Vc tissue. The immunoactivity of Cdk5 and p35 were determined by Western blot after the concentration of antigen was tested. Finally the immunoprecipitation and protein activity of Cdk5 were analyzed.4. Results1. Detection of the expression of Cdk5 and p39 protein by Western blotThe expression of p39 in Vc tissues changes significantly in various developmental stages in rat. The expression of p39 in new-born rat was less, the expression increased after one week and was at the highest levels in three and four weeks. After that the expression level was going to decreased, finally it was at the same level in adult as in new-born. While the expression of Cdk5 in the whole developmental stages showed no significant changes.2. Statistical analysis of immunoprecipitation and enzyme activity analysis of Cdk5 The activity of Cdk5 in Vc tissues in various developmental stages decreased rapidly, in the new-born was in the highest level, and after then it was going to decrease gradually, in adult, was at the least level. Cdk5 activity in new-born rats was about six times more than in adult.5. Conclusion(1)In various developmental stages, there was no significant relationship between the expression and activity of Cdk5(2) After birth in rat tissues of different developmental stages of Vc, p35 and p39 expression were significantly different. expression of p35 in the new birth, one week, two weeks were most obvious, and expression of p39 in two weeks, three weeks were the most obvious, expression of p39 is later than the expression of p35 1W.(3)In the early development stage, the activity changes of Cdk5 in rat Vc tissues has closely relationship with the past reports. In the later development stage, the the activity changes of Cdk5 in rat Vc tissues has closely relationship with expression of p39.
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