Differential expression of hsa-miR-126 and hsa-miR-518b in esophageal squamous carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify the differentially expressed microRNAs (miRNAs) between esophageal squamous carcinoma (ESC) and adjacent non-tumorous tissue (NT).</p><p><b>METHODS</b>The expression levels of the miRNAs were detected in 3 fresh ESC and NT samples by hybridization with miRNAs microarray chip. Real-time quantitative RT-PCR was performed to confirm the results of the microarray analysis. The expressions of hsa-miR-126 and hsa-miR-518b in ESC were validated by real-time quantitative RT-PCR in another independent 15 matched samples.</p><p><b>RESULTS</b>A total of 11 miRNAs exhibited differential expressions in ESC samples as compared to their expressions in the NT samples, including a 1 up-regulated miRNA and 10 down-regulated miRNAs. Compared with normal esophageal samples, the ESC tissues showed up-regulated hsa-miR-126 and down-regulated hsa-miR-518b expression.</p><p><b>CONCLUSION</b>hsa-miR-126 and hsa-miR-518b are differentially expressed in ESC, and they might play important roles in the carcinogenesis and progression of ESC.</p>

Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system.</p><p><b>METHODS</b>The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody.</p><p><b>RESULTS</b>The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII.</p><p><b>CONCLUSION</b>An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.</p>

Construction of a prokaryotic expression vector for apoptin and preparation of polyclonal antibody of apoptin / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a prokaryotic expression vector for apoptin and prepare polyclonal antibody of apoptin.</p><p><b>METHODS</b>Apoptin gene amplified from pGEM-T/Apoptin plasmid by PCR was cloned into pET-28a (+). E.coli BL21 (DE3) was transformed by the recombinant plasmid, and apoptin protein expression induced by IPTG was analyzed by SDS-PAGE. BALB/c mice were immunized with the protein and the titer of the antibody was determined using indirect enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Apoptin gene was successfully cloned into pET-28a (+), and the expression of a protein with relative molecular mass of about 17 000 was identified by SDA-PAGE. After 5 immunizations of the mice with the protein, the blood antibody titer reached 1:5x10(5).</p><p><b>CONCLUSION</b>The prokaryotic expression vector for apoptin is successfully constructed and the polyclonal antibody of apoptin is obtained, which allows further functional study of apoptin.</p>

A Meta-Analysis of the Accuracy of Prostate Cancer Studies Which Use Magnetic Resonance Spectroscopy as a Diagnostic Tool

OBJECTIVE: We aimed to do a meta-analysis of the existing literature to assess the accuracy of prostate cancer studies which use magnetic resonance spectroscopy (MRS) as a diagnostic tool. MATERIALS AND METHODS: Prospectively, independent, blind studies were selected from the Cochrane library, Pubmed, and other network databases. The criteria for inclusion and exclusion in this study referenced the criteria of diagnostic research published by the Cochrane center. The statistical analysis was adopted by using Meta-Test version 6.0. Using the homogeneity test, a statistical effect model was chosen to calculate different pooled weighted values of sensitivity, specificity, and the corresponding 95% confidence intervals (95% CI). The summary receiver operating characteristic (SROC) curves method was used to assess the results. RESULTS: We chose two cut-off values (0.75 and 0.86) as the diagnostic criteria for discriminating between benign and malignant. In the first diagnostic criterion, the pooled weighted sensitivity, specificity, and corresponding 95% CI (expressed as area under curve [AUC]) were 0.82 (0.73, 0.89), 0.68 (0.58, 0.76), and 83.4% (74.97, 91.83). In the second criterion, the pooled weighted sensitivity, specificity, and corresponding 95% CI were 0.64 (0.55, 0.72), 0.86 (0.79, 0.91) and 82.7% (68.73, 96.68). CONCLUSION: As a new method in the diagnostic of prostate cancer, MRS has a better applied value compared to other common modalities. Ultimately, large scale RCT (randomized controlled trial) randomized controlled trial studies are necessary to assess its clinical value.

Intercellular imaging by a polyarginine derived cell penetrating peptide labeled magnetic resonance contrast agent, diethylenetriamine pentaacetic acid gadolinium / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance (MR) contrast agent. Cell penetrating peptide (CPP) is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) in molecular imaging in vitro.</p><p><b>METHODS</b>Fluorescein-5-isothiocyanate (FITC) and Gd-DTPA respectively labeled with CPP (FITC-CPP, Gd-DTPA-CPP) were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T(1)WI signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide reduction assay (MTT).</p><p><b>RESULTS</b>The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry (FITC-CPP MW = 2163.34, Gd-DTPA-CPP MW = 2285.99), were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T(1) weighted imaging (T(1)WI) signal, and that the T(1)WI signal intensity was increasing in a time-dependent manner (r = 0.972, P = 0.001), while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different (P = 0.225). By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant (F = 0.006, P = 1.000) effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group.</p><p><b>CONCLUSIONS</b>The newly constructed CPP based on polyarginine can translocate cells by carrying FITC and MR contrast agent Gd-DTPA, and the intracellular concentrations are readily detectable by MR imaging, suggesting a new way for MR molecular imaging.</p>