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Objective To prepare mouse monoclonal antibodies against the ectodomain of E2 (E2ecto) glycoprotein of Western equine encephalitis virus (WEEV). Methods A prokaryotic expression plasmid pET-28a-WEEV E2ecto was constructed and transformed into BL21 (DE3) competent cells. E2ecto protein was expressed by IPTG induction and presented mainly as inclusion bodies. Then the purified E2ecto protein was prepared by denaturation, renaturation and ultrafiltration. BALB/c mice were immunized with the formulated E2ecto protein using QuickAntibody-Mouse5W as an adjuvant via intramuscular route, boosted once at an interval of 21 days. At 35 days post-immunization, mice with antibody titer above 1×104 were inoculated with E2ecto intraperitoneally, and spleen cells were fused with SP2/0 cells three days later. Hybridoma cells secreting specific monoclonal antibodies were screened by the limited dilution method, and ascites were prepared after intraperitoneal inoculation of hybridoma cells. The subtypes and titers of the antibodies in ascites were assayed by ELISA. The biological activity of the mAb was identified by immunofluorescence assay(IFA) on BHK-21 cells which were transfected with eukaryotic expression plasmid pCAGGS-WEEV-CE3E2E1. The specificity of the antibodies were evaluated with E2ecto proteins from EEEV and VEEV. Results Purified WEEV E2ecto protein was successfully expressed and obtained. Four monoclonal antibodies, 3G6G10, 3D7G2, 3B9E8 and 3D5B7, were prepared, and their subtypes were IgG2c(κ), IgM(κ), IgM(κ) and IgG1(κ), respectively. The titers of ascites antibodies 3G6G10, 3B9E8 and 3D7G2 were 105, and 3D5B7 reached 107. None of the four antibody strains cross-reacted with other encephalitis alphavirus such as VEEV and EEEV. Conclusion Four strains of mouse mAb specifically binding WEEV E2ecto are successfully prepared.
Subject(s)
Animals , Mice , Horses , Encephalitis Virus, Western Equine , Ascites , Immunosuppressive Agents , Antibodies, Monoclonal , Immunoglobulin MABSTRACT
@#【Objective】 To reveal the integral in vivo polypharmacokinetics (PPK) similarity or difference between Jinyinhua (Lonicerae Japonicae Flos, LJF) and Shanyinhua (Lonicerae Flos, LF), and provide reference for their clinical application. 【Methods】 The PPK model and its total quantum statistical moment similarity (TQSMS) method were used to compare the integral PPK profiles of nine components with anti-inflammatory efficacy (rutin, caffeic acid, chlorogenic acid, cryptochlorogenic acid, dispsacoside B, macranthoidin B, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C) of LJF and LF. A total of 54 Specific Pathogen Free (SPF) grade Kunming (KM) mice were randomized into LJF group and LF group (n = 27), and each group was divided into nine subgroups (n = 3) according to different time points. Subsequently, mice model of p-xylene-induced ear edema was constructed by oral administration of LJF and LF. The concentrations of the nine anti-inflammatory components in plasma samples of the mice were determined by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). And the pharmacokinetics (PK) parameters of single component and the integral PPK parameters [total quantum statistical moment (TQSM) and TQSMS] of multiple components were calculated by Drug And Statistics (DAS) software and home-brew programs with Excel, respectively. 【Results】 There were significant differences in single-component PK parameters between LJF and LF (P < 0.05). Whereas, no significant differences were found in multi-component TQSM parameters, including total quantum zero moment (AUCT0-t, AUCT0-∞) and total quantum first moment (MRTT0-t, MRTT0-∞) for the total quanta (P > 0.05). Accordingly, single-component TQSMS varied from 0.220 4 to 0.968 9, and that for the total quanta was 0.828 4, suggesting no significant differences in the speed and extent of bioavailability between LJF and LF. Furthermore, in light of high TQSMS (0.828 4), the integral PPK profiles of the nine anti-inflammatory components of LJF and LF were similar under 90% confidence intervals. 【Conclusion】 The PPK model and its TQSMS method are appropriate and efficient to compare the similarity or difference of integral PPK profiles of multi-component herbal medicines. It is suggested in this research that LJF can be replaced with LF or vice versa for anti-inflammatory treatment.
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Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.
Subject(s)
Animals , Mice , Humans , Adenoviruses, Human/genetics , Escherichia coli/genetics , HEK293 Cells , Isopropyl Thiogalactoside , Blotting, Western , Immunoglobulin G , Antibodies, Monoclonal , Antibody Specificity , Mice, Inbred BALB CABSTRACT
To date, there have been three common methods for sampling the cerebral ischemic border zone in a rat model of transient middle cerebral artery occlusion (tMCAO): the "two o'clock method", the "diagonal method", and the "parallel line method". However, these methods have their own advantages and limitations. Here, we propose a modified technique (the "rectangular method") for sampling the ischemic border zone. A rat tMCAO model was prepared under the support of a compact small animal anesthesia machine. Cerebral blood flow was monitored by high-resolution laser Doppler to control the quality of modeling, and 2,3,5-triphenyl tetrazolium chloride (TTC) staining was used for cerebral infarction location assessment. Superoxide dismutase 2 (SOD2), cysteinyl aspartate specific proteinase (caspase)-3, caspase-9, and heat shock protein 70 (HSP70) were used to verify the reliability and reproducibility of the rectangular method. The expression of biomarkers (SOD2, caspase-3, caspase-9, and HSP70) in the traditional (two o'clock method after TTC staining) and modified (rectangular method) groups were increased. There were no significant differences between the groups. The rectangular method proposed herein is based on a modification of the diagonal method and parallel line method, which could provide a directly observable infarct borderline and a sufficient sampling area for subsequent experimental operations regardless of the cerebral infarct location. The assessed biomarkers (SOD2, caspase-3, caspase-9, and HSP70) demonstrated the reliability and reproducibility of the rectangular method, which may facilitate inter-laboratory comparisons.
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Objective To investigate the effect of statin pretreatment on collateral circulation and prognoses of patients with cardiac large artery occlusive stroke.MethodsFifty-three patients with cardiac large artery occlusive stroke admitted to our hospital from January 2016 to July 2019 were selected. All patients had unilateral middle cerebral artery occlusion. DSA was used to evaluate the collateral flows, and the differences of collateral flows and prognoses in patients took statins before onset were compared with those did not take statins.ResultsAs compared with patients did not take statins, patients took statins had higher incidences of diabetes and coronary heart disease, lower content of low density cholesterol, higher proportion of patients with good collateral circulation (grading 3 to 4), and lower modified Rankin scale scores 3 months after surgery, with statistically significant differences (P< 0.05). Multivariate Logistic regression analysis showed that percentage of patients took statins was independent factors affecting collateral flow in patients with cardiac arterial occlusive stroke(OR=5.000, 95％CI: 1.136-22.006,P=0.033).ConclusionIn patients with cardiac large artery occlusive stroke, statin pretreatment could improve collateral flow and clinical prognoses.
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OBJECTIVE@#To evaluate the surgical technique and clinical effect of arthroscopic pullout suture repair of posterior root tear of the medial meniscus via the double tibial tunnels.@*METHODS@#From May 2014 to May 2017, 22 patients with posterior root tear of medial meniscus were treated by pullout suture repair via the double tibial tunnels, including 8 males and 14 females, aged 34 to 53 years old, with a mean of averaged(45.7±4.7) years old. The patients were followed up for 12 to 24 months, with a mean of (16.4±5.2) months.@*RESULTS@#The Lysholm score of knee joint before operation was 61.8±4.3, IKDC score before operation was 59.9±2.9, Lysholm score at the latest follow-up was 89.1±3.0, and IKDC score was 89.0±2.5. The difference was statistically significant.@*CONCLUSIONS@#Arthroscopic pullout suture repair via the double tibial tunnelsis an effective treatment for symptomatic posterior root tear of medial meniscus, and it can significantly improve the knee functional outcome.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Arthroscopy , Menisci, Tibial , Suture Techniques , Sutures , Tibial Meniscus Injuries , General SurgeryABSTRACT
Current studies have demonstrated that SLC38A1 proteins play a causal role in neoplastic cell transformation.The twofold aim of this study was to provide insight into whether a variance in the expression of SLC38A1 exists between human colorectal cancer and healthy human tissues and to determine how silencing or overexpressing the SLC38A1 gene could affect the proliferation,viability and migration of colorectal cancer cells.Immunohistochemical staining was used to analyze the expression of SLC38A1 in colorectal cancer tissues and adjacent normal mucosa in 77 patients who underwent surgical resection.The expression of SLC38A1 in colorectal cancer tissues and cell lines was detected using RT-PCR and Western blotting.Two colorectal cancer cell lines SW480 and HCT116 were used to examine whether silencing SLC38A1 with siRNA and overexpressing SLC38A1 with shRNA could affect cell viability and migration.As a result,the SLC38A1 protein was very low or undetectable in the normal colon mucosa.In contrast,strong staining of SLC38A1 protein was found in the cytoplasm in 79.2％ colorectal cancer samples.More pronounced SLC38A1 expression in colorectal cancer tissues was significantly associated with tumor node metastasis (TNM) stage.Inhibition of SLC38A1 reduced tumour growth and suppressed proliferation and migration of SW480 cells.In contrast,overexpression of SLC38A1 had the opposite effects on HCT116 cells.S LC38A1 is overexpressed in colorectal cancer,which suggests that it is associated with tumour progression.These results encourage the exploration of SLC38A1 as a target for intervention in colorectal cancer.
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The Grainyhead-like 3 (GRHL3) is involved in epidermal barrier formation,neural tube closure and wound repair.Previous studies have suggested that GRHL3 has been linked to many different types of cancers.However,to date,its effects on human colorectal cancer (CRC) has not been clarified yet.Our microarray analysis has indicated predominant GRHL3 expression in CRC.The purpose of this study was to investigate the expression and significance of GRHL3 in CRC tumorigenesis using CRC tissues and paired paracancerous tissues,as well as using distinct CRC cell lines (HT29 and DLD1).We observed increased GRHL3 expression at both mRNA and protein levels in CRC tissues and CRC cell lines using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting.Moreover,silencing GRHL3 with siRNA could suppress CRC cell proliferation,viability and migration in vitro.We also found that knockdown of GRHL3 could promote cell cycle arrest at G0/G1 phase in HT29 cells and DLD1 cells,and induce cell apoptosis in HT29 cells.Together,our study revealed the down-regulation of GRHL3 in vitro could inhibit CRC cell activity and trigger cell cycle arrest at G0/G1 phase and apoptosis.
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The sialyl Lewis X (SLex) antigen encoded by the FUT7 gene is the ligand of endotheliam-selectin (E-selectin).The combination of SLex antigen and E-selectin represents an important way for malignant tumor metastasis.In the present study,the effect of the SLeX-binding DNA aptamer on the adhesion and metastasis of hepatocellular carcinoma HepG2 cells in vitro was investigated.Reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence staining were conducted to detect the expression of FUT7 at both transcriptional and translational levels.The SLex expression in HepG2 cells treated with different concentrations of SLeX-binding DNA aptamer was detected by flow cytometry.Besides,the adhesion,migration,and invasion of HepG2 cells were measured by cell adhesion assay,and the Transwell migration and invasion assay.The results showed that the FUT7 expression was up-regulated at both mRNA and protein levels in HepG2 cells.SLeX-binding DNA aptamer could significantly decrease the expression of SLex in HepG2 cells.The cell adhesion assay revealed that the SLeX-binding DNA aptamer could effectively inhibit the interactions between E-selectin and SLex in the HepG2 cells.Additionally,SLeX-binding DNA aptamers at 20 nmol/L were found to have the similar effect to the monoclonal antibody CSLEX-1.The Transwell migration and invasion assay revealed that the number of penetrating cells on the down-side of Transwell membrane was significantly less in cells treated with 5,10,20 nmol/L SLeX-binding DNA aptamer than those in the negative control group (P＜0.01).Our study demonstrated that the SLeX-binding DNA aptamer could significantly inhibit the in vitro adhesion,migration,and invasion of HepG2 cells,suggesting that the SLeX-binding DNA aptamer may be used as a potential molecular targeted drug against metastatic hepatocellular carcinoma.