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ObjectiveNecrotizing fasciitis (NF) has a high rate of deterioration and rapid progress, and the mortality rate is still high even after receiving treatment. The study was to explore evaluation value of serum procalcitonin (PCT), C-reactive protein (CRP) and plasma albumin (ALB) and prealbumin (PA) in the prognosis of patients with perianal necrotizing fasciitis (NF).MethodsAll data and detection data of serum PCT, CRP and plasma ALB, PA at admission from 41 perianal NF patients admitted to Hefei first people's Hospital Group from May 2009 to August 2019 were retrospectively collected. The death status was statistically analyzed. The patients in this group were divided into survival group and non-survival group, and the predictive value of various indicators on the prognosis of NF was evaluated. ResultsThe ratio of male to female was 9.25∶1. There were multiple complications, and diabetes mellitus was most common (41.46%). The patients with mixed infection and negative bacterial culture accounted for 12.20% and 17.07%, respectively. There were many kinds of bacteria detected out. Klebsiella pneumoniae (20.45%), Escherichia coli (15.91%) and streptococcus (15.91%) were common. All underwent one or more surgical treatments, with mortality of 17.07%. There was no significant difference between survival group and death group in gender, complications, microbiological test results, operation status within 6h or and nutrition support (P>0.05), while there were significant differences in time from onset to admission, proportion of patients in ICU, serum PCT and CRP, plasma ALB and PA and proportion of patients undergoing vacuum sealing drainage (VSD) (P<0.05). Logistic multivariate regression analysis showed that long duration from onset to admission, staying in ICU, high serum PCT and CRP, low plasma ALB and PA, and no conducting VSD were risk factors of poor prognosis (P<0.05). Serum PCT, CRP and plasma ALB, PA predict the prognosis receiver operating characteristic(ROC) curve, the area under the curve is 0.859, 0.811, 0.802, 0.747; the area under the curve of the four indicators combined prediction is 0.926.ConclusionThere are certain features in terms of gender, complications and microorganisms in perianal NF patients. Death risk is high. In addition, serum PCT and CRP, plasma ALB and PA are also related with prognosis, which can be applied as biochemical indexes for prognosis evaluation.
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Objective: Using yeast surface presentation technology, secreted anti-PD-L1 single-chain antibody fragment ( sc Fv), then purify the sc Fv that specifically binds PD-L1 antigen. The sc Fv antibody gene sequence was synthesized based on the single chain antibody gene sequence. We express this sc Fv-mFc protein by using p Fuse eukaryotic expression vector to study its affinity and in vitro and in vivo inhibition of lung adenocarcinoma cells ( A549). Methods: Recombinant plasmid p Fuse-scFv was constructed by gene engineering. The recombinant plasmid p Fuse-scFv was transfected into 293 F ( human embryonic kidney cells) and cultured in serum-free Pro293 a-CDM for 72 hours, then the fusion protein was collected, and use the Rapid Protein Liquid Phase Separation and Purification System to purify the sc Fv-mFc fusion protein. Then the fusion protein and the tumor cells were detected by immunohistochemistry; the affinity of fusion protein and tumor cells was analyzed by flow cytometry; ADCC was used to determine the proliferation of tumor cells in vitro. The nude mice inoculated with lung adenocarcinoma cells, and use the fusion protein to verify its anti-tumor effect in vivo. Results: sc Fv-mFc fusion protein was secreted into serum-free culture medium by recombinant plasmid transfection into the 293 F cells; immunohistochemistry and flow cytometry showed that the fusion protein was highly expressed with the surface of PD-L1 protein;ADCC showed that the fusion protein inhibited the proliferation of tumor cells in vitro; the results of tumor-bearing mice showed that the fusion protein inhibited the growth of the tumor. At the dose of 5 mg/kg, The tumor volume growth rate decreased from 14. 90％ to3. 72％, the two independent samples t test P＜0. 05, the difference was statistically significant. Conclusion: The fusion protein containing single chain antibody was successfully prepared, which had good binding ability to A549 cells and inhibited the proliferation of tumor cells in vitro and in vivo, and provided the laboratory basis for the development of targeted anti-tumor drugs.
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OBJECTIVE@#To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer.@*METHODS@#The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed.@*RESULTS@#The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer.@*CONCLUSION@#The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.
Subject(s)
Animals , Mice , Cell Differentiation , Cell Line , Cell Separation , Embryonic Stem Cells , Feeder Cells , Green Fluorescent Proteins , Leukemia Inhibitory FactorABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of endomorphin-1 (EM-1) on the maturation phenotype, cytokine secretion, T cell proliferation and TLR4 expression in human peripheral blood dendritic cells (PBDCs) stimulated and induced by high glucose, and to explore the regulatory mechanism of EM-1 on DC immune function.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMNCs) were induced into immature dendritic cells (imDCs). The high glucose was used as the stimulating factor, and the EM-1 was used as the interventional factor. Then, the experiments were divided into normal glucose group (NG group), high glucose group (HG group), high glucose plus EM-1 group (EM group) and high glucose plus EM-1 and naloxone group (Nal group), respectively. The PBDC's phenotype changes were detected by flow cytometry; ELISA was used to detect the changes of cytokines secreted by PBDCs co-cultured with autologous lymphocytes; CFSE was used to detect the proliferation of T lymphocytes. TLR4 expression on PBDC surface was detected by RT-PCR.</p><p><b>RESULTS</b>Compared with HG group, the expression of PBDC surface molecules CD86, CCR7 and CD36 was up-regulated in EM group (P<0.01), while the change of CD83 expression was not statistically significant. However, IL-12 and IL-10 secreted by PBDCs and the proliferation index of T-lymphocytes stimulated by PBDCs were both decreased in EM group. Compared with EM group, the expression of CD86, CCR7 and CD36 was decreased in Nal group (P<0.01), while the expression of CD83 was almost unchanged (P>0.05). T-lymphocyte proliferation index was increased very significantly in Nal group (P<0.01). The gray ratio of TLR4 in HG group was higher than that in NG group, while the gray ratio in EM group's was very significantly lower than that in HG group's (P<0.01). These results indicate that the high glucose can promote the expression of PBDC TLR4, while the EM-1 inhibits the expression of TLR4.</p><p><b>CONCLUSION</b>EM-1 up-regulates the expression of PBDC surface molecules CD86, CCR7 and CD36 stimulated and induced by high glucose, but inhibites the induction of PBDC to maturity by high glucose. And the secreted inflammatory cytokines IL-12 and IL-10 inhibites the proliferation of T lymphocytes derived from PBDCs, while naloxone inhibites the effect of EM-1. EM-1 inhibites the expression of TLR4 on PBDC surface induced by high glucose.</p>
Subject(s)
Humans , Cell Differentiation , Cells, Cultured , Cytokines , Dendritic Cells , Glucose , Oligopeptides , Toll-Like Receptor 4ABSTRACT
Biochip technology will bring a tremendous revolution to life science and medical research in 21 century. Microarray assays represent an essential technical advance in biomedical research. Recently, the demand for microarray assay technology has spring up. Therefore, low cost and flexible techniques are needed to meet specific requirements for increasingly integrated biochips. Also performance must be improved in terms of speed and sensitivity. To this end, promising approaches, mainly based on micro and nanotechnologies, have been developed. In this paper, the design and microfabrication of a novel type of micro-cantilever probe are introduced. These probes were fabricated using silicon dioxide by Micro-electromechanical System (MEMS) techniques, and they featured one micron split gap, microchannels and self-replenishing reservoirs. All fabricated micro-cantilever probe were tested on Nanoarrayer instrumentation. Cy3-streptavidin was loaded as biological sample and patterned on DSU gold surface. Results showed these probes were capable of generating high quality biological arrays with routine spot sizes of 2 - 3 microns and could deposit at least three thousand spots without reloading. The spot size could potentially achieve sub-micron when probe size was further shrunk down by the high-resolution lithography technique or more precise microfabrication technologies, such as E-beam lithography. To further improve sample loading efficiency, it is needed to modify the cantilever surface in order to better confine sample inside the microchannel and reservoir, which will be researched in the future.
Subject(s)
Microarray Analysis , Methods , Microelectrodes , Molecular Probe Techniques , Nanotechnology , Methods , Silicon Dioxide , ChemistryABSTRACT
<p><b>OBJECTIVE</b>To confirm whether human MHC class I chain-related A (MICA) induces the amplification of V delta 1 gamma delta tumor-infiltrating lymphocytes (TILs) in vitro and to identify the cytotoxicity of MICA-reactive V delta 1 gamma delta TILs towards epithelial tumor cells.</p><p><b>METHODS</b>MICA protein was prokaryoticly expressed and purified by molecular cloning technology. The purified recombined MICA (rMICA) was used to induce V delta 1 gamma delta T cells from tumor tissues in vitro and the cytotoxicity of these V delta 1 gamma delta TILs were tested by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT).</p><p><b>RESULTS</b>The rMICA was expressed in prokaryocyte with pET30 as a vector. The immobilized rMICA protein could markedly induce the amplification of V delta 1 gamma delta T cells from tumor tissue in vitro. These V delta 1 gamma delta T cells showed strong cytolytic activities towards tumor cell lines expressing MICA.</p><p><b>CONCLUSION</b>The MICA-reactive V delta 1 gamma delta T cell may be a candidate for adoptive cellular therapy of tumors.</p>