| Objective: To identify whether acidic peptide exists in different kinds of animals' brain, determine the content of AP and that of Glu in the big mouse's brain and explore the effect of extra-originated AP on the endogenous AP in the big mouse' brain.Methods: The marker including three kinds of standard small peptide (SP) or amino acide: Glu, AP and GSH, were isolated and located by using filter paper electrophoresis method. Then the existence of AP in the brain of bovine, sheep, hen, duck, rabbit, big mouse and other animals were identified by using this marker. The density of mixed solution which included standard Glu, AP, GSH were 0.5mg/ml, 1.0 mg/ml, 2.0mg/ml, 3.0mg/ml, 4.0mg/ml. 4μl of each density was taken out and put on the filter paper respectively, then were separated by electrophoresis method. The power was shut off after 1 hour and the paper was put into the drying oven for 10 minutes, and then the spot was cut and put into the eluting fluid to elute for 15min. The OD values of different density of AP or Glu were determined by spectrophotometric method. Take the OD value as the y-coordinate, the density or the content as the x-coordinate. The standard curve of Glu and GSH were drew up respectively. Mice were divided into six groups (10 in each group). One of the group without filling stomach was beheaded as the control group. Five groups' stomach was filled with AP at dose of 100mg/kg, and after lh, 2h, 3h, 4h, and 5h respectively, the mice were beheaded, their brains were taken out from their skull and homogenized and extracted with the normal saline whose volume was 4 times the weight of theirbrains respectively. Then the homogenized brains were centrifuged by 3000w/m for 10min, the clear solution were extracted and heated in boiling water for 5min, then centrifuged for 10min by 5000w/m after cooling, the extracted solution is for the examination. Bind electrophoresis method with spectrophotometric method to analyze the OD values of the Glu and AP in the mouse brain, and then find out the density or the content according to the standard curve respectively. Observe the change of the density or content of AP and Glu at the point of different time respectively.Results: The mixed solution including Glu, AP, and GSH can be isolated by filter paper electrophoresis successfully. The standard curves of Glu and AP were made by binding electrophoresis method with spectrophotometric method. The regression equation of standard curve of Glu is y=0.0125x+0.0005, r=0.9996.The regression equation of standard curve of AP is y=0.0096x+0.0025, r=0.9985. The correlations are both good. Compared with that of control group, the content of Glu had a significant increase after 1h, 2h, 3h(P<0.01), and the content of AP also increased after 2h, 3h (P<0.01).Conclusions: 1. Small peptides (SP) can be isolated by using paper electrophoresis method. 2. AP consists in the animal's brain generally, and its content is higher than the glutamic acid. 3. Extra-originated AP can penetrate the animal's blood brain barrier (BBB) to the brain, and execute its biological function. |
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