| Objective:Currently,the Ficoll centrifugation method is widely used in the extraction of Buffy coat,but this method cannot be used clinically due to the cytotoxicity of Ficoll.Therefore,the main objective of this experiment is to develop a simple and affordable method for extraction of Buffy coat that can be used clinically.Methods:20 healthy New Zealand white rabbits(16 weeks old,male,weighing 3.0 to 3.5 kg)were selected for the experiment.4 ml of bone marrow was extracted from the anterior superior iliac spine of rabbits using a syringe containing 2 ml of sodium citrate.The collected bone marrow was placed in a tube containing Ficoll and centrifuged as Ficoll group(F)and another 4ml of bone marrow was placed in a test tube without Ficoll and centrifuged as New method group(N);Centrifuge the F group and N group at 2000 rpm at 4? and a centrifugal force of 1000 g for 30 minutes.After the centrifugation,there was a unique upper serum between Ficoll and serum in the F-tube,under which was a light yellow liquid(Buffy Coat)between Ficoll and serum,and the bottom was red blood cells;there was a thin layer of Buffy Coat between the serum and red blood cells at the top of the N-tube,about 1 ml of sample was extracted from the bottom of the serum and the top of red blood cells together with Buffy Coat,it was placed in a 1 ml syringe for a second centrifugation and the time was reduced to 15 minutes.After centrifugation,a clear upper serum was visible,under which was a thick pale liquid(Buffy Coat)and the bottom red blood cells.Under a high powered microscope the number of MSCs in the Buffy Coat isolated from group F and group N was counted.The amount of fibroblast colony forming unit fibroblast(CFU-f)was established after 12 days of mesenchymal stem cell(MSC)culture in MSC medium containing a-MEM containing 10%FBS and 1%antibiotic-antifungal solution.The transforming growth factor-?1(TGF-?1),platelet derived growth factor(PDGF),fibroblast growth factor(FGF),insulin-like growth factor-1(IGF-1),endothelial growth factor(EGF)and vascular endothelial growth factor(VEGF)growth factors and interlukin-1?(IL-1?),interlukin-6(IL-6)and tissue necrosis factor-a(TNF-a)and other inflammatory factors were detected by ELISA.Results:1.There was no significant difference in MSC counts between the two groups(2766±345.75 vs.2757.25±344.66,P>0.05).2.In groups N and F the mean values of TGF-?1 were 1.29 ng/ml and 1.28 ng/ml,respectively;PDGF was 1.49 ng/ml and 1.42 ng/ml respectively;FGFs were 1.34 ng/ml and 1.33 ng/ml,respectively.-1 each was 240.94 pg/ml and 220.96 pg/ml;EGF was 164.81 pg/ml and 161.91 pg/ml respectively;VEGF was 37.26 pg/ml and 35.42 pg/ml respectively,there was no significant difference in the above growth factors between the two groups(n=16,all P>0.05).3.In groups N and F the mean values of IL-1 were 866.55 pg/ml and 870.78 pg/ml respectively;IL-6 were 218.64 and 259.78 respectively;TNF-a were 1073.29 pg/ml and 929.84 pg/ml respectively,there was no significant difference in the above growth factors between the two groups(n=16,all P>0.05).Conclusion:1.The bone marrow-derived Buffy coat extracted by the new method has no cytotoxicity,and the number of MSCs,various growth factors and inflammatory factors are almost similar to the Buffy coat extracted by Ficoll centrifugation.2.The new method can replace the old Ficoll method.The experimental results provide experimental basis for the clinical application of bone marrow Buffy Coat and research in the field of tissue engineering. |
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