Real-time fluorescent quantitative reverse transcription polymerαse chain reaction in detecting expression of ERα, PR and Her2 mRNA in breast cancer tissues / 第二军医大学学报

Objective To evaluate the clinical value of real-time fluorescent quantitative reverse transcription polym ERαse chain reaction (qRT-PCR) in detecting expression of estrogen receptor alpha (.ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HerZ) genes in breast cancer tissues. Methods Totally 48 breast cancer tissues and 28 benign breast tumor tissues (control) were obtained from patients undergoing surgery in our hospital during Mar. 2010 and Oct. 2010. The expression of ERα, PR and Her2 protein was examined by immunohistochemistry (IHC) in breast cancer tissues and the expression levels of ERα, PR and HerZ mRNA were detected by real-time qRT-PCR in breast cancer tissues and benign breast tumor tissues. The values of these two methods in diagnosis of breast cancer were evaluated. Results The expressions of ERα and HerZ mRNA were significantly higher in the breast cancer tissues than in the controls (P0. 05). Real-time qRT-PCR in detecting ERα, PR and Her2 mRNA expression had similar capability with IHC method in evaluating the sensitivity, specificity of endocrine thERαpy; moreover, the two methods also had a consistent pathological diagnosis rates (P>0. 05). Conclusion ERα, PR and Her2 genes are important predictive markers for endocrine thERαpy or targeted thERαpy of breast cancer. Real-time qRT-PCR method can be used for clinical detection and research of ERα, PR and Her2 mRNA.

Panning and identification of humanized antinuclear antibody Fab fragment / 第二军医大学学报

Objective: To prepare humanized antinuclear antibody Fab fragment. Methods: The reconstructed humanized antinuclear antibody (ANA) Fab phage display library was enriched by 4 rounds of panning and was identified by indirect ELISA method. Phasmid DNA isolated from positive clones was deprived of g III gene. After self-ligation the recombinant plasmid was used to transform E. coli. XL1-Blue, then XL1-Blue was induced by IPTG to product soluble human antinuclear antibody Fab fragment. Finally, soluble human antinuclear antibody Fab in the supernatant was identified by indirect ELISA method and immunofluorescence. Results: The eluted phages were enriched by more than 200 folds after 4 rounds of panning. Two positive clones were isolated from the ANA Fab library. Electrophoresis after Xho I digestion proved that the self-ligation was successful after deletion of g III gene. The results of indirect ELISA indicated that the 2 positive clones of Fab had specific anti-dsDNA activity. Indirect immunofluorescence showed homogeneous fluorescence within nuclei of Hep2 and monkey hepatic cells and in the Crithidia kinetoplast. Conclusion: We have successfully prepared soluble, specific human antinuclear antibody Fab fragment, which pares a way for preparation of high affinity antinuclear antibody Fab fragment.

Effects of Poly I:C in inducing growth inhibition and apoptosis of human hepatocellular carcinoma cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effect and mechanism of Poly I:C in inducing growth inhibition and apoptosis of human hepatocellular carcinoma SMMC-7721 cells.</p><p><b>METHODS</b>SMMC-7721 cells were treated with different doses of Poly I:C for 24, 48, and 72 h, and the cell growth inhibition rate was analyzed with CCK-8 assay. The cell cycle and the apoptosis were analyzed using flow cytometry with Annexin-V and PI staining, and quantitative RT-PCR analysis were used to detect the expression of TLR3, TRIF, and IFN-beta mRNA in cells.</p><p><b>RESULTS</b>In the cells exposed to Poly I:C at low, moderate, and high doses, the inhibitory rates was the highest in high-dose Poly I:C group, and at a given Poly I:C dose, prolonged exposure resulted in significantly increased cell growth inhibition rate (P<0.05). Flow cytometry showed that Poly I:C induced cell apoptosis in a time- and dose-dependent manner and significantly increased the percentage of G1-phase cells as compared with that in the control group. The mRNA level of TLR3, TRIF, and IFN-beta were also increased following Poly I:C treatment in comparison with the control group.</p><p><b>CONCLUSION</b>Poly I:C can induce significant growth inhibition and apoptosis of SMMC-7721 cells in a dose- and time-dependent manner possibly by causing cell cycle arrest and TLR3 signaling pathway activation that leads to IFN-beta production and cell apoptosis.</p>

A study on the relationship between interleukin-10 promoter polymorphism and autoimmune liver disease / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether three biallelic polymorphisms at the position -592, -819 and -1082 in the promoter region of the IL-10 gene were associated with the incidence of autoimmune liver disease.</p><p><b>METHODS</b>The IL-10 -592 and IL-10-1082 polymorphisms were genotyped by polymerase chain reaction-restriction fragment length polymorphisms analysis (PCR-RFLP), while polymerase chain reaction- sequence specific primer (PCR-SSP) assay was used to detect IL-10 -819 polymorphisms.</p><p><b>RESULTS</b>Among 54 Chinese patients with AIH or 77 Chinese patients with PBC versus healthy controls, the frequency of AA, GA genotypes at IL-10 gene promoter -1082 position was 87.0% or 83.1% versus 90.0%, 13.0% or 16.9% versus 10.0%, respectively (P > 0.05), the GG genotype in Chinese populations is absent; the frequency of CC, CT, TT genotypes at IL-10 gene promoter -819 position was 11.11% or 9.1% versus 8.1%, 44.4% or 53.3% versus 45.0%, 44.4% or 37.7% versus 46.9%, respectively (P > 0.05); the frequency of CC, CA, AA genotypes at IL-10 gene promoter -592 position was 4.9% or 14.3% versus 10.0%, 51.2% or 53.3% versus 51.9%, 43.9% or 32.5% versus 38.1%, respectively (P > 0.05). No alleles differed significantly in each groups.</p><p><b>CONCLUSION</b>There were no association between IL-10 gene polymorphisms and autoimmune liver disease</p>