Variation of 24 plasma amino acid metabolite levels in patients with gastric cancer / 第二军医大学学报

Objective To study whether plasma amino acid metabolites in patients with gastric cancer can be used as biomarkers for the diagnosis of gastric cancer. Methods The levels of 24 kinds of plasma amino acids were detected by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), and were compared between patients with gastric cancer and normal controls, between patients with different stages of gastric cancer, and between gastric cancer patients before and after operation. Results The levels of 18 kinds of plasma amino acids including alanine (Ala), glycine (Gly) and glutamic acid (Glu) in gastric cancer patients were significantly lower than those in the normal controls, while the levels of valine (Val), arginine (Arg) and serine (Ser) were significantly higher than those in the normal controls (all P0.05); there were no significant differences in symmetric dimethylarginine (SDMA), kynurenine (Kyn) or hippuric acid (HA) levels between the two groups (P0.05). The plasma level of Arg in patients with III-stage of gastric cancer was significantly higher than that in the-Ⅱstage, while the plasma glutamine (Gln), Glu, methionine (Met) and phenylalanine (Phe) levels were significantly lower than those in the-Ⅱstage (all P0.05). The levels of plasma leucine (Leu), Arg and citrulline (Cit) in patients after operation were significantly lower than those before operation, while the plasma Gln, lysine (Lys), Glu and Phe levels were significantly higher than those before operation (all P0.05). Conclusion Amino acids metabolites in plasma of patients with gastric cancer such as Gln and Arg play important roles in the early prediction of gastric cancer.

Three kinds of mesenchymal stem cells:differentiating into neuron-like cells / 中国组织工程研究

BACKGROUND:Stem cells,characterized with convenient collection,wide sources and great potential,have been developed in the cell therapy.The most commonly used stem cells include bone marrow mesenchymal stem cells,placental mesenchymal stem cells and umbilical cord blood mesenchymal stem cells.OBJECTIVE:To compare the biological changes in bone marrow mesenchymal stem cells,placental mesenchymal stem cells and umbilical cord blood mesenchymal stem cells after co-cultured with nerve cells.METHODS:Human bone marrow mesenchymal stem cells,human placental mesenchymal stem cells and human umbilical cord blood mesenchymal stem cells were isolated and cultured in vitro.The morphological changes of mesenchymal stem cells differentiated into nerve cells were observed by co-culture with Transwell culture plates.The expression of neuron-specific enolase was detected after induction.RESULTS AND CONCLUSION:In the co-culture system,these three kinds of mesenchymal stem cells gradually formed a star-like shape and were interconnected.Moreover,all the cells were positive for neuron-specific enolase.These findings reveal that the microenvironment provided by nerve cells can induce and promote the differentiation of three kinds of mesenchymal stem cells into neuron-like cells.

Correlation between HLA-DB1 genes and susceptibility to echinococcosis in Tibetan population in Tibetan Plateau / 中国血吸虫病防治杂志

Objective To determine the susceptibility genes and resistance genes in HLA-DRB1 alleles in Tibetan patients with cystic and alveolar hydatid diseases,so as to provide the references for the research of the genetic characteristics and infec-tion mechanism of Tibetan hydatid diseases. Methods The case control method was applied. The Tibetan patients with cystic and alveolar hydatid diseases(63 and 73 cases respectively)in Yushu and Guoluo Tibetan Autonomous Prefecture,and unrelat-ed healthy people(60 cases)in this area were selected as the study subjects. The polymerase chain reaction-sequence based typ-ing(PCR-SBT)technique was applied for genotyping of HLA-DRB1,and the comparison of the gene frequency. Results The frequency of HLA-DRB1*04 in the alveolar/cystic echinococcosis group was lower than that in the control group(χ2 =4.71, 4.31,both P<0.05). Conclusion HLA-DRB1*04 genotypes may be associated with the resistance of cystic and alveolar echi-nococcosis and its resistance genes.

Quantitative Analysis of Immuno-fluorescence of Nuclear Factor-κB Activation / 生物医学工程学杂志

Immuno-fluorescence technique can qualitatively determine certain nuclear translocation, of which NF-κB/ p65 implicates the activation of NF-κB signal pathways. Immuno-fluorescence analysis software with independent property rights is able to quantitatively analyze dynamic location of NF-κB/p65 by computing relative fluorescence units in nuclei and cytoplasm. We verified the quantitative analysis by Western Blot. When we applied the software to analysis of nuclear translocation in lipopolysaccharide (LPS) induced (0. 5 h, 1 h, 2 h, 4 h) primary human umbilical vein endothelial cells (HUVECs) , we found that nuclear translocation peak showed up at 2h as with calculated Western blot verification results, indicating that the inventive immuno-fluorescence analysis software can be applied to the quantitative analysis of immuno-fluorescence.

Progress in understanding of molecular mechanisms of pathogenesis and drug-resistance in ovarian clear cell adenocarcinoma / 肿瘤

Ovarian clear cell adenocarcinoma (OCCA) is a special epithelial ovarian cancer with distinct clinical and molecular characteristics from other subtypes. What may contribute to the pathogenesis of OCCA are specific gene mutation in AT-rich interactive domain 1A (ARID1A) and phosphatidyl inositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK 3 CA) as well as aberrant phosphatidyl inositol-3-hydroxy kinas (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR)/hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) signaling pathways. Within the pathways, some specifically expressed molecules, such as hepatocyte nuclear factor-1β (HNF-1β), insulin-like growth factor-binding protein 1 (IGFBP-1) and Napsin A, could be used as potential diagnostic signatures for OCCA. The underlying mechanisms of poor prognosis and resistance to conventional platinum-based chemotherapy are closely related to tumor angiogenesis, low proliferation capacity, DNA repair system and high expression of Stathmin. Developing optimized therapeutic strategies aiming at novel targets, such as mTOR, would provide new insight in the treatment of OCCA.

Selenium dioxide inducing apoptosis of cervical cancer cells by regulating apoptosis-related proteins / 重庆医学

Objective To investigate the inducing effects of selenium dioxide(SeO2 ) on the apoptosis in human cervical carcino-ma cell line Hela and its influence on the expression of apoptosis-related proteins caspase-3 and P53 .Methods Hela cells were trea-ted with different concentrations of SeO2 for 24 h in vitro ;the morphological changes of Hela cells were observed by the optical mi-croscope;the influence of SeO2 on the cell proliferation and vitality was examined by the MTT assay ;the flow cytometry was em-ployed to detect the cell apoptosis rate ;the expressions of caspase-3 and P53 proteins in Hela cells were determined by the Western blot analysis .Results Under the optical microscopy ,SeO2 generated the obvious influence on the cell growth morphology ,a large number of cells became rounded and shrunken ,and lost the normal form ,while the adherence cell number was evidently decreased and the proliferation was slowed down ;the MTT results showed that SeO2 markedly inhibited the cell proliferation and viability in a dose-dependent manner ,in which ,the cell apoptosis rates induced by the 0 ,1 .875 ,3 .750 ,7 .500 ,15 .000 and 30 .000 μmol/L con-centrations of SeO2 were 3 .12% ,30 .56% ,33 .42% ,37 .50% ,45 .43% and 69 .38% respectively ,which revealing the obviously in-creasing trend;the Western blot assay revealed that SeO2 could up-regulate the caspase-3 and P53 levels ,and reached the peak value at the concentration of 7 .500μmol/L .Conclusion SeO2 could induce the cervical cancer cell apoptosis possibly by up-regulating the expressions of caspase-3 and p53 in Hela cells .

Regulatory effects of lanthanum chloride on the activation of nuclear factor kappa B inhibitor kinase beta induced by tumor necrosis factor alpha / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the regulatory effects of lanthanum chloride (LaCl3) on the activation of nuclear factor kappa B inhibitor (IκB) kinase beta (IKKβ) induced by tumor necrosis factor alpha (TNF-α).</p><p><b>METHODS</b>(1) Hela cells were cultured routinely in vitro. One portion of cells were collected and divided into TNF-α group (cultured with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min), low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, LaCl3 group (cultured with serum-free RMPI 1640 medium containing 100 µmol/L LaCl3 for 30 min), and control group (cultured with serum-free RMPI 1640 medium for 30 min) according to the random number table. Cells in low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group were first cultured with serum-free RMPI 1640 medium containing 5, 25, 100 µmol/L LaCl3 for 4 h, and then stimulated with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min. There were 3 samples in each group. Cells were collected for detection of intracellular location of NF-κB/p65 protein by immunofluorescence staining. (2) Another portion of cells were collected and divided into TNF-α group, low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, and control group with the same treatment as above. There were 3 samples in each group. The protein levels of NF-κB/p65 in nuclei, and the protein levels of IκBα, phosphorylated IκBα (p-IκBα) as well as IKKβ and phosphorylated IKKβ (p-IKKβ) in cytoplasm were determined by Western blotting. The binding activity between NF-κB/p65 in the nuclear and target gene was determined by NF-κB/p65 transcription factor kit (denoted as absorption value). Data were processed with analysis of variance or LSD-t test.</p><p><b>RESULTS</b>(1) High expression of NF-κB/p65 was observed in cytoplasm of control group. High expression of NF-κB/p65 was observed in nuclei of TNF-α group. The expression of NF-κB/p65 in cytoplasm of LaCl3 group was lower than that of control group. In groups treated with LaCl3 and TNF-α, NF-κB/p65 expression levels in nuclei and cytoplasm were decreased along with the increase in the concentration of LaCl3, which were all lower than those in TNF-α group. (2) There was certain amount of NF-κB/p65 protein expressed in nuclei of control group. The expression of NF-κB/p65 protein in nuclei of TNF-α group was higher than that of control group. In groups treated with LaCl3 and TNF-α, the expressions of NF-κB/p65 protein in nuclei were decreased along with an increase in the concentration of LaCl3. The level of IκBα in TNF-α group was significantly decreased but that of p-IκBα increased as compared with those in control group. Along with the increase in the concentration of LaCl3, the levels of IκBα gradually increased and the levels of p-IκBα gradually decreased in groups treated with LaCl3 and TNF-α. There were no statistical differences in expression levels of IKKβ among the 5 groups. The expression of p-IKKβ could be hardly observed in control group, but it was obviously increased in TNF-α group. The expression levels of p-IKKβ in groups treated with LaCl3 and TNF-α were gradually decreased along with the increase in the concentration of LaCl3. The absorption value in TNF-α group was 0.39 ± 0.03, which was higher than that in control group (0, t = -7.23, P<0.01). The absorption values in low-dose LaCl3 +TNF-α group, moderate-dose LaCl3 + TNF-α group, and high-dose LaCl3 +TNF-α group were respectively 0.17 ± 0.03, 0.15 ± 0.03, and 0, which were obviously lower than that in TNF-α group (with t values respectively -6.54, -5.92, -7.23, P values all below 0.01).</p><p><b>CONCLUSIONS</b>LaCl3 can block the activation of NF-κB signaling pathway by blocking the phosphorylation of IKKβ of Hela cells.</p>

Construction of TK Gene-deleted PRV SH StrainContaining a Single LoxP Site / 中国生物工程杂志

Pseudorabies virus (PRV) is a swine herpesvirus of the Alphaherpesvirinae subfamily and a pathogen of swine resulting in devastating disease and economic losses worldwide. Cre/loxP site-specific system has the character of site specific, time specific, tissue specific and high efficiency in recombination, which makes this system universal in vivo and in vitro recombination of bacteria, fungus, plants, insects and mammals. A recombinant PRV which contain a loxP site in TK locus by using Cre/LoxP recombinant system was construsted. A pair of primers were synthesized according to the pEGFP-C1 sequence published on GenBank, and were used to amplify the EGFP gene expression cassette with two loxP sites flanking each side. This target gene was cloned into pSKLR, the resulting transfer vector pSKLR-GFP-loxP was then cotransfected into 293T cells with PRV SH strain genomic DNA. The recombinant virus rPRV1 was selected and purified in TK-143 cells by choosing fluorescent expressing plaques. Cre expression vector pOG231 was cotransfected into 293T cells with rPRV1 genomic DNA. The second recombinant virus rPRV2 was obtained, which contains only one loxP site in TK locus. Sequencing results of rPRV2 TK gene indicated that 34bp loxP site was inserted into rPRV2 genome and there were 270bp deletion in TK gene. PCR amplifying different generations of rPRV2 TK gene showed that the mutant was stable when passages in RK-13 cells. TCID_ 50 assay indicated that rPRV2 grows well on RK-13 cells. The LD_ 50 test results on BALB/C mice suggested that the virulence of rPRV2 was reduced. As a conclusion, the report gene GFP expression cassette was removed successfully from rPRV1 genome and only one LoxP site was leaved in rPRV2 genome by using Cre/LoxP recombinant system.