RESUMO
Objective To establish the methodology of flow cytometry for detecting human cells in human/goat chimerisra.Methods Human hemopoietic stem/progenitor cells (CD+34 cells) or MIG-tranadueed-GFP CD+34 cells were transplanted into the peritoneal cavity of fetal goats in utero to obtain human/goat chimera modeL The peripheral blood cells from the chimeras were labeled with multiple mouse anti-human antibodies and the monoelonal antibodies that were specific for human but had not or only minimal cross-reaction with goat were screened as the primary antibodies for routine analysis in flow cytometry.Human cord blood was proportionally (25% ,50% ,75%,100%) added into the blood of the untransplanted goats and the cells were labeled with CD+34 monoclonal antibody.The region and size of the "gate" were chosen based on to the distribution of CD+34 cells or human cord blood.One human/goat chimera marked with GFP (MIG goat) was sacrificed and the substantial liver cells from its perfused liver were analyzed for the GFP+cells percentage and DNA contents by flow cytometry.Results CD7,CD15,CD38,CD45CD20CD34CD14and GPA monoclonal antibodies were chosen as the primary antibodies in rou tine detection by flow cytometry.The size and area of the "gate" were also defined.29.1% (29100/100 000 ) of the substantial liver cells from the MIG goat expressed GFP.DNA content analysis showed that the GFP+ cells obtained from the liver of MIG goat mainly manifested two peaks that were correspond to those of human.Conclusions Flow cytometry is rapid,simple and effective for the investigation of differentiation,homing and biological characteristics of stem cells in vivo.The selections of suitable surface antibodies and the "gate" are very important for detecting human cells accurately in the human/goat chimerism.
RESUMO
<p><b>OBJECTIVE</b>To identify the human hematopoietic stem cells from the human/goat xenogeneic model with molecular techniques.</p><p><b>METHODS</b>DNA and total RNA were extracted from 11 transplanted goat peripheral blood cells. Human CD(34), GPA and SRY genes were amplified with PCR in these samples, and CD(34), GPA mRNA transcripts were detected using RT-PCR in 5 and 6 goat peripheral blood cells, respectively. Southern blot analysis was performed in 8 goat DNAs to detect the human specific alpha-satellite sequence. Meanwhile FISH was also performed to detect the human cells in goat blood with a probe of human Y chromosome.</p><p><b>RESULTS</b>Human CD(34) and GPA genes could be detected with PCR in all the 11 goats, and SRY gene did in 5 goats transplanted with hematopoietic stem cells derived from male human babies. Southern blot showed that human specific alpha-satellite sequence was present in 8 goats. By RT-PCR, human CD(34) mRNA was detected in 5 experimental goats, GPA mRNA was found in the other 6 experimental goats and FISH assay showed that some peripheral blood cells of the human/goat xenogeneic model were positive.</p><p><b>CONCLUSION</b>Existence of human cells in the recipient goats was identified by molecular detection, which was feasible for the examination of human/goat xenogeneic models.</p>