Study And Analysis Of Differentially Expression Genes In Jurkat T Cell Line Induced By Arsenic Trioxide

Arsenic is a metalloid distributed widely in the environment. Epidemiological evidence has demonstrated that exposure to arsenic at relatively high does results in acute and chronic arsenism, which is not only associated with various tissues and organs injury, even increase risk of cancers, but also impart immunological function. So far as, the molecular mechanism of arsenic toxicity is not clear, The accumulated research evidences show that the toxic effects of arsenic on tissues and cells results from various genes imparted or associated signal pathway disturbed. In order to understand the molecular mechanism of arsenic toxicity to human T lymphocytes, the normal human T lymphocytes and Jurkat T cells as experimental samples, in vitro, were used to observe the effect of arsenic inhibiting growth of human T lymphocytes and Jurkat T cells, in vitro. Jurkat T cells exposure to arsenite oxidation (As2O3) as experimental sample were used and suppressive subtraction hybridization were applied to construct the subtracted cDNA library from Jurkat T cells treated with As2O3 for detecting differentially expression genes. Reverse Northern Blot was applied for screening differentially expression genes.PART I: The Effect of Arsenic on Inhibiting Growth of Human T Lymphocytes and Jurkat T Cells, in vitro.Arsenic has effects of immunosupression and immunoto-xicity, but the mechanism of immunosupression and immunoto- xicity of arsenic is not clear. In order to understand the molecular mechanism of arsenic toxicity to human T lympho- cytes, the human T lymphocytes from health donors and Jurkat T cells as experimental samples were treated with As2O3 for 24 hours in different concentrations (0, 0.5, 1, 2, 5, 10, 20, 50, 100uM/L, respectively). The results showed that As2O3 could markedly inhibit growth of human T lymphocytes and Jurkat T cells in the manner of dose-response relationship after exposure to As2O3. The 1C50 of As2O3 for human T lymphocytes and Jurkat T cells were 8.09uM and 8.9 uM, respectively. Transmit- ting electron microscope (TEM) was also used to observe the morphologic alteration of Jurkat T cells treated with As2O3 in concentration of 5 uM for 24 hours. The results of observations by TEM showed that many cells in Jurkat T cells treated with As2O3 underwent apoptosis that displayed small nuclear condensation, chromatin margination, plasma membrane blebbing. The chromatins of some apoptotic cells had split into several small masses, that is chromatinorrhexis. Comparing with untreated cells, part of cells treated with As2O3 showed active function because increasing numbers of mitochondrion. Most of the treated cells have integrated nuclear and cellular membranes, but many larger vesicles in cellular plasma, which indicate that As2O3 (at 5uM, treated 24h) affects on more mitochondrion then otherorganelles. The results of observation by TEM suggested that one of the arsenic toxic effect on inhibiting growth of jurkat T cells be contributed to inducing apoptosis. PART II. Construction of Forward Subtracted cDNA Library From Jurkat T Cell Induced by As2O3 .The main objective of this study was to detecting differential expression of mRNA in Jurkat T cell. After treated with As2C>3 (5uM, 24H) it is used that suppressive subtraction hybridization (SSH) which is key steps in brief following (1) extracting mRNAs from treated and Untreated Jurkat T cells, (2) reverse transcripting mRNAs into double strain cDNA (ds cDNA), (3) digesting ds cDNA with Rsa I nuclease, (4) subdivided into two portions and each is ligated with a different cDNA adaptor, (5) two hybridizations and nest PCR performed, (6) after cDNA getting from PCR, cDNAs were inserted into PGEM-T easy vector. The 35positive clones had been selected from LB agar plates, then identified them contain 250條OOOObp cDNA by PCR. Sequencing results showed that there were 30 clones were identified, another 5 clones could not be identified because without positive results or some of bases were not clear. After comparing with public database (GenBank/EMBL...