A Study On Effect And Mechanism Of TGF-β Isoforms On Proliferation Of HL-60 Cell Line

Transforming growth factor (TGF-β) family is a group of multipeptides which are relative in construction and similar in biological activity. They play a negative role in regulation of hematopoiesis to keep balance of proliferation and differentiation of blood cell with positive factors. There are more than 30 members in this family but only three isoforms in human-- TGF-β₁, TGF-β₂, and TGF-β₃. These isoforms play different role in regulating proliferation and differentiation of cell. It is reported that TGF-β₁ inhabit the growth of leukemia cells, but there is rare report about effect of TGF-β₂ and TGF-β₃ on leukemia cell.And there are little knowledge about whether there are some kind of relationship among these three isoforms. ATRA and ATO are main medicines to cure AML-M3. There are little reports about autocrine cytokines, especial TGF-β₁ has attend the mechanism of these two medicines.Our study included three parts as following:1. Study the effect of TGF-β₁, TGF-β₂ and TGF-β₃ on HL-60 cell by growth curve, MTT, cloning of cells, NBT, DNA ladder, FACS; Analyse expression of some different genes associated with apoptosis in this progress by relative quantitive RT-PCR.2. Study expression of TGF-β₁ in leukemic cells and different kind of blood cells by real time qRT-PCR and ELISA;3. Construction of siRNA plasmid of TGF-β₁ aiming at different sites and invstigate expression of TGF-β₂ and TGF-β₃ of HL-60 cell deficient in TGF-β₁ by real time qRT-PCR and ELISA; Study the effect of ATRA and ATO on HL-60 cells defficient in TGF-β₁ by NBT, and FACS;Our main results and conclusions are as following:1. TGF-β₁, TGF-β₂, and TGF-β₃ have negative dose-dependent effect on growth of HL-60 cell. They can induce differentiation of cell at low concentration and apotosis of cell at high concentration. These three isoforms can regulate the level of expression of some genes associated with apoptosis, which may be one of mechanisms of apoptosis induced by them. The effect of TGF-β₁ is strongest among the three isoforms which may be because of different degree of regulation of these genes.2. TGF-β₁ and its isforms express in CD₃₄⁺ cells, mononulear cell and platelet. The level of mRNA and protein of TGF-β₁ are higher in BMMC cells than that in CD₃₄⁺ cells but TGF-β₂ and TGF-β₃ lower. TGF-β₁ and its isforms express also in acute leukemia cells, but the level of mRNA and protein of TGF-β₁ is lower than that of in CD₃₄⁺ cells, while TGF-β₂ and TGF-β₃ are higher than latter. Expression of TGF-β₂ and TGF-β₃ drop in Chronic myelogenous leukemia (CML) cells, which indicates down-regulation of expression of endogenous TGF-β₂ and TGF-β₃ may be one of the machenism of pathogenesis of CML.3. Successful construction of siRNA plasmid of TGF-β₁ which can inhibit the gene expression to 73.2%. Expression of TGF-β₂, and TGF-β₃ did not rise in such cells while TGF-β₁ was silenced, which may show TGF-β₂ and TGF-β₃ have their own biological functions. The effect of ATRA and ATO on HL-60 defficient in TGF-β₁ attenuates which indicates autocrine TGF-β₁ may join the machenism of function of these two medicines on leukemia cells.