Identification of role of PI3K in mediating necroptosis of L929 cells induced by tumor necrosis factor alpha / 军事医学

Objective To identify the role of phosphatidylinositol-3-kinase(PI3K) in mediating necroptosis induced by tumor necrosis factor alpha (TNFα) and the involved mechanism.Methods Knockdown of p110α,receptor-interacting protein 1(RIP1) or both p110αand RIP1 was mediated by the specific short hairpin RNA (shRNA) lentivirus and verified by RT-PCR or Western blotting .In addition , Western blotting was used to detect phosphorylation of mixed lineage kinase domain-like protein(MLKL) and protein kinase B(AKT) or tetramerization of MLKL.Cell death was measured by micros-copy and flow cytometry.Results AKT phosphorylation and TNFα-induced necroptosis of L929 cells were suppressed by the inhibitors of PI3K or AKT, as well as p110αknockdown.Moreover, RIP1 knockdown did not inhibit L929 cell death induced by TNFαplus Z-VAD, but the RIP1-independent necroptosis was inhibited by p 110αknockdown.In addition, p110αknockdown suppressed MLKL phosphorylation and tetramerization induced by TNFαwith Z-VAD in L929 cells. Conclusion PI3K mediates necroptosis of L929 cells induced by TNFαby activating AKT and MLKL, respectively.

Construction of spider draggling silk protein MaSp1 prokaryotic expression vector and its expression and purification in Escherichia coli / 军事医学

Objective To establish a key technological system for spider fibroin gene code tandem connection , vector construction , prokaryotic expression and purification using genetic engineering in order to achieve MaSp 1 heterologous ex-pression in Escherichia coli and its separation and purification .Methods Isocaudarner ligation method was used to connect synthetic spider fibroin gene monomer code in tandem , and a recombinant clone concatemer was obtained .The identified recombinant clones were connected with prokaryotic expression vector pET 28a(+), and then transformed into E.coli BL21 (DE3).After being induced by IPTG for 6 hours, the expression product was identified by SDS-PAGE and Western blot-ting.Engineering bacteria were fermented in high density , and the obtained protein was purified through ammonium sulfate fractionation.Results and Conclusion The expression plasmids of MaSp1concatemers were successfully constructed , and the induced expression genetic engineering MaSp 1 protein was of the expected relative molecular mass .In addition, the pu-rity of the purified protein was above 80%.This study has developed crucial technologies for mass production of genetic en-gineering spider silk proteins .

Influence of electroporation on immunogenicity of the DNA vaccine pVAX-tG250FcGB / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the influence of electroporation on the immunogenicity of the DNA vaccine pVAX- tG250FcGB.</p><p><b>METHODS</b>The DNA vaccine pVAX-tG250FcGB was constructed by inserting the coding gene of tG250 fusion genes into the expression vector pVAX. The DNA vaccine was delivered in BALB/c mouse by electroporation or intramuscular injection, and the induced antigen specific immune responses were compared.</p><p><b>RESULTS</b>The vaccine delivered by electroporation and intramuscular injection both induced immune responses in BALB/c mouse, but electroporation produced an obviously stronger effect than intramuscular injection.</p><p><b>CONCLUSION</b>Electroporation-mediated DNA vaccine delivery can produce strong immune response in mice and is an effective means for studying the immunogenic effect of DNA vaccine pVAX-tG250FcGB.</p>

Identification of novel metastasis associated genes MAG-1 and MAG-2 / 中国肺癌杂志

<p><b>BACKGROUND</b>To isolate and identify the genes related to cancer metastasis by comparison of two cell strains with different metastasis potentials subcloned from human lung giant cell carcinoma cell line.</p><p><b>METHODS</b>Suppression subtractive hybridization (SSH) was used to compare the levels of gene expression between the two cell strains and SSH library was constructed. After screening the library by gene chip, the expressed sequence tags (ESTs) with different expressing level were sequenced and blasted with GenBank.</p><p><b>RESULTS</b>Seventy-nine genes were obtained that were expressed much higher in PLA-801D than in PLA-801C, including two full-length cDNA. GenBank Accession numbers of the two cDNA, named MAG-1 and MAG-2, were BC006236 and BC002420, the 8.5 kb MAG-1 gene was composed of four exons and located on the chromosome of 4q21. The MAG-2 gene, which was made up by 9 exons, had a length of 5.2 kb and its location was 2q35. Both sequences had open reading frames (ORF) and promoters before the theoretical transcription start points. Using special software, the secondary structure of theoretical products of the two cDNAs was prognosticated, α-helix was the main proportion, but β-pleated sheet and random coil were also included.</p><p><b>CONCLUSIONS</b>The expression of MAG-1 and MAG-2 has significant differences in these two cell strains, so they might impact tumor metastasis in some ways that are still uncharted.</p>

Mitochondrial DNA deletion on the growth and invasiveness of human lung cancer cells / 第三军医大学学报

Objective To explore the relationship between mitochondrial DNA deletion and malignant phenotypes of human lung cancer cells. Methods Two rho? derivatives of 95C and 95D were generated by treating the cultured cells with ethidium bromide. Agarose colony formation assays and Transwell invasion assays were carried out to detect the phenotypes of colony formation and invasiveness of the cultured cells, respectively. Cell growth was determined by MTT. Results The partially mtDNA-deleted cells exhibited stronger capacity of colony formation and invasiveness, and faster growth rates than their respective parental cell lines. Conclusion Mitochondrial DNA deletion might play a role in the formation of malignant phenotypes of human lung cancer.

Expression of chimeric antigen of HCV with multiple immunodominant epitopes in E .coli and its application for serological tests / 中华传染病杂志

Objective ToexpresschimericantigenofHCVwithmultipleimmunodominantepitopes inE .coliforimprovingthequalityofreagentsforHCVscreening .Methods Thegenefragmentencoding HCVchimericantigenwasobtainedbymolecularcloningmethodandclonedintopQE 30 plasmidforex pressioninE .coliM 15 .TheexpressedchimericantigenwaspurifiedbyNi NTAandcoatedonELISA platestoanalyzeitssensitivityandspecificity .Results Thechimericantigenof 5 30 0 0washighlyex pressed .ELISAassayofserumsamples (including 18HCVpositiveand 17negativesera)indicatedthatthe HCVchimericantigenhadhighsensitivityandspecificity .Conclusions ChimericantigenofHCVwith typicalimmunodominantepitopescanbeusedtodevelopgoodreagentsforHCVimmunoassay .

Cloning and sequencing of partial gene from SENV isolate in China / 军事医学科学院院刊

Objective:To investigate the presence of SENV infection among patients in China,and analyze partial nucleotide sequence of SENV isolated from a patient with non A-G hepatitis.Methods:A nested polymerase chain reaction (PCR) assay with primers from ORF1 of SENV genome was established to detect SENV DNA.The PCR product was cloned and sequenced.Results:SENV DNA was positive in 2 of 7 patients with non A-G hepatitis and TTV negative.Partial gene of a SENV isolate was compared with the corresponding region of SENV isolate(AX025730)from Italy and was found that the nucleotide homology was 90%.Conclusions:The results of this study confirmed the presence of SENV infection in China.The development of a PCR assay for SENV DNA detection and the cloning,sequencing of the SENV isolate have important implication for the diagnosis and epidemiological investigation on SENV infection.

Combined application of MS-275 and curcumin in inhibiting cell survival signaling pathways and inducing apoptosis of prostate cancer cells / 解放军医学杂志

Objective Curcumin and MS-275,an inhibitor of histone deacetylase (HDAC),are promising anti-tumor agents. The aim of present study was to investigate the mechanisms of apoptosis induced by combined use of MS-275 in low dosage and curcumin in prostate cancer cell line DU145. Methods MTT assay was used to evaluate the lethal effect on DU145 cells by solitary use of MS-275 and or combined with curcumin. The changes in cell life cycle was detected by flow cytometry. The expressions of the survival signaling pathways were determined by Western blotting. Results Solitary application of MS-275 or curcumin may inhibit the growth of DU145 cells in a time and dose-dependent manner. The combination of MS-275 (1?mol/L) and curcumin (20?mol/L) exhibited obvious cytotoxic effect on cell viability,which was only 45.9% 48h after the combined treatment. Cell cycle assay showed that the combination of MS-275 and curcumin resulted in obvious appearance of sub-G1 phase in DU145 cells,implying that the cell apoptosis had been induced. The results of Western blotting showed that after treatment of MS-275 or curcumin singly,the phosphorylation level of Akt and ERK kinase declined slightly,however,when MS-275 and curcumin were used together,there appeared a prominent inhibitory effect on Akt and ERK kinase,indicated by a sharp decline of their phosphorylation level,and at the same time,the level of cleaved PARP,a hallmark of apoptosis,was increased in DU145 cells. Conclusion Combined use of MS-275 and curcumin may exert a synergistic cytotoxic effect on the viability of DU145 cells,and exhibit an inhibitory activity on Akt and ERK kinases to induce apoptosis.