Experimental Study On Gene Therapy Of Butyrate In The β-globin Disorders

Objictive A in vitro model system, which was set up to study the molecular mechanisms regulating the expression of globin genes, was used to investigate the induction of the expression of globin genes by butyrate at the levels of cell, protein and mRNA, and to provide a base for the study of gene therapeutic mechanism of butyrate and for the screen of new inducers in Chinese medicine in the future. Methods A simple and convenient and practical in vitro model system was selected for this experiment firstly after studying the BFU-E culture and K562 human cell line culture respectively. At the levels of cell, cells, used as a in vitro model system, were stained with benzidine to detect hemoglobin production after treatment with different concentration of butyrate and with different butyrate regimen to compare the percentage of benzidine-positive cells(BZ%) in untreated and butyrate-treated cells to investigate the erythroid differentiation of the cells induced by ~?Lk~ 2 0 0 1 butyrate. The change of Hb concentration per cell was determined by the protein absorption at 414nm using spectrophotometer after 72 hours incubation in the presence of 0 mM ,0.5 mM ,l.0 mM and 1.5 mM of butyrate. The change of the different type of hemoglobin product within butyrate-treated and untreated cells was detected by cellulose acetate gel electrophoresis. 7 globin mRNA primers were designed and the technique of RT-PCR was applied to amplify 7 globin mRNA in untreated and butyrate-treated cells in the same condition to observe the selective action of the drug in the levels of mRNA of globin genes. Hydroxyurea was used as positive control and its induction was detected at the levels of protein and mRNA at the same time with butyrate. Result The growth and differentiation of erythriod progenitor in semisolid culture is more near that in vivo, but it抯 culture has some disadvantages, for example, difficult culture, long circle, and less cell numbers, et al. It is more simple and convenient to culture the K562 cells than to erythriod progenitor, and the culture system of K562 cells is more stable. K562 cell line was used as the model of this study. Butyrate can induced inhibition of the growth of the K562 cells, and at 0.5 mM of butyrate it produced the maximal differentiation but without cytotoxicity. The BZ% increased 4----6 fold after 3 days of treatment with differential concentration of butyrate. The increase of benzidine-positive cells was gradual and reached a peak of l9%拁?8% on 3 or 4 days with once treatment of butyrate which time of incubation was I 2h,24h,36h,48h,72h respectively ,followed by a subsequent rapid fall of BZ% ,and at about 7d---- 9d it decreased to the BZ% level of untreated K562 cells. There was no correlation between the during of incubation in presence of butyrate and increase and sustained time of benzidine-positive cells. The change of BZ% in continuous treatment of butyrate was similar to that of in once pulse treatment. While BZ% reached a peak on 72h, the hemoglobinization of K562 cells was still maintained between 20% to 30% by pulse butyrate regimen until incubation of 3 circles in presence of butyrate was finished. ?? 2 0 0 1 The Hb production per cell increased 9??14 fold after 3 days of treatment with butyrate. Butyrate selectively increased fetal...