Influence of immunosuppressive therapy on expression of TNF-α/IFN-γ in cytoplasm of peripheral blood lymphocytes of patients with aplastic anemia / 中国实验血液学杂志

The purpose of this study was to investigate the influence of immunosuppressive therapy on the expression of TNF-α/IFN-γ in cytoplasm of peripheral blood lymphocytes of patients with aplastic anemia (AA). The expression of TNF-α and IFN-γ in cytoplasm of peripheral CD3(+) lymphocytes were measured by flow cytometry in 25 cases of de novo AA patients and 20 cases of AA after immunosuppressive therapy. The results showed that the positive rates of CD3(+)/TNF-α(+) and CD3(+)/IFN-γ(+) in de novo AA patients were (5.97 ± 6.78)% and (15.20 ± 11.28)% respectively, and (1.56 ± 0.87)% and (1.76 ± 0.87)% in normal controls respectively. There was significant difference between de novo AA patients and normal controls (p < 0.05). The positive rates of CD3(+)/TNF-α(+) and CD3(+)/IFN-γ(+) in immunosuppressive therapy group were (1.67 ± 1.26)% and (4.35 ± 4.33)% respectively. The difference between immunosuppressive therapy group and de novo AA group was statistically significant (p < 0.05). It is concluded that the levels of intracellular TNF-α and IFN-γ in AA patients are higher than those in normal controls. Immunosuppressive therapy significantly reduces the expression of intracellular TNF-α and IFN-γ. Its relationship with the clinical treatment is worth further observing.

Screening of proteins binding to FXR1P using yeast two-hybrid technique / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen the proteins interacting with FXR1P for functional investigation of FXR1P.</p><p><b>METHODS</b>The yeast strain AH109 transformed with the recombinant expression vector pGBKT7/FXR1 was mated with the yeast strain Y187 pretransformed with human fetal brain cDNA library. The positive clones were screened and identified by sequence analysis.</p><p><b>RESULTS</b>The recombinant expression vector pGBKT7/FXR1 was constructed successfully. Five proteins binding to FXR1P were screened from human fetal brain cDNA library using the yeast two-hybrid system, including CMAS, FTH1, GOLGA4, HSD17B1 and CSH1.</p><p><b>CONCLUSIONS</b>These results provide new clues for investigating the biological functions of FXR1P and the pathogenesis of Fragile X syndrome.</p>

Preparation and identification of monoclonal antibody against human thrombomodulin / 生理学报

To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.

Gene expression profile of peripheral blood monocyte in patients with fulminant hepatitis B by cDNA microarray / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate peripheral blood monocyte (PBMC) gene expression profile in patients with fulminant hepatic failure (FHF) by cDNA microarray.</p><p><b>METHODS</b>Microarrays consisting of 8,192 human cDNAs and labelled cDNAs prepared from PBMC in both 10 FHF patients and 10 asymtomatic surface antigen carriers (ASC) were applied to analyze gene expression. Relative ratios of gene expression in individuals were obtained by comparing the hybridization results, by GenePix 4000B scanning and by ImaGene3.0 software analysis, of Cy5-labelled cDNA from FHF patients with those of Cy3-labelled cDNA from ASC.</p><p><b>RESULTS</b>249 genes out of 8,192 were identified differently, at least two times. Most of the genes (79%) involved in cell signaling transduction, cell cycles, metabolism, inflammatory response and apoptosis, whose mRNAs were differently regulated.</p><p><b>CONCLUSIONS</b>These results suggest that HBV infection alters a broad range of cellular genes expression during developing into FHF and provide a framework for future functional study on the genes expressed differently.</p>

Effect of 3' exonuclease activity of polymerase on extension of phosphorothioate-modified primers / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine whether 3'phosphorothioate-modified-2 terminal mismatched primers can turn off DNA polymerization mediated by Exo(+) polymerase.</p><p><b>METHODS</b>Two-directional primer extension was performed using polymerase with and without 3' exonuclease activity. The effects of unmodified primers and 3' phosphorothioate-modified primers on primer extension were evaluated.</p><p><b>RESULTS</b>Exo(-) polymerase yielded products from matched and mismatched primers regardless of their modification. However, 3' phosphorothioate-modified primers with a single base mismatch at -2 position worked similarly to the terminal (-1) mismatched primers in triggering the novelly reported "off-switch" of Exo(+) polymerase.</p><p><b>CONCLUSION</b>These data suggested that the "off-switch" can be of enormous application in the diagnosis of single gene diseases and in the association studies by single nucleotide polymorphism screening.</p>