Objecfive To investigate the effects of treatment for cerebrovascular disorder patients with heparin and low molecular weight heparin(LMWH) on serum PAPP-A concentrations and provide the basis for evaluating the clinical significance of PAPP-A in the following study.Methods Forty cases with cerebrovascular disease from Qilu Hospital from November 2009 to May 2010 were collected in this study.Blood samples were taken before and after drug administration.All cases were divided into four groups according to situation of medication.Group A consisted of 10 patients who received subcutaneous LMWH anticoagulation therapy, and blood samples were collected before LMWH injection, three hours after subcutaneous LMWH anticoagulation therapy in the first day, the second day and the seventh day and 24 hours after the last injection. Group B consisted of 10 patients who did not receive LMWH therapy, and blood samples were collected immediately after admission, the first day, the second day and the seventh day after admission. Group C consisted of 10 patients with percutaneous carotid intervention who received intravenous heparin at the beginning of stenting, and blood samples were collected from the arterial sheath just before angiography and heparin administration, and at 3, 5, 15, 40 and 100 min after heparin administration. Group D consisted of 10 patients who received carotid angiography but LMWH-free therapy,and blood samples were collected from the arterial sheath just before and after angiography. Serum PAPP-A concentrations were analyzed by ELISA to evaluate the differences of intra-groups and differences at different time points of inter-groups. Results In group A, PAPP-A concentrations were time dependent and elevated gradually from 12. 36 (9. 90-14. 32) mIU/L before LMWH injection to 21.80 (23.50-19.73) mIU/L at the seventh day after injection (M=38. 72, P < 0.01 ). In group C, there was a rapid increase of PAPP-A concentration from 12. 86 ( 9. 67-14. 05 ) mIU/L to 51.56 ( 44. 20-66. 00 ) mIU/L within 5 min after intravenous heparin injection (M=46. 06, P <0. 01 ). The PAPP-A concentration of one week after LMWH administration in group A was 21.80 (23.50-19.73) mIU/L, significantly higher than that in group B [11.81 (9. 21-12. 89) mIU/L] (U<0. O01, P<0.01). The PAPP-A concentration at 15 min after heparin administration in group C was 43.70 (37.70-54. 30) mIU/L, significantly higher than that after angiography in group D [14. 18 (11.25-15. 86) mIU/L] ( U<0. 001, P <0. 01 ). The peak level of blood PAPP-A after subcutaneous LMWH injection was significantly lower than that after intravenous heparin injection. The concentrations in group A and C were 21.80 ( 23.50-19. 73 ) and 51.56 (44. 20-66. 00) mIU/L respectively, and had a significant difference ( U=0. 999, P < 0. 01 ) . Conclusions Both intravenous heparin and subcutaneous LMWH administration induce an increase in serum PAPP-A concentration. The effect of drug should be considered when PAPP-A is selected as an evaluation indicator.

Objective To explore the application value of plasma sHLA-G in diagnosis of CIN and cervical cancer. Methods The plasma sHLA-G levels were detected by ELISA in 102 cases with cervical cancer( FIGO Ⅰ stage 32 cases, Ⅱ stage 28 cases, Ⅲ stage 25 cases and Ⅳstage 17 cases; tumor size:＜4 cm 63 cases and ≥4 cm 39 cases; squamous cell carcinoma 78 cases and adenocarcinoma 24 cases;cell differentiation:well 57 cases, moderate 29 cases and poor 16 cases; lymph nodes metastasis negative64 cases and positive 38 cases ), 72 cases with CIN( Ⅰ grade 21 cases, Ⅱ grade 25 cases and Ⅲ grade26 cases ) and 20 cases of healthy controls. The diagnostic value of sHLA-G and its correlations with clinical parameters were analyzed. Results The plasma levels of sHLA-G were 193.6( 151.3-287.4 ) kU/L in cervical cancer group, 48.3( 34.6-57.2 ) kU/L in CIN Ⅰ group, 91.3( 68.2-118.6 ) kU/L in CIN Ⅱ group, 106.4( 73.8-165.7 ) kU/L in CIN Ⅲ group and 45.2( 38.0-55.5 ) kU/L in health control group.The level of sHLA-G was significantly higher in cervical cancer group than that in CIN Ⅰ group, CIN Ⅱ group, CIN Ⅲ group and healthy control group( U value of 8.832, 6.456, 4.017, 9.873, P ＜ 0.05,respectively ). The level of sHLA-G was significantly higher in CIN Ⅱ group and CIN Ⅲ group than that in CIN Ⅰ group and health control group( U value of 4.361,4.892, 5.139, 5.485, P ＜0.05, respectively ).The levels of SCC Ag in healthy control group, CIN Ⅰ group, CIN Ⅱ group, CIN Ⅲ group and cervical cancer group were 0.43( 0.38-0.69 )μg/L, 0.47( 0.35-0.72 )μg/L, 0.65( 0.53-0.81 )μg/L, 0.82( 0.54-1.03 )μg/L and 1.02( 0.62-1.87 )μg/L. The level of SCC-Ag was significantly higher in cervical cancer group than that in CIN Ⅰ group, CIN Ⅱ group and healthy control group( U value of 7.926, 4.877, 8.132,P ＜0.05, respectively ). The level of SCC-Ag was significantly higher in CIN Ⅲ group than that in CIN Ⅰ group and health control group( U value of 6.574, 6.763, P ＜0.05, respectively ). The levels of CA125 in healthy control group, CIN Ⅰ group, CIN Ⅱ group, CIN Ⅲ group and cervical cancer group were 14.38 ( 6.14-21.82 ) kU/L, 15.42( 6.25-23.53 ) kU/L, 21.34( 9.82-32.58 ) kU/L, 25.69( 14.47-38.71 )kU/L and 27.72( 14.29-43.87 ) kU/L. The level of CA125 was significantly higher in cervical cancer group than that in CIN Ⅰ group, CIN Ⅱ group and healthy control group( U value of 7.564, 4.522, 7.429, P ＜0.05, respectively ). The level of CA125 was significantly higher in CIN Ⅲ group than that in CIN Ⅰ group and health control group( U value of 5.871, 5.435, P ＜0.05, respectively ). ROC curve analysis showed AUC for sHLA-G was 0.828( 95% CI:0.768-0.879 ), which was high as compared with the AUC of SCC-Ag [ 0.727( 95% CI:0.658-0.788 );Z = 2.294, P ＜ 0.05 ] and the AUC of CA125 [ 0.705( 95% CI:0.636-0.769 );Z =2.842 ,P ＜0.05 ]. There was no significant difference of diagnostic efficiency between SCC and CA125( Z =0.672, P ＞ 0.05 ). When cutoff value of sHLA-G was 109.6 kU/L, the diagnostic sensitivity,specificity, positive predictive value, negative predictive value and accuracy rate were 86.3%, 76.1%,80.0%, 83.3%, and 78.4%, respectively. The levels of sHLA-G in cervical cancer patients were significantly correlated with FIGO stages and lymphoid node metastasis ( U value of 6.085, 4.451, P ＜0.05, respectively ), while there were no significant differences between the levels of sHLA-G and age,tumor size, histological type and cell differentiation( U value of 1.274, 1.956, 1.268, 2.719, P ＞0.05,respectively ). Conclusions sHLA-G can be used for the early screening of cervical cancer and its precancerous lesion. It could also be used as an index for judging progression and lymphoid node metastasis.

Objective To establish a direct reverse transcription real-time fluorescence quantitative polymerase chain reaction ( RT-qPCR-D ) method for detecting serum circulating B cell-specific moloney murine leukemia virus integration site-1 (Bmi-1) mRNA, and analyze the levels of serum circulating Bmi-1 mRNA in colorectal cancer patients by using of this method for exploring its diagnosis value in colorectal cancer.Methods Methodology establishment.RNA was extracted from colorectal cancer HT 29 cell line, and detection standard curves of Bmi-1, ubiquitin C ( UBC), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) mRNAs were established , then the amplification efficiencies were calculated.Bmi-1 mRNA level was directly detected in serum and preparation buffer mixture , then the specificity of assay was evaluated by melting curve, and detection limit was observed through diluted serum samples.The serum circulating Bmi-1 mRNA levels were detected by ELISA in 158 cases with colorectal cancer , of which there were 26 cases of tumor node metastasis ( TNM)Ⅰstage, 53 cases of TNMⅡ, 47 cases of TNMⅢ, 32 cases of TNMⅣand 53 cases of controls with normal colonoscopy collected from January 2008 to January 2009 in Qilu Hospital of Shandong University.Comparisons of groups were determined by applying Mann-Whitney U test or Kruskal-Wallis test, and receiver operating characteristic ( ROC) curves were established to illustrate the diagnostic performance.Results The log values of Bmi-1, UBC and GAPDH showed good linear correlations with quantification cycle (Cq) values(R2 =0.990, 0.990, 0.991, all P <0.001), and the amplification efficiencies were 0.875, 0.917 and 0.935, respectively.Using the established RT-qPCR-D method, the peak of melting curve of Bmi-1, UBC and GAPDH mRNAs were single, the detection limit was up to 1.25μl.The levels of serum circulating Bmi-1 mRNA detected by RT-qPCR-D were 0.138 ( 0.078-0.228 ) in colorectal cancer stage Ⅰ patients, 0.163(0.067 -0.287) instage Ⅱ patients, 0.217(0.072-0.267) instage Ⅲpatients, 0.273(0.139 -0.419) in stage Ⅳ patients and 0.021(0.008 -0.029) in health controls, a significant difference was found among groups ( H =89.5, P <0.001 ).The levels of serum circulating Bmi-1 mRNA in each stage colorectal cancer were all significantly higher than that in control group(U=58.0, 287, 246, 72.5,all P<0.001).The levels in Ⅳstage patients were significantly higher than those in other stages patients (U=247, 590, 540,P=0.008, 0.020, 0.035), while no significant differences among Ⅰstage,Ⅱstage and Ⅲstage patients(U=633, 514, 1170,all P>0.05).ROC curve analysis showed area under the ROC curve ( AUC) for serum circulating Bmi-1 mRNA was 0.921(95%CI=0.876-0.953), which was significantly superior to the AUC of CEA (0.745, 95%CI=0.680-0.802, Z=4.697, P<0.001 ).When cutoff value was 0.034, the diagnostic sensitivity and specificity was 89.2%(141/158) and 90.6%(48/53), while 41.8%(66/158) and 73.6%(39/53) using CEA.The AUC for combination of circulating Bmi-1 mRNA and CEA was 0.933(95%CI=0.890-0.963 ) , which was no statistical significance when compared with the AUC of circulating Bmi-1 mRNA(Z=4.697, P>0.05).Conclusions The study establishes a higher sensitive, specific for detecting serum circulating Bmi-1 mRNA. Based on this method , serum circulating Bmi-1 mRNA is found to be increased in colorectal cancer , and is superior to traditional tumor marker CEA in diagnosis of colorectal cancer, which may become a potential detection index for early detection of colorectal cancer.

Objective Next Generation Sequencing(NGS) platform was used to study the characteristics of hot gene mutations in non-small cell lung cancer (NSCLC). The distribution, type and frequency of mutation sites were systematically analyzed to evaluate the pathogenicity of mutation sites . Methods A total of 94 NSCLC tissue samples were included in this study including paraffin-embedded (FFPE) samples and fresh tissue samples, which were collected from July 2015 to April 2017 at the Qilu Hospital of Shandong University. The patient's age ranged from 35 to 82 years with a median age of 61 years. There were 63 males and 31 females. 22 hot genes in NSCLC were selected as the detection panel, including KRAS, EGFR, BRAF, PIK3CA, AKT1, ERBB2, PTEN, NRAS, STK11, MAP2K1, ALK, DDR2, CTNNB1, MET, TP53, SMAD4, FBXW7, NOTCH1, ERBB4, FGFR1 and FGFR2. Mutation detection was performed using the Ion AmpliSeq Colon and Lung Cancer Panel of the Thermo fisher's Ion Torrent sequencing platform. The sequencing data was analyzed using Ion Torrent suite v4.4.2 software. Results Among the 22 mutant genes commonly found in NSCLC, the mutation frequency of TP53 was the highest, accounting for 46.9％ of all mutations, followed by the EGFR mutation (28.1％); A total of 89 mutations were detected, including 63 hot spot mutations (reported mutations) and 26 new mutations (unreported mutations). The most frequently detected mutation was the frameshift deletion of exon 19 of EGFR, followed by the mutation of exon L858R;Analysis of the mutation in targeted drug sites of EGFR showed that the frameshift deletion of exon 19 of EGFR was the most frequently detected, followed by the mutation of exon L858R on chromosome 21. Bioinformatics software was used to analyze the pathogenicity of 26 new mutation sites. Results showed that in addition to ATK1:c. 47-12G>A and TP53: c. 214 C>G, the remaining 24 new mutation sites had at least one major impact on the gene function in three aspects, including gene conservation, amino acid sequence change and protein structure influence. Conclusion In this study, NGS was used to conduct combined detection of mutation sites of multiple hot genes, which might cover more comprehensively genetic variation and provide a basis for screening the most suitable targeted therapy groups. The pathogenicity prediction of new mutations and the changes in tumor-related signaling pathways involved provide a reference for further study of the pathogenesis of NSCLC.

Exosomes are nano-scale double-layer membrane structure vesicles that can be actively secreted by cells. They carry a large amount of biologically active substances and can serve as carriers for material transfer and information exchange between cells. In recent years, exosomes have become a frontier hotspot in biomedical research, and show broad application prospects in the field of laboratory diagnosis and clinical treatment of diseases. However, in general, most exosomes-related researchs are still in the laboratory research stage, and there are still many problems and challenges in separation and enrichment, technical operation specifications, and quality control. At the same time, it is urgent to carry out multi-center, large-sample clinical trials to provide evidence for exosomes from the laboratory to the clinical application.

Objective:To screen the characteristic gut microbiota and fecal metabolites related to the efficacy of oxaliplatin-based neoadjuvant chemotherapy in patients with colorectal cancer liver metastasis, to analyze the relationship between gut microbiota and fecal metabolites, and to evaluate the predictive value of relevant markers for the efficacy of neoadjuvant chemotherapy in patients with colorectal cancer liver metastasis.Methods:This is a case-control study, 34 patients with colorectal cancer liver metastasis who were treated in Qilu Hospital of Shandong University from October 2021 to July 2022 were selected as the research objects, and were divided into chemotherapy effective group (20 cases) and chemotherapy ineffective group (14 cases) according to the efficacy evaluation criteria. Logistic regression was used to construct a prediction model to screen the microbiota and metabolic markers capable of predicting the effect of chemotherapy, and the receiver operating characteristic (ROC) curve and survival analysis curve were plotted to evaluate the predictive effect of related microbiota and metabolites on the efficacy of neoadjuvant chemotherapy.Results:There was no significant difference in the α and β diversity of gut microbiota between the patients in the chemotherapy effective group and in the ineffective group (all P>0.05). In terms of species, the relative abundance of 5 species was up-regulated and 10 species were down-regulated in the chemotherapy-effective group compared with the chemotherapy-ineffective group, and the difference was statistically significant (all P<0.05), among which Prevotella salivae could effectively predict the chemotherapy effect (AUC=0.750, P=0.007), with a sensitivity of 80.0% and a specificity of 71.4%. The overall survival of patients with high abundance (17 cases) was lower than that of patients with low abundance (17 cases) ( χ 2=5.239, P=0.022). In terms of metabolites, 20 metabolites were up-regulated and 4 metabolites were down-regulated in the chemotherapy-effective group compared with the chemotherapy-ineffective group, and the difference was statistically significant (all P<0.05), among which threonine and prostaglandin F2α-1-ethanolamide could distinguish between patients who responded to chemotherapy and those who did not respond to chemotherapy (AUC=0.743, 0.707, all P<0.05), and the overall survival of patients with high levels of relative abundance (17 cases) was higher than that of patients with low levels (17 cases) ( χ 2=4.748, 5.407, all P<0.05). The Logistic regression model of Prevotella salivae and prostaglandin F2α-1-ethanolamide was obtained through screening analysis, and the ROC curve results showed that the model had a good predictive value (AUC=0.836, sensitivity: 90.0%, specificity: 78.6%), and the overall survival of patients with high predict probability (17 cases) predicted by the model was higher than that of patients with low predict probability (17 cases) ( χ 2=9.260, P=0.002). Conclusion:Prevotella salivae and prostaglandin F2α-1-ethanolamide can be used as predictive biomarkers of neoadjuvant chemotherapy for colorectal cancer liver metastasis, and the model has good clinical reference value for prognosis assessment of patients in this cohort.

Objective:To explore the antibacterial performance and mechanism of reticulated cruciform DNA nanomachines mimicking neutrophil extracellular traps (NETs).Methods:In this study, a cross-shaped DNA nanostructure was synthesized using the one-step method to form a network under the polymerization of magnesium ions. The network was used as a template for inlaying reduced Cu 0 to form reticulated DNA-templated copper nanoclusters (CuNPs). The cruciform DNA formation process was characterized by polyacrylamide gel electrophoresis (PAGE). The reticulated cruciform DNA nanomachine was characterized by atomic force microscopy (AFM) and energy dispersive spectrometer (EDS). The optimal concentrations of Cu 2+, Mg 2+and DNA in the reticulated cruciform DNA nanomachine was determined by plate coating experiments. The antibacterial performance of the reticulated cruciform DNA nanomachine against Escherichia coli ( E. coli), Staphylococcus aureus and Klebsiella pneumoniae was verified by plate coating experiments. The aggregation effect of E. coli was evaluated by crystal violet staining, and the changes on the membrane surface of E. coli were observed by scanning electron microscopy (SEM), in order to explore the antibacterial mechanism of the reticulated cruciform DNA nanomachine. Results:PAGE showed that the band migration distance of the four DNA strands was the smallest after co-incubation. AFM showed that the DNA structure was reticulated and evenly distributed, and the Cu, Mg, and P elements coexisted in the structure. The optimal concentrations of Cu 2+, Mg 2+, and DNA required for the synthesis of the reticulated cruciform DNA nanomachine were 1 mmol/L, 100 mmol/L, and 20 μmol/L, respectively. The reticulated cruciform DNA nanomachine had the ability to inhibit E. coli, Staphylococcus aureus and Klebsiella pneumoniae, with survival rates of 39.72%, 43.56% and 60.57%, respectively. The reticulated cruciform DNA nanomachine had an aggregation effect on E. coli. The surface of the bacterial film exhibited shrinkage and fractures. Conclusion:The reticulated cruciform DNA nanomachine constructed in this study can aggregate E. coli, leading to the shrinkage and fracturing of the bacterial film, and exhibiting antibacterial effects.

Objective:To understand the epidemiological characteristics and post-exposure prophylaxis (PEP) situation of rabies exposure population in the animal injury outpatient department of Lishui Central Hospital, so as to provide reference for rabies prevention and control in this region.Methods:The epidemiological data of 6 471 rabies-exposed persons in Lishui Central Hospital from 2021 to 2023 were retrospectively analyzed, including the gender and age of exposed persons, the month and location of injury, the species of injured animals, the exposure grade, rabies vaccination, rabies passive immunization agents, etc.Results:From 2021 to 2023, a total of 6 471 cases of rabies exposure were treated in the animal injury outpatient department of Lishui Central Hospital. From 2021 to 2023, 1 133 cases, 2 135 cases and 3 203 cases were treated respectively. April to November was the peak period of exposure. The population of 21 to 30 years had the highest rate of treatment, reaching 27.79% (1 798/6 471), and was a high-risk group of exposure. The age composition ratio of rabies exposure in the three years was statistically significantly different ( χ2=43.82, P<0.001); the male to female ratio was 1∶1.14 ( χ2=1.63, P=0.442); 3 317 cases (51.26%, 3 317/6 471) were injured by cats, and 2 614 cases (42.16%, 2 614/6 471) were injured by dogs, cats and dogs were the main injured animals ( χ2=18.63, P=0.098). The upper limbs (4 131/6 471, 63.84%) and lower limbs (1 848/6 471, 28.56%) were the most exposed sites, and there was a statistically significant difference in the exposure composition ratio of each site in three years ( χ2=105.79, P<0.001). Grade II exposure accounted for 31.79% (2 057/6 471), grade III exposure accounted for 62.31% (4 032/6 471). Among grade III exposure individuals, those who used passive immune preparations accounted for 55.13% (2 232/4 032). Conclusions:The number of rabies-exposed patients in the animal injury outpatient department of Lishui Central Hospital has been increasing year by year, and the population of patients injured by cats and dogs is particularly prominent. The utilization rate of passive immune preparations for grade III exposure patients still needs to be further improved.