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【Objective】 To identify two ABO discrepancy samples and explore the molecular mechanism. 【Methods】 The serological phenotype of the proband was determined with standard serological methods. ABO genotype was determined by polymerase chain reaction-sequence specific primer (PCR-SSP). Exon 6 and 7 of the ABO gene were amplified with PCR and sequence-based typing (SBT). The amplicon of exon 6 and 7 was also cloned and sequenced. Pymol software was used to simulate the 3D structural model and predict the effect of GTB protein mutation on the structure. The sample were collected from proband’s father and analyzed. 【Results】 The proband’s erythrocytes were detected with B antigens, along with the presence of anti-B in serum. The genotype O1/B of the proband was identified by PCR-SSP. Direct sequencing of the proband revealed 261delG/G, 297A/G in exon 6 and 526C/G, 646A/T, 657C/T, 681A/G, 703A/G, 771C/T, 796A/C, 803C/G, 829A/G, 905A/G, 930A/G, 1096A/G heterozygote in exon 7, which was assigned as Bx02/O02 genotype. Clone sequencing showed that a 905 A>G mutation in the ABO*B.01 allele. The 3D structure simulation suggested that Asp302Gly may cause the change of GTB enzyme activity or function. 【Conclusion】 Two cases of Bx02 allele were identified. Combined detection of serological and genotyping methods is important for identification of ABO blood group.
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Objective:To explore the profile and gene functional changes of gut microbiota (GM) in women with postmenopausal osteoporosis (PMOP) in Northwest China, and the correlations between GM and bone mineral density (BMD).Methods:From November 2018 to October 2019, postmenopausal women were screened on their initial visits to our hospital, and 24 new osteoporosis (OP) patients, 30 new osteopenia patients and nine negative controls were recruited. Fecal samples were collected for GM DNA extraction, and Illumina platforms were used for high-throughput sequencing of 16S rRNA and metagenome. Species annotation, GM profile and gene functions were viewed and analyzed.Results:GM profiles were significatly different in different groups, and the LDA scores of Peptostreptococcaceae, Romboutsia, unidentified Clostridiales, Megamonas, Erysipelatoclostridium, Klebsiella and Erysipelatoclostridium ramosum were more than 3 in OP group. Metagenomic sequencing analysis indicated that gene numbers were positively correlated with BMD, and metabolism, carbohydrate metabolism, starch and sucrose metabolism and oxidative phosphorylation were negatively correlated with BMD. Receiver operating characteristic curve(ROC) showed that carbohydrate metabolism, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, oxidative phosphorylation, respectively, could identify OP with preferable sensitivity and specificity (areas under curve were 0.70, 0.72, 0.73 and 0.75, respectively). Conclusions:High-throughput sequencing had great potential for GM analysis of postmenopausal women with OP, providing evidence of the correlations between GM and BMD.
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ObjectiveTo investigate the clinical features of patients with severe alcoholic hepatitis (AH) with different underlying liver diseases and the influencing factors for short-term prognosis. MethodsA retrospective analysis was performed for the clinical data of 170 patients with severe AH who were admitted to Tianjin Third Central Hospital from August 2004 to August 2018, and according to the underlying liver disease, they were divided into group A (27 patients without liver cirrhosis), group B (52 patients with compensated liver cirrhosis), and group C (91 patients with decompensated liver cirrhosis). Related scores were calculated, including Maddrey’s discriminant function (MDF) score, Chronic Liver Failure-Sequential Organ Failure Assessment (CLIF-SOFA) score, Model for End-Stage Liver Disease (MELD) score, age-bilirubin-international normalized ratio-creatinine (ABIC) score, and Glasgow alcoholic hepatitis score (GAHS). An analysis of variance or the Kruskal-Wallis H test was used for comparison of continuous data between multiple groups, and the chi-square test was used for comparison of categorical data between multiple groups. Univariate and multivariate Cox regression analyses were used to screen out the independent influencing factors for the short-term prognosis of patients with severe AH. The Kaplan-Meier method was used to plot survival curves, and the log-rank test was used for comparison of survival rate between groups. The receiver operating characteristic (ROC) curve was used to calculate the area under the ROC curve (AUC) and 95% confidence interval (CI), sensitivity, and specificity for each predictive model, and the DeLong method was used for comparison. ResultsThe 28-day survival rates of patients in groups A, B, and C were 88.9%, 80.8%, and 51.6%, respectively, with a significant difference between the three groups (χ2=1983, P＜0.001). The AUCs (95% CIs) of MELD score, MDF score, GAHS score, ABIC score, and CLIF-SOFA score were 0.584 (0.493-0.676), 0.696 (0.605-0.786), 0.644 (0.554-0.735), 0.745 (0.662-0.827), and 0.795 (0.726-0.863), respectively, in predicting 28-day mortality rate, and there were significant differences between CLIF-SOFA score and MDF, MELD, and GAHS scores (all P＜0.05); CLIF-SOFA score had a sensitivity of 79.0% and a specificity of 67.9% at the optimal cut-off value of 850 points in predicting 28-day mortality rate. Different underlying liver diseases (hazard ratio ［HR］=2.296, 95% CI: 1.356-3887, P=0.002) and hepatic encephalopathy (HR=1.911, 95% CI: 1.059-3.449, P=0.031) at disease onset were risk factors for 28-day prognosis. ConclusionPatients with severe AH with different underlying liver diseases have different clinical features and short-term prognoses. Different underlying liver diseases and hepatic encephalopathy at disease onset are closely associated with the 28-day prognosis of patients with severe AH. CLIF-SOFA score can predict the 28-day prognosis of patients with severe AH.
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Objective@#To investigate the effects of cerebral cavernous malformation 3 (CCM3) gene knockout on the lead exposure-induced blood-brain barrier malfunction in mice brain, and the relationship between CCM3 knockout and the Alzheimer's disease (AD).@*Methods@#Wide type (WT) mice and CCM3+/- mice were divided into 4 groups, control group and lead exposed group in WT as well as CCM3+/- mice. Lead exposed groups were treated with 0.05% lead acetate in drinking water for 12 weeks, while control group drink deionized water freely. Blood lead and brain lead levels in each group were detected by graphite furnace atomic absorption spectrometry. The brain tissue of each group was made into paraffin sections, whose morphology were observed by HE staining. The expression of Aβ1-42 in brain tissue was detected by immunohistochemistry and the brain capillaries were labeled by VRGFR2. The protein expression of Claudin-5, ZO-1, and p-Tau was detected by Western blot. The brain tissue RNA was extracted and the relative expression of LRP-1 mRNA was detected by qRT-PCR.@*Results@#The levels of blood lead WT (216.07±84.16) and CCM3+/- (189.64±101.86) μg/L in lead exposed group were higher than those in control group WT (19.52±11.46) and CCM3+/- (11.79±8.20) μg/L, the difference was statistically significant (t=4.18, P=0.006; t=3.79, P=0.016). The levels of brain lead WT (1.78±0.69) and CCM3+/- (1.74±0.66) μg/L were higher than those in control group WT (1.06±0.87) and CCM3+/- (0.97±0.64) μg/L, the difference was statistically significant (t=3.67, P=0.018; t=3.88, P=0.015). The HE staining showed no obvious lesions in the brain of each group of mice. The results of immunohistochemistry showed that there was no Aβ1-42 deposition in the brain of mice in each group. The numbers of microvessels in the brain of CCM3+/- mice in the lead exposed group were decreased. Compared with the relative expression levels of Claudin-5 (WT: 1.30±0.03, CCM3+/-: 1.07±0.08) in control group mice brain, the relative expression of Claudin-5 (WT: 0.96±0.04, CCM3+/-: 0.59±0.01) was decreased with statistical significance (F=199.27, P<0.001). The relative expression level of LRP-1 gene mRNA in brain of lead exposed group (WT: 0.32±0.10, CCM3+/-: 0.06±0.01) was higher than that of unexposed group (WT:1.00±0.06, CCM3+/-:2.12±0.18), the difference was statistically significant (F=288.29, P<0.001). The relative expression level of LRP-1 gene mRNA in brain of CCM3+/- mice exposed to lead was lower than that of WT mice ((0.06±0.01)vs(0.32±0.10), t=26.90, P<0.001).@*Conclusion@#The mice did not show significant AD-like lesions under low-does lead exposure, but resulted in early damage of brain blood-brain barrier and early changes of AD-like lesions in mice, with CCM3+/- mice being sensitive to lead exposure stronger than that of WT mice, suggesting that deletion of CCM3 gene may be one of the potential risk factors for accelerating the development of AD in mice exposed to lead.
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Objective@#To establish the immortalized mouse brain microvascular pericytes model and to apply to the cerebrovascular toxicants screening study.@*Methods@#Brain pericytes were isolated from 3 weeks of mice by tissue digestion. Immortalized pericyte cell line was constructed by infecting with LT retrovirus. Monoclone was selected to purify the immortalized pericyte cell line. The pericyte characteristics and purity were explored by immunocytochemistry. Cell proliferation was measured by using the Pomega MTS cell Proliferation Colorimetric Assay Kit. Pericytes were treated with 0, 160, 320, 640, 1 280, 2 560 μmol/L lead acetate, 0, 5, 10, 20, 40, 80 μmol/L cadmium chloride and 0, 5, 10, 20, 40, 80 μmol/L sodium arsenite in 24 hours. Cell toxicity of each group was determined by MTS assay, median lethal dose (LD50) was calculated in linear regression.@*Results@#Mouse brain pericytes were successfully isolated by tissue separation and enzyme digestion method. After immortalized by LT retroviruses, monoclone was selected and expanded to establish pericyte cell line. The brain pericytes exhibited typical long spindle morphology and positive staining for α-SMA and Vimentin. The proliferation of brain pericytes cell lines was very slowly, and the doubling time was about 48 hours. The proliferation of immortalized brain pericytes cell lines was very quickly, and the doubling time was about 24 hours. After lead acetate, cadmium chloride and sodium arsenite treatment for 24 hours respectively, gradual declines in cell viability were observed. The LD50 of lead acetate was 2 025.0 μmol/L, the LD50 of cadmium chloride was 36.6 μmol/L, and the LD50 of sodium arsenite was 33.2 μmol/L.@*Conclusion@#The immortalized mouse brain microvascular pericyte model is established successfully by infecting with LT retrovirus, and can be applied to screen cerebrovascular toxicants. The toxicity of these toxicants to immortalized mouse brain microvascular pericyte is in sequence: sodium arsenite,cadmium chloride, lead acetate.
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Background:Chronic constipation is a major cause of impaired quality of life in modern society. Reasonable and effective management of chronic constipation could be achieved based on the principle of evidence-based medicine and the modern concept of constipation,and this is a challenge facing the clinicians. Aims:To investigate the role of barium-based colonic transit detection in diagnosis and treatment of chronic constipation. Methods:Fifty patients with chronic constipation from Apr. 2013 to Oct. 2014 at the First Hospital of Shanxi Medical University were recruited and randomly allocated into two groups,control group and individualized treatment group. Patients in individualized treatment group received 20 barium markers orally and abdominal plain radiography was performed 48 and 72 hours later,respectively for calculating the colonic transit index. According to the type of colonic transition and the characteristics of colonic motility estimated by colonic transit index and clinical manifestations,an individualized therapeutic regimen was formulated and the efficacy was evaluated. Patients in control group were treated empirically according to the clinical manifestations. Results:Mosapride and lactulose or polyethylene glycol were administered orally in control group;when abdominal pain or abdominal distension was predominant,pinaverium bromide or trimebutine was used respectively instead of mosapride. Barium-based colonic transit detection revealed that 9 patients in individualized treatment group were slow transit constipation,6 were outlet obstructive constipation and 8 were the mixed type. After 2 weeks of empirical or individualized treatment,the defecation rates of the two groups were 24. 0%(6 / 25)and 52. 2%(12 / 23)within 24 hours and 64. 0%(16 / 25)and 87. 0%(20 / 23)within 48 hours,respectively(P all < 0. 05). Conclusions:Barium-based colonic transit detection is a simple,economical and practical modality for guiding the individualized treatment in patients with chronic constipation.
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Objective To investigate the expression of unfolded protein response (UPR) gene glucose regulated protein 78(GRP78) and X-box binding protein 1(XBP1) in esophageal squamous cell carcinoma, its effect on activation of the endoplasmic reticulum stress (ERS) and its mechanism in the growth of esophageal squamous cell carcinoma. Methods The expressions of GRP78 and XBP1 were detected by immunohistochemistry in 30 samples of esophageal squamous cell carcinoma and 30 samples of normal esophageal squamous epithelium. The correlation between expressions of both proteins and prognosis was analyzed. Results GRP78 positive rate was 83.3 %(25/30) in esophageal carcinoma, while the proportion was 20.0 %(6/30) in normal esophageal (χ2=25.833, P<0.05). XBP1 positive rate was 70.0 % (21/30) in esophageal carcinoma, while the proportion was 26.7%(8/30) in normal esophageal(χ2=20.872, P<0.05). The positive rates of GRP78 and XBP1 in invasive muscular layer of esophageal squamous cell carcinoma were significantly higher than those in invasive mucous layer. Conclusion GRP78 and XBP1 are highly expressed in esophageal squamous cell carcinoma, which may involve the occurrence and development of the esophageal carcinoma.
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Objective To investigate the drug resistance and homology status of Multi-drug Resistant AcinetobacterBauman-nii (MDR-AB)in orthopaedic hospital.Methods 34 strains MDR-AB were isolated from 2016.1~2016.7 for DNA extrac-tion and were typed by repetitive extragenic palindromie-polyrnerase chain reaction (REP-PCR).Results The resistance rate of MDR-AB were ≥70% to 15 of 17 antimicrobials,except to cotrimoxazole (14%)and levofloxacin (61%),34 strains of MDR-AB were divided into three types by REP-PCR including typeⅠ,typeⅡ and typeⅢ.Conclusion Drug resistance of MDR-AB was severe and mainly composed of the same genotype (typeⅠ).Rational use of antimicrobial and regular mo-nitoring of drug resistance is necessary to reduce the nosocomial transmission.
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Objective To observe the expression of cytokeratin 14,15 (CK14,CK15) expression level in normal esophageal tissues and esophageal squamous cell carcinoma tissues of different differentiation degree and to analyze the relationship between occurrence,development of esophageal squamous cell carcinoma and CK14,CK15 expression level.Methods Esophageal squamous epithelial tissue from 55 cases of carcinoma tissues and 55 cases of adjacent tissues were collected.Immunohistochemical method was used to compare CK14,CK15 and PCNA expression levels in esophageal squamous carcinoma.Results Expression positive rates of CK14,CK15 and PCNA in esophageal squamous carcinoma were 72.7 ％ (40/55),63.6 ％ (35/55) and 65.5 ％ (36/55),respectively,and PCNA expression was correlated with CK14 or CK15 expression (C =0.585,P ＜ 0.001; C =0.405,P ＜ 0.001).CK14 and CK15 levels were higher in high differentiation carcinoma tissue than those in low differentiation carcinoma tissue,and PCNA expression level was increased in low differentiated carcinoma tissue.CK14,CK15 and PCNA were expressed located in base layer of esophageal squamous epithelial adjacent to carcinoma tissue,and their expression positive rates were 56.4 ％ (31/55),52.7 ％ (29/55) and 56.4 ％ (31/55).CK14 and CK15 levels were higher in esophageal squamous epithelial tissues of far-cut edge than those in tissues of near-cut edge (intraepithelial neoplasia).There were no associations between CK14,CK15 expression and the clinical parameters (P ＞ 0.05).Postoperative survival time in patients with CK14 or CK15 positive expression was shorter than that of patients with negative expression (P ＜ 0.05).Conclusions CK14 or CK15 positive expressions localized to base layer of esophageal squamous epithelial adjacent to carcinoma tissue may play some roles in generation and differentiation of esophageal squamous cell cancer.CK14 or CK15 positive expression in esophageal squamous carcinoma involves in differentiation process.Joint detection of CK14 and CK15 expression has clinical application value for early diagnosis,the degree of differentiation and prognostic judgment in esophageal squamous carcinoma.
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Objective To observe the inhibitory effect of xiaoaiping injection and octreotide on H22 tumor-bearing mice and find the best drug concentration,then to explore its mechanism.Methods Establish a mouse H22 subcutaneous tumor model.After tumor the experiment animals were divided into normal control group,model group,Xiaoaiping low,medium and high dose group,octreotide group,and the group of XAP low,medium and high dose groups were combined with OCT.Calculate the tumor's volume and draw the tumor growth curve.Intraperitoneal injection for 14 days,Inhibitory rate was calculated; To observe its pathological changes by light microscope; The ratio.of CD3 + NK1.1-T cells,CD3-NK1.1 + NKcells,CD3 + NK1.1 + NK-Tcells in peripheral blood were measured by flow cytometry.Results Compared with the control group,H22 liver cancer in different treatment group had a certain inhibition effect on growth,The inhibitory effect of the combination group was better than single-agent group,High-dose Xiaoaiping + octreotide was best,Tumor model group compared with normal control group,The ratio of T cells,NK cells and NKT cells was significantly lower(P ＜0.05) ; T cells,NK cells and NKT cells after treatment in each group had some enhancement,High-dose Xiaoaiping + octreotide was the most obvious,the ratio of T cells,NK cells and NKT cells of the combination group was significantly more than the single-drug group and the same concentration of octreotide monotherapy Xiaoaiping group(P ＜ 0.05).Conclusion High-dose Xiaoaiping + octreotide is the best drug for the inhibitory drug concentration.The inhibition of tumor growth may pass to improve the tumorbearing mice with immune status and enhance the body's anti-tumor capacity.
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Objective To study the effects of XiaoAiping (XAP) with different concentrations and action time on the proliferation and apoptosis of hepatocellular carcinoma cells Hepa1-6. Methods The hepatocellular carcinoma cell line Hepal-6 was treated with XAP at doses of 30 mg/ml(the high concentration group), 20 mg/ml (the moderate concentration group) and 10 mg/ml (the low concentration group) for 24 h, 48 h and 72 h, respectively. The MTT assay was used to detect the inhibitory effects of XAP on cell growth. The cell morphological alteration was observed after HE staining, and the apoptosis rates were assayed by flow cytometry. Results XAP produced an obvious time-and-dose-dependent inhibitory effect on the Hepa1-6 cells (P<0.01), and the cell differentiation trend was benign on light microscopy. After 48 h Hepa1-6 cells were incubated by XAP, there was an apoptosis peak, and the apoptosis rate was increased statistically in XAP group with the increasing XAP concentration [(7.65±0.40)%, (11.26±1.09)% and (26.71±0.85)% in low, moderate and high concentration group, respectively]compared with that in the controls (2.88 ±0.30)%, P < 0.01). Conclusion XAP produced obvious time-and-dose-dependent inhibitory effects on Hepa1-6 cells. Inhibiting DNA synthesize and inducing apoptosis of tumor cells may involve in the mechanisms of antineoplastic effect.
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To compare the activity of different promoter in baculovirus-insect system, a series of recombinant baculoviruses were generated harboring the E-GFP reporter gene under the control of one of 5 promoters, including the ie1 promoter of shrimp white spot syndrome virus (WSSV), the truncated ie1 (mie1) promoter, the ETL promoter of the baculovirus, the elongated ETL (mETL) promoter, and the polyhedron promoter (P(PH)) of the baculovirus. The expression efficiency of the E-GFP reporter gene in the recombinant baculovirus-infected Sf9 cells was determined by flow cytometry. The results showed that both ie1 and mETL promoters had a strong promoter activity at early phase, while P(PH) showed a strong promoter activity at late phase. The ie1 promoter suggested the strongest promoter activity. The homologous region 1 (hr1) was also found to enhance the ETL promoter activity.
Subject(s)
Animals , Baculoviridae , Genetics , Metabolism , Green Fluorescent Proteins , Genetics , Metabolism , Insecta , Genetics , Metabolism , Penaeidae , Virology , Promoter Regions, Genetic , Genetics , Recombinant Proteins , Genetics , Transfection , White spot syndrome virus 1 , GeneticsABSTRACT
Six recombinant plasmids co-expressing the wild-type GP5 gene or the codon-optimized GP5 gene (containing pan-DR epitope) of porcine reproductive and respiratory syndrome virus (PRRSV) and the E2 gene of classical swine fever virus (CSFV) or the E2 fused with the UL49 of pseudorabies virus (PrV) were constructed based on the suicidal DNA vaccine pSFV1CS-E2 described previously. Expression of GP5 and E2 was confirmed by indirect immunofluorescence assay. The immunogenicity of six plasmids was evaluated in BALB/c mouse model. For the six plasmids, low-level of E2 and GP5 protein specific antibodies could be detected in the sera of the immunized mice. Specific lymphoproliferative responses to the PRRSV or CSFV stimulation were induced in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. Antigen specific IFN-gamma and L-4 secretion was detected in the splenocytes of some immunized mice by cytokine ELSIA. Fusion with the PrV UL49 in the suicidal vaccines induced significantly higher lymphoproliferative responses and cytokine secretion. Taken together, the suicidal DNA vaccines co-expressing GP5 and E2 could induce PRRSV and CSFV specific humoral and cell-mediated immune responses.
Subject(s)
Animals , Female , Mice , Antibodies, Viral , Blood , Antibody Formation , Cytokines , Blood , Immunity, Cellular , Lymphocytes , Allergy and Immunology , Mice, Inbred BALB C , Random Allocation , Vaccines, DNA , Allergy and Immunology , Viral Envelope Proteins , Genetics , Allergy and Immunology , Viral Structural Proteins , Genetics , Allergy and Immunology , Viral Vaccines , Allergy and ImmunologyABSTRACT
Objective To observe the effect of Jiangzhi Yigan Chongji (JYC) on the nonalcoholic fatty liver tissue PPAR? and Trx mRNA expression,and explore the mechanism of treating fatty liver. Methods The model was made by feeding high-fat diet and the rats were divide into 3 groups:normal group,model group and treated group. Result Expression of liver tissue PPAR? and Trx mRNA in the model group were both decreased. JYC can increase their expression of liver tissue of model rats. Conclusion It is likely to be one of the important mechanisms for JYC in treating nonalcoholic fatty liver.
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Objective To discuss the preschool children after hepatitis B vaccines injected,the variation trend of hepatitis B surface antibody (HBsAb)'s positive rate- Methods 4149 preschool children's HBsAb were examined ,by means of enzyme linked immunosorbent assay(ELISA). Results The HBsAb's positive rate among the age from 0～2,2.1～3.0,3.1～4.0,4. 1～5.0,5.0～6.0,for each age period,children's HBsAb positive rates was 64.65% ,68.64% ,71.55% ,74.21%,68.38% respectively. The statistical analysis showed that there was statistcal significance(x2=19.77, P