Evaluation of Storage Performance of Preserving Bags for Manually Separated Platelets / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate the storage performance of the domestically made platelet storage bags (experimental group) and the United States Trima set platelet storage bags (control group).</p><p><b>METHODS</b>The manually separated platelets were divided in two equal parts, which was added to control blood bags and experimental blood bags respectively, all samples were stored at a 22 °C ± 2 °C. The platelet count, mean volume, aggregation activity (ADP, THR), pH, glucose, lactate concentration, lactate dehydrogenase concentration, hypotonic shock reaction, CD62P and phosphatidic acid serine content were detected at day 0, 3, 5 and 7 of storage.</p><p><b>RESULTS</b>There was no significant difference of platelet quality at day 5 after storage between the experimental group and the control group (T-test, P > 0.05).</p><p><b>CONCLUSION</b>Two kinds of platelet storage bags have the similar storage performance.</p>

Influence of cytokine combinations on proliferation and differentiation of umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro / 中国实验血液学杂志

In order to investigate the influence of cytokine combinations on proliferation and differentiation of human umbilical cord blood CD34(+) cells into megakaryocytes/platelets in vitro, the CD34(+) cells from human umbilical cord blood were amplified in serum-free medium StemSpan(SFEM) supplemented with several cytokine combinations by three-phase culture system. The effects of the cytokine combinations were compared. The results showed that at day 14 of the first culture phase, the CD34(+) cells cultured with cytokine combinations SCF + TPO + FL + IL-3 were amplified (11 000 ± 1 000) times, which were significantly higher than that of cells cultured with SCF + TPO + FL, but were not significantly different from that of cells cultured with SCF + TPO + IL-3 or SCF + TPO + FL + IL-3+ hydroxyl-corticosteroids. At day 7 of the second culture phase, the CD34(+) cells cultured with cytokine combination SCF + TPO + FL + IL-11 were amplified by (204666.7 ± 11718.9) times, which were significantly higher than that of cells cultured with SCF + TPO + FL + IL-3, but were not significantly different from that of cells cultured with SCF + TPO + FL + IL-11 + BMP4 + VEGF. At day 3 and day 6, the CD34(+) platelet-like cells accounted for about (39.8 ± 1.9)%, (39.7 ± 2.6)% and (25.5 ± 1.4)%, (23.1 ± 3.5)% cultured with SCF + TPO + FL + IL-11 and SCF + TPO + FL + IL-11 + BMP4 + VEGF, and significantly higher than that of the cells cultured with SCF + TPO + FL + IL-3. It is concluded that the cytokine combination of SCF + TPO + FL + IL-3 is most suitable cytokines combination for the amplification of CD34(+) hematopoietic progenitor cells. The cytokine combination of SCF + TPO + FL + IL-11 is preferred for the proliferation and differentiation of megakaryocytes, this study lays an experimental basis for investigating the proliferation and differentiation of CD34(+) into megakaryocytes/platelets in vitro.

Gene cloning of cdtB from Actinobacillus actinomycetemcomitans and bioactivity test of the recombinant protein CdtB in vitro / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To construct the prokayotic expression vector pET-15b-cdtB containing the cdtB gene from Actinobacillus actinomycetemcomitans (Aa) and to test the bioactivity of this recombinant CdtB in vitro.</p><p><b>METHODS</b>The toxic cytolethal distending toxin (CDT) subunit encoding gene cdtB was amplified by PCR. Through restriction endonuclease digestion, gene cdtB and vector pET-15b were ligated to form pET-15b-cdtB expression system which was transformed into competent cells Escherichia coli BL21 (DE3). Protein expression was induced by isopropyl-beta-D-thiogalactoside and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Supercoiled plasmid pET-32a DNA was incubated with purified recombinant CdtB protein in vitro to view any changes in the electrophoretic mobility of the plasmid pET-32a DNA band.</p><p><b>RESULTS</b>PCR testing results of pET-15b-cdtB transformed cells demonstrated that all strains contained cdtB gene. The DNA sequence was blast with cdtB gene from GenBank and 99% homology was obtained. Both of SDS-PAGE and Western blotting confirmed that recombinant CdtB was obtained. After incubated with the purified recombinant CdtB in vitro, the supercoiled plasmid pET-32a DNA was observed relaxing by 1% agarose gel electrophoresis test.</p><p><b>CONCLUSIONS</b>The recombinant plasmid pET-15b-cdtB was successfully constructed and the recombinant CdtB protein which has the DNaseI-like activity was obtained.</p>