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The Influence of Acidic Neuropeptide on Nerve Nutrition Factor in Newborn Rat Astrocytes in Vitro

Author: MaHongXia
Tutor: AnYuHui
School: Zhengzhou University
Course: Biochemistry and Molecular Biology
Keywords: acidic peptide astrocyte LDH NGF BDNF
CLC: R749.16
Type: Master's thesis
Year: 2005
Downloads: 101
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Abstract

Background and purpose:Biopetides refer to the petides that have beneficial or physiological effect on organism. In recent years , the research on biopeptides made rapid progress. It has been shown that biopeptides have the function such as immunomodulation, endocrine modulation, enzyme inhibition , antibiotic, antiviral, anticancer and flavour adjusting.Therefore, Biopeptides can be widely used in medicine and food industry.Neuropeptides are some important substance that have nerve signal transmit and regulation action. They distribute widely in the central neural system, participating in a variety of functional regulations. An Yuhui isolated some small neuropeptides from bovine brains . Experiments in animal model have proved that : (1) acidic peptide has not mutation, but has antimutation. Its mechanism of antimutation is antioxidation action , enhancing II xiang reaction; eradicting mutation action; and avoiding normal cell transform into mutation cell. (2) It can regulate neural system by increasing amino acid and affecting the contents in cGMP of brains. (3) By increasing the synthesis of protein and the antioxidants defences of brain and improving the energy metabolise of brain and decreasing the synthesis of NO in the brain, acidic peptide have good treatment on model of AD(Alzheimer’s disease)rats and VD(Vascular dementia)mice. (4)Acidic peptides is a small molecular neuropeptide without antigenicity, and can permeate blood brain barrier.Alzheimer’s disease is a nerve retrograde disease which has memorydecrudescence and recognition function damage .Its pathology display SP(senile plaques) and neurofibrillary. But because of not knowing its noxa and pathogeny , nowsdays there is not still effective remedy . Consequencely, AD’S clinical therapy is still an unsolved difficult medicine problem. Nowadays the drug therapies about AD mainly are as follows: (D cholinesterase inhibitor; (2) excitocatabolic drug; (3) hormonal drug; ? anti-inflammatory analgesic; (§) anti-free radical ; ? prevent amyloid protein synthesis and sludging’drug ; ?Ca + antagonistic drug ; (D excitatory amino acids receptor antagonistic drug; (§) NGF(nerve growth factor) and NGF’s gene therapy . Induction NGF form is a new research idea. It was reported that some molecular and receptor excitomotor can induce the formation of NGF. In order to research acidic peptide that can or cannot induce the formation of nerve nutrition factor, we do this study.Astrocyte can secrete nerve nutrition factor automaticaly.Our aim is to observe the effect of acidic peptide on nerve nutrition factor expression in astrocyte in vitro, and to observe the produce and secretion increasing or not increasing of nerve nutrition factor, and to provide theory basis for acidic peptide’s therapy on AD.Method:The cerebral cortex was extracted from the SD big rat newborn 2 days to throw PBS on the condition of aseptic technique, and culled carefully midbrain, bulbus olfactorius, hippocampus, meninges, capillary and so on ,and sheared cortex into l-2mm3 dice, and hitted for about 1 minute by straw, and digested for 10 minutes, then collected sediment and suspended by DMEM culture medium. Suspended cells were filtered by colatorium, and regulated its density to 1.0xl06/ml.Cell survival rate was confirmed justo 95% by trypan blue. Cells were inoculated to 25cm2 Culture bottle. Cells were changed culture medium after 24 hours, then changed once every 3 days. When occupied 70% bottle bottom, cells were passaged. After two passages, astrocyte was confirmed justo 95%.The third passage astrocyte were digested to inoculate to 12 fenestra culture plate lml every fenestra. After 24 hours, culture liquid was changed. Cell culture liquid wasdivided into four groups: 20% serum group; no-serum group; positive control group, experiment group. 20% serum group and no-serum group were not added to experiment element. Positive control group was lOOOU/ml IFN-a. Acidic peptide was added to experiment group by 0.15mg/ml,0.075mg/ml,0.0375mg/rnl. Control group and experiment groups reacted 24 hours,48 hours,72 hours separately.When experiment was terminated, supernate was strawed to aseptic EP tube,and was centrifuged at lOOOr/min for five minutes.Then supernate was strawed to aseptic EP tube again,and was put into -20 °C. A little 0.25% trypsin was put into culture plate .when digested for about fifteen minutes in CO2 culture box, cells became cell suspension by straw hit. Complete culture medium terminated disgestion, and added to lml, and counted cell after mixture at once. A drop of 0.4% trypan blue work liquid and 9 drops cell suspensions were put into a small bottle, and after mixture droped on the blood cell count plate. And counted blue cells and totle cells.Cell survival rate = (total cell quantity- blue cell quantity)/total cell quantity xl00%LDH content in the culture supernate was assayed by Rili 7170 fully automatic biochemistry analyzer.The third generation astrocyte were digested by 0.25% trypsin , which were diluted by 10% fetus cattle serum DMEM culture medium for O.5xlO5/ml cell concentration .Cells were inoculated to a culture bottle where polysine was pre-overspread. Cells were transformed culture medium every other two days. When cells occupied 70% culture bottle volume. Cells were passaged to 96 hole culture plate for 24 hours. Cells were changed culture medium. According to experiment groups, acidic peptide group and posive control IFN-a group were diluted by no-serum culture medium. Cells were cultured for 72 hours. Cell form and growth were observed under the inversion microscope. When experiment was terminated, 96 hole culture plate was extracted from CO2 cell culture box. 200ul/hole MTT was added to every hole (MTT concentration, 5mg/ml), and culture plate was put into CO2 culture box for 4 hours. Cell culture liquid was extracted, and 150ul/hole DMSO was added to 96 hole cell culture plate. Culture plate was shaken for 10 minutes. Thenculture plate was detected for OD value at 492 nm wavelength. All data were statisticed by t-test.Cell proliferation rate = (experiment group OD value - control group OD value)/ control group OD value x 100%The OD value of NGF,BDNF located in cell culture supernate was assayed with ELISA. The OD value may reflect NGF, BDNF synthesis and secretion level indirectedly.Result:1. Astrocyte itself can synthesize and secrete NGF. NGF is increasing with timeextention.Different concentration acidic peptide can synthesize and secrete different quantity of NGF in different time.The quantity of NGF is increasing compared with no-serum control group, and has some effect on quantity and time.2. Astrocyte itself can synthesize and secrete BDNF. BDNF is decreasing with timeextention.Different concentration acidic peptide can synthesize and secrete different quantity of NGF in different time.The quantity of BDNF is increasing compared with no-serum control group,3. 20% serum group do some harm to astrocyte .LDH leakage quantity is increasing, and cell survival rate is low compared with no-serum acidic peptide group.4. Acidic peptide can make astrocyte in vitro proliferate in quantity and increase in survival rate.Conclude:1. Acidic peptide does not harm astrocyte in vitro, and has some nutrition and protection action on the growth and survival of astrocyte and neuron.2. Acidic peptide can promote astrocyte in vitro synthesizing and secreting NGF, BDNF . Acidic peptide has some therapy and relief action on nerve retrograde disease, for example , Alzheimer’s disease. It has nerve nurtition factor action, but it avoids the difficult and restriction of nerve nurtition factor,and has a better development prospect.3. This study provides some theroy basis for acidic peptide’s therapy action on Alzheimer’s disease, and has great theroy and society significance.

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