Objective To test the infeciton efficiency of recombinant adenovixal vector carrying HBsAg-HSP70 chimeric gene and to abserve its biological characteristics. Methods Peripheral blood mononuclear cells (PBMC) were separated from healthy blood donor and they were infected by Ad-HSP70-HBsAg on the first day of isolation. DCs were induced in medium with cytokines IL-4, GM-CSF and TNF-α in vitro. The biological characteristics of DC induced were analyzed by inverted fluorecent microscope, RT-PCR, flow cytometer(FACS), and mixed lymphocyte reaction (MLR). Results The traced gene-GFP were abserved in DCs by inverted fluorecent microscope and HSP70-HBsAg gene mRNA expression was detected by RT-PCR after the Ad-HSP70-HBsAg infection. FACS analysis shown that the expression of CD1a,CDSO,CD86 and HLA-DR on surfece of two groups of DCs were similar. MLR showed that there are not a statitie difference of stimulated index (SI) between two groups.Conclusion Results indicated that Ad-HSP70-HBsAg can effectively infected DCs without affecting its biological characteristics.

OBJECTIVETo induce the differentiation of K562/MDR1 cells into dendritic cells (DC) with multidrug resistance property.

METHODSK562/MDR1 cells and K562 cells were cultured in the presence of GM-CSF and IL-4 to generate DC and matured by TNF-α. On d14 K562/MDR1-DC and K562-DC cells were harvested and the expressions of CD1a, CD83, CD80, CD86, HLA-ABC and HLA-DR were assessed by flow cytometry (FCM). The antigen presentation function of K562/MDR1-DC and K562-DC was determined by allogenic mixed lymphocyte reaction (Allo-MLR). The expression of P-glycoprotein and the intracellular accumulation of daunorubicin (DNR) were detected by FCM. The sensitivity of K562/MDR1-DC and K562-DC cell to vincristine, adriamycin was measured using MTT assay.

RESULTSBoth K562/MDR1 and K562 cells were differentiated into dendritic cells in the presence of cytokine cocktails, showing the morphologic and immunophenotypic characteristics of DC. K562/MDR1-DC more markedly enhanced proliferation of allogeneic lymphocytes in MLR than K562-DC. High level expression of P-glycoprotein and efflux of DNR were demonstrated in K562/MDR1-DC. K562/MDR1-DC showed multidrug resistance property, with higher IC(50) to VCR and ADM than that of K562-DCs.

CONCLUSIONK562/MDR1 cells can be differentiated into DC with the presence of cytokines, the induced K562/MDR1-DC cells express high level of P-glycoprotein and acquire the multidrug resistance property.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; Cell Differentiation ; drug effects ; Dendritic Cells ; cytology ; Drug Resistance, Multiple ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; K562 Cells ; cytology ; Transfection ; Tumor Necrosis Factor-alpha ; pharmacology

Adult ; Female ; Humans ; Myxoma ; diagnosis ; pathology ; surgery ; Vulvar Neoplasms ; diagnosis ; pathology ; surgery

BACKGROUNDThe capsule associated protein 10 gene (cap10) is indispensible for the formation of the polysaccharide capsule, and is important in maintaining virulence of the Cryptococcus (C.) neoformans. In this study, we aimed to construct an short hairpin RNA (shRNA) expression vector targeting C. neoformans cap10 gene expression and confirm its biologic relevance.

METHODSA pair of oligonucleotides targeting the cap10 cDNA sequence was designed and synthesized. It was cloned into the plasmid psilencer4.1-CMV neo to construct an eukaryotic shRNA expression vector. The vector was transfected into C. neoformans cells using the LiAc method. The expression of cap10 was assessed by real-time fluorescence quantitative PCR. Groups of C. neoformans cells were incubated with murine macrophage-like J774A.1 cells, and the phagocytic indexes and ratios were determined by the microscopic observation method.

RESULTSThe expression of cap10 in C. neoformans cells transfected with ps4.1 neo-cap10 ((175,535.00 ± 47,004.00) copies/µl) was lower than that of cells transfected with the empty vector ((512,698.89 ± 32,318.02) copies/µl) and mock transfected cells ((562,931.66 ± 65,928.41) copies/µl). The average phagocytic ratio and phagocytic index of J774A.1 cells following incubation with C. neoformans were higher for cells transfected with ps4.1 neo-cap10 (0.21 ± 0.02, (19.06 ± 1.66)%) than for the control experimental group (0.08 ± 0.02, (6.57 ± 1.23)%) and the blank experimental group ((0.07 ± 0.01), (5.89 ± 1.07)%) (P < 0.05).

CONCLUSIONSThe cap10 shRNA vector was successfully prepared and transfected into C. neoformans cells. The effect of RNA interference on the expression of the C. neoformans cap10 gene is effective, and it can induce phagocytosis of C. neoformans.

Animals ; Cell Line ; Cryptococcus neoformans ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Genetic Vectors ; genetics ; Phagocytosis ; Plasmids ; genetics ; Polymerase Chain Reaction ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

Metabolomics is a new science and technology, which it refers to a holistic analytical approach to all the low molecular weight metabolites in an organism or a cell. In this paper, the definition and objective of metabolomics are provided, and the current application of metabolomic research in malignant tumors (diagnosis and therapy) are summarized.
Animals ; Biomarkers, Tumor ; genetics ; metabolism ; Genomics ; methods ; trends ; Humans ; Metabolism ; Models, Biological ; Neoplasms ; genetics ; metabolism ; Proteomics ; methods ; trends ; Transcription, Genetic

OBJECTIVETo construct the adenovirus vector containing recombinant human catalase (CAT) and to express the recombinant gene in vitro.

METHODSTotal RNA was extracted from human leukocytes and full-length human CAT cDNA was obtained with RT-PCR method. The CAT gene was cloned into pcDNA3.1(+) vector and pcDNA3.1(+)CAT was constructed. The positive clones were confirmed by the restriction enzyme digestion and gene sequencing. The CAT gene was cloned into the entry vector pENTR1A, and pENTR1A-CAT vector was constructed. By LR reaction pENTR1A-CAT and pAd/CMV/V5-DEST was recombined in vitro, and the recombinant adenovirus pAd/CMV/V5-DEST-CAT was obtained. The positive pAd/CMV/V5-DEST-CAT was confirmed by sequencing and transfected into 293A cells with Pac I linearization and Lipofectamine 2 000, and the recombinant virus particles were packaged and amplified in the cells. The expression of CAT protein and CAT enzyme activities of the recombinant virus were determined by Western blot and 240 nm UV absorption methods.

RESULTHigh expression of recombinant adenovirus was obtained and the expressed human catalase had high enzyme activity.

CONCLUSIONAd/CMV/V5-DEST-CAT vector containing human catalase gene has been constructed successfully; and the expressed enzyme in 293A cells has high activity.

Adenoviridae ; genetics ; Catalase ; genetics ; metabolism ; Cell Line ; Genetic Vectors ; Humans ; Transfection

OBJECTIVETo explore the mechanisms of Chinese herbal medicine Sanqi Oral Liquid, composed of Astragalus membranaceus and Panpax notoginseng, in alleviating renal injury by observing its effect on the expressions of CD4(+), CD8(+) and CD68(+) cells in 5/6 nephrectomized rats with chronic renal failure.

METHODSA total of 102 SD rats were randomly divided into six groups: three treatment groups were administrated with high, medium and low dosage of Sanqi Oral Liquid respectively by gavage; a normal group, a 5/6 nephrectomized model group, and a group treated with coated aldehyde oxygenstarch were used as controls. Following oral administration of Sanqi Oral Liquid for 12 weeks, the general condition and renal pathological changes were observed, and the renal function, platelet count (PLT) and the expressions of CD4(+), CD8(+) and CD68(+) cells were determined for each group.

RESULTSThere were proliferation of mesangial matrix, renaltubularnecrosis and obvious tubulointerstitial fibrosis in the model group, and they were much milder in the treatment groups. Compared with the model group, the amounts of blood urea nitrogen (BUN), serum creatinine (Scr) and PLT in the treatment groups decreased (P<0.05 for all); and in the group administrated of medium dosage of Sanqi Oral Liquid, the expression of CD4(+) cells was up-regulated and those of CD8(+) and CD68(+) cells were down-regulated (P<0.05 for all), leading to an increased ratio of CD4(+)/CD8(+)(P<0.01).

CONCLUSIONSanqi Oral Liquid has a significant effect on regulating lymphocyte subsets, reducing the infiltration of macrophages in renal tissues and alleviating tubulointerstitial fibrosis, and this may be one of mechanisms of Sanqi Oral Liquid in delaying the progression of chronic kidney diseases.

Administration, Oral ; Animals ; Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Astragalus membranaceus ; chemistry ; CD4-Positive T-Lymphocytes ; drug effects ; pathology ; physiology ; CD8-Positive T-Lymphocytes ; drug effects ; pathology ; physiology ; Drug Evaluation, Preclinical ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Kidney Failure, Chronic ; drug therapy ; immunology ; pathology ; surgery ; Lymphocyte Count ; Male ; Nephrectomy ; Panax notoginseng ; chemistry ; Rats ; Rats, Sprague-Dawley ; Solutions

We constructed prokaryotic expression vectors for different domains of TACE gene and expressed the fusion proteins, so as to explore their effects on the proliferation, adhesion and invasion potential of tumor cells in vitro. The total RNA was isolated from THP1 cell. TACE cDNA was amplified by RT-PCR and subcloned into pMD18-T vector to construct pMD-18T-TACE vector. The different cDNA fragment of TACE were amplified from plasmid pMD-18T-TACE and then cloned into pET-28a( + ) to construct expression vector pET28a( + )- 300, pET28a( + )-T800, and pET28a( + )-T1300, which respectively transformed into E. coli BL21 (DE3). The expression of His-tagged fusion proteins were induced with IPTG and purified through BBST NTA resin. The proliferation ability was examined by MTT assay. The adhesive and invasive ability were examined by plated adhesion model and Transwell assay. The protein pET28a( + )-T300 and pET28a( + )-T1300 can reduce the proliferation, adhesion and invasion ability of human lung carcinoma cell A549 in vitro, but otherwise the protein pET28a( + )-T800 had not shown the inhibitive function. The fusion protein of disintegrin domain of TACE have the similar biological function to other disintegrins, which can be used for further research on function of TACE in inflammation and tumor.
ADAM Proteins ; biosynthesis ; genetics ; pharmacology ; ADAM17 Protein ; Adenocarcinoma ; pathology ; Cell Adhesion ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Lung Neoplasms ; pathology ; Neoplasm Invasiveness ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology

BACKGROUND: Hyaluronic acid is an important component of extracellular matrix, has good biocompatibility, unique rheological properties and a variety of physiological functions. Therefore, it has wide application prospect in the tissue engineering. OBJECTIVE: To review the latest research progress in hyaluronic acid scaffold materials in different directions of tissue engineering. METHODS: Using the keywords of "hyaluronic acid, tissue engineering, regeneration, scaffold" in English and Chinese, we retrieved relevant articles published from January 2013 to September 2016 in the databases of PubMed, ScienceDirect and CNKI. RESULTS AND CONCLUSION: The application of hyaluronic acid tissue engineering scaffold materials mainly focuses on the regeneration of bone, cartilage, cardiovascular and nerve. The researchers optimize the composition, ratio of different materials, and modification or cross-linking methods to obtain the ideal scaffold materials to match the mechanical properties and physiological activity of the target tissue. However, there are not enough data on the influence of the molecular weight of hyaluronic acid and the original source of other raw materials on the scaffold properties. Further investigations are needed on the clinical transformation of hyaluronic acid tissue engineering products.

BACKGROUND:Intervertebral disc degeneration is an important cause of low back pain.At present,there are many modeling methods for disc degeneration in China and abroad,but there is not a model for low back pain due to disc degeneration. OBJECTIVE:To compare the effect of mechanical puncture combined with tumor necrosis factor α and complete Freund's adjuvant with a conventional disc mechanical puncture alone. METHODS:A total of 18 male adult Sprague-Dawley rats were randomly divided into 3 groups,with 6 animals in each group.No treatment was given in the blank group.Animal models of intervertebral disc degeneration were made in the L4-5 segments of rats in the control using conventional mechanical puncture.In the experimental group,on the basis of mechanical puncture,tumor necrosis factor α+complete Freund's adjuvant was injected into the L4-5 intervertebral discs using a microinjector to establish a model of disc degeneration induced by mechanical puncture combined with inflammatory factors.Four weeks after surgery,the pain threshold of rats was measured by the hot plate method for assessing the perception of heat injury in rats with intervertebral disc degeneration.MRI examination was performed to observe the disc degeneration in each group.ELISA was used to detect the levels of serum tumor necrosis factor α,interleukin 1β,interleukin 6 and prostaglandin E2.Hematoxylin-eosin and Safranin O-fast green staining were used to observe the morphological changes of the disc. RESULTS AND CONCLUSION:In terms of pain,the behavioral pain threshold of the experimental group was continuously decreased,and the levels of serum inflammatory factors were significantly higher compared with the control group.In terms of morphology,the MRI results showed that the L4-5 nucleus pulposus signal completely disappeared in the experimental group.Histopathological results showed that in the control group,the nucleus pulposus was intact,more notochord cells were visible,and some fiber rings were ruptured,while in the experimental group,there are fewer notochord cells and the structure of the nucleus pulposus and fibrous ring is disturbed,with the boundary disappearing.To conclude,mechanical puncture combined with tumor necrosis factor alpha and complete Freund's adjuvant can successfully establish a discogenic low back pain model in rats.This operation is simple and economical to achieve obvious disc degeneration and low back pain,with greatly shortened molding cycle.This model can be used as a reference for studying discogenic low back pain models.