Upregulation Endogenous TGF-β₁ Expression In HL-60 Cells By Tetracycline Inducible System And Its Effects On Cell Proliferation, Differentiation And Apoptosis

Transforming growth factor-β₁(TGF-β₁)is a pluripotent cytokine that controls multiple cellular responses including the induction of cell growth inhibition,differentiation,cellular senescence,wound healing, inflammation repair,host immunity and Apoptosis,The effect of TGF-β₁ in tumor is complex,TGF-β₁ is considered as a tumor suppressor gene in early tumor and growth inhibition by theTGF-β₁ has been extensively studied in many cell types.However,TGF-β₁ can enhance growth in progressive tumor and possible mechanisms for these growth enhancing effects,including the induced mmunosuppression,enhanced angiogenesis,and increased peritumoral stroma formation.Although TGF-β₁ is proposed to act as a potent endogenous negative regulator of hematopoiesis,its role in leukemogenesis remains largely unknown until recently.Development of the tetracycline(Tet)inducible gene expression system allows genes to be controlled remotely from outside a living organism.In this system,tetracycline or its analogue doxycycline is used as an effective "genetic switch".The successful construction of these systems offers an implementation in which the exogenous gene transcription in organisms can be precisely switched on/off in an exact time,at a certain level or in a specific tissue by administration or removal of Tet.This raises the feasibility of wide application of gene therapy.In this study,Based on the tetracycline regulatory of the Tet-on system,eukaryotic expression plasmids pcDNA4- TGF-β₁ which express TGF-β₁ was constructed by use recombinant DNA technique.The stable transferction of HL-60 with recombinant plasmid expressing hTGF-β₁ was established by using Lipofectamine 2000,and the biological characteristics of transfected HL-60 cell was investigated.The subjects of the thesis were focused on the following aspects:1.hTGF-β₁ cDNA was inserted into eukaryotic expression vector pcDNA4/TO to construct pcDNA4- TGF-β₁,Then The recombinant pcDNA4- TGF-β₁ and pcDNA6/TO was co-transferred into HL-60 cell line with lipofectamine 2000.HL-60 cells stably transfected with pcDNA6/TR and pcDNi4- TGF-β₁ were acquired by drug selection with Blasticidin and/or Zeocin.2.Study the effect of endogenous hTGF-β₁ that can be induced by continuous Tet administration on HL-60 cells stably transfected with pcDNA6/TR and pcDNA4- TGF-β₁ by growth curve,MTT,cloning of cells,NBT, DNA ladder,FACS;Analyze expression of some different genes and protein associated with apoptosis in this progress by relative quantitive RT-PCR and Western-Blot.3.The nude mice tumor xenograft models were established and antitumor effect of endogenous TGF-β₁ that could be induced by continuous Tet administration was analyzed in vivo.Tumor growth inhibition in these nude mice was used to evaluate the anticancer activity of drugs.The main results are as following:1.The eukaryotic expression vector of hTGF-β₁(named pcDNA4/TGF-β₁)was constructed successfully.The recombinant pcDNA4- TGF-β₁ and pcDNA6/TO was co-transferred into HL-60 cell line with lipofeetamine 2000.HL-60 cells stably transfected with pcDNA6/TR and pcDNA4- TGF-β₁(named TGFβ₁—HL₆₀ cells)were acquired by drug selection with Blasticidin and/or Zeocin.The expression of TGF-β₁ mRNA and protein in TGFβ₁-HL₆₀control by Tet were assayed by RT-PCR or ELISA.2.The biological characteristics of transferred cells TGFβ₁—HL₆₀were investigated.Endogenous hTGF-β₁ could be induced by continuous Tet administration on TGFβ₁—HL₆₀cells.TGFβ₁—HL₆₀cells could be induced differentiation and apoptosis by administration of Tet.RT-PCR and/or Western-Blot showed the expressions of Bcl-2,c-myc,h-TERT,fas in treated Hl-60/ TGF-β₁ cells decreased,but the expressions of Bax increased.3.Xenograft of HL-60 or TGFβ_—HL₆₀cells was established by subcutaneous implantation of cultured HL-60 cells or TGFβ₁—HL₆₀cells in nude mice. Endogenous hTGF-β₁ induced by Tet was able to inhibit potently the growth of the tumor cells in vivo.Tet-ip resulted in significant reduction of the tumor mass and apoptosis of the tumor cells as shown by transmission electron microscopy(TEM)assay.The inhibitory rate of tumor weight of high dose of Tet test group(10mg/kg)and low dose of Tet test group(0.4mg/kg)were 50.0%(P＜0.01)and 20.7%(P＜0.05)respectively.