Hun Su Chu | Search PubMed

PubMed2006

A precise mRNA quantification method using CE-based SSCP.

Even though mRNA quantification provides significant information for biological analysis, current methods such as Northern blot analysis and real-time PCR are known to be laborious and lacking in precision. In this study, we demonstrate a new precise mRNA quantification method using CE based on SSCP (CE-SSCP) coupled with reverse transcription. mRNA samples could be simply analyzed for the quantification directly with reverse transcript obtained from a single reaction. This helps to avoid considerable errors generated by a series of the tedious manual steps. Also, unlike real-time PCR, reverse transcripts can be directly quantified by CE-SSCP in this method without further data estimation. Reproducibility and accuracy of CE-SSCP for mRNA quantification was examined using enhanced green fluorescent protein (eGFP) mRNA transcribed in vitro. Specific reverse transcription primer was determined for the accurate quantification of eGFP mRNA from total RNA obtained from the recombinant Escherichia coli. Using elongation factor Tu mRNA as an internal standard, it was shown that sample-to-sample variation could be minimized. Expression kinetics at both mRNA level and protein level was studied and the potential of CE-SSCP in expression analysis was demonstrated by comparison with the eGFP activity assay.

Electrophoresis, Capillary↗

PubMed2006

Enhancement of in vivo endothelialization of tissue-engineered vascular grafts by granulocyte colony-stimulating factor.

Successful reconstruction of large-diameter blood vessel in humans has been demonstrated using the tissue engineering technique, but improvement in patency of small-diameter bioartificial vascular graft remains a great challenge. This study reports that granulocyte colony-stimulating factor (G-CSF) can enhance in vivo endothelialization of tissue-engineered vascular grafts, which could be used to improve patency of small-diameter vascular graft. Vascular grafts were tissue engineered with decellularized canine abdominal aortas and canine autologous bone marrow-derived cells. Prior to cell seeding onto decellularized graft matrices, bone marrow-derived cells were induced to differentiate into endothelial cells and smooth muscle cells. The cell-seeded vascular grafts were implanted into the abdominal aortas of bone marrow donor dogs. Before and after graft implantation, G-CSF was administered subcutaneously to the dogs (n = 3). The grafts implanted into the dogs not receiving G-CSF were used as controls (n = 3). Eight weeks after implantation, grafts in both groups showed regeneration of vascular tissues including endothelium and smooth muscle. Importantly, endothelium formation was more extensive in the G-CSF-treated grafts than in the control grafts, as assessed with reverse transcription polymerase chain reaction, western blot, and immunohistochemistry. In addition, intimal hyperplasia was significantly reduced in the G-CSF-treated grafts compared to the control grafts. This study suggests that G-CSF administration could be applied to improve patency of small-diameter tissue-engineered vascular grafts.

Animals↗