Objective To develop a chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine rash-and fever-causing pathogens,namely,Measles virus,Rubella virus,Enterovirus type 71, Varicella zoster virus,Dengue fever virus,Human small FDNA virus B19,Coxsackie virus type A16,A-βStreptococcus pyogenes (hemolytic streptococcus)and Salmonella typhi.Methods Primers and probes were designed based on the specific sequence in the conserved region of genomes of the nine pathogens.The nucleic acids of the nine pathogens were amplified and labelled by multiplex PCR method.The multiplex PCR amplification products were hybridized with specific probes of microarray that was scanned after washing and chemiluminescence coloration to identify the nine pathogens.After the optimization of the multiplex PCR system,hybridization and chemiluminescence imaging,the specificity,sensitivity and reproducibility of the chip were evaluated.The serial diluted nucleic acid of Enterovirus type 71 was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods.Results Nine specific primers and eleven specific probes were selected.The microarray demonstrated high sensitivity and specificity.The minimum detection limit of plasmid DNA and in vitro transcribed RNAs was 3 ×103 copies per reaction.The detection sensitivity of this microarray was 10 percent of that by the real-time PCR method.The rate of sensitivity and specificity of clinical sample detection was 95% and 85.7% respectively,and the rate of accuracy was 93.2%.Conclusion A chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine pathogens that cause rash and fever illnesses is established successfully,which can serve as a new high throuthput screening method for clinical diagnosis and epidemiological investigation of rash and fever illnesses.

Objective To observe the effect of Astragalus injection on the expressions of inflammatory cytokines in human primary macrophages stimulated by lipoteichoic acid (LTA) and lipopolysaccharide (LPS),and investigate its effects on inflammatory reactions of Gram-positive (G+) and Gram-negative (G-) bacteria sepsis and its mechanisms.Methods Percoll density gradient centrifugation was used to isolate the human peripheral blood mononuclear cells,then they were purified by immune Anti-Biotin Microbeads with magnetic character and under the induction of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF),the cells were cultivated for 12 days in vitro,eventually the human monocyte-derived macrophage was formed.The cultured human macrophages were inoculated in 96-well plates (each group 3 wells) and 6-well plates (each group 3 wells).The cells were divided into control group (200 μL DMEM added in each well),LTA 1 mg/L group,LPS 0.1 mg/L group and low astragalus injection (0.1 mg/L) and high astragalus injection (0.2 mg/L) dose groups.After the incubator plates were put in an incubator for 24 hours,the protein content of IL-8 and IL-10 in supernatant were detected by enzymelinked immunosorbent assay (ELISA),and the mRNA expression levels of IL-8 and IL-10 were detected by real time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR).Results LTA and LPS all can obviously up-regulate the expression levels of pro-inflammatory factor IL-8 and anti-inflammatory factor IL-10 of macrophage.The expressions of IL-8 and IL-10 protein and mRNA in LTA group and LPS group were significantly higher than those in control group after cuhure for 8 hours and 24 hours,the degrees of increment were more significantly at 24 hours [LTA stimute group:IL-8 protein (ng/L,× 103):41.57± 1.90 vs.1.58 ±0.24,IL-8 mRNA (A value):21.49±8.05 vs.1.00±0.16;IL-10 protein (ng/L):5.90±3.02 vs.2.91 ± 1.54,IL-10 mRNA (A value):1.35±0.34 vs.0.95±0.14;LPS stimute group:IL-8 protein (ng/L,× 103):345.00±22.80 vs.5.60±0.31,IL-8 mRNA (A value):29.84 ± 8.93 vs.1.00 ± 0.16,IL-10 protein (ng/L):122.37 ± 39.26 vs.44.79 ± 3.67,IL-10 mRNA (A value):7.38 ± 1.58 vs.1.35 ± 0.34,all P ＜ 0.05].The Astragalus injection could regulate LTA and LPS to stimulate the macrophage to decrease the expression levels of pro-inflammatory factor IL-8 protein and mRNA and increase the expression levels of anti-inflammatory factor IL-10 protein and mRNA in the macrophage;the changes of regulatory effect in the 24 hour-culture of Astragalus injection high dose group was the most significant [LTA stimute group:IL-8 protein (ng/L,×103):22.63±1.91 vs.41.57±1.90,IL-8 mRNA (A value):12.10±1.93 vs.21.49±8.05,IL-10 protein (ng/L):14.03±2.22 vs.5.90±3.02,IL-10 mRNA (A value):10.37±6.08 vs.1.35±0.34;LPS stimute group:IL-8 protein (ng/L,× 103):167.75 ± 19.90 vs.345.01 ±22.80,IL-8 mRNA (A value):15.61 ± 3.63 vs.29.84±8.93;IL-10 protein (ng/L):243.22±14.41 vs.122.37±39.26,IL-10 mRNA (A value):16.14±4.10 vs.7.38± 1.58,all P ＜ 0.05].Conclusions In the process of inflammatory response,the pro-inflammatory and anti-inflammatory factors co-exist simultaneously.Astragalus injection can inhibit the expression levels of pro-inflammatory factor gene and protein in the inflammatory response of G+ and G-bacteria sepsis and in the mean time,it can promote the expression levels of anti-inflammatory factor gene and protein,thus the immune mechanism of sepsis is affected,achieving the balance between pro-inflammation and anti-inflammation.

This study is to explore the effects of quercetin (QUE) on the 3 week-old mice ovarian development and relative hormone levels. The 3 week-old mice were exposed to QUE (45, 25, and 5 mg x kg(-1) x hd(-1)) by gavage for 50 days. The estrous cycle during 50 days and the changes of hormone level such as FSH, LH, etc were monitored. Moreover, the ovaries were removed after sacrifice. The organ index was measured, and the ratios of different stages of follicles were analyzed by HE staining. Furthermore, the proportion of PCNA positive cells during all stages was detected by immunohistochemistry. The results showed that QUE could increase body weight of mice and reduce the anogenital distance (AGD) to some extent, and was able to disrupt mice's estrous cycle, but it could not extend or reduce the cycle regularity. It increased ovarian organ index with a dose-dependent manner. The proportion of the primordial follicle and secondary follicles rose obviously, and that of mature follicles', atretic follicles' and corpus luteums' reduced, while primordial follicle had no change. Immunohistochemistry analysis showed that QUE could effectively increase the percentage of proliferating cells in all kinds of follicles. Serum hormone assay showed that there were significant changes of FSH and LH levels. In summary, QUE showed an estrogen-like effect on mice's ovarian development. The weight of ovary, the proportion of all kinds of follicles, the development of ovarian cells and the level of plasma hormone in mice were altered obviously by oral administration of QUE.

Objective: To investigate the effects of Fufang Jiangzhi No. 3, a compound traditional Chinese herbal medicine, on cholesterol-bile acid metabolism in rabbits with hypercholesterolemia and to explore the mechanism. Methods: Twenty-four male New Zealand white rabbits were randomly assigned into normal control group, untreated group and Fufang Jiangzhi No. 3 group, with 8 rabbits in each group. Rabbits in the untreated group and Fufang Jiangzhi No. 3 group were fed high cholesterol diet to induce hypercholesterolemia. After 4-week treatment, serum total cholesterol and bile acid contents were assessed. Activity of cholesterol 7alpha-hydroxylase (CYP7A1) in liver tissues was measured by enzyme-linked immunosorbent assay. The expressions of CYP7A1, bile salt export pump (BSEP) and small heterodimer partner (SHP) mRNAs in liver tissues were observed by real-time fluorescent quantitative polymerase chain reaction. Results: Compared with the normal control group, serum total cholesterol and bile acid contents in the untreated group were increased (P<0.01). Activity of CYP7A1 and expression of CYP7A1 mRNA were decreased and expressions of BSEP and SHP mRNAs were increased in liver tissues in the untreated group as compared with the normal control group (P<0.01). Serum total cholesterol level, and expressions of BSEP and SHP mRNAs in the Fufang Jiangzhi No. 3 group were lower than those in the untreated group (P<0.01). The CYP7A1 activity and expression of CYP7A1 mRNA in the Fufang Jiangzhi No. 3 group were increased as compared with the untreated group (P<0.01), however, there was no significant difference in bile acid between the Fufang Jiangzhi No. 3 group and the untreated group. Conclusion: Fufang Jiangzhi No. 3 can up-regulate the expression of CYP7A1 mRNA, raise the activity of CYP7A1, and inhibit the expressions of BSEP and SHP mRNAs to regulate the metabolism of total cholesterol in rabbits.

BACKGROUND:Posterior stabilized femoral knee prosthesis needs additional condyle osteotomy to accommodate the tibial post and femur fossa structures. Intercondylar fossa on both sides connected at the femoral body with concentrated stress is a place easily affecting fractures. Differences in bone mass between different models of different brands did not have specific data, which was not convenient to select prosthesis for clinicians.
OBJECTIVE: To compare the difference of intercondylar osteotomy data among clinical commonly used posterior stabilized knee prostheses (six imported and domestic brands), and to provide basis for the selection and application of the prostheses.
METHODS:The current commonly used posterior stabilized knee prostheses (six imported and domestic brands) were used, including Zimmer NexGen LPS, Stryker Scrorpio NRG Knee-Flexed, Depuy PFC Sigma, Smith & nephew Genesis-2 PS, United-U1 and Wego GKPS. According to the osteotomy template, the osteotomy-surfaces consisting of femoral condyle starting section and cross section, distal section of femoral condyle, and back-oblique section were identified. The corresponding femoral prosthesis diameter lines included condylar ambilateral and anteroposterior diameters, width and depth of femoral intercondylar fossa. The above data were compared and measured.
RESULTS AND CONCLUSION:The six kinds of knee femoral prostheses were different in ratio of ambilateral diameter and anteroposterior diameter, bone resection of intercondylar fossa, and geometry. Imported prostheses carry shorter diameters in femoral starting and cross sections, so it can catch more posterior condylar osteotomy. With increasing prosthesis sizes, the ratio of bone loss causing by width of intercondylar osteotomy is decreased among six brands. In al sizes, Stryker Scrorpio NRG Knee-Flexed catches shorter width of intercondylar osteotomy. Knee prosthesis osteotomy among six brands is different. The result of this study is not sufficient to evaluate the pros and cons between different prostheses, but as reserving bone is concerned, the design of less intercondylar osteoomy catches more advantages.

Objective To evaluate antimicrobial effect and mechanism of meropenem in the model of PA infection by C.elegans.Methods To evaluate drug effects of PA infection with caenorhabditis elegans by different concentrations of culture medium, determinate the lethal rate of C.elegans.Western blot detected mitogen activated protein kinase ( Mitogen-activated protein kinase MAPK ) activity change, and PCR detected antimicrobial peptide genes expression in C.elegans after PA infection,the effect of meropenem on MAPK activity change and antimicrobial peptide genes expression.Results Compared with the control group (OP-50), the death rate of C.elegans in PA infection group changed significantly (P<0.01). Meropenem showed protective effect after C.elegans infection ( P <0.01 ) .Detection of MAPK kinase activity showed that PA infection caused PMK-1 kinase activation, further study showed that antibiotics meropenem did not affect the activation of PMK-1 kinase (no significant difference).C.elegans antimicrobial peptide gene Lys-1, clec-85, F55G11.7, K08D8.5 activity increased in PA infection (P<0.01).Meropenem promoted the expression of the antimicrobial peptide gene increased (P<0.01),with synergistic effects.Conclusion Our results show that a C.elegans pathogenicity model can be applied screening drug susceptible to pathogens infection quickly and easily.

Objective To investigate the relationships between the C6 glioma haemodynamic response and basal blood perfusion parameters.Methods Ten Wistar rats with C6 gliomas implanted subcutaneously have undergone Carbogen-induced steady state free precession functional MRI (SSFP fMRI)examination,followed dynamic contrast enhancement MRI (DCE-MRI)examination in the normal air breathing.For each glioma,5 regions of interest(ROI)were drawed randomly on SSFP fMRI images,and the percentage of signal change(PSC)of each ROI was calculated with the analysis of functional neuroimages (AFNI)software.The dynamic signal intensity of ROI in DCE-MRI on the same space and location was fitted by Hoffmann model in R language software,thus to obtain basal blood perfusion parameters (Akep and kel).Distribution-free and small-sample-size-oriented permutation test was used to investigate the relationships between PSC and basal blood perfusion parameters.Results 50 ROI were drawed randomly from 10 glioma. There were no statistically relationship between PSC and Akep,or between PSC and kel (F=0.629 8,P=0.431 3;F=1.859,P=0.179 1).Conclusion The glioma basal blood perfusion could not predict the PSC of SSFP fMRI.

Objective To study the relationship of serum 25-hydroxy vitamin D [25-(OH) D] and vitamin D binding protein (DBP) in premature infants with bronchopulmonary dysplasia (BPD) and their clinical significance.Method From March 2017 to September 2018,the premature infants with gestational age (GA)＜32 weeks admitted to the neonatal department of our hospital were prospectively studied.All the premature infants were given 800 IU/d vitamin D supplement from one week after birth.Venous blood sample were collected at birth and 28 d after birth to measure 25-(OH) D aud DBP levels.The infants were evaluated for BPD at 28 d after birth and then assigned into the BPD group and the non-BPD group.The differences of 25-(OH) D and DBP levels were compared.Result A total of 170 premature infants (GA＜32 weeks) were included,including 56 cases in the BPD group and 114 cases in the non-BPD group.The BPD group had 34 males,the GA was (29.8±1.2) weeks,the birth weight (BW) was (1 198± 157) g.The non-BPD group had 95 males,the GA was (30.2± 1.5) weeks,the BW was (1 243± 146) g.No significant differences existed in GA,BW and male gender proportion between BPD group and non-BPD group (P＞0.05).The BPD group had a lower levels of serum 25-(OH) D at birth [(27.8±5.9) nmol/L vs.(30.4±1.1) nmol/L,P＜0.05].The levels of serum 25-(OH) D in moderate/severe BPD group were significantly lower than mild BPD group [(25.3±4.9) nmol/L vs.(29.7±5.9) nmol/L,P＜0.05];25-(OH) D in BPD group was still lower than the non-BPD group at 28 days after birth (after vitamin D supplement) [(77.5±11.7) nmol/L vs.(83.8±11.6) nmol/L,P＜0.05].Comparison of serum DBP levels between the two groups showed that,DBP at 28 d after birth in BPD group were significantly lower than the non-BPD group,and DBP in moderate/severe BPD group were significantly lower than the mild BPD group [(373.9± 19.1) μg/ml vs.(391.4±23.6) μg/ml],the differences were both statistically significant (P＜0.05).Multivariate analysis showed that the high serum 25-(OH)D level at birth (OR=0.827,95％CI0.693～0.987) was protective factors for BPD,while neonatal pneumonia (OR=4.331,95％CI 1.269～14.784) and neonatal sepsis (OR=4.020,95％CI 1.153～14.015) were risk factors for BPD.Conclusion The high serum 25-(OH) D level at birth in preterm infants was protective factors for BPD,while neonatal pneumonia and sepsis were the risk factors for BPD.Moreover,low serum 25-(OH) D level at birth and low serum DBP level at 28 d after birth maybe useful indicators for the severity of BPD.

Objective:To evaluate the therapeutic effect of electroacupuncture on acute gastrointestinal injury (AGI) in patients with severe traumatic brain injury (sTBI).Methods:A prospective randomized controlled trial was conducted. 126 consecutively hospitalized patients with AGI after sTBI admitted to intensive care unit (ICU) of the First Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine from January 2018 to December 2019 were enrolled. The patients were divided into observation group and control group by random number table. All the patients of two groups were given conventional treatment of western medicine for consecutive 7 days, including the treatments of primary diseases, indwelling nasogastric tube to extract gastric contents every 6 hours to determine gastric residual volume (GRV). When vital signs were basically stable, enteral nutrition (EN) was implemented and EN feeding amount and speed were adjusted according to GRV. On the basis of conventional western medicine treatment, the observation group was treated with electroacupuncture at Zusanli, Tianshu, Shangjuxu, Xiajuxu and Zhongwan, once in the morning and once in the evening, 30 minutes each time. The gastrointestinal function parameters including intra-abdominal pressure (IAP), serum diamine oxidase (DAO) and gastrointestinal failure (GIF) scores were observed before treatment and at day 3 and day 7 of treatment. The incidence of ICU hospital-acquired pneumonia (HAP-ICU), duration of mechanical ventilation (MV), length of ICU stay, 28-day mortality and adverse reactions of electroacupuncture were also observed in the two groups. Kaplan-Meier method was used for 28-day survival analysis.Results:During the 7-day treatment and observation, 26 cases of 126 patients withdrew from the study, and 100 cases were actually enrolled, 50 cases in the observation group and 50 cases in the control group. IAP and DAO at day 3 of treatment in both groups were significantly lower than those before treatment [control group: IAP (cmH 2O, 1 cmH 2O = 0.098 kPa) was 13.75±2.76 vs. 18.11±3.97, DAO (U/L) was 129.88±24.81 vs. 158.01±22.64; observation group: IAP (cmH 2O) was 13.56±2.19 vs. 18.50±3.54, DAO (U/L) was 129.11±29.32 vs. 159.36±28.65; all P < 0.01]. The gastrointestinal function parameters of the two groups improved gradually with the extension of treatment time, and the IAP, DAO and GIF scores at day 7 of treatment in the observation group were significantly lower than those in the control group [IAP (cmH 2O): 11.28±3.61 vs. 12.68±3.23, DAO (U/L): 49.69±17.56 vs. 57.27±20.15, GIF score: 2.02±0.74 vs. 2.40±0.70, all P < 0.05). The duration of MV and the length of ICU stay in the observation group were significantly shorter than those in the control group [duration of MV (days): 15.72±4.60 vs. 18.08±4.54, length of ICU stay (days): 16.76±4.68 vs. 19.26±5.42, both P < 0.05], and the incidence of ICU-HAP and 28-day mortality were significantly lowered (12.0% vs. 30.0%, 22.0% vs. 32.0%, both P < 0.05). Survival analysis showed that the 28-day cumulative survival rate in the observation group was significantly higher than that in the control group (86.4% vs. 76.1%; Log-Rank test: χ 2 = 37.954, P < 0.001). The patients in the observation group had no significant adverse reaction of electroacupuncture treatment. Conclusion:Electroacupuncture at corresponding acupoints can effectively improve gastrointestinal function in patients with AGI after sTBI, which is beneficial to shortening the length of ICU stay, promoting the recovery of the patients, and reducing the 28-day mortality.

Objective:To compare the clinical effects of microwave ablation (MWA) and surgical resection in the treatment of small hepatocellular carcinoma(SHCC).Methods:Sixty five SHCC patients with intact clinical data, treated in the Center of Hepatobiliary Surgery, Peking University People's Hospital between Feb 2005 and Aug 2012, were enrolled in this study. Among them, 30 patients were treated by MWA, and the other 35 by hepatectomy. Follow-up was conducted from Mar 2013 to Feb 2021. The differences in long-term survival, intraoperative blood loss, operative time, postoperative complications, performance status (PS), and postoperative hospital stay were compared between the two groups.Results:The survival probability at 1, 3, 5 and 10 years was 93.2%, 82.5%, 55.6% and 41.2%, respectively, in the MWA group, and 97.1%, 82.6%, 67.2% and 48.3%, in the resection group ( P=0.347). The MWA group had less perioperative complications, less blood loss, shorter operation time, better PS score and better hospital stay than the surgical resection group (all P<0.001).There was no statistically significant difference in the survival rate between BCLC stage 0~A1 and A2~A4 patients( P=0.773, 0.536). Conclusions:Microwave ablation in the treatment of small hepatocellular carcinoma can achieve similar results as hepatectomy with less traumatic,better postoperative PS score and shorter postoperative hospital stay.