ABSTRACT
A complementary DNA (cDNA) fragment library from SH-SY5Y cells is constructed using a restriction display polymerase chain reaction (RD-PCR) technique. Messenger RNA (mRNA) is extracted from SH-SY5Y cells and single-strand cDNA synthesised using an anchored oligo primer (dT18). The second strand is produced by nick translation. The double strands are cleaved with the restriction enzyme Sau3AI and the fragments ligated with universal linker. The products are amplified with universal primers and selected primers, ligated into the pMDI8-T vector, and then sequenced. The library constructed contained 136 subgroups, each comprising seven to 12 cDNA fragments. RD-PCR proved a simple, effective way to construct a cDNA library, and this will contribute to the investigation of gene expression in the neuron in future microarray studies.
Subject(s)
DNA, Complementary/genetics , DNA, Neoplasm/genetics , Gene Library , Humans , Polymerase Chain Reaction/methods , Tumor Cells, CulturedABSTRACT
AIM: To illustrate the expression of hypermethylation p15 gene in 53 non-Hodgkin’s lymphoma (NHL). METHODS: The methylation of p15 gene in 53 cases of non-Hodgkin’s lymphoma was detected by using methylation-specific PCR (MSP) technique. RESULTS: 18.9% (10/53) in NHL were methylation in p15 gene. p15 gene was frequently in high malignant NHL patients (27.3%) compared with in low malignant patients (0%). CONCLUSION: The result suggested that the hypermethylation of p15 may play an important role in non-Hodgkin’s lymphoma.
ABSTRACT
AIM: IL-12 acts upon T lymphocytes and activates its receptor complexes of ?1/?2 ,and so IL-12 can regulate TH1/TH2 balance. Our study is aimed at IL-12-inducing apoptosis of T cells and expression and signal transduction of Fas/FasL during T cell apoptosis. METHODS: The apoptosis of T cells was detected by Annexin V staining cytometry and the expression of Fas/FasL under different inhibitors were detected by semi-quantitative PCR. RESULTS: IL-12 induced the human leukemic T cell line(TIB-152) and the human lymphoma T cell line(HTB-176) and the normal human T cells to undergo apoptosis. The FasL expression at 6 hours after treatment with IL-12 increased apparently, and reached the max at 24 hours, and FasL expression induced by IL-12 was inhibited by PKC inhibitor. But IL-12 did not influence Fas expression. CONCLUSIONS: IL-12 can induce T cells to undergo apoptosis which is characterized by early membrane changes, the inducing effect is correlated with the concentration of IL-12 and the maturation of T cells. FasL participates in the progression of T cell apoptosis as a apoptosis mediator, and the effect of IL-12 on FasL expression may be related with PKC pathway.
ABSTRACT
In order to explore the role of p15(INK4B) gene with highly methylated CpG island in the pathogenesis of leukemia, the expression levels of p15(INK4B) gene was detected in patients with AML and CML. Methylation-specific PCR (MSP) assay was employed in the experiments. The methylation incidence was 83.9% (26/31) in AML and 0% (0/28) in CML. The results showed that methylation of p15(INK4B) gene was the one of the major ways for inactivation of the gene, and the methylation could be appeared in clinical development of the disease and patients condition worsened. Methylation of p15(INK4B) did not occur and its function probably is normal in CML.
ABSTRACT
AIM: To study the effect of IL-12 on T lymphocytes apoptosis, the expression of Fas/FasL and TNFR/TNF?. METHODS: Terminal dUTP nick end labeling(TUNEL) and Annexin V assay were used. Anti-TNFR were labeled with FITC, anti-CD95 was labeled with PE and Anti-FasL with biotin. Three kinds of T cells (HTB176,TIB152 and human normal T cells) were analysed through flow cytometry. RESULTS: At 1st hour after being treated with IL-12, the expression of FasL protein and FasL mRNA in HTB176 and TIB152 began to increase and reached peak value in 24 hours. In the normal T cells, FasL just began to increase in 1 hour and maintained stability in 6, 12 and 24 hours through the later experiment period. All three kinds of T cells displayed no change in the expression of CD95 and TNFR/TNF? under the stimulation of IL-12. CONCLUSION: Expression of such apoptosis regulating factors were different in the apoptosis of T cells induced by IL-12.
ABSTRACT
AIM: IL-12 acts upon T lymphocytes and activates its receptor complexes of ?1/?2 ,and so IL-12 can regulate TH1/TH2 balance. Our study is aimed at IL-12-inducing apoptosis of T cells and expression and signal conduction of Bcl-2 during T cell apoptosis. METHODS: The apoptosis of T cells was detected by Annexin V staining cytometry and the expression of Bcl-2 under different inhibitors were detected by the method of semi-quantitative PCR. RESULTS: IL-12 can induce the human leukemic T cell line(TIB-152) and the human lymphoma T cell line(HTB-176) and the normal human T cells to undergo apoptosis. The Bcl-2 expression at 6 hours of treatment with IL-12 increased aparently,and reached the max at 24 hours. But IL-12 did influence Bcl-2 expression. IL-12 can induce T cells to undergo apoptosis which is characterized by early membrane changes. CONCLUSION: The inducing effect is correlated with the concentration of IL-12 and the maturation of T cells. Bcl-2 takes part in the progression of T cells' apoptosis as a apoptosis mediator.
