Real-time quantitative RT-PCR for the detection of bcl-2 mRNA expression / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a real-time quantitative RT-PCR method for the detection of expression of bcl-2 mRNA.</p><p><b>METHODS</b>The vector containing bcl-2 gene as standard template was constructed with T-A cloning technique. The fluorogenic probe (i.e.,Taq man probe) was used to establish a real-time RT-PCR. bcl-2 mRNA expression level in Burkitt's lymphoma pre-and post-treated with bcl-2 antisense phosphothioate oligodeoxynucleotides (AS-PS-ODN) was determined with real-time quantitative RT-PCR, also with semi-quantitative RT-PCR.</p><p><b>RESULTS</b>The expression of bcl-2 mRNA in Burkitt's lymphoma treated with bcl-2 AS- PS-ODN decreased significantly and no changes of bcl-2-mRNA expression in group treated with nonsense ODN were noticed. The semi-quantitative RT-PCR results also demonstrated that bcl-2 level varied as detected with real-time fluorogenic quantitative RT-PCR, but less sensitive and accurate.</p><p><b>CONCLUSION</b>Detection of bcl-2 mRNA expression with the fluorogenic real-time quantitative RT-PCR is more accurate and sensitive than semi-quantitive RT-PCR.</p>

Effects of TGF-?_1 and TNF-? antisense PS-ODNS on e x vivo expansion of hematopoietic stem/progenitor cells(HSPC) / 中国病理生理杂志

AIM: To study the effect of TGF-? 1 and TN F-? antisense PS-ODNS on ex vivo expansion of hematopoietic stem/progenitor cell s (HSPC). METHODS: CD 34 + cells were purified from fres h umbilical cord blood by immunomagnetic beads, and mononuclear cells were purifi ed from bone marrow by Ficoll-hypaque . The effects of TGF-? 1 and /or TNF-? an tisense PS-ODNS on ex vivo expansion of CD 34 + cells、CFU-GEMM、CFU-G M、CFU-E and BFU-E were detected by using liquid and semi-solid culture systems . RESULTS: TGF-? 1 antisense PS-ODNS cooperated with cytokines increased the number of CD 34 + cells,CFU-GEMM,CFU-GM,CFU-E and BFU-E , which was as 4,2.6,2.7,1.8,2.1 times as that of the control (the cytokine s combination), respectively. TNF-? antisense PS-ODNS cooperated with cytokines respectively increased the number of CD 34 + cells, CFU-GEMM, CFU-GM, CF U-E and BFU-E by 4, 2 9, 2 6, 1 7, 1 8 times as that of the control. The ab ove two antisense PS-ODNS cooperated with cytokines could respectively increased the number of CD 34 + cells, CFU-GEMM, CFU-GM, CFU-E and BFU-E by 5 3,2 1, 2 7, 1 9, 1 8 times as that of the control. CONCLUSION: I nhibition of endogenous TGF-? 1 and TNF-? by antisense PS-ODNS will be one of the effective methods to expand HSPC ex vivo.