Objective: To study the functional effects of Hsa-miR-411-3P on gastric cancer cells and related molecular mechanisms. Method: The effect of Hsa-miR-411-3P on the proliferation of gastric cancer cells SGC-7901 and AGS was detected by MTT; The effect of Hsa-miR-411-3P on the cycle and apoptosis of gastric cancer cells SGC-7901、 AGS was detected by flow cytometry; Three online tools: RNAhybrid2.1.2, Miranda3.3a, TargetScan7.0 predicted Hsa-miR-411-3P target genes, and combined with mRNA sequencing results, the intersection genes were considered reliable target genes, and then performed GO, KEGG analysis; Real-time PCR was used to detect the expression of Hsa-miR-411-3P target genes VAV3, ROCK2, PLD1, and PTCH1. Result: Overexpression of Hsa-miR-411-3P inhibited the proliferation of gastric cancer cells SGC-7901 and AGS, promoted the apoptosis of gastric cancer cells SGC-7901, AGS, arrested the cell cycle of SGC-7901 in S phase, and arrested AGS cell cycle in G1 phase. Three online tools: RNAhybrid2.1.2, Miranda3.3a, TargetScan7.0, predicted Hsa-miR-411-3P target genes, and combined with mRNA sequencing results, the intersection genes were considered to be reliable target genes, and a total of 235 intersection targets were obtained. Take 235 intersection target genes as target gene sets for subsequent analysis. GO and KEGG analysis of 235 cross-target genes found that their molecular function was concentrated in enzyme binding, protein binding, transferase activity, etc. Biological processes was concentrated in metabolic processes, assembly of cellular components, development of anatomical structures, and biogenesis of cellular components, etc.(P<0.05). KEGG signaling pathway was concentrated in insulin signaling pathway, cAMP signaling pathway, AMPK signaling pathway, FOXO signaling pathway, etc. (P<0.05). Insulin signaling pathway, AMPK signaling pathway, FOXO signaling pathway, and cAMP signaling pathway are all closely related to gastric cancer. The Hsa-miR-411-3P target genes VAV3、 ROCK2、 PLD1 and PTCH1, which are highly correlated with gastric cancer, all participate in the cAMP signaling pathway.VAV3、 ROCK2、 PLD1 and PTCH1 may have a negative regulatory relationship with Hsa-miR-411-3P, because in the sequencing results, the expression of Hsa-miR-411-3P was up-regulated after berberine treatment of SGC-7901 cells, but the expression of VAV3、 ROCK2、 PLD1 and PTCH1 was down-regulated. For this, verification was performed by using Real-time PCR. It was found that overexpression of Hsa-miR-411-3P would decrease the expression of VAV3 and ROCK2 mRNA in SGC-7901、 AGS cells; overexpression of Hsa-miR-411-3P would increase the expression of PLD1 mRNA in SGC-7901 cells and decrease the expression of PLD1 mRNA in AGS cells;overexpression of Hsa-miR-411-3P would decrease the expression of PTCH1 mRNA in SGC-7901 cells and increase the expression of PTCH1 mRNA in AGS cells. It was indicated that overexpression of Hsa-miR-411-3P could down-regulate the expression of target genes VAV3 and ROCK2, because the expression of PLD1 and PTCH1 was differently in two gastric cancer cells, which was controversial and need to be further studied by other gastric cancer cells. Conclusion: Overexpression of Hsa-miR-411-3P will decrease the expression of target genes VAV3、 ROCK2 to affect cAMP signaling pathway, inhibit the proliferation of SGC-7901, AGS cells, promote apoptosis, and arrest SGC-7901 cell cycle in S phase, AGS cell cycle in G1 phase.
Objective: Single chain fragment variable(ScFv) is the smallest functional structural unit with the antigen-binding specificity of the parent antibody. Due to its affinity and low immunogenicity, ScFv has broad application prospects in medical treatment and diagnosis. To reduce the host immune rejection during clinical treatment with heterologous murine monoclonal antibody (McAb) in canine, ScFv were prepared against canine parvovirus (CPV) using a prokaryotic expression system. Methods: Total RNA was extracted from hybridoma cell lines specific for CPV,amplification of the antibody heavy chain variable region (VH) gene and the light chain variable region (VL) gene from the reverse transcription cDNA into the expression vector pOPE101. The recombinant plasmid was transformed into E. coli for expression, and the expressed protein was identified by Western blot. The activity of the ScFv was detected by ELISA, and the ScFv purified by affinity chromatography was identified by virus neutralization test. Results: The recombinant plasmid pOPE101-ScFv was successfully constructed. The correct expression of single-chain antibody in E. coli was determined by western blot. The ability of the fusion protein to specifically bind to the virus was verified by ELISA and virus neutralization test. The potency was 1∶40 (0.014 μg/ml). Conclusion: A ScFv with neutralizing activity was obtained using an E. coli expression system, which provides a basis for clinical immunotherapy for CPV disease.
Objective: To investigate the effect of miR-5195-3p on the proliferation, migration and invasion of human cervical cancer cell line SiHa. Methods: The expression levels of miR-5195-3p in human cervical cancer cells SiHa and normal epithelial cells HaCaT were detected by real-time quantitative PCR.The miR-5195-3p mimic was transfected into SiHa cells to construct exogenous overexpressed cell lines,while the negative control group was transfected with NC mimic and the transfection efficiency was verified by real-time quantitative PCR;MTT and colony formation experiments were utilized to assess the proliferation capacity; The competence of transversal migration was examined by Wound healing experiment;Transwell assay was performed to evaluated the longitudinal migration and invasion ability;The mRNA transcription and protein expression levels of E-cadherin, Vimentin and snail were analyzed by real-time quantitative PCR and Western blot. Results: the expression level of miR-5195-3p in SiHa was lower than HaCaT (P<0.05).Compared with the negative control group, the level of miR-5195-3p in SiHa cell which transfected the miR-5195-3p mimic was significantly increased (P<0.01);Moreover, their proliferation,migration and invasion in vitro were distinctly reduced(P<0.001);Meanwhile, the expression levels of E-cadherin were up-regulated while those of Vimentin and snail were down-regulated. Conclusion: overexpression of miR-5195-3p may inhibit the proliferation, migration and invasion of cervical cancer cell SiHa by blocking EMT pathway.
Objective: To develop a continuous flow sucrose density gradient centrifugation purification process for freeze-dried human rabies vaccine (chicken embryo fibroblasts). Methods: The effects of concentration of initial sucrose solution and loading speeds on the purification of Rabies virus (RABV) were compared to determine the purification process. The sample collection range was determined by multi-batch experiments. The impurity removal and antigen recovery were compared under different concentration multiples to determine the appropriate concentration ratio of the harvest liquid. The impurity removal rate and repeatability of the different batch samples after purification were compared to determine the stability of the purification process. Results: When the initial sucrose concentration is 60%, the 10-fold concentrated virus could be effectively purified at 150-200 ml/min. The removal rates of ovalbumin, bovine serum albumin and gentamicin reached respectively 99%, 95% and 95%. Conclusions: The continuous flow sucrose density gradient centrifugation technique developed in this study can be used as an industrial purification process for freeze-dried human rabies vaccine (chicken embryo fibroblasts).
Objective: A rapid colloidal gold immunochromatography assay (GICA) for the detection of Streptococcus pneumoniae was explored by using the ribosomal protein L7/L12 as detection marker. Method: The gene sequence of ribosomal protein L7/L12 was analyzed, the prokaryotic expression vector was constructed, the recombinant protein was expressed and purified, and the monoclonal antibody was prepared by immunizing BALB/c mice; The affinity of antigens and antibodies was detected by a labelless molecular interaction instrument based on BLI technology; The colloidal gold immunochromatographic test strip was developed based on the principle of double antibody sandwich, and its specificity, sensitivity and stability were evaluated. Result: Two hybridoma cell strains that can efficiently secrete anti-monoclonal antibody were acquired by means of screening. Purified monoclonal antibodies all had high affinity and no competition with antigen. Pairing and detection were made on colloidal gold immunochromatography platform. The minimum detection limit of the test strips was 1.0×105 CFU/ml; It had a specific reaction with Streptococcus pneumoniae, but did not cross-react with other 9 kinds of common respiratory pathogens such as Haemophilus influenzae and Moraxella catarrhalis; Test strips were kept at 25°C for 12 months and still had good repeatability and stability. Conclusions: RP-L7/L12 can be used as a detection marker for Streptococcus pneumonia. The colloidal gold immunochromatographic test strips prepared by its monoclonal antibody can be used for rapid detection of streptococcus pneumoniae.
Objective: To determine a stable and efficient electroporation protocol to construct a high-capacity phage display antibody library. Methods: Explored the effects of voltage, pulse time, quality and concentration of phagemid DNA, growth phase of TG1 E.coli, buffer for resuspension and washing and medium optimization on the electrotransformation efficiency of TG1 E.coli. Results: Using electroporation cuvettes with electrode spacing of 2 mm, the parameters of the electrorotator were set to 3kV, 25μF, 5ms, and 200 Ω. The exogenous DNA was purified and added to the competent bacterial suspension to make the final concentration of DNA 1ng/μl. 20mmol/L MgCl2 was added to the medium, and the growth phase of TG1 was adjusted to OD600=0.8. The cells were resuspended and washed with sterile ultrapure water, and the concentration of competent cells was adjusted to 4×1010 cells/ml. Under such conditions, the electrotransformation efficiency can reach 4.9×109 CFU/μg DNA. Conclusion: Electrotransformation efficiency has been improved through multiple condition optimization, which established the basis for constructing a high-capacity phage display antibody library.
Epoxy group, a very active group, can react with biomolecules such as enzymes, proteins, nucleic acid and form covalent bonds, which is beneficial for the immobilization of biomolecules. Enzymes immobilized on carriers via covalent binding exhibit better stability and reusability than free enzymes. The extracellular proteases of marine bacterium Bacillus sp. DL-2 were immobilized by covalent bonding onto epoxy resin ES-103B. After the the optimization of immobilization conditions, pH 8.0 extracellular proteases solution, 25 g/L epoxy resin ES-103B, and 45 ℃ for 8 h were determined as the optimal conditions for immobilized proteases to asymmetrically hydrolyze (±)-1-phenylethyl acetate, which generated (R)-1-phenylethanol with the e.e.p being 97.5% and the yield being 45.0%, and generated (S)-1-phenylethyl acetate with the e.e.s being 99.2% and the yield being 83.9%, respectively. The the immobilized enzyme could be reused to resolve (±)-1-phenylethyl acetate for eight times and the e.e.p still retained over 90%. Additionally, the immobilized extracellular proteases showed good storage stability at 4℃.
Chronic obstructive pulmonary disease (COPD) is a common chronic airway inflammatory disease with high morbidity and mortality in China, and it has caused heavy social and economic burden. It is reported that the incidence of COPD is closely related to genetic and environmental factors. Animal model is an important tool to study its pathogenesis, prevention, treatment and identify potential therapeutic targets and biomarkers. With the development of genetic engineering technology and the continuous discovery of related targets and genes of COPD, gene modified animal models are increasingly established and used in COPD research. In this paper, we searched published papers in PubMed to analyze the animal species and modeling methods of previous models of COPD. Then we data-mined the susceptibility genes of COPD and reviewed the susceptibility genes of different species by literature and database tool analysis. Finally, we integrated and listed the information and research progress of COPD genetic engineering mouse and rat models. Those informations are convenient for researchers and clinicians to reference and use. Thereafter, they can facilitate the research of pathogenesis and prevention methods of COPD.
The failure of chemotherapy caused by tumor multi-drug resistance (MDR) is still a difficult point in tumor treatment. Although three generations of inhibitors have been successfully developed for MDR, targeting ATP binding cassette transporter (ABC), there are also effective methods to reverse MDR, such as MDR regulator or chemical sensitizer, multifunctional nanocarrier and RNA interference, but MDR is still a difficulty in tumor treatment due to the complexity of tumor multi-drug resistance mechanism. In this paper, we will focus on the abnormal expression of ABC transporter, The changes of DNA injury repair and apoptosis, autophagy induction and drug resistance, tumor stem cells and drug resistance were reviewed in order to provide ideas and methods for the study of MDR.
In recent years, although nanoparticles have made great progress in biomedical research, they rarely enter clinical trials. This is mainly due to the lack of understanding of the interaction between nanoparticles and the physiological environment and the limited understanding of the biological characteristics of nanoparticles after they enter the body. In the physiological environment, proteins adsorb on the surface of nanoparticles and form protein corona. The formation of this nanoparticles-protein corona complex seriously affects the biological characteristics of nanoparticles and restricts the clinical application of nanoparticles. Therefore, the interaction between protein corona and nanoparticles should be further studied. At present, the research on nanoparticle-protein corona complex is a relatively new field. This review summarizes the research status of protein corona, and focuses on the impact of the interaction between protein corona and nanoparticles. It also introduces methods to prevent and reduce the formation of protein corona, providing ideas for further research and development of nanoparticles.
Streptococcus suis (SS) is an important pathogen causing respiratory diseases in pigs, causing huge economic losses to the pig industry around the world. At the same time, SS can infect people through wounds, respiratory tracts and other channels. Accurate, sensitive and rapid SS detection methods help to understand the prevalence of SS in the herd, and then take appropriate prevention, treatment and comprehensive prevention and control measures. In this paper, the detection methods of SS, including selective culture medium, PCR technology, immunology and molecular typing methods, were reviewed. The advantages and disadvantages of these methods and their application were compared, which provided reference for the establishment of standard diagnostic methods of SS.
Due to the biocompatibility, degradability, and similarity to the structure of natural extracellular matrix, hydrogel has become a research hotspot and focus of tissue engineering. Based on in-situ formation and injectability, and compatibility with existing processing technologies (3D printing, electrospinning), photocrosslinked hydrogels are widely used in the field of tissue engineering. In this paper, recent advances in the field of tissue engineering in photocrosslinked hydrogels are reviewed, including new advances in cartilage, bone, adipose and periodontal tissues. The paper reviews the photocrosslinked hydrogels and provides relevant references for future research.
Objective: Compare the development status and trends of mesenchymal stem cells (MSCs) in basic research, patent application and clinical trial registration between China and the US. Find out the main characteristics of MSCs research in these two countries and provide suggestions for Chinese MSCs development. Methods: The paper data in SCI database, the patent data in DII database and the clinical trial data in Clinical Trials database are retrieved. The quantitative analysis is carried out by Excel and DDA. SWOT analysis is used to discuss the advantages and disadvantages, opportunities and threats of Chinese MSCs development. Results: Although China started late on MSCs research, it has developed rapidly in recent years. The number of papers since 2014 and patent applications in 2016 exceeds that of the United States, and the number of clinical trials has reached more than 200. China has advantages in the research fields of osteoporosis and spinal cord injury and some clinical trials such as endocrine system diseases and autoimmune diseases. However, there is still room for improvement in the study of MSCs in China. Conclusion: China should give full play to its existing advantages and strengthen the strategic layout; attach importance to the enterprise-led new drug development path; make efforts to enhance its international competitiveness and influence; increase capital investment and support of industrial policies and improve the regulatory mechanism and evaluation system.
