The Expression Of Hoxb8 In The Development Of Granulocyte Progenitor In Vitro And Intervention Study

objective:The objective is to observe the expression of Hoxb8 on the differentiation and proliferation of hematopoietic stem cell to CFU-G in vitro and the progress affected by ATRA and/or As2O3. Aimed to explore the effect of all-trans-retinoic acid and/or arsenic trioxide on the expression of Hoxb8gene in the developement of Granulocyte Progenitor。Methods:1.36 cases cord blood sample were offered by obstetric of the affiliated hospital, all samples were collected from fetal placenta umbilical vein.2. Groups:①Normal CFU-G culture was used as blank control.②ATRA (6×10-8mol/l) was added into Normal CFU-G colonies culture system as ATRA group.③As2O3 (6×10-8mol/l) was added into Normal CFU-G colonies culture system as As2O3 group.④ATRA and As2O3 were added into culture system together, final concentration as above 3. By the colony culture in vitro, the impact of As2O3 and/or ATRA on the CFU-G colony formation were observed, then detect the expression of Hoxb8 genes on the differentiation progress of Hematopoietic Stem Cell (HSC) to CFU-G intervented by As2O3 and/or ATRA on the third, seventh and twelfth day.4. The component of the colonies of granulocyte progenitor was determined by Wright staining.5. The total RNA was extracted by Trizol reagent in all samples from all the above groups respectively at day3,7,12. Total RNA was electrophoresed in 1% formaldehyde denaturalized agarose gel in order to validate integrity of RNA; The nucleoprotein was extracted by RIPA lysis buffer in all samples from all the above groups respectively at day3,7,12.Make an assay of protein density by BCA assay kit.6.The total RNA from different time and group was transcribed into cDNA by using oligonucleotide primer, The expression of Hoxb8 mRNA on the proliferation granulocyte progenitor in vitro were detected with FQ-RT-PCR in different group.The expression of Hoxb8 protein on the proliferation granulocyte progenitor in vitro was detected by western-blot.7.The electrophoresis of Hoxb8 genes by experiment of electrophoresis were obtained respectively at day 3,7,12. The grading diluted positive reserve transcribed production of Hoxb8 gene and ACTB gene were recorded and used to make their standard curves; determination of content about protein-antibody compound by chemiluminescent, in the gel imaging system imaging and measuring the expression intensity of specific band, the same meeting, the proceeds of Hoxb8 and ACTB electrophoresis bands of gray value compared to obtain the sample Hoxb8/ACTB ratio of gray, every group measuring 3 samples.8. Statistical methods:The results were showed by means plus or subtracting standard deviation we compared means between groups by LSD;The software SPSS 17.0.for windows was used to analyze the data. Results:1. The colony forming was identified Granulocyte progenitor cells by Wright staining.2. Total RNA electrophoresed by 1% formaldehyde denaturalized agarose gel showed that the 5S,18S,28S RNA strips were all explicit and intact, and had no obvious degradation.3. The cDNA of the Hoxb8 and the ACTB gene were reservedly transcribed by RT-PCR, and compared with the standard DNA Marker. They were 96 and 111 bp long respectively. Which were in consonance with anticipation.4. Homeobox genes do have a regulatory function in the differentiation process of hematopoiesis. The expression of Hoxb8 mRNA appeared on regularly changes during the differentiation and proliferation of hematopoietic stem cell to colony forming unit-granulocyte in vitro, which increased slightly on 3rd day, and were obviously higher on 7th day, but became lower in 12th day.5. The differentiation progress affected by ATRA. Compared with the expression of HoxbB8 mRNA and protein of Normal group and As2O3 group and ATRA associated with As2O3 group, the expression of HOXB8 of the group ATRA were up-regulated remarkably (p<0.05). Conclusion:1. The expression of Hoxb8 mRNA and protein expressed in the stages of HSPC's proliferation into granulocyte progenitor cells in human cord biood in vitro. It reaveals there is some relationship between Hoxb8 genes and granulocyte progenitor hematopoiesis.2. ATRA(6×10-8mol/1) can up-regulate the expression of Hoxb8 mRNA and protein.3. As2O3 (6×10-8mol/1) on the expression of Hoxb8 gene had no significant regulatory role.4. ATRA(6×10-8mol/l) associated with As2O3 (6×10-8mol/l) on the expression of HoxbB8 gene had no significant regulatory role.