| Velvet antler s not full ossification young angle with densely hairy owned by cervidae like sika deer (Cervus Nippon Temminch) or red deer (Cervus elaphus Linnaeus), which has been used as a medicina substance stretches back more than two thousand years of history. well-documented in Shen Nong’s Herbal Classic of the Han Dynasty, Commonly used as traditional Chinese medicine, deer antler do nourishing care medicine such as warming and recuperating kidney,promote the formation of red blood cell hemoglobin, strong gluten bone and other effects been using until now. Modern Medicine study shows that antler contains a large number of inorganic elements, amino acids, proteins, lipids and carbohydrates compounds and other ingredients, especially young deer antler as a special kind of cartilage tissue. is a natural rich in a variety of active factors in cytokine library. The current study found that the peptide and protein plays an important role in life activities, which is one of the main medicinal components of antler velvet medicinal function. Till now, however, it has been long time spent on seeking whether there are any unique biologically active substance in antler, resulting in antler growth-promoting, promoting metabolism, anti-inflammatory, immune regulation of brain, the blood of anti-aging, health, and promote wound and fracture healing pharmacological role has not an appropriate explanation in pharmacological composition aspect. As the country gradually attaching increasingly importance to the modernization of Chinese medicine, deer antler, which is rich in control of the organs and tissues to grow and develop a variety of peptide cell growth factor, is therefore very meaningful. We chose the PAP for wound healing and cellular immunoregulatory tests in the following research.Weng et al (2001) reported new findings from the antler of a polypeptide with a molecular weight size3.2kDa stimulate the activity of the mouse epithelial cells and rabbit costal cartilage cell growth and there is a certain dose relationship. Guan et al (2006) get a size similar to other homologous peptides with similar biological activity isolated from red deer antler. However, due to the low-yield limiting can not be carried out in vivo biological activity tests instead crude extracts or secondary extract. Although it has been observed to the expected activity, but is not yet clear what specific single component plays a major role or a variety of peptide composition result of the cooperation. In order to solve the problem, we focus on the following work:First, with the basis of previous studies, the potential biological activity of the PAP component was carried out with extraction, separation, purification, identification and then the single PAP gene was remodeling by the use of recombinant technology, and try its expression in a prokaryotic expression system and purified in vitro. Finally, the recombinant3.2kDa velvet antler polypeptide (rVAP32) and its natural polypeptide (nVAP32) were comparatively researched in wound-healing test and the cell immunomodulatory roles tests in vivo and in vitro.Experiment result:1. Fresh plum antler raw materials, duplication and improve the process of preparation of the monomer PAP:by colloid homogenate,65%ethanol precipitation, freeze drying method of preparation of antler polypeptide; further use by Tricine-SDS-PAGE and laser desorption ionization time of flight mass spectrometry and other preliminary physical and chemical properties, the purpose of total polypeptide production has increased to65.6%, active monomer peptide yield rate of1.6mg/kg with the final purity of98.5%. The target peptide primary structure is32amino acid peptides N-terminal:VLSATDKTNVLAAWGK V G G N A P A F G A. E-A L E R M, we named the natural velvet antler polypeptide (nVAP32).2. Reconstruction of a structure of3.2kDa polypeptide amino acid sequence and reference of E. coli preference codon table, the gene sequence and prokaryotic expression vector PET-22b-VAP32completed in E. coli BL21(DE3) pLysS expression and purification. Recombinant PAP N-terminal His tag fusion protein expression, and mainly in the form of inclusion bodies, the yield of about66mg/L, which was named the rVAP32.Wistar rats were used in wound-healing test, suffered from homemade rectangular iron in the rat back for mading a length×width:6cm×1cm area of burn-wound model along the long axis direction of the torso. By twice daily administration of5mg homemade ointments contained nVAP32or rVAP32(0.02%,0.05%and0.1%) until the skin is healing well. The results showed that the high dose group treatment is effective. Specific detection of indicators:(1) The surface of skin wound repair test results:When the administration rose to0.05to0.1%(w/w) concentration nVAP32rVAP32test groups showed a significant effect very similar to the results of the positive control group, but significantly different results with the negative control group. Dosing concentration of0.1%(w/w) nVAP32mouse skin repair effects of the test group (15.83±1.81days) is a little better in rVAP32(16.13±1.47days) experimental group. (2) Skin tension test results:test in group nVAP32and rVAP32compared with the control group tension values (kg/cm2) when administered concentration of0.05and0.1%(w/w) significantly increased;(3) Skin hydroxyproline content of the test results:When the drug treatment for burns animal model test to12days,0.05and0.1%(w/w), nVAP32test group mice regeneration of skin tissue hydroxyproline content of37.12±0.45μg/g and46.15±0.53, higher than the equivalent dose rVAP32the value of the experimental group (36.38±0.57μg/g and40.48±0.49μg/g) The group and the two test the value was significantly higher than measured values of hydroxyproline content control.4. nVAP32and rVAP32pairs of mice in vitro immunomodulatory effects test, using BALB/c mice, the extract of spleen cells, NK cell proliferation and cytokine secretion levels determination, with this indicator as follows:(1) On the proliferation of splenocytes to ConA and LPS (0,5,25,50,200μg/ml) for the control,50-100μg/ml nVAP32, rVAP32are able to effectively stimulate spleen cell proliferation.(2) The phagocytic activity of mouse NK cells:nVAP32and rVAP32in25-100μg/ml declared to enhance the phagocytic activity of NK cells.(3) The impact on the level of secretion of various cytokines:nVAP32and rVAP3225-100μg/ml when the cell sub-IL-2, IL-12and TNF-alpha and IFN-γ regulation of cell factor IL-4and IL-10have a significant downward effect, and significant dose effect relationship was observed.Conclusion:(1) An in vivo test results show that,0.05%,0.1%(w/w) rVAP32dose showed significant damage to the skin repair effect, and no dose effect relationship when the dose is lower than0.05%, so the relatively high yield rate rVAP32potential clinical tissue repair real basis of drug development.(2) The administration of concentration for25-100μg/ml rVAP32could significantly increase NK cell phagocytosis ability, and at the molecular level to increase IL-2, IL-12, IFN-γ and TNF-alpha factor and lowered IL-4and IL-10factor, indicating that the peptide is likely to enhance the immune response of the overall level of Th1cells as target cells, which might play a leading role in cellular immune function. |
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