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Objective:: To investigate the effects of pilose antler polypeptide on the abilities of proliferation and collagen secretion of mouse embryonic fibroblasts NIH/3T3, and to clarify the relevant mechanisms. Methods: The NIH/3T3 cells were treated with different doses 1.56, 3.13, 6.25, 12.50, 25.00, 50.00, 100.00, and 200.00 mg • L_ 1) of pilose antler polypeptide as experimental groups, the cells treated with 0 mg • L_ 1 pilose antler polypeptide were used as blank control group, and the cells treated with 50.00 fig • L-1 basic fibroblast growth factor (bFGF) were used as positive control group. MTT assay was used to detect the survival rates of NIH/3T3 cells in various groups. ELISA assay was used to detect the collagen secretion of NIH/3T3 cells in various groups. Wound healing assay was used to detect the migration abilities of NIH/3T3 cells. Western blotting method was performed to detect the expression levels of p-ERK 1/2 in the NIH/3T3 cells in various groups. Immunofluorescence method was used to detect the expression levels of transforming growth factor-fil (TGF-J31) in the NIH/3T3 cells in various groups. Results: Compared with blank control group, the survival rates of NIH/3T3 cells in positive control group and 6.25, 12.50, 25.00, 50.00, 100.00, 200.00 mg • L_ 1 pilose antler polypeptide groups were markedly increased (P < 0 . 05 or P < 0 . 01). Compared with blank control group, the levels of type I collagen protein in the culture solution of the NIH/3T3 cells in positive control group and 6. 25, 12. 50, 25. 00, and 50. 00 mg • L_ 1 pilose antler polypeptide groups were markedly increased (P < 0 . 05 or P < 0 . 01), and the levels of type IE collagen protein in the culture solution of the NIH/3T3 cells in positive control group and 12. 50 and 25.00 mg • L_ 1 pilose antler polypeptide groups were markedly increased (P < 0 . 05). Compared with blank control group, the scratch healing rates of NIH/3T3 cells, and the expression levels of p-ERK 1/2 in the NIH/3T3 cells, and the expression levels of TGF-J31 in the NIH/3T3 cells in positive control group and 12. 50 mg • L_ 1 pilose antler polypeptide groups were markedly increased (P < 0 . 05 or P < 0 . 01). Conclusion: Pilose antler polypeptide can promote the proliferation, and collagen secretion of NIH/3T3 cells and increase the migration ability, which may be achieved by activating the phosphorylation of ERK 1/2 and increasing the expression of TGF-J31.
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Objective:To explore the effect and mechanism of pilose antler different components on the bone tissue of ovariectomized osteoporosis model rats and ascertain the material basis of pilose antler. Method:fifty-six SD rats were divided randomly into seven groups:normal group,model group,Xianling Gubao group(468 mg·kg-1),Bujiale group(80 mg·kg-1),polysaccharide group(50 mg·kg-1),polypeptides group(175 mg·kg-1),polysaccharide and polypeptide mixture group(50 mg·kg-1+175 mg·kg-1). Osteoporosis mode was established through ovary resection of female rats,meanwhile,the rats were given different components of pilose antler for consecutively 12 weeks. Subsequently, using absorptiometry to measure the rats' bone mass density. The activities of bone alkaline phosphatase(BALP),osteocalcin (OT),bone morphogenetic protein2(BMP-2),Smad1,Smad5,Runt-related transcription factor 2 (RUNX2) were detected by enzyme-linked immuno sorbent assay (ELISA). The expression of BMP-2,Smad1,Smad5,Runx2 protein was examined by Western blot and Real-time polymerase chain reaction (Real-time PCR). Morphological assay for bone tissue were detected by htoxylin eosin(HE) staining. Result:After 12 weeks, Compared with the normal group, the osteoporosis model group showed significantly decrease in bone mineral density(PPPConclusion:Pilose antler different components has therapeutic effect on ovariectomized osteoporosis model rats.The mechanism may be related to up-regulat the expression of BMP-2/Smad1,Smad5/Runx2 signal pathways.
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Objective:To investigate the effect of pilose antler polypeptide combined with Schwann cells modified by glial cell line-derived neurotrophic factor (GDNF)gene on the proliferation of human bone marrow mesenchymal stem cells (BMSCs)in vitro .Methods:According to the conventional method,the bone marrow (10 mL)was extracted from the healthy volunteers and was inoculated into the culture flask.The primary cultured cells were completely fused.The BMSCs were harvested at the 3rd generation and the cells were adjusted to 5 × 106 mL-1 . 4 μL GDNF gene modified Schwann cells was added into GDNF group,4 μL (10 mg· L-1 )PAP combined with GDNF gene modified Schwann cells was added into combination group,and only same amount of medium was added into control group.The proliferative activities,cell nuclear antigen (PCNA)levels and apoptotic rates of BMSCs in various groups were detected by enzyme-linked immunosorbent assay,ELISA method and Annexin Ⅴ-FIFC/PI cell apoptosis detection kit,respectively.Results:After primary culture for 48 h,most of the cells adhered to the wall,and the morphology of the cells changed into polygonal shape and few of them showed spindle.The passaged cells showed spindle spindle,the cells were confirmed as BMSCs,and all of them were non-hematopoietic stem cells.Compared with control group,the proliferative activities and the PCNA level of the BMSCs in GDNF group and combination group were increased (P <0.05)and the apoptotic rates were decreased (P <0.05);compared with GDNF group,the proliferative activity and the PCNA level of the BMSCs in combination group were increased (P < 0.05 )and the apoptotic rate was decreased (P < 0.05 ).Conclusion:PAP combined with Schwann cells modified by GDNF gene can promote the proliferation of human BMSCs in vitro .
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10.3969/j.issn.2095-4344.2013.25.013