Effects Of Nerve Growth Factor From Snake Venom (Naja Naja Atra) On Expressions Of Opioid Receptors In PC12 Cells

Objectives: Rat adrenal gland pheochromocytoma cell (PC12) was used to determine NGF's activity generally. Under the induction of NGF, differentiated PC12 cells did like neurons in morphology, physiology, and biochemistry and function aspects. These cells produced neurites and synapsis conjunctions, and were stable in vitro more easily. So PC12 cell was a practical model to research neurons'differentiation at molecular levels. But its differentiated mechanism was yet completely unclear. Some results indicated that when mouse NGF induced PC12 cells, the endogenous opioid receptors were changed. This hinted that opioid receptor may be related to PC12 differentiated mechanism. Thus, we examined the effects of NGF from snake venom (Naja Naja Atra) (nNGF) on cells'morphology and expressions of opioid receptor genes and proteins in PC12 cells, probe the molecular mechanism of PC12 cell differentiation induced by nNGF, and provide an experimental foundation for further study on nNGF.Methods: (1) Using microscope to observe the effects of 50 ng/ml nNGF on the morphology of PC12 cells in vitro culture. (2) The effects of nNGF at different doses (25 ng/ml, 50 ng/ml, 100 ng/ml nNGF) on expressions ofδ,κopioid receptors mRNA in PC12 cells, the expression patterns ofδ,κopioid receptors mRNA in PC12 cells at five different time phases after the induction of nNGF, and the effects of Morphine and Naloxone on the induction of nNGF were all examined by semi-quantitative RT-PCR method. (3) The effects of nNGF at different doses (25 ng/ml, 50 ng/ml, 100 ng/ml nNGF) on expressions ofκopioid receptor proteins in PC12 cells, the expression patterns ofκopioid receptor proteins in PC12 cells at five different time phases after the induction of nNGF, and the effects of Morphine and Naloxone on the induction of nNGF were all examined by Western blot method.Main results and conclusions: (1) PC12 cells were sensitive to nNGF. nNGF could promoted PC12 cells to differentiate highly, produced some nervous fibers and their morphology were similar to neurons.(2) After dealing with nNGF, the expressions ofδandκopioid receptors mRNA in PC12 cells presented the dosage effect of nNGF. With the raising of nNGF concentration, the expressions ofκopioid receptor mRNA in PC12 cells were down-regulated. The expressions ofκopioid receptor mRNA most significantly were down-regulated at the concentration of 50 ng/ml nNGF. However, the expressions ofδopioid receptor mRNA in PC12 cells were up-regulated with the raising of nNGF concentration.(3) After addition of 50 ng/ml nNGF, the expressions ofδandκopioid receptor mRNA in PC12 cells were opposite to each other from the 1st day to the 10th day. The expressions ofκopioid receptor mRNA were down-regulated; however the expressions ofδopioid receptor mRNA were up-regulated.(4) nNGF increased the expressions ofδopioid receptor mRNA, the effect of nNGF could be reversed by Naloxone, and the effects of Naloxone could be reversed by Morphine; nNGF decreased the expressions ofκopioid receptor mRNA, the effects of nNGF could be reversed by Naloxone or Morphine respectively.(5) After dealing with nNGF, the expressions ofκopioid receptor proteins and its mRNA in PC12 cells had the same tendency. With the raising of nNGF concentration, the expressions ofκopioid receptor proteins in PC12 cells were down-regulated and most significantly 50 ng/ml nNGF down-regulated the expressions ofκopioid receptor proteins.(6) After addition of 50 ng/ml nNGF, the expressions ofκopioid receptor proteins in PC12 cells were down-regulated from the 1st day to the 10th day.(7) nNGF could down-regulated the expressions ofκopioid receptor proteins, the effect of nNGF could be reversed by Naloxone or Morphine respectively.(8) nNGF could influenced the expressions of the opioid receptors. Opioid systems may take part in the PC12 cells'differentiation under the induction of nNGF.