Objective:To construct the extracellular region of the human TRAIL cDNA expression vector and express and purify the extracellular region of the TRAIL protein. Methods: The mRNA of TRAIL was extracted from CD3 activated normal human PBMC and used as a template for reverse transcription. After PCR amplification, a 730 bp fragment including extracellular region was obtained and cloned into pGEX-2T.The recombinant vector was named pGEX/TRAILex. The pGEX/TRAILex vector was transformed into E.coli DH5a. After IPTIG induced at lower temperature, the collection of the sonicated extract was purified by using the GST agarose 4B. The purified fusion protein was identified by Western blotting with anti-TRAIL McAb.Results:The pGEX/TRAILex was constructed. After IPTG induced,a high level expression of the extracellular region of the TRAIL protein was obtained, SDS-PAGE analysis showed that the recombinant E. coli could express a 54 kD GST fusion protein which accounted for about 28% of the total cellular protein. The study of solubility of expression protein indicated that GST-Tex was expressed predominantly in the soluble form.The purified production was obtained 2.2 mg/L of culture media and the purity of the GST-Tex was more than 95%. GST/TRATLex protein could be recognized by anti-TRAL McAb in Western blot. Conclusion:The expression of recombinant extracellular domain of the human TRAIL protein may be useful for the study of biological functions of TRAIL and it's biotheraphy in tumor.

Objective:To observe the effect and safety of the inhalation of Lanqin oral solutions combined with interferon in the treatment of children herpes angina. Methods:The children with herpes angina were randomly divided into two groups. On the basis of support treatment, the control group (57 cases) was intravenously treated with ribavirin, and the observation group (58 cases) was treated with the combined inhalation of interferon and Lanqin oral solutions. The time of transference cure, total efficiency and adverse drug reactions in the two groups were observed and compared. Results:The time of transference cure of the observation group was sig-nificantly shorter than that of the control group (P<0. 05) with higher total efficiency (P<0. 05). The adverse drug reactions were few and mild in the two groups. Conclusion:The inhalation of Lanqin oral solutions combined with interferon in the treatment of chil-dren with herpes angina is effective with short transference cure time, convenience of use and promising security, which is worthy of clinical application.

AIM: To investigate the role of hydrogen sulfide (H_2S) in the cholecystokinin octapeptide (CCK-8) attenuating lipopolysaccharide (LPS)-induced lung injury. METHODS: A rat model of lung injury induced by intravenous injection of LPS was developed. Male Wistar rats were divided into normal control group, LPS group, LPS+CCK-8 group and CCK-8 group. Six hours after LPS injection, partial pressure of oxygen in the arterial blood (PaO_2), H_2S content and cystathionine-γ-lyase (CSE) activity in lung tissue were detected. The mRNA expression of CSE in lung tissue was determined by RT-PCR;the structure of lung tissues was observed under optical microscope. RESULTS: Compared to normal control rats, the LPS-treated rats had significantly decreased PaO_2 level, increased index of quantitative assessment (IQA) score, while H_2S content, CSE activity and the mRNA expression of CSE in lung tissue were significantly increased (all P<0.05). Administration of CCK-8 into LPS-treated rats increased the PaO_2 level and alleviated the degree of lung injury (measured by IQA score). In addition, CCK-8 decreased H_2S content, CSE activity, and the mRNA expression of CSE (all P<0.05). No significant difference of the above-mentioned parameters between CCK-8 group and normal control group was observed. CONCLUSION: CCK-8 reduces LPS-induced lung injury through inhibiting the generation of endogenous H_2S.

OBJECTIVEThis work was conducted to investigate the expression of matrix metalloproteinase 9 (MMP 9) gene in cancer cells by fibronectin adhesion and the underlying mechanism of cell invasion.

METHODSFollowing adhesion of ovarian cancer cells A2780 to fibronectin, mRNA expression of MMP cells were assayed by reverse transcription-polymerase chain reaction (RT-PCR). MMP9 promoter was cloned from genomic DNA of HT1080 cells with PCR. The MMP-9-pGL2 reporter gene vector was constructed and then transiently transfected into A2780 cells.

RESULTSAdhesion induced the increase of cellular MMP9 mRNA content in A2780 cells, not affecting the expression of MMP2 or TIMP-1 gene. The stimulation was enhanced with the increase adhesion time. When the transfected cells were allowed to adhere and spread on FN-coated surface, the promoter activity of MMP9 gene was also enhanced dramatically.

CONCLUSIONCell-ECM adhesion may stimulate the expression of MMP9 gene through stimulating the promoter activity, thereby enhancing cancer cell invasion and metastasis.

Cell Adhesion ; physiology ; Female ; Gene Expression ; Humans ; Matrix Metalloproteinase 9 ; genetics ; Ovarian Neoplasms ; genetics ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured

OBJECTIVE To investigate the metabolic changes in MEG-01 megakaryocytes after treatment with linezolid （LZD） from metabolomic perspective and its impact on the the proliferation of cells and generation of platelet precursors. METHODS MEG-01 cells were seeded in proliferation medium and divided into blank control group （untreated）， solvent control group （4‰DMSO）， and 100， 200， 400， 800 μg/mL LZD groups. The proliferation status of cells in each group was observed under the microscope； cell proliferation and viability were assessed. Cells were also seeded in differentiation medium and divided into blank control group （untreated）， solvent control group （4‰DMSO）， and 400 μg/mL LZD group； after 14 days of culture， platelet precursor generation was observed under the microscope； immunofluorescence staining was performed to count the proportion of cells producing pseudopodia， the relative length of pseudopodia was measured， and the expression levels of CD41 and CD42b mRNA were assessed. Cells from the solvent control group and the 400 μg/mL LZD group， cultured in differentiation medium for 14 days， were extracted and subjected to non-targeted metabolomics and targeted energy metabolomics analysis using liquid chromatography-tandem mass spectrometry. The relative content of pyruvate in cells， after being cultured for 14 days with the addition of pyruvate （10 mmol/L） or LZD （400 μg/mL）+pyruvate （10 mmol/L）， was measured and observed， as well as its effects on cell proliferation and platelet precursor generation. RESULTS 400 μg/mL LZD could significantly inhibit the proliferation of MEG-01 cells and the generation of platelet precursors （P＜0.01）. Non-targeted metabolomic analysis of MEG-01 cells after 400 μg/mL LZD treatment revealed significant changes in energy metabolism-related pathways such as mTOR signaling pathway， alanine， aspartate and glutamate metabolism， and central carbon metabolism in cancer. Targeted energy metabolomic analysis further showed that the relative contents of adenosine triphosphate， guanosine triphosphate， and pyruvate in MEG-01 cells were significantly reduced （P＜0.01）， while the relative contents of lactate were significantly increased （P＜0.01）. Compared with the LZD group， the relative content of pyruvate， cell count， the proportion of cells producing pseudopodia and the relative length of pseudopodia were significantly increased in the LZD+pyruvate group （P＜0.01）. CONCLUSIONS LZD may reduce pyruvate production by inhibiting mitochondrial energy metabolism， thereby suppressing megakaryocyte proliferation and platelet precursor production， ultimately leading to the occurrence of thrombocytopenia.