ABSTRACT
The DNA extraction method of animal medicine material is difficult and un-unified,which limits the application of molecular identification to identify animal medicines.In this study,based on the DNA extraction theory of SDS,we assessed the effects of three elements including different EDTA concentrations (0.025 mol·L-1,0.25 mol· L-1,and 0.5 mol· L-1) and whether containing NaCl and Triton X-100 in the lysis buffer on the quality of DNA extracted from different kinds of animal medicine.The optimized lysis buffer was used to extract DNA from 121 commercial animal medicines for original and species identification.The results showed that the lysis buffer of 1％ SDS,0.03 mol· L-1 Tris-HCl,0.25 mol· L-1 EDTA and 0.2 mol· L-1 NaCl had the optimum effect on DNA extraction.This lysis buffer can obtain DNA from animal medicine which is difficult to extract,such as Cicadae periostracum.The DNA extractions of 121 commercial animal medicines by optimized lysis buffer can satisfy the experimental requirements for molecular identification.All samples of commercial animal medicines can be accurately identified to the level of species.It was concluded that optimized lysis buffer can be used in the DNA extraction of different kinds of animal medicines except shells,secretions and processed products.This method provides technique support for the molecular identification of animal medicines.
ABSTRACT
BACKGROUND:Natural colagen is considered to have low immunogenicity and good biocompatibility relatively. OBJECTIVE:To evaluate the immunogenicity of animal-based colagenin vitro. METHODS:Type I colagen was extracted from bovine tendon after immunogenicity removal. The colagen purity was detected by high performance liquid chromatography and residual DNA was measured quantitatively by fluorescence staining. Fifty BALB/c mice were randomly divided into five groups: subcutaneous injection of normal saline solution (negative control), bovine colagen (positive control), 33.4, 66.8, 133.4 mg/kg of bovine tendon colagen, respectively, once a day. After 12 days of continuously subcutaneous injection, lymphocyte proliferation, and cel classification and NK cel kiling function of mice were detected; after 3 weeks of continuous injection, the spleen, liver, spleen and lung tissue of mice were taken for histological examination. RESULTS AND CONCLUSION:Compared with the standard type I colagen, the purity of purified type I bovine colagen reached more than 99%, but the residual DNA was below 1 mg/L which was far less than the residue level of conventional cel-free DNA in the matrix (dry weight: 50-100 μg/g). After 12 days of continuous injection, there were no changes in lymphocyte proliferation, NK cel kiling function and the proportion of lymphocyte subsets. After 3 weeks of injection, the spleen and lymph sheath of mice around the smal artery became thickened in the 66.8 and 133.6 mg/kg bovine tendon colagen groups, which could cause accidental liver injury and lung injury, but the splenic corpuscle germinal center area had no change. These findings indicate that continuously subcutaneous injection of animal-based colagen can cause the lower lymphocyte immune response to the spleen of BALB/c mice, which may cause accidental liver and lung injuries.
ABSTRACT
BACKGROUND:Colagen sponges are applied for hemostatic use, wound healing, and residual cavity filing, which have great values in clinical application and scientific research. OBJECTIVE:To investigate the biological properties, biocompatibility and biodegradability of colagen spongesin vivo. METHODS: The spatial structure, pore diameter and porosity of colagen sponges were characterized by scanning electron microscopy. Transmission electron microscopy was used to observe the conformation of colagen sponges. The secondary structure and thermal denaturation temperature of colagen sponges were analyzed by circular dichroism spectrum. Colagen sponges were implanted intramuscularly into the spinal cord of New Zealand rabbits to observe the degradation and absorption and histological changesin vivo. RESULTS AND CONCLUSION: Colagen sponges had porous structure with varying pore sizes ranging 40-150 μm, the mean pore size of 100 μm, the thickness wal of 1 μm, and a porosity of approximately 95.8%. Colagen sponges had a typical porous structure and periodic light and dark zones. The solution of colagen sponges had a weak positive band near 220 nm and an intense negative band near 206 nm, which indicated a classic triple helix. And the secondary structure and thermal stability of colagen sponges were similar to that of liquid colagen. Colagen sponges began to degrade at 4 weeks, and remained 20% at 12 weeks. These sponges had been associated with foreign body response and inflammation within 2 weeks after implantation. With wound healing, inflammatory reactions gradualy reduced and disappeared. During the implantation and degradation of sponges, no significant fibrous capsule formed and no tissue necrosis occurred at implantation site, indicating that colagen sponges have good performance in bioactivity, biocompatibility and degradation.