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Objective:To evaluate the effectiveness and safety of intravitreal injection of different doses of aflibercept for polypoidal choroidal vasculopathy (PCV) with serous pigment epithelial detachment (PED) resistant to ranibizumab.Methods:A non-randomized controlled clinical study was conducted.Seventy-three eyes of 73 patients with PCV and serous PED resistant to ranibizumab were enrolled at the First Affiliated Hospital of Zhengzhou University from January 2019 to December 2020.All patients were treated by intravitreal injection of 2 mg or 4 mg aflibercept according to patients' willingness.2 mg aflibercept or 4 mg aflibercept was intravitreally injected monthly for three consecutive months following pro re nata (PRN) regimen in 2 mg aflibercept group (38 eyes) and 4 mg aflibercept group (35 eyes), respectively.PED height and central macular thickness (CMT) were measured by optical coherence tomography, and the best corrected visual acuity (BCVA) was examined with a visual acuity chart and converted to logarithm of the minimum angle of resolution (LogMAR) unit before injection and 1 month, 2, 3, 6 months from the first injection.Intraocular pressure and treatment-related adverse events were recorded.This study adhered to the Declaration of Helsinki and was approved by an Ethics Committee of The First Affiliated Hospital of Zhengzhou University (No.2021-KY-1252).Written informed consent was obtained from each patient prior to entering study cohort.Results:Thirty-three patients (86.84%) in 2 mg aflibercept group and 30 patients (85.71%) in 4 mg aflibercept group finished the treatment and follow-up, respectively. The PED, BCVA and CMT before treatment and at the end of follow-up were (379.24±95.50) and (280.09±120.50)μm, 0.68±0.27 and 0.51±0.19, (393.96±100.81) and (291.70±44.09)μm in 2 mg aflibercept group, respectively, showing statistically significant differences (all at P<0.05).The PED, BCVA and CMT before treatment and at the end of follow-up were (393.07±93.76) and (278.63±145.07)μm, 0.66±0.31 and 0.48±0.22, (377.43±79.61) and (284.67±84.88)μm in 4 mg aflibercept group, respectively, with statistically significant differences (all at P<0.05).The CMT value in 4 mg aflibercept group was significantly lower than that in the 2 mg aflibercept group in one month after injection ( P<0.05).No severe ocular and systemic adverse events were found during the follow-up, such as retinal detachment, endophthalmitis, cataract, and persistent high intraocular pressure. Conclusions:Both 2 mg and 4 mg aflibercept can effectively treat ranibizumab-resistant PCV with serous PED, and improve the anatomical structure of retina and BCVA.4 mg aflibercept can accelerate the recovery of PED and CMT.
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Objective:To observe the efficacy of conbercept in the treatment of different types of diabetic macular edema (DME).Methods:A retrospective clinical study. From March 2019 to March 2021, 136 eyes of 136 patients with DME diagnosed in Department of Ophthalmology of Xi'an No.3 Hospital were included in the study. Among them, there were 65 males and 71 females; the average age was 56.65±8.65 years. All patients underwent best corrected visual acuity (BCVA), optical coherence tomography (OCT) examination, and glycosylated hemoglobin level (HbA1c) examination. Early Treatment Diabetic Retinopathy Study visual acuity chart was used for BCVA examination, which was converted into the logarithmic minimum angle of resolution (logMAR) visual acuity during statistics. An OCT instrument was used to measure the central retinal thickness (CRT) of the macula. According to the characteristics of OCT, DME was divided into diffuse retinal thickening (DRT) type, cystoid macular edema (CME) type, serous retinal detachment (SRD) type, mixed type, and grouped accordingly, respectively, about 30, 38, 33, 35 eyes. There was no significant difference in age ( F=1.189), sex ratio ( χ2=1.331), and HbA1c level ( F=3.164) of the four groups of patients ( P>0.05). All eyes were treated with intravitreal injection of 10 mg/ml conbercept 0.05 ml (including conbercept 0.5 mg) once a month for 3 consecutive times, and then treated as needed after evaluation. BCVA and OCT examinations were performed 1, 3, and 6 months after treatment with the same equipment and methods as before treatment. The changes of BCVA and CRT before and after treatment were compared and observed. For measurement data subject to normal distribution, one-way analysis of variance was performed for comparison between groups; χ2 test was performed for comparison of count data. Results:Before treatment, the logMAR BCVA of the eyes in the DRT group, CME group, SRD group, and mixed group were 0.68±0.11, 0.69±0.15, 0.71±0.12, 0.73±0.14, and CRT was 631.4±50.7, 640.6±55.7, 652.3±63.4, 660.4±61.8 μm. Compared with before treatment, 1, 3, 6 months after treatment, DRT group (BCVA: t=8.139, 11.552, 11.672; CRT: t=16.163, 21.653, 25.855), CME group (BCVA: t=8.923, 9.995, 13.842; CRT: t=16.163, 21.653, 25.855), SRD type group (BCVA: t=5.171, 7.315, 6.051; CRT: t=9.099, 13.731, 21.306), mixed type group (BCVA: t=5.072, 6.939, 7.142; CRT: t=6.920, 15.352, 17.538) The BCVA of the affected eyes was significantly increased, and the CRT was significantly decreased, and the difference was statistically significant ( P<0.05). At 6 months after treatment, the differences in logMAR BCVA and CRT of the 4 groups of eyes were statistically significant ( χ2=58.478, 64.228; P<0.05). The average number of injections in the eyes of the DRT group, CME group, SRD group, and mixed group were 3.37±1.35, 3.68±1.38, 4.18±1.40, 4.13±1.50 times, respectively. Compared with the average number of injections in the eye, the difference was statistically significant ( χ2=9.139, P=0.028). Conclusions:Conbercept can effectively reduce CRT and increase BCVA in eyes with different types of DME. Compared with SRD type and mixed type, DRT and CME type eye are more effective in improving vision, CRT reduction degree is greater, and the number of injections is less.
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Objective:To observe the relationship between the response to anti-vascular endothelial growth factor (VEGF) drug treatment and single nucleotide polymorphism (SNP) genotype in patients with wet age-related macular degeneration (wAMD).Methods:A retrospective clinical study. From August 2019 to September 2020, 103 eyes of 103 wAMD patients diagnosed in Tianjin Medical University Eye Hospital were included in the study. Among them, there were 59 males (57.28%, 59/103) and 44 females (42.72%, 44/103); the average age was 68.74±7.74 years. The standard logarithmic visual acuity chart was used to detect the Best Corrected Visual Acuity of the affected eye and converted to the logarithmic minimum angle of resolution (logMAR) visual acuity during statistics. Optical coherence tomography was used to detect the central retinal thickness (CRT) of the affected eye. At the same time, the patient's high-density lipoprotein cholesterol (HDL-C) was tested. All eyes were treated with intravitreal injection of anti-VEGF drugs once a month for 3 months. Before the initial treatment, peripheral venous blood from the patient were collected. Interleukin-8 ( IL-8), complement C3 gene ( C3), complement factor H ( CFH), liver lipase ( LIPC), cholesterol ester transfer protein ( CETP), ATP binding cassette subfamily a member 1 ( ABCA1), lipoprotein lipase ( LPL), fatty acid desaturation gene cluster ( FADS1) SNP. According to gene frequency, genotypes are divided into wild type and mutant type were detected. Qualitative data such as the frequency difference of the genotype distribution in the clinical phenotype and the Hardy-Weinberg equilibrium of the genotype distribution were compared with the Chi-square test or Fisher's exact test. Results:There were fewer CRT responders in IL-8 rs4073 mutant (TA+AA) patients than wild-type (TT) [odds ratio ( OR)=0.310, 95% confidence interval ( CI) 0.106-0.910, P<0.05). Among them, after the drug stratification test, the proportion of patients with IL-8 rs4073 locus TT genotype in the conbercept treatment group was less CRT non-responders ( OR=0.179, 95% CI=0.034-0.960, P=0.033). Patients with LIPC rs2043085 mutant (CT+TT) with BCVA increased ≥0.2 logMAR are more likely than wild-type (CC) ( OR=3.031, 95% CI 1.036-8.867, P<0.05); HDL-C level was significantly lower Compared with wild type (CC), the difference was statistically significant ( t=2.448, P=0.016). There was no significant difference in logMAR BCVA and CRT between IL-8 rs4073, LIPC rs2043085 mutant and wild-type patients before treatment ( IL-8 rs4073: Z=-0.198, -1.651; P=0.843, 0.099; LIPC rs2043085: Z=-0.532, -0.152; P=0.595, 0.879). C3 rs 225066, CFH rs800292, CETP rs708272, ABCA1 rs1883025, FADS1 rs174547, LPL rs12678919 have no correlation with anti-VEGF drug treatment response. Conclusions:Patients with wAMD are treated with anti-VEGF drugs. Those with IL-8 rs4073 locus A genotype may be less responsive to CRT. LIPC rs2043085 locus T genotypes may be relatively more responsive to BCVA.
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Objective:To observe the short-term effects of intravitreal injection of aflibercept (IVA) for initial treatment and dressing change on exudative age-related macular degeneration (eAMD).Methods:A retrospective clinical study. From June 2018 to February 2021, forty-nine eAMD eyes of 38 patients who underwent IVA treatment in Department of Ophthalmology of Central Theater Command Hospital of People’s Liberation Army were included in the study. Among them, there were 24 males with 29 eyes and 14 females with 20 eyes; the average age was 66.82±8.71 years. All affected eyes were treated with IVA. The initial loading dose was 2.0 mg, which was injected once a month for 3 consecutive months, followed by monthly review and treatment as needed. Of the 49 eyes, 26 eyes were initially treated (initial treatment group), they were diagnosed within 3 months of the first onset and followed by IVA treatment, and no intraocular drugs and surgery were performed from the onset to the first diagnosis. Twenty-three eyes were treated with drug exchange therapy (dressing change group), they received intravitreal injection of ranibizumab and/or conbercept more than 4 times 6 months before the replacement therapy, during which there was persistent interlaminar cystoid edema and/or subretinal fluid (SRF) in the macular area and no improvement in pigment epithelial detachment (PED). Before IVA treatment, there were no statistically significant differences in the best corrected visual acuity (BCVA), foveal thickness (CMT), PED height (PEDH), and PED volume (PEDV) of the two groups of eyes before IVA treatment ( P>0.05). The same equipment and methods as before treatment were used for related examinations, and the changes of BCVA, CMT, PEDH, PEDV and complications of the two groups of eyes were recorded in 1, 3, and 6 months after treatment. The comparison of BCVA, CMT, PEDH, and PEDV between the two groups were used repeated measures analysis of variance. Results:Six months after treatment, the number of IVA injections in the eyes of the initial treatment group and dressing change group were 4.15±0.73 and 4.39±0.72 times, respectively, and the difference was not statistically significant ( t=-1.141, P=0.260). The BCVA, CMT, PEDH, and PEDV of the the initial treatment group ( F=5.345, 22.995, 6.764, 5.425) and the dressing change group ( F=12.519, 15.576, 8.843, 9.406) were significantly improved compared before treatment with 1, 3, and 6 months. All were statistically significant ( P<0.05). There was no significant difference in BCVA, CMT, PEDH, and PEDV between the initial treatment group and the dressing group at each time point after treatment ( F=1.741, 0.069, 0.876, 3.455; P>0.05). During the follow-up period, none of the affected eyes had complications such as persistent intraocular pressure increase, endophthalmitis, and retinal pigment epithelial tear. Conclusions:IVA can improve eyesight of patients with eAMD and reduce CMT, PEDH, and PEDV. The initial treatment and dressing change have the same effect.
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With the development of programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) inhibitors,the second generation of combined immunosuppressive agents emerge as the times require.As a bifunctional anti-PD-L1/transforming growth factor-β (TGF-β) fusion protein,M7824 can antagonize PD-L1 pathway and trap TGF-β at the same time,which can effectively enhance the immune response and reduce the occurrence of immune escape and drug resistance.The drug has achieved remarkable results in many preclinical studies,however,the indications,safety and efficacy still need to be confirmed by large-scale clinical research data.
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Objective To evaluate the effect of recombinant human tumor necrosis factor receptor type Ⅱ:IgG Fc fusion protein (rhTNFR:Fc,trade name Etanercept) combined with methotrexate on levels of interleukin-17A (IL-17A) and tumor necrosis factor-α (TNF-α) in the serum and mononuclear cells of patients with moderate to severe plaque psoriasis.Methods A total of 30 patients with moderate to severe plaque psoriasis were enrolled from Department of Dermatology of Tenth People's Hospital of Tongji University between August 2014 and February 2016,and then were randomly and equally divided into Etanercept group and Etanercept + methotrexate group.The treatment lasted 24 weeks.Fifteen healthy blood donors served as healthy control group.Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR were performed to measure the serum levels and mRNA expression of IL-17A and TNF-α,respectively,in the patients of the above two groups before and after the treatment.Results Before the treatment,the serum levels of IL-17A and TNF-ct,as well as the mRNA expression of IL-17A and TNF-α in peripheral blood mononuclear cells (PBMCs),were all significantly higher in all the patients than in the healthy controls (all P ＜ 0.05).After the treatment,compared with the Etanercept group,the Etanercept + methotrexate group showed significantly lower serum levels of IL-17A (142.67 ± 14.82 vs.163.54 ± 23.18,P ＜ 0.05) and TNF-α (70.07 ± 25.02 vs.91.98 ± 14.62,P ＜ 0.05),as well as lower mRNA expression of IL-17A (1.12 ± 0.33 vs.1.56 ± 0.77,P ＜ 0.05) and TNF-α in PBMCs (2.50 ± 1.04 vs.3.61 ± 2.14,P ＜ 0.05).Conclusion Etanercept combined with methotrexate is superior to Etanercept alone in the treatment of psoriasis,and can reduce treatment duration and improve therapeutic effect,likely by down-regulating the expression of IL-17A and TNF-α.
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Objective To study the inhibitory effect of antimicrobial peptide LL-37 on Candida albicans through its ability to promote the secretion of immune factors by vaginal epithelial cells. Methods (1) LL-37 prokaryotic expression vector pET-Duet/LL-37 was constructed and its expression was induced in Escherichia coli M15. The expressed LL-37 fusion protein was purified and identified by western blot. Antifungal activity of the purified protein was initially identified by Kirby-Bauer (K-B) method. (2) Purified LL-37 protein was added to human vaginal epithelial cells co-cultured with Candida, and inhibitory effect on Candida growth was determined by the glucose consumption method. Interferon γ (IFN-γ), interleukin 10 (IL-10) concentration and IFN-γ/IL-10 ratio were measured by ELISA at different time points. Results (1) LL-37 fusion protein was purified to 96% purity at a concentration of 433.92 μg/ml, and was shown to possess anti-fungal activity confirmed by the K-B method. (2) A Candida-vaginal epithelial cells co-culture system was successfully constructed. LL-37 recombinant protein inhibited the growth of Candida with absorbance values significantly higher in the treatment group compared to the control group at all measured time points (12-hour:3.008±0.003 versus 2.967±0.003, 24-hour:2.941±0.003 versus 2.601±0.003, 48-hour:2.893 ± 0.004 versus 2.409 ± 0.003; all P<0.01). Furthermore, the rate of decrease was also much slower compared to the control group. In both control and experimental groups, IFN-γ and IL-10 secretion levels were observed to rise at first peaking at 24 hours and subsequently decrease. For each time period, IFN-γconcentration in the experimental group was significantly higher at 24 hours compared to the control group [(104.00 ± 1.07) versus (85.17 ± 0.28) pg/ml, P<0.01]. In contrast, IL-10 concentrations were significantly lower than the control group at all time points (P<0.01). IFN-γ/IL-10 ratio was also observed to be significantly higher than the control group at all measured time points (P<0.01). Conclusions (1) Recombinant protein LL-37 could significantly inhibit the growth of Candida. (2) By influencing the secretion of immune factors such as IFN-γ, IL-10, etc, recombinant protein LL-37 is able to adjust vaginal epithelial cells local immunity, and enhance resistance to Candida infection.
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Objective To evaluate the effect of heme oxygenase-1 (HO-1) protein transduction on acute lung injury in septic rats.Methods Eighteen healthy male Sprague-Dawley rats,aged 7-9 weeks,weighing 210-260 g,were randomly allocated into 3 groups (n =6 each) using a random number table:sham operation group (group Sham),sepsis group (group Sep),and fusion protein PEP-1-HO-1 group (group HO).Sepsis was produced by cecal ligation and puncture (CLP).In group HO,PEP-1-HO-1 fusion protein 0.6 mg was injected via the left iliac vein at 1 h before CLP and 5 h after CLP.The equal volume of normal saline was given instead of PEP-1-HO-1 in Sham and Sep groups.At 12 h after CLP,blood samples were collected from the right common carotid artery for measurement of serum tumor necrosis factoralpha (TNF-α) and interleukin-6 (IL-6) concentrations.The rats were then sacrificed,and lungs were removed for microscopic examination and for determination of wet/dry lung weight ratio (W/D ratio) and malondialdehyde (MDA) content (by thiobarbituric acid colorimetric method).Results Compared with group Sham,the W/D ratio and MDA content were significantly increased,the serum TNF-α and IL-6 concentrations were significantly increased (P＜0.05),and the pathological changes were significantly aggravated in Sep and HO groups.Compared with group Sep,the W/D ratio and MDA content were significantly decreased,the serum TNF-α and IL-6 concentrations were significantly decreased (P＜0.05),and the pathological changes were significantly attenuated in group HO.Conclusion HO-1 protein transduction can attenuate acute lung injury in septic rats,and the mechanism is probably related to inhibition of lipid peroxidation in lung tissues and systemic inflammatory responses.
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TEM8 was a number of tumor endothelial markers (TEMs),though it was overexpressed in the vasculature of human tumors and in several tumor cells,was not or less expressed in normal vessels or tissue,which suggested that TEM8 was very important in tumor angiogenesis and growth.
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Objective:To design the small ubiquitin modification-fibroblast growth factor receptor 4 (SUMO-FGFR4) fusion gene and construct the expression vector pET22b-SUMO-FGFR4, to optimize the expression conditions. Methods:The SUMO-FGFR4 fusion gene was obtained by Overlap PCR and was connected to pET22b;the recombinant expression vector pET22b-SUMO-FGFR4 was obtained. The influence of lactose concentration, induction time,induction temperature,induction point and adding mode of lactose in the expression levels was observed,and the best induction condition was determined; then the solubility of recombinant protein was analyzed.Results:The SUMO-FGFR4 fusion protein was highly expressed,the molecular weight of the fusion protein was about 40 000 and it could bind with FGFR4 specific antibody.When the lactose concentration was 1.0 g·L-1 ,the induction time was 3 h,the induction temperature was 37℃,the value of A (600)was 0.8,the expression level was highest;but adding mode of lactose had no remarkable effect on the protein expression.The expression level of recombinant protein induced by lactose was higher than IPTG.SUMO-FGFR4 protein existed in a form of inclusion body.Conclusion:The SUMO-FGFR4 fusion protein is expressed successfully in this study while lactose is used as inducer and the best expression conditions are confirmed.
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Objective To investigate the inhibitory effects of IBI302 on experimental choroidal neovascularization (CNV).Methods Affinity of IBI302 to vascular endothelial growth factor (VEGF) family cytokines (including VEGF-A165,VEGF-A121 and placental growth factor PlGF) and complements (C3b,C4b) was determined by enzyme-linked immunosorbent assay (ELISA).The antagonist effect of IBI302 on VEGF was measured by proliferation,migration and tube formation tests of human umbilical vein endothelial cells (HUVEC).The anti-complement activity of IBI302 was measured by hemolysis test mediated by complement classical pathway and alternative pathway.Rhesus laser-induced CNV model was divided into 5 groups including model control group,bevacizumab group,IBI302 0.25 mg group,IBI302 0.50 mg group and IBI302 1.25 mg group.Fluorescein angiography and optical coherence tomography were performed on these monkeys at 14 and 28 days after drug delivery to observe the fluorescein leakage area and retinal thickness.The aqueous VEGF concentration was measured at 29 days after drug delivery.Results IBI302 showed good affinity to VEGF-A165,VEGF-A121 and PlGF,as well as C3b and C4b.IBI302 significantly inhibited the proliferation,migration and tube formation of HUVEC induced by VEGF-A165.IBI302 inhibited the hemolysis induced by complements obviously.At 14 and 28 days after drug delivery,the area of fluorescein leakage and retinal thickness in IBI302 0.25 mg group,IBI302 0.50 mg group,IBI302 1.25 mg group were reduced.The differences of the area of fluorescein leakage and retinal thickness in three IBI302 groups were not significant (P＞0.05).At 29 days after drug delivery,the VEGF concentration in the aqueous of rhesus monkey in bevacizumab group [(38.644 ± 6.521) pg/ml] was decreased than that in model control group [(94.203± 17.360) pg/ml],the difference was significant (P＜ 0.05).The VEGF concentration in the aqueous of rhesus monkey in three IBI302 groups were less than 31.300 pg/ml.Conclusion IBI302 inhibited experimental CNV through blocking the activity of VEGF and complement.
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Objective To establish a cell model expressing the Langerhans cell-specific C type lectin receptor Langerin in vitro.Methods The cDNA of Langerin was obtained by PCR and cloned into a eukauotic green fluorescent protein (EGFP) expression vector pEGFP-C 1 with EGFP located in the N terminal region of the Langerin gene.Then,the recombinant plasmid was transfected into a human embryonic kidney carcinoma cell line HEK293T.Subsequently,laser confocal microscopy was performed to observe the expression of EGFP-Langerin fusion protein,and flow cytometry to measure the expression of Langerin.Laser confocal microscopy was also conducted to visualize the recognition and endocytosis of dust mite antigen (nDer p 2) by Langerin.Results PCR and Western blot confirmed the successful transfection of HEK293T cells with the recombinant plasmid as well as the expression of Langerin in the transfected cells.As flow cytometry revealed,the expression level of Langerin in transfected HEK293T cells was increased by 43％ compared with untransfected cells.Laser confocal microscopy showed that green fluorescein-labeled Langerin was successfully expressed,and could bind to and endocytose the red fluorescein-labeled antigen nDer p 2.Conclusions The fusion protein EGFP-Langerin expressed in HEK293T cells showed the distribution characteristic of cell surface receptors,and could bind to and endocytose allergens.
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Objective: To investigate the inhibition effect of trans-activator of transcription (TAT)-ovarian cancer specific binding peptide (OSBP)-mitogen-activated protein kinase kinase 6 mutant (E) [MKK6(E)] on human ovarian xenograft tumor in nude mice and its possible mechanism. Methods: The subcutaneous xenograft tumor model of human ovarian cancer HO8910 cells in nude mice was established. Then the tumor-bearing nude mice were randomly divided into three groups: treatment group [TAT-OSBP-MKK6(E) was administered intraperitoneally], negative control group (TAT-OSBP was administered intraperitoneally) and the blank control group (the saline was administered intraperitoneally). The tumor growth, tumor volume and body weight of nude mice of three groups were observed. The apoptosis and the expressions of proliferating cell nuclear antigen (PCNA) and p38 of xenograft tumors were examined by TUNEL method, immunohistochemistry and Western blotting, respectively. Results: As compared with the negative control and the blank control groups, the growth rate of xenograft tumor in the treatment group was decreased significantly (P 5). The apoptosis index (AI) value in the treatment group was higher than those in the negative control and the blank control groups (P 0.05). Conclusion: TAT-OSBP-MKK6(E) can inhibit the growth and the expression of PCNA in subcutaneous xenograft tumor of ovarian cancer in nude mice. This effect may be related to promoting apoptosis.
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Objective: To express the fusion protein-thioredoxin-apoptosisinducing factor-defected mitochondria localization sign (Trx-DMLS-AIF) in prokaryotic cells and to detect its effect on cell-free system of leukemia K562 cells. Methods: The recombinant plasmid expressing Trx-DMLS-AIF fusion protein was established and transformed into E.coli BL21. The expression of Trx-DMLS-AIF fusion protein was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and purified by Ni-NTA Spin Columns. The cell-free system of leukemia K562 cells was established. The effect of Trx-DMLS-AIF fusion protein on cell-free system of K562 cells was detected by acridine orange (AO) staining. The effect of plasmosin on the ability of Trx-DMLS-AIF fusion protein entering into nuclei of K562 cells was detected by immunofluorescent staining. Results: The expression of Trx-DMLS-AIF fusion protein was successfully induced by IPTG, and the purified Trx-DMLS-AIF fusion protein was obtained. The result of AO staining showed that Trx-DMLS-AIF fusion protein could induce the nucleus apoptosis in cell-free system of K562 cells. The result of immunofluorescent staining showed that the plasmosin of K562 cells could prevent Trx-DMLS-AIF fusion protein from entering into the nuclei of K562 cells. Conclusion: The expression of Trx-DMLS-AIF fusion protein is successfully induced. TrxDMLS-AIF can induce the nucleus apoptosis of K562 cells, and the plasmosin of K562 cells can prevent Trx-DMLS-AIF fusion protein from entering into the nuclei of K562 cells.
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Objective To construct the prokaryotic expression vector for HD-5 and purify the recombinant HD-5 protein then analyze its antifungal activity. Methods The HD-5 gene was cloned by PCR, then was inserted into prokary-otic expression plasmid pQE-30Xa to construct pQE-30Xa/HD-5. After sequencing, pQE-30Xa/HD-5 was transformed in-to E.coli M15 cells. Its expression was induced by IPTG and confirmed by SDS-PAGE. The recombinant protein was purified through Ni-NTA affinity purification system. The antifungal activity was tested by disk diffusion method. Results HD-5 gene and pQE-30Xa/HD-5 vector were obtained successfully. E.coli M15 strains was used to express HD-5 fusion protein. After purification, the fusion protein was confirmed by Western blot. The disk diffusion test confirmed that the fusion pro-tein can inhibit Candida albicans. Conclusion Expression vector pQE-30Xa/HD-5 was successfully constructed. The HD-5 fusion protein was expressed in E.coli successfully, which showed a certain degree of antifungal activity.
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Objective To construct eukaryotic Flag (DYKDDDDK) expressing recombinant plasmids, pCMV-N-Flag-SND1-No1/2, which contain the coding sequence of human SND1-No1(from 1st AUG)or SND1-No2 (from 2nd AUG), and perform the cellular localization analysis of Flag-tagged SND1-No1/2 under stress condition to study the function of the two AUG in the SND1 containing stress granules formation. Methods The gene fragments of SND1-No1/2 were amplified by PCR from the whole SND1 transcript and inserted into pCMV-N-Flag expressing vector through BamHI/EcoRI double en-zyme digestion and T4 DNA Ligase connection. The recombinant pCMV-N-Flag-SND1-No1/2 plasmids were transfected in-to HeLa cells and the expression of Flag-SND1-No1/2 fusion proteins was examined by Western blotting assay. Immunofluo-rescence assay was performed to detect the co-localization of Flag-SND1-No1/2 with endogenous SND1 granule. Results The pCMV-N-Flag-SND1-No1/2 were sequenced and digested correctly by restriction single/double enzyme. The Flag-tagged SND1-No1/2 fusion proteins were also detected in transfected HeLa cell by Western blotting assay. Both of them showed the co-localization with endogenous SND1 granule. Conclusion The recombinant eukaryotic plasmids of pCMV-N-Flag-SND1-No1/2 were constructed successfully and expressed effectively. The depletion of 1st AUG failed to af-fect the formation of SND1 containing stress granules.
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Objective To prepare monoclonal antibody specifically against carcinoembryonic antigen glypican-3 (GPC3) and its preliminary application. Methods GPC3 was cloned with PCR to pET16b vector and expressed in E. co-liBL21. Spleen cells were obtained from Balb/c mice embedding immunized with purified antigen intraperitoneally, and fused with Sp2/0 cells. Hybridoma cells were screened by indirect ELISA, and identified by Western blot assay using puri-fied protein after the cell fusion. The indirect immunofluorescence method was used to detect the GPC3 expression in HepG2 cell line. Results The prokaryotic expression vector of GPC3 was successfully constructed, and GPC3 was stably expressed in E. coliBL21. A mouse hybridoma cell line secreting monoclonal antibody to GPC3 was obtained. Western blot analysis showed that monoclonal antibody specifically recognized recombinant protein. Monoclonal antibody could be used to detect GPC3 protein expression in HepG2 cell line by indirect immunofluorescence. Conclusion The monoclonal antibody against GPC3 was successfully obtained.
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Objective To investigate the effects of heme oxygenase-1 (HO-1) protein transduction mediated by cell penetrating peptide PEP-1 on intestinal injury in a rat model of sepsis induced by cecal ligation and puncture (CLP).Methods Twenty-four healthy male Sprague-Dawley rats,aged 7-9 weeks,weighing 210-260 g,were randomly divided into 4 groups (n =6 each) using a random number table:sham operation group (group S),group CLP,low-dose fusion protein PEP-1-HO-1 + CLP group (group P1) and high-dose fusion protein PEP-1-HO-1 + CLP group (group P2).Fusion protein PEP-1-HO-1 0.3 mg was administrated via the left iliac vein at 1 h before CLP and 5 h after CLP in group P1.Fusion protein PEP-1-HO-1 0.6 mg was administrated via the left iliac vein at 1 h before CLP and 5 h after CLP in group P2.The equal volume of normal saline was given instead of PEP-1-HO-1 in the other groups.The animals underwent laparotomy,but the caecum was not ligated or punctured in group S.Blood samples were collected at 12 h after CLP from the right common carotid artery for measurement of serum TNF-α and IL-6 levels.The rats were then sacrificed and intestines were removed for microscopic examination and for determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in intestinal tissues.Results Compared with group S,the serum TNF-α and IL-6 levels,and MDA content in intestinal tissues were significantly increased,while SOD activity in intestinal tissues was decreased in CLP,P1 and P2 groups.Compared with group CLP,the serum TNF-α and IL-6 levels,and MDA content in intestinal were significantly decreased,while SOD activity in intestinal tissues was increased in P1 and P2 groups.Compared with group P1,the serum TNF-α and IL-6 levels,and MDA content in intestinal tissues were significantly decreased,while SOD activity in intestinal tissues was increased in group P2.The pathological changes of intestines were significantly mitigated in P1 and P2 groups as compared with group CLP.Conclusion HO-1 protein transduction attenuates intestinal injury induced by sepsis in rats,and the mechanism is related to inhibition of systemic inflammatory responses and lipid peroxidation in intestinal tissues.
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Objective To establish a cellular model for the expression of the C-type lectin dendritic cellspecific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN),and to provide a basis for the functional analysis of DC-SIGN.Methods The cDNA of DC-SIGN was obtained via PCR,and cloned into the eukaryotic expression vector porcine cytomegalovirus-enhanced green fluorescent protein (PCMV-EGFP) with EGFP at the N terminal of DC-SIGN.Then,the recombinant PCMV-EGFP-DC-SIGN plasmid was transfected into HEK293T cells followed by the detection of DC-SIGN expression using PCR,Western blot and flow cytometry.Confocal microscopy was performed to localize the expression of DC-SIGN-EGFP and visualize the recognization and internalization of the Derp2 allergen by DC-SIGN.Results The recombinant fluorescent fusion protein-expressing plasmid was successfully constructed.Both PCR and Western blot confirmed the expression of DC-SIGN.Flow cytometry showed that the expression of DC-SIGN was increased by approximately 50％ in HEK293T cells transfected with the recombinant expression plasmid compared with those untransfected.As confocal microscopy showed,the green fluorescence-labelled DC-SIGN was located on the cell membrane,which could bind to the red fluorescence-labelled antigen Derp2 and internalize it into the cells.Conclusions The recombinant DC-SIGN-EGFP fusion protein is characteristically located on the surface of 293T cells,which can be recognized by the DC-SIGN-specific antibody and is capable of internalizing the allergen Derp2,and may serve as an ideal cell model for further studies on molecular function of DC-SIGN.
ABSTRACT
Objective To investigate the effect of transduction of heme oxygenase-1 (HO-1) protein on oxygen-glucose deprivation and restoration (OGD/R)-induced injury to hippocampal neurons in rats.Methods Plasmid 11R-HO-1 was constructed using plasmid pET-21a(+)-p53-11R (plasmid 11R) and 11R-HO-1 fusion protein was identified and collected.Hippocampal neurons obtained from newborn Wistar rats (＜ 48 h) were cultured for 7 days in vitro and then the neurons were randomly divided into 5 groups (n =171 each) using a random number table:OGD/R group,normal saline group (group NS),plasmid 11R group (11R group),300 nmol/L 11R-HO-1 group (H1 group),and 1 500 nmol/L 11R-HO-1 group (H2 group).In NS,11R,H1 and H2 groups,the neurons were incubated for 2 h with 300 nmol/L normal saline,300 nmol/L plasmid 11R,300 nmol/L 11R-HO-1 fusion protein,and 1 500 nmol/L 11R-HO-1 fusion protein,respectively,and then OGD/R was performed.The neurons were incubated in deoxygenated glucose-free DMEM medium and sealed under 5 ％ CO2-95 ％ N2 in an anaerobic chamber equilibrated to 37 ℃ for 45 min.OGD was terminated by replacement of the medium with high glucose DMEM medium and by returning the cultures to a standard incubator maintained at 37 ℃ in 5 ％ CO2-95 ％ air and the neurons were then incubated for 24 h.Immediately after OGD/R was established,the cell survival rate (by MTT assay),apoptosis rate (using TUNEL),and expression of HO-1 and caspase-3 protein (by using Western blot) were measured.Results Compared with group OGD/R,the cell survival rate was significantly increased,the apoptosis rate was decreased,the caspase-3 expression was down-regulated,HO-1 protein expression was up-regulated in H1 and H2 groups (P ＜ 0.05),and no significant change was found in the parameters mentioned above in NS and 11R groups (P ＞ 0.05).Compared with group H1,the cell survival rate was significantly increased,the apoptosis rate was decreased,the caspase-3 expression was down-regulated,and HO-1 protein expression was up-regulated in group H2 (P ＜ 0.05).Conclusion Transduction of HO-1 protein can reduce OGD/R-induced injury to hippocampal neurons of rats.