The Reseach Of The Expression Of Homeobox (Hox) Genes On The Differentiation And Proliferation Of The Hematopoietic Stem Cell To The Colony Forming Unit-T Lymphocyte

objective: The objective is to observe the expression of Homeobox gene(C4,C6,B4,B6) on the proliferation and differentiation of hematopoietic stem cell to colony Colony Forming Unit-T Lymphocyte in vitro. And the differentiation progress was affected by ATRA to observe the influence of ATRA on Homeobox genes in this experiment. Methods: 1. 10 cases cord blood sample were offered by obstetrics of the affiliated hospital, all samples were collected from fetal placenta umbilical vein. 2.Groups:①Normal CFU–TL culture was used as normal group.②ATRA (6×10-8mol/l) was added into normal CFU- TL colonies culture system as ATRA group. 3. By the colony culture in vitro, the impact of ATRA on the Hematopoietic Stem-Progenitor Cell(HSPC) were surveyed, then observe the groups of CFU-TL on the differentiation progress of Hematopoietic Stem Cell (HSC) to CFU-TL induced by PHA on the third, seventh , and twelfth day. 4. The component of the colonies of lymphocyte progenitor was determined by Giemsa-Wright staining. 5. The total RNA was extracted by Trizol reagent in all samples from the two groups respectively at day3,7,12. Total RNA was electrophoresed in 1% formaldehyde denaturalized agarose gel in order to validate integrity of RNA. 6.The total RNA from different time and group was reverse transcribed into cDNA by using random primer, The variable HOX gene was chosen to amplify in vitro with its specific primer by FQ-RT-PCR, at the same time, the expression of HOXC4,HOXC6,HOXB4,HOXB6 genes on the proliferation and differentiation of lymphocyte progenitor cell in vitro were detected with fluorescence quantitive real time polymerize chain reaction (FQ-RT-PCR)in different group. 7.The electrophoresis graph of HOXC4,HOXC6,HOXB4 and HOXB6 genes by experiment of electrophoresis were obtained respectively at day 3,7,12. 8. Statistical methods: The results were showed by means plus or subtracting standard deviation,we compared means between groups by LSD . The expression level Comparison of every genes between two groups by Paired-samples T test .All data was processed and statistical analysis was done by SPSS15.0. Results: 1 .The cultural colony forming was identified lymphocyte progenitor cells by Giemsa-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 HOXC4,HOXC6,HOXB4,HOXB6 and the GAPDH gene were reservedly transcribed by RT-PCR, and compared with the standard DNA Marker. they were 254bp,212bp,138bp,119bp and 141bp long respectively,which were in consonance with anticipation. 4.Homeobox genes do have a regulatory function in the differentiation progress of Hematopoietic Stem Cell (HSC) to CFU-TL. Compared with the expression of HOXC4,HOXC6,HOXB6 genes on day 3 , the quantity of HOXC4,HOXC6 amd HOXB6 genes was obviously higher on day 7 and lower on day 12 respectively in each group,while the expression of hoxb4 gene was diminished gradually. 5. Compared with the expression of HOXC4,HOXC6,HOXB4 and HOXB6 genes of normal group, the ones of the ATRA group were up-regulated remarkably (p<0.05). Conclusion:1. HOXC4,C6,B4,B6 genes-mRNA expressed regularly in the differentiation and proliferation of the hematopoietic stem cell into CFU-TL in human cord blood in vitro,which shows that there has a positive relationship between HOX genes and the differentiation and proliferation of the hematopoietic stem cell into CFU-TL. 2.During the differentiation and proliferation of the hematopoietic stem cell into CFU-TL, the expressions of HOX C4,C6,B4,B6 are all regular in time .3. ATRA can up-regulate the expression of HOXC4,C6,B4,B6 genes .