Expression And Modulation Of Endogenous TGF-β₁ And TNF-α In Blood Cells And Their Effects On Expansion Of Hematopoietic Stem/Progenitor Cells

Expression and modulation of endogenous TGF- 3 iand TNF- a in bloodcells and their effects on expansion of hematopoietic stem/progenitor cellsPostgraduate student: Meijuan Huang Tutor: Prof. Yuanzhong ChenFujian Institute of Hematology, Fujian Medical University, P. R. ChinaHematopoiesis is a complicate biological process with many stimulating factors and inhabiting factors interacting with microenviroment and matrix cells to modulate deliberately. Among them, endogenous TGF- ?i and TNF-a and their application in ex vivo expansion of hematopoietic stem/progenitor cells(HSPC) is now one of the most popular research issues.We hypothesized that endogenous TGF- i and TNF- a played an important role during proliferation, differentiation and apoptosis of blood cells. To investigate this possibility, we studied expression of positive and negative factors and their receptors during different stages of blood cells by means of RT-PCR, expression of cytokines and their receptors mRNA and level of TGF-13 1 protein in supernatant during apoptosis of HL-60 cell lines induced by AS2O3 or AS2O3 plus TGF- ?l and/or TNF- a antisense PS-ODNS by means of DNA ladder, TUNEL > ELISA and RT-PCR, and also investigated the effect of TGF- 1 and/or TNF- a antisense PS-ODNS on ex vivo expansion of HSPC by use of liquid culture and clone analysis.Our major results were obtained as follows: l.CD34+ cells could simultaneously express mRNA for IL-l, IL-l, IL-3, IL-6, GM-CSF,G-CSF and their receptors, and the receptors of SCF(SCFR), MPO(MPL) aswell. Peripheral mononucleat cells could only express IL-6, GM-CSF and their receptors > SCFR and G-CSFR. Granulocytes only express IL-6, GM-CSF> IL-6R and G-CSFR. Megakaryocytes and platelets could only express IL-3R> IL-6> IL-6R and MPL, While negative factors such as TGF-1, TNF- a and their receptors occurred in normal cells of various stages. 2. During apoptosis of HL-60 cell line induced by ASiOs, up-regulation of TGF-, TNF- a and down-regulation of bcl-2 mRNA were detected. TGF- ?i and TNF- a antisense PS-ODNS could alleviate this kind of apoptosis by regaining of bcl-2. 3. TGF- ?i and TNF- a antisense PS-ODNS cooperated with cytokines could dramatically increase the number of CD34+ cells ^ CFU-GEMM, CFU-GM, CFU-E and BFU-E.From above mentioned, we conclude that positive autocrine factors are progressively lost while negative autocrine factors exist continuously during proliferation and differentiation of normal blood cells, which infers that endogenous TGF- 3 i and TNF- a played an important role in this process. We also conclude that endogenous TGF- ?i and TNF- a may initiate the apoptosis of blood cells and TGF- ?i and TNF- a antisense PS-ODNS plus maybe one of the effective methods to expand HSPC ex vivo.