Objective To establish a stable rat model of non-obese polycystic ovary syndrome(PCOS)with clinical characteristics.Methods Dehydroepiandrosterone(DHEA)was used to establish a PCOS rat model by subcutaneous injection.Three-week-old female SD rats were divided into a normal group,6 mg/kg DHEA model group,and 60 mg/kg DHEA model group.The model groups were subcutaneously injected with the corresponding dose of DHEA daily,while the normal group was subcutaneously injected with glycerol daily for 21 consecutive days.The model was evaluated with ovarian histopathology as the gold standard to determine the optimal dosage of DHEA to induce a PCOS rat model.On this basis,the optimal DHEA modeling dose was selected,and stop and continue modeling groups were set up to observe the model for 28 days and evaluate its maintenance.The stop modeling group was no longer given DHEA,and the continued modeling group was subcutaneously injected with 60 mg/kg DHEA every 48 h.The evaluation indicators included body mass,estrous cycle,fasting blood glucose,serum insulin,histopathologic morphology of the ovaries,and serum sex hormone levels.Results(1)Compared with the normal group,the 6 mg/kg and 60 mg/kg DHEA model groups showed no significant difference in body mass,and their estrous cycles were irregular.There were more cystically dilated large follicles in the ovaries;fewer mature follicles;reduced layers of granulosa cells,which were arranged in a sparse and disorganized manner;and fewer lutea in the 6 mg/kg and 60 mg/kg DHEA model groups than the normal group.Furthermore,serum T and E2 levels were significantly higher in the 60 mg/kg DHEA model group(P＜0.05)than the normal group.(2)The stop modeling group(A2 group)resumed regular estrous cycles after 2 weeks,various growth follicles and corpora lutea were observed in the ovarian tissues,the number of cystic follicles was reduced,the number of granulosa cell layers increased,mature follicles were visible,oocyte morphology was locally intact,and the levels of E2 and AMH were reduced compared with the normal group(A1 group)(P＜0.05).(3)The continue model group(B2 group)was in the late stage of estrous cycle for a long period,and there were more large follicles with cystic dilatation,fewer mature follicles,fewer layers of granulosa cells with a sparse and disordered arrangement,and significantly fewer corpus lutea in the ovaries compared with the normal group(B1 group).The levels of serum LH,LH/FSH,and T were elevated(P＜0.05).Conclusions Subcutaneous injection of 60 mg/kg DHEA for 21 consecutive days can be used to successfully construct a non-obese PCOS rat model that possesses clinical characteristics.Subcutaneous injection of 60 mg/kg DHEA every 48 hours maintains the stability of the model.

Obesity and related metabolic syndromes have been recognized as important disease risks,in which the role of adipokines cannot be ignored.Adiponectin(ADP)is one of the key adipokines with various beneficial effects,including improving glucose and lipid metabolism,enhancing insulin sensitivity,reducing oxidative stress and inflammation,promoting ceramides degradation,and stimulating adipose tissue vascularity.Based on those,it can serve as a positive regulator in many metabolic syndromes,such as type 2 diabetes(T2D),cardiovascular diseases,non-alcoholic fatty liver disease(NAFLD),sarcopenia,neurodegenerative diseases,and certain cancers.Therefore,a promising therapeutic approach for treating various metabolic diseases may involve elevating ADP levels or activating ADP receptors.The modulation of ADP genes,multimerization,and secretion covers the main processes of ADP generation,providing a comprehensive orientation for the development of more appropriate therapeutic strategies.In order to have a deeper understanding of ADP,this paper will provide an all-encompassing review of ADP.

Objective:To analyze the characteristics of drug resistance genes in a Klebsiella pneumoniae strain coproducing carbapenemases KPC-2 and NDM-5. Methods:Klebsiella pneumoniae KPN-hnqyy was separated from the stool specimen of a patient in the Hematology Department of Affiliated Cancer Hospital of Zhengzhou University. The strain was identified with a BD Phenix-M50 automated microbiology system and the minimum inhibitory concentration against the strain was measured as well. The genotypes of the carbapenemases were tested by enzyme immunochromatographic assay and PCR method. The transferability of related plasmids was analyzed by conjugation test. Whole-genome sequencing of the strain was conducted using PacBio and Illumina platforms. The MLST type, resistance gene and plasmid type of the strain were retrieved in BacWGSTdb. The genome and open reading frame sequence of the strain were compared using Easyfig_2.2.3. Visual cycle graphs were generated using BRIG v0.95. Results:Klebsiella pneumoniae KPN-hnqyy was resistant to carbapenem antibiotics. It belonged to ST11 and carried two carbapenemase genes of blaKPC-2 and blaNDM-5. The conjugant only harbored the blaKPC-2 gene. Whole-genome sequencing revealed that the strain contained one chromosome and three plasmids. Its chromosome genome shared more than 99.9% similarity with that of Klebsiella pneumonia KP69 and KP19-2029. Moreover, a similar IncR and IncFⅠ resistance gene fusion region was contained in different types of plasmids carried by them: the blaKPC-2 gene was located in a structure—which evolved from the Tn3-△Tn4401-Tn1721/Tn1722 sequence—inside this fusion region with its ends inserted into the transposase IS26 gene; the blaNDM-5 gene was located on a transposon containing the special plasmids of the insertion fragment in phages, with its ends inserted into the transposase IS26 gene too. Conclusions:The IncR and IncFⅡ resistance gene fusion region of blaKPC-2 carried by Klebsiella pneumoniae ST11 might be widely coexistent with the chromosomal genome. The blaNDM-5 gene carried by special plasmids might be accidentally obtained through gene recombination mediated by transposable element IS26. The wide transmission of Klebsiella pneumoniae ST11 carrying the blaKPC-2 gene in China and its ability to obtain other carbapenemase genes through transposable element IS26 were well worth attention.

Objective:To evaluate the clinical value of percutaneous transhepatic one-step biliary fistulation(PTOBF)lithotomy plus laparoscopic cholecystectomy(LC) in the treatment of choledocholithiasis combined with cholecystolithiasis.Methods:From Jul 2012 to Jun 2018, 44 patients with cholecystolithiasis and choledocholithiasis were treated by PTOBF + LC ( n=20) vs laparoscopic common bile duct exploration(LCBDE)+ LC( n=24). Results:The success rate of one-step operation in both groups was 100%.The average intra-operative hemorrhage and the average hospital stay after operation were higher in LCBDE+ LC group (all P<0.05). The post-operative complication rate of PTOBF lithotomy + LC group was 10.0% (2/20), recurrence rate of observation period was 10.0% (2/20), while that of LCBDE+ LC group was 8.3% (2/24), and 12.5% (3/24), the difference was not statistically significant (all P>0.05). Conclusion:PTOBF lithotomy combined with LC is a safe, effective and feasible minimally invasive method for the treatment of choledocholithiasis combined with gall stones.

Background: An in vitro study on rapid culturing method of human gingival fibroblast cells (HGFCs) was established to investigate the potential use of the leukocyte-platelet rich fibrin (L-PRF) in tissue engineering technology, different medical fields, including periodontology and implantology. Methods: Eight biopsies were obtained from eight different donors and a modified culturing technique was developed to obtain HGFCs. The modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay was used to compare the cell viability when the modified culturing method was used in comparison to the standard method. Blood samples were collected from the same patients and L-PRF was isolated using a standard protocol. The releases of platelet-derived growth factor-AA and transforming growth factor-beta1 at various time intervals were observed using enzyme-linked immunosorbent assay (ELISA) kit. The proliferative effect of L-PRF on HGFCs was assessed by the cell counting kit—8 assay. Results: A simple and rapid modified method for in vitro HGFC culture yielded a cellular monolayer within three to nine days after cell culture. L-PRF with three-dimensional polymer fibers released growth factors that peaked during the first three hours and continued to produce up to 10 days. The L-PRF presented a dose-dependent effect on HGFCs proliferation where HGFCs proliferation increased with an increase in L-PRF concentration. Conclusion The modified technique for the culture of HGFCs might be useful for the development of future experimental and clinical studies, besides L-PRF has great therapeutic potential in oral surgery fields.

Objective:To investigate the key mechanism of transforming growth factor-β (TGF-β) inducing the expression of interleukin-17D (IL-17D) in lung cancer-associated fibroblast (CAF) and promoting the recruitment of myeloid-derived suppressor cells (MDSCs).Methods:C57BL/6 mice were established for B16 lung melanoma metastasis model (tumor model group), and control group was set up, 6 mice in each group. Flow cytometry (FACS) was used to detect the lung CAF and the changes of its ability to secrete IL-17D and the proportion of MDSCs in tumor mice. The changes of TGF-β level in lung tumor were examined by ELISA and quantitative real-time PCR (RT-qPCR). Lung fibroblasts were screened by FACS, and the effects of TGF-β on the secretion of IL-17D, C-C motif chemokine ligand (CCL)2 and CCL7 in fibroblasts were detected by RT-PCR. The migration of MDSCs under the condition of TGF-β stimulating fibroblasts was detected by Transwell.Results:The proportion of CAF (CD45 -CD326 -CD31 -) in the tumor model group was higher than that in the control group [(28.02±2.23)% vs. (7.35±2.14)%, t=9.956, P<0.001]. The ability of CAF to secrete IL-17D in the tumor model group was significantly higher than that in the control group [(38.27±2.93)% vs. (19.04±3.16)%, t=5.995, P=0.001]. The proportion of MDSCs in the tumor model group was significantly higher than that in the control group [(12.93±1.27)% vs. (8.21±1.40)%, t=4.804, P=0.009]. Compared with the control group, the protein and transcription levels of TGF-β in lung of the tumor model group were significantly increased [(1 685.07±135.61) ng/L vs. (1 047.98±68.50) ng/L, t=5.051, P=0.002; 2.17±0.03 vs. 1.00±0.05, t=51.237, P<0.001]. In vitro, lung fibroblasts were stimulated with different concentrations of TGF-β (0, 5 and 10 μg/L) for 24 hours, the relative expressions of IL-17D mRNA secreted by stimulated fibroblasts were 0.42±0.01, 0.67±0.01 and 0.84±0.04 respectively, the relative expressions of CCL2 mRNA in each group were 0.89±0.08, 1.08±0.04, 1.19±0.01 and CCL7 were 0.53±0.05, 0.65±0.04, 0.74±0.03 respectively. With the increase of TGF-β concentration, the expression levels of IL-17D, CCL2 and CCL7 in fibroblasts were significantly increased ( F=57.384, P<0.001; F=15.802, P=0.004; F=14.544, P=0.005). In addition, compared with the control group (0 μg/L TGF-β), fibroblasts treated with 10 μg/L TGF-β for 24 hours could promote the migration of MDSCs in spleen of tumor mice [(9.59±0.21)% vs. (2.14±0.24)%, t=6.585, P<0.001]. Conclusion:TGF-β can induce high expression of IL-17D in lung CAF, which is an important factor in promoting the expressions of CCL2 and CCL7 and the migration of MDSCs in tumor microenvironment.

Background: An in vitro study on rapid culturing method of human gingival fibroblast cells (HGFCs) was established to investigate the potential use of the leukocyte-platelet rich fibrin (L-PRF) in tissue engineering technology, different medical fields, including periodontology and implantology. Methods: Eight biopsies were obtained from eight different donors and a modified culturing technique was developed to obtain HGFCs. The modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay was used to compare the cell viability when the modified culturing method was used in comparison to the standard method. Blood samples were collected from the same patients and L-PRF was isolated using a standard protocol. The releases of platelet-derived growth factor-AA and transforming growth factor-beta1 at various time intervals were observed using enzyme-linked immunosorbent assay (ELISA) kit. The proliferative effect of L-PRF on HGFCs was assessed by the cell counting kit—8 assay. Results: A simple and rapid modified method for in vitro HGFC culture yielded a cellular monolayer within three to nine days after cell culture. L-PRF with three-dimensional polymer fibers released growth factors that peaked during the first three hours and continued to produce up to 10 days. The L-PRF presented a dose-dependent effect on HGFCs proliferation where HGFCs proliferation increased with an increase in L-PRF concentration. Conclusion The modified technique for the culture of HGFCs might be useful for the development of future experimental and clinical studies, besides L-PRF has great therapeutic potential in oral surgery fields.

Objective:To investigate the effects of body mass index (BMI) levels at different baseline on the risk of new-onset acute pancreatitis (AP).Methods:The subjects were from the Kailuan Study Cohort and divided into 3 groups according to baseline BMI levels: BMI<24 kg/m 2, normal weight; BMI 24-28 kg/m 2, overweight; BMI≥28 kg/m 2, obesity. The incidence of new-onset AP in these three groups was analyzed. The survival curve was plotted by Kaplan-Meier method, the cumulative incidence was calculated and tested by log-rank method. Multivariate Cox proportional hazards regression model was used to calculate HR of baseline BMI levels for AP. Results:A total of 123 841 subjects were included and followed up for (11.94±2.13) years, during which, 395 cases were found with AP. The incidence of AP was 2.67 per 10 000 person years in total population, and the incidences of AP were 2.20, 2.72 and 3.58 per 10 000 person-years in the normal, overweight and obesity groups, respectively. The cumulative incidences of AP was 0.32%, 0.40% and 0.49% in normal, overweight and obesity groups, respectively, which showed a significant inter-group difference by log-rank test ( χ2=13.17, P<0.01). The results of multivariable adjusted Cox proportional hazards regression model analysis indicated that obesity group ( HR=1.45, 95% CI: 1.10-1.92) had a higher risk for AP compared with the normal BMI group. The subgroup analyses by age and sex showed that compared with the normal weight group,the HRs for AP in the obesity group was 1.58(95% CI:1.14-2.19) and 1.40(95% CI:1.03-1.90) among subjects younger than 60 years old and male subjects, respectively. After excluded onset AP within two years from baseline,with a control group from normal weight,the results of multivariate Cox proportional hazards regression model analysis indicated that the AP in the obesity group was 1.60 (95% CI: 1.18-2.15). Conclusion:Obesity may increase the risk of developing AP, particularly among young and middle-aged men.

Objective To study the efficacy and safety of percutaneous transhepatic one-step biliary fistulation (PTOBF) with rigid cholangioscopic lithotripsy for treatment of complicated hepatolithiasis under the ultrasonic navigation technique.Methods In this retrospective study,PTOBF lithotripsy surgery was performed in 94 patients with hepatolithiasis under general anesthesia with tracheal intubation,and with percutaneous transhepatic puncture of targeted bile duct under ultrasonic navigation in The First Affiliated Hospital of Guangzhou Medical University.Biliary expanders were used along a guidewire to expand the sinus gradually until 14Fr to establish a fistulous channel.Lithotripsy was then performed through the channel by rigid cholangioscopy.The operation-related data were collected and analyzed,including puncture and fistula establishment success ratio,complication rate,intraoperative blood loss,residual and recurrence hepatolithiasis rates.Results 94 patients (total 122 patient-times) underwent PTOBF lithotripsy.There was no perioperative mortality.The overall puncture success rate was 100％,and the fistula/puncture rate was 97.5％ (119/122).In 118 patients success was achieved in 2 time (96.7％).The complication rate was 9.6％ (9/94).The average intraoperation blood loss were (24.9 ± 21.3)ml.The residual calculus rate after therapy was 13.8％ (13/94).All patients were followed-up for a period that ranged between 18 and 30 months.The recurrence rate was 14.9％ (14/94).Conclusions Ultrasonic navigation technique plays an important role in bile duct puncture,sinus expansion and rigid cholangioscopic lithotripsy for treatment of complicated hepatolithiasis.PTOBF lithotripsy is a safe and effective procedure,which provides a new way in mini-invasive treatment for hepatolithiasis.It is worth generalizing.

Objective:To analyze the characteristics of plasmids in KPC-2-producing Serratia marcescens ( S. marcescens) isolates. Methods:Four carbapenem-resistant S. marcescens strains were isolated from four patients admitted to the hepatobiliary ward of Affiliated Cancer Hospital of Zhengzhou University in 2016. BD Phenix-100 was used to identify the strains and detect the minimum inhibitory concentrations (MICs). Homology analysis was performed using pulsed-field gel electrophoresis (PFGE). The modified Hodge test was used to detect the phenotypes of carbapenemase. PCR and gene sequencing were used to detect the types of carbapenem resistance genes. The transferability of plasmids was detected by conjugation test. The characteristics of plasmids were analyzed by genomic alignment method after whole genome sequencing. DNAMAN V9 software was used to compare the amino acid sequences of the replication initiation proteins. A phylogenetic tree was constructed with neighbor-joining method using MEGA7.0. Results:All of the four S. marcescens strains were resistant to carbapenem antibiotics. They were highly homologous according to PFGE. Hodge test results were all positive and the carbapenemase genotype was blaKPC-2. Conjugation test results were positive. The plasmid was a circular DNA of 42 742 bp in length. It had the similar skeleton of incX6 plasmid and the similar amino acid sequence of replication initiation protein. Moreover, it and incX6 plasmid were at the same node in the phylogenetic tree. The blaKPC-2 was located in the core of drug resistance, which was composed of insertion elements including Tn3 family transposons, recombinant enzyme genes, △ISKpn6 and ISKpn27. Conclusions:The plasmid was incX6-like. The blaKPC-2 gene was located in the transposon of △Tn6296. More attention should be paid to the bacteria carrying KPC-2 in incX plasmids.