Differentiation of in vitro induced human pluripotent stem cells into hematopoietic stem/progenitor cells / 中国实验血液学杂志

This study was aimed to explore the differentiation of in vitro induced human pluripotent stem cells (iPSC) into hematopoietic stem progenitor cells. The human iPSC were induced to differentiate into hematopoietic stem/progenitor cell by co-culturing with OP9 bone marrow stromal cells. The expression of hematopoietic stem/progenitor cell surface markers were detected by flow cytometry. The regulation gene expressions of iPSC and hematopoietic stem/progenitor cells were measured by real-time PCR. The CD34(+) hematopoietic stem/progenitor cells were isolated by using immunomagnetic beads, and were used for colony formation assay. The results showed that after iPSC were co-cultured with OP9 cells for 4 days, the morphological changes of iPSC could be observed. Hematopoietic stem/progenitor cell surface markers CD34 and CD43 could be detected by flow cytometry after differentiation. The pluripotent marker gene OCT4 expression gradually decreased and blood-related transcription factor Gata-2 expression gradually increased, while Runx-1 expression was wavily changed, CD34 expression gradually increased. The erythroid colony(CFU-E), granulocyte colony(CFU-G), megakaryocytic colony(CFU-M), granulocyte-megakaryocytic colony(CFU-GM), and mixed colony(CFU-GEMM) were obtained after cultures for 14 d. It is concluded that the human iPSC cells can be induced to differentiate into hematopoietic stem/progenitor cells in vitro by co-culture with OP9 cells.

Experimental study on HDV ribozyme in vitro cleaving the HBV derived RNA fragment / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the possibility of transacting hepatitis D virus (HDV) ribozyme cleaving in vitro the hepatitis B virus (HBV) mRNA fragments.</p><p><b>METHODS</b>According to the established pseudoknot-like structure, its' H1 domain was changed to design the transacting HDV ribozyme Rc1 and Rc2, which targeted the 701-713 site and 776-788 site of HBV C domain. After the chemically synthesised cDNA of the ribozyme was cloned into the vector PGEM-4Z, the transacting HDV ribozyme was transcriped using in vitro transcription technology. The in vitro cleavage characteristics of the ribozyme were studied and the kinetic parameters (Kcat and Km) were determined by Eadie Hofstee plotting.</p><p><b>RESULTS</b>Both the two ribozymes had the ability to cleave the substrate, the cleavage percentage at 37 degrees for 90 minutes were 50% and 51%. According to the Eadie Hofstee plot, the Km of the Rc1 and Rc2 were 0.61 micromol and 0.58 micromol, the Kcat were 0.64 x min(-1) and 0.60 x min(-1),respectively.</p><p><b>CONCLUSIONS</b>The cleaving ability of trans-acting HDV ribozyme on non-HDV RNA fragment was tested. The results showed a new potential of the antisense antisense regent for HBV gene therapy.</p>