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

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.

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.

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.

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, n=19) and non-hypernatremia group (donor serum sodium < 150 mmol/L, n=54). Serum alanine aminotransferase(ALT), aspartate aminotransferase (AST), model for end-stage liver disease (MELD) score, albumin, total bilirubin (TB), serum creatinine, prothrombin time and hepatocyte growth factor (HGF) in the recipients were detected at 1, 3, 7, 14 and 21 d after liver transplantation. The time of postoperative use of liver-protecting drugs in the recipients, the length of intensive care unit (ICU) stay, the average length of hospital stay and the incidence rate of postoperative complications were statistically compared and analyzed. Results Compared with the non-hypernatremia group, the serum levels of TB, ALT, AST, HGF and MELD scores of the recipients in the hypernatremia group at the postoperative 1, 3 and 7 d were significantly higher (all P < 0.05), whereas the serum albumin level was significantly decreased (P < 0.05). The prothrombin time in the hypernatremia group was significantly longer than that in the non-hypernatremia group at 3 and 7 d after operation (both P < 0.05). In the hypernatremia group, the time of postoperative use of liver-protecting drugs and the length of ICU stay were 9 (7-13) d and 11 (8-13) d, significantly longer than 4 (3-9) d and 7 (3-9) d in the non-hypernatremia group (both P < 0.05). The average length of hospital stay, serum creatinine level and incidence rate of postoperative complications did not significantly differ between two groups (all P>0.05). All recipients were recovered and discharged. Conclusions The hypernatremia in donors exert no significant effect on the perioperative liver function of the recipients, whereas it can prolong the postoperative recovery time of liver function of the recipients.

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.

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.

Objective To establish a nude mouse model of type 2 diabetes mellitus(T2DM)and pancreatic cancer that allows dynamic observation of tumor formation process and facilitates in vivo research.Methods At first,human pancreatic cancer PANC-1 cells were transfected with lentiviral vector GV260 to construct the pancreatic cancer cell line PANC-1-Luc with stable expression of firefly luciferase.Then,36 specific pathogen-free nude mice were randomly divided into control group with 12 mice and model group with 24 mice(nude mice with T2DM and pancreatic cancer).The mice in the control group were fed with breeding diet and were then given ectopic subcutaneous implantation of PANC-1-Luc cells,and those in the model group were first given high-fat diet and intraperitoneal injection of 1%STZ,followed by ectopic subcutaneous implantation of PANC-1-Luc cells.The fluorescence in vivo imaging system and the manual measurement method were used for simultaneous and dynamic monitoring of the growth of pancreatic cancer in nude mice in the two groups,and the tumor growth curve was plotted to investigate the correlation between fluorescence value and tumor volume.Subcutaneous tumors and pancreatic islets were observed under a microscope to verify whether the model was successfully established,and immunohistochemistry was used to measure the expression of Ki-67 in tumor tissue to investigate the influence of hyperglycemia on the growth of pancreatic cancer in nude mice.The independent-samples t test was used for comparison of normally distributed continuous data between groups,and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between groups.Results The optimal virus titer was determined as 5×107 TU/mL for the stable transfection of lentiviral vector in PANC-1 cells,and the optimal concentration selected with puromycin was 20 μg/mL,with an optimal selection time of 9 days.The fluorescence value of PANC-1-Luc cells was linearly and positively correlated with the number of cells,with the linear equation of y=42.56x-42 504(r=0.977,P=0.004).The blood glucose value of T2DM nude mice was 23.05(19.25-26.40)mmol/L,with a blood glucose level of＞11.1 mmol/L in each nude mouse,and there was a significant difference in blood glucose value between the T2DM nude mice and the control nude[6.15(5.20-7.30)mmol/L](Z=-8.45,P＜0.001).Compared with the control group,the model group had reductions in the number and volume of pancreatic islets,with irregular shapes and unclear boundaries,and pathological examination confirmed that the xenograft tumor was pancreatic cancer tissue,which showed that the model was established successfully.In the model group,there was a linear positive correlation between subcutaneous tumor size and fluorescence values,with the linear equation of y=232 348 691x-8 258 608(r=0.911,P=0.031).The model group had a significantly higher positive rate of Ki-67 than the control group(50.333%±7.808%vs 15.917%±4.055%,t=13.55,P＜0.001),suggesting rapid tumor proliferation in the model group.Conclusion The T2DM nude mouse model of pancreatic cancer established in this study can simulate the pathological process of the development and progression of pancreatic cancer in the context of T2DM and dynamically observe the influence of hyperglycemia on the growth of pancreatic cancer cells in vivo,thereby providing a new experimental vector for the in vivo study of the development and progression of pancreatic cancer in the context of T2DM.

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 (HR) = 1.613, 95% confidence interval (95%CI) was 1.338-1.943, P < 0.001] and 1-year mortality (HR = 1.573, 95%CI was 1.296-1.908, P < 0.001). After adjustment for demographic factors (age, gender and ethnicity), severity of illness (SOFA and SAPS Ⅰ scores), clinical complications (hypertension, COPD, diabetes with or without complications, congestive heart failure, liver disease, renal failure, etc.), and influence of medications (sedatives and catecholamines), the night-day heart rate ratio was also correlated with 28-day mortality (HR = 1.256, 95%CI was 1.018-1.549, P = 0.033) and 1-year mortality (HR = 1.249, 95%CI was 1.010-1.545, P = 0.040). ② According to the SOFA score (median value of 5), the patients were divided into two subgroups, in which 5 478 patients with SOFA score ≤ 5 and 3 961 patients with SOFA score > 5. Cox regression subgroup analysis showed that circadian heart rate variation was related with higher 28-day mortality (HR = 1.430, 95%CI was 1.164-1.756, P = 0.001) and 1-year mortality (HR = 1.393, 95%CI was 1.123-1.729, P = 0.003) in patients with SOFA score > 5. After adjustment for covariates, the 28-day mortality (HR = 1.279, 95%CI was 1.032-1.584, P = 0.025) and 1-year mortality (HR = 1.255, 95%CI was 1.010-1.558, P = 0.040) also increased with the increasing of night-day heart rate ratio in patients with SOFA score > 5. However, the relationships did not exist in patients with SOFA score ≤ 5. Conclusion In ICU patients, the 28-day mortality and 1-year mortality increase with the higher circadian heart rate variation, which indicates that the circadian heart rate variation in ICU patients is positively correlated with the short-term and long-term mortality, especially in patients with relatively severe illness.

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 (SAPS I), 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 SAPS I 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 (HR) = 1.613, 95% confidence interval (95%CI) was 1.338-1.943, P < 0.001] and 1-year mortality (HR = 1.573, 95%CI was 1.296-1.908, P < 0.001). After adjustment for demographic factors (age, gender and ethnicity), severity of illness (SOFA and SAPS I scores), clinical complications (hypertension, COPD, diabetes with or without complications, congestive heart failure, liver disease, renal failure, etc.), and influence of medications (sedatives and catecholamines), the night-day heart rate ratio was also correlated with 28-day mortality (HR = 1.256, 95%CI was 1.018-1.549, P = 0.033) and 1-year mortality (HR = 1.249, 95%CI was 1.010-1.545, P = 0.040). (2) According to the SOFA score (median value of 5), the patients were divided into two subgroups, in which 5 478 patients with SOFA score ≤ 5 and 3 961 patients with SOFA score > 5. Cox regression subgroup analysis showed that circadian heart rate variation was related with higher 28-day mortality (HR = 1.430, 95%CI was 1.164-1.756, P = 0.001) and 1-year mortality (HR = 1.393, 95%CI was 1.123-1.729, P = 0.003) in patients with SOFA score > 5. After adjustment for covariates, the 28-day mortality (HR = 1.279, 95%CI was 1.032-1.584, P = 0.025) and 1-year mortality (HR = 1.255, 95%CI was 1.010-1.558, P = 0.040) also increased with the increasing of night-day heart rate ratio in patients with SOFA score > 5. However, the relationships did not exist in patients with SOFA score ≤ 5. CONCLUSIONS In ICU patients, the 28-day mortality and 1-year mortality increase with the higher circadian heart rate variation, which indicates that the circadian heart rate variation in ICU patients is positively correlated with the short-term and long-term mortality, especially in patients with relatively severe illness.
Circadian Clocks ; Critical Care ; Heart Rate/physiology* ; Humans ; Intensive Care Units ; Mortality/trends* ; Organ Dysfunction Scores ; Prognosis ; Retrospective Studies