1.The cloning and sequencing of H-2Kk gene cDNA of 615 mice.
Longjiang LI ; Hao GONG ; Yuming WEN ; Junjie CHEN ; Ruohan WANG
West China Journal of Stomatology 2002;20(5):313-315
OBJECTIVEThe purposes of this study were to clone and sequence the major histocompatibility complex type I (MHC I) molecular antigen recognizing gene (H-2Kk) of 615 mice, and to provide the functional gene for transgenic therapy.
METHODSThe 1.4 kb full-length fragment of H-2Kk gene complementary DNA (cDNA) was amplified from the total RNA of 615 mouse liver by using reverse transcription polymerase chain reaction (RT-PCR). The cDNA was inserted into PGEM3Zf(+) vector directionally, and the competent E. coli JM109 was transformed with the ligated product. The recombinant PGEM3Zf(+)-H-2Kk cDNA plasmid was obtained using restricted enzyme analysis of the transfectants. The complete sequence of 615 mouse H-2Kk cDNA was determined by using Sanger's method.
RESULTSThe sequences of 615 mouse H-2Kk cDNA were 99% similar with those of H-2Kk cDNA which were reported by other researchers, and the sequences encoding antigen recognizing regions (ARS) were identical with each other.
CONCLUSIONThe authors cloned the MHC I molecular antigen recognizing gene (H-2Kk) of 615 mice successfully and got the functional gene of MHC I.
Animals ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genes ; genetics ; Genes, MHC Class I ; genetics ; Genetic Therapy ; H-2 Antigens ; genetics ; Mice ; Mice, Inbred C57BL ; Point Mutation ; Sequence Analysis, DNA ; Transgenes
2.Eight-arm polyethylene glycol nanoconjugates for brain targeted delivery of photosensitizer
Junbo XIN ; Fang LI ; Ruohan LI ; Ran ZHANG ; Furong PAN ; Xin MING
Journal of China Pharmaceutical University 2019;50(3):299-307
The aim of this study was to prepare the nanoconjugates for targeted photodynamic therapy of brain cancer by using eight-arm polyethylene glycol(8PEG)as the carrier and cRGD as the targeting ligand, and to investigate the antitumor effect and its mechanism. UV-Vis spectra and confocal microscopy were used for characterization and cellular uptake behavior of nanoconjugates respectively. Alamar Blue assay and Calcein AM/PI staining were applied to investigate the cytotoxocity of nanoconjugates against tumor cells, and tumor spheroid growth curve was used to assess the tumor growth suppression effect. In addition, the generation of reactive oxygen species(ROS), apoptosis and spheroid permeability test was used to reveal the antitumor mechanism of nanoconjugates. The results showed that cRGD-8PEG-IR700 was taken up efficiently by integrin overexpressed U87MG cells, while almost no uptake was found in integrin free NIH/3T3 cells. Remarkable photokilling effect against U87MG cells was only shown in cRGD-8PEG-IR700 group due to the light-induced ROS generation and apoptosis, whereas growth suppression effect was also observed in U87MG spheroids treated with cRGD-8PEG-IR700 plus light owing to the superior penetration ability of targeted nanoconjugates. Hence, tumor-targeted PEG nanoconjugates may provide a promising drug delivery system for photodynamic therapy of cancers.
3.Early growth response gene-1 regulates host cell autophagy in HTLV-1 infection
Qingsong HUANG ; Zhiguo NIU ; Weidong ZHAO ; Ziqiang DING ; Menglin WU ; Xiaomei HOU ; Ruohan LYU ; Lushuang MAO ; Ze LI ; Xinxiang HUANG ; Hui WANG
Chinese Journal of Microbiology and Immunology 2017;37(7):492-496
Objective To investigate the influence of early growth response gene-1 (EGR1) on the autophagy of host cells following infection with human T cell leukemia virus type 1 (HTLV-1).MethodsA HTLV-1-positive cell line MT2 was co-cultured with HeLa cells for 24 h to construct the virus early infection model.Immunoblotting assay was used to detect the expression of HTLV-1 core protein p19 and EGR1.Luciferase reporter gene analysis was used to detect the transcriptional activity of 5′-regulatory sequence of EGR1 at different time points after co-culturing.An effective small interfering RNA (siRNA) targeting EGR1 was screened out and transfected into HeLa cells by Lipofectamine 2000.Then the transfected HeLa cells were co-cultured with the HTLV-1-positive cell line MT2 for 24 h.Immunoblotting assay was used to detect HTLV-1 core protein p19, EGR1 and autophagy-related protein LC3.Real-time PCR was performed to detect viral load.Autophagosome was analyzed by immunofluorescence after co-culturing.Results The expression of EGR1 and the transcriptional activity of pEGR1-luc gradually increased after co-culturing HeLa cells with MT2 cells for 8 h (P<0.01).The expression of EGR1 was positively correlated with host cell autophagy following HTLV-1 infection.The effective siRNA for silencing the expression of EGR1 was obtained and named as siE2.The viral load, the expression of HTLV-1 core protein p19 and the proportion of LC3B/LC3A in the co-culture model were markedly down-regulated by RNA interference with siE2, which was concomitant with a persistent decrease of intracellular autophagosome (P<0.01).Conclusion EGR1 is associated with host cell autophagy and viral replication in HTLV-1 infection.
4.Expression and significance of miRNA-31 in peripheral blood mononuclear cells of rheumatoid arthritis patients
Ruohan YU ; Yuequan ZHU ; Lin SUN ; Leilei JIAN ; Jinxia ZHAO ; Xiangyuan LIU ; Changhong LI
Chinese Journal of Rheumatology 2018;22(1):4-8
Objective To investigate the expression of miRNA-31 in peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients,and the relationship between miRNA-31 and disease activity of RA.Methods After obtaining the informed consent,peripheral blood samples of 56 RA patients,12 systemic lupus erythematosus (SLE) patients,6 Sj(o)gren's syndrome (SS) patients and 30 healthy controls were collected from the Department of Rheumatology,Peking University Third Hospital.RNA was extracted from the PBMCs which were separated by Ficoll-Paque PLUS.The expression of miRNA-31 in the PBMCs of RA patients,SLE patients,SS patients and healthy controls was detected by real-time Polymerase Chain Reaction (PCR).Furthermore,according to the RA disease activity score (DAS28),RA patients were divided into high,moderate and low disease activity groups and remission group,and miRNA-31 expression was compared between different groups.Data were analyzed using t test or Mann-Whitney U test.Results The expression of miRNA-31 in PBMCs of RA patients was 7.25 times (P=0.003 8) higher when compared with that of the control group.To be specific,the expression of miRNA-31 was 10.63 times in PBMCs of high activity RA group (P=0.01) and 8.95 times in moderate activity RA group (P=0.000 3) when compared with that of the control group,and there was no significant difference between low activity,remission groups and control groups in terms of miRNA-31 expression.Furthermore,the expression of miRNA-31 in PBMCs of SLE patients was not significantly different from the control and miRNA-31 expression in PBMCs of SS patients was 1.64 times (P=0.02) higher than that of the RA patients,but the average level of miRNA-31 was much less than that of RA patients.The increased miRNA-31 may serve as a diagnostic marker for disease activity of RA.
5.The expression of oxidized low density lipoprotein and scavenger receptor that binds phospha-tidylserine and oxidized lipoprotein in patients with rheumatoid arthritis
Leilei JIAN ; Ruohan YU ; Lin SUN ; Jinxia ZHAO ; Xiangyuan LIU ; Changhong LI
Chinese Journal of Rheumatology 2018;22(2):76-81
Objective To investigate the expression level of oxidized low density lipoprotein (ox-LDL) and its scavenger receptor scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SRPSOX) in patients with rheumatoid arthritis (RA),and to explore the relationship between ox-LDL and disease activity.Methods The serum ox-LDL in RA patients and healthy control group were detected by enzymelinked immunosorbent assay (ELISA),as well as the fluidox-LDL in RA,osteoarthritis (OA) and inflammatory arthritis (IA).The expression of SR-PSOX in mixed cells of RA and IApatients was detected by western blot.The expression of serum ox-LDL between RA groupand the control group was analyzed by t-test and non-parametric test.The correlation of serum ox-LDL expression levels in RA patients with C-reactive protein (CRP),erythrocyte sedimentation rate (ESR) and other inflammatory factors and disease activitywas analyzed by Pearson linear regression.Results The expression of ox-LDL in the serum of RA patients was significantly higher than that of normal control group [(3 076±131) mU/ml,(2 334±84) mU/ml,t=4.242,P<0.01].The expression of ox-LDL in synovial fluid of RA patients was significantly higher than that of the OA group [(4 963±354) mU/ml],(3 956±347) mU/ml,t=2.372,P<0.05).The expression of SR-PSOX in synovial fluid mixed cells of RA patients was higher than that of the IA group [(4.92±0.18) vs (0.24±0.04),t=33.53,P<0.01].The expression of ox-LDL in serum of RA patients was negatively correlated with ESR,CRP and overall disease activity DAS28 (r=-9.42,P=0.009;r=-0.35,P=0.029 7;r=0.42,P=0.008 4).The expression of ox-LDL in the serum of RA patients with moderate disease activity was significantly higher than those patients with high disease activity [(3 302±138) mU/ml vs (2 464±228) mU/ml,t=3.335,P<0.01],however,those with low disease activity and disease remission had higher serum ox-LDL expression but without statistical significant differences.After treated with anti-rheumatic drugs (DMARDs),serum ox-LDL of RA patients had a trend of slight increasing butwithout sign-ificant difference.The ox-LDL/LDL-C or ox-LDL/HDL-C was negatively not correlated with disease activity score in 28 (DAS28),ESR,CRP.Conclusion In RA patients,the expression of ox-LDL in the serum and synovial fluid is high and the SR-PSOX expressionin synovial fluid is also high.The serum ox-LDL levels are negatively correlated with ESR,CRP and DAS28,which are related to disease activity of RA.These findings suggest that the ox-LDL and the receptor SR-PSOX may play a role in RA pathogenesis,but needs further study.
6.Effect of hypernatremia in donors on perioperative liver function of recipients undergoing liver transplantation
Bo WANG ; Xiao LI ; Pengcheng ZHANG ; Ruohan ZHANG ; Kaishan TAO
Organ Transplantation 2019;10(3):313-
Objective To evaluate the effect of hypernatremia in donors on perioperative recovery of liver function in the recipients undergoing liver transplantation. Methods Clinical data of 73 liver transplant recipients were analyzed retrospectively. According to the serum levels of sodium in donors, all recipients were divided into hypernatremia group (donor serum sodium ≥150 mmol/L,
7.Oxidized low density lipoprotein promotes proliferation and mRNA expression of inflammatory factors in fibroblast like synoviocytes of rheumatoid arthritis
Zhenzhen MA ; Leilei JIAN ; Xinyu WANG ; Ruohan YU ; Jinxia ZHAO ; Xiangyuan LIU ; Changhong LI
Chinese Journal of Rheumatology 2020;24(2):95-100
Objective:To investigate the effects of oxidized low density lipoprotein (Ox-LDL) on cell proliferation and mRNA expression of inflammatory factors in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).Methods:Tissue culture was used to isolate and 4-6 generation cultured RA-FLS cells were used for subsequent experiments. RA-FLS were stimulated for 24 hours with different con-centr-ations of human Ox-LDL, then the MTS cell proliferation and toxicity test kit were used to detect the prolifer-ation of RA-FLS. Real time-polymerase chain reaction (RT-PCR) was used to test the expression of inflamm- atory factors like interleukin (IL)-6, transforming growth factor (TGF)-β, IL-8, tumor necrosis factor (TNF)-α and receptors like CD36 and scavenger receptor binds phosphatidylsed neoxidized lipoprotein (SR-PSOX) inRA-FLS. T test and F test were used in this study. Results:Ox-LDL (10, 25, 50 μg/ml) could obviously promote the proliferation of RA-FLS, and theabsorbance values (490 nm) were (1.04±0.15), (1.05±0.14), and (1.00±0.10), respectively, all higher than the control group (0.81±0.04) and the difference was statistically significant ( F=4.737, P<0.01). In addition, 50 μg/ml and 100 μg/ml Ox-LDL also promoted the expression of IL-6 mRNA ( F=14.709, P<0.01) and inhi-bited the expression of TGF-β mRNA ( F=299.074, P<0.01), but there was no obvious effect on the expression of IL-8 and TNF-α. Ox-LDL stimulation could obviously promote the expression of SR-PSOX receptor on RA-FLS ( F=68.636, P<0.01) and inhibit the expression of CD36( F=18.085, P<0.01). After the transfection of siRNA, SR-PSOX mRNA level was significantly inhibited and the mRNA expression of IL-6 was significantly decreased after Ox-LDL stimulation of RA-FLS ( t=3.875, P<0.01), while TGF-β mRNA expres-sion was not significantly changed( t=-0.193, P>0.05). Conclusion:Ox-LDL may play a role in promoting the activation of RA-FLS proliferation and the expression of IL-6 mRNA by increasing the SR-PSOX receptor of RA-FLS, suggesting that Ox-LDL is involved in the synovial inflammation of RA.
8. Effects of circadian heart rate variation on short-term and long-term mortality in intensive care unit patients: a retrospective cohort study based on MIMIC-Ⅱ database
Yanni LUO ; Jingjing ZHANG ; Ruohan LI ; Ya GAO ; Yanli HOU ; Jiamei LI ; Xiaochuang WANG ; Gang WANG
Chinese Critical Care Medicine 2019;31(9):1128-1132
Objective:
To investigate the effect of circadian heart rate variation on short-term and long-term mortality in intensive care unit (ICU) patients.
Methods:
A retrospective cohort study was conducted. A total of 32 536 ICU patients were recorded from 2001 to 2008 published by Multiparameter Intelligent Monitoring in Intensive Care Ⅱ (MIMIC-Ⅱ v2.6) in April 2011. The circadian heart rate variation was defined as the ratio of mean nighttime (23:00 to 07:00) heart rate to mean daytime (07:00 to 23:00) heart rate. The 28-day mortality and 1-year mortality were defined as outcome events. The information such as age, gender, ethnicity, first sequential organ failure assessment (SOFA) score, first simplified acute physiology score Ⅰ (SAPSⅠ), usage of sedatives and catecholamines within 24 hours admission of ICU, clinical complications [hypertension, chronic obstructive pulmonary disease (COPD), diabetes with or without complications, congestive heart failure, liver disease, renal failure, etc.], and the complete heart rate records within 24 hours after ICU admission were collected. Cox proportional risk regression models were used to investigate the association between circadian heart rate variation and 28-day mortality and 1-year mortality in ICU patients. Besides, subgroup analysis was also performed in patients with different first SOFA scores.
Results:
Totally 15 382 ICU patients in MIMIC-Ⅱ database were enrolled, excluding the patients without heart rate records or death records, using pacemaker with arrhythmia, without SOFA or SAPSⅠ score records. Finally, 9 439 patients were enrolled in the study cohort. ① Cox regression analysis of the whole patient showed that the higher circadian heart rate variation was correlated with the increased 28-day mortality [hazard ratio (
9.Pathogenic microorganism monitoring in the food processing of school canteens
NIU Huimin,JIAO Qiang,LI Ruohan,LIU Zhiyong
Chinese Journal of School Health 2024;45(6):895-898
Objective:
To analyze the key control points of pathogenic microbial contamination during the food processing of school canteens, so as to provide a basis for effectively preventing the occurrence of campus food safety incidents.
Methods:
In March 2023, 10 school canteens from Zhengzhou City, Henan Province were selected by convenient sampling method. A total of 300 samples were collected from the storage equipment of raw materials, production tools, staff hands, infrastructure, dining utensils and other facilities in the school canteens during the food processing process.The rapid detection method of foodborne pathogens based on isothermal multiple selfmatchinginitiated amplification (IMSA) technology was used to analyze the possible risk of pathogenic microorganism contamination. Fisher exact probability method was applied for the statistical analysis.
Results:
Among the 300 samples collected, pathogenic microorganisms including Staphylococcus aureus, Salmonella and Listeria monocytogenes were detected in 5 samples, distributed in the food preparation utensils such as cutting boards, operating tables, knives and staff hands, while no pathogenic microorganisms were detected in dining utensils, raw material storage equipment and canteen infrastructure.There was no statistically significant difference between the IMSA rapid detection method and the national standard method for testing Staphylococcus aureus, Salmonella and Listeria monocytogenes(P>0.05). Compared to international method, the positive coincidence of pathogenic microorganism detected by IMSA rapid inspection system was 83.3%(5/6), the negative coincidence was 100%(n=294), and the overall consistency rate was 99.7%.
Conclusions
The key control points of pathogenic microbial contamination in the food processing process of school canteens mainly are the utensils in the food production process, such as cutting boards, operating tables, knives and staff hands in the food processing process of the school canteen, which could be accurately identified by the rapid detection method of pathogenic microorganism established based on IMSA technology. The quality and efficiency campus food safety supervision should be promoted to prevent the food safety events in school canteen.
10.Abnormal changes of white matter structure in temporal lobe epilepsy patients with sleep disorders based on diffusion kurtosis imaging
Min GUO ; Yanjing LI ; Boxing SHEN ; Hong LUO ; Ruohan YUAN ; Jie HU ; Xing TANG
Journal of Practical Radiology 2024;40(1):1-5
Objective To investigate the microstructural changes of temporal lobe epilepsy(TLE)in patients with sleep disorders based on diffusion kurtosis imaging(DKI).Methods This research prospectively included 38 TLE patients(case group)and 20 healthy controls(HC)(HC group).Participants used sleep questionnaires to evaluate their sleep status.All TLE patients were divided into groups with and without sleep disorders according to the diagnostic criteria and scale scores of sleep disorders.The mean kurtosis(MK),mean diffusivity(MD),and fractional anisotropy(FA)of the relevant region of interest(ROI)were measured by DKI sequence.The differences of sleep quality scores and DKI parameters between groups were further compared via independent samples t-test and one-way analysis of variance.Results The Epworth sleepiness scale(ESS),Athens insomnia scale(AIS),and Pittsburgh sleep qual-ity index(PSQI)scores of TLE patients with sleep disorders were significantly higher than those of HC group(P<0.05).The FA and MK values in TLE patients were significantly lower than those in HC group,while the MD value of TLE patients were substan-tially higher than that of HC group(P<0.05).The values of MK and FA in left TLE patients with sleep disorders were significantly lower than those of without sleep disorders(P<0.05),while there was no significant difference in MD value between the two groups(P>0.05).MK value of right TLE patients with sleep disor-ders was significantly lower than that of without sleep disorders(P<0.05),however,there were no significant differences in MD and FA values between the two groups(P>0.05).Conclusion Quantitative DKI analysis revealed differences in DKI parameters in TLE patients combined with sleep disorders,inferring a specific white matter fiber damage in this group and providing imaging data to support the personalized treatment and prognostic assessment of these patients.