Objective To obtain the Cyclin D1,the Cyclin D1 gene was cloned and expressed in Pichia pastoris.Methods The total RNA was extracted from liver cancer tissue.The Cyclin D1 cDNA was obtained by RT-PCR.The Cyclin D1 cDNA was sequenced.And sub-clonedto the Ppic9k,the Pichia pastoris expressed was used to obtain the Cyclin D1.Results The 483 bp Cyclin D1 cDNA was obtained.And the sequence of Cyclin D1 was corrected.The 36KD Cyclin D1 was obtained by Pichia pastoris expression.Conclusion The Cyclin D1 cDNA was cloned and Cyclin D1 was expressed in Pichia pastoris.

Objective To establish the rheumatoid arthritis (RA) mouse model induced by glucose-6-phosphate isomerase (GPI),and explore the mechanism of GPI in RA.Methods Totally 36 DBA/1 mice were randomly divided into three groups:test group (injection GPI),positive group (injection of bovine collagen Ⅱ),and negative group (saline).The rates and changes of weight were observed.The score of the arthritis,the ankle histopathological changes and serum GPI content were detected.Results Toes swollen slightly,joint swelling,deformity and accompanied by block were appeared at 35th day in the test group.Compared to the control group,the rates and changes of weight in test group showed a significant difference (P ＜ 0.05).The score of arthritis was showed by x ± s.Compared to the negative group,the test group and positive group were showed significant difference (P ＜ 0.05).A lot of lower synovial lining exudate macrophages,fibroblasts,and other inflammatory cells were increased in the test group.The GPI content in the test group [(0.39 ±0.11)μg/ml] was significantly higher than the negative group [(0.10± 0.06) μg/ml,P ＜ 0.05].Conclusions GPI could induce rheumatoid arthritis in mice.It provides the experimental basis to diagnose RA.

Objective To investigate the clinical application of fecal calprotectin in inflammatory bowel disease (IBD).Methods Colonoscopy took 79 patients with IBD that were diagnosed with pathology,including 47 cases of ulcerative colitis (UC) patients,32 cases of Crohn's disease (CD).Moreover,42 cases of IBD patients without abdominal pain,diarrhea and other intestinal inflammation were used as disease control group,and 34 cases of healthy people were used as healthy control group.The level of fecal calprotectin in each group was detected by enzyme-linked immunosorbent assay (ELISA).Results The positive rate of fecal Calprotectin in IBD group,disease control group and the healthy control group was 57.0％,19.0％,and 0,respectively; each positive rate in IBD group was significantly higher than the other two groups (P ＜ 0.05).The serum concentration of fecal calprotectin in IBD group [(493.86 ±204.18) μg/g] was significantly higher than the disease control group [(71.46 ± 60.51) μg/g] and the healthy control group [(36.19 ± 13.46) μg/g] (P ＜ 0.05) ; IBD active calprotection [(1015.23 ± 324.96) μg/g] was significantly higher than resting [(52.69 ±34.71) μg/g] (P ＜0.01).Conclusions Fecal calprotectin test benefits early diagnosis of IBD,and may be taken as the diagnostic index of IBD activity.It has extensively clinical value.

Objective To obtain the Cyclin D1 through cloning and prokaryotic expression of Cyclin D 1 gene.Methods The total RNA was extracted from liver cancer tissue .The Cyclin D1 cDNA was obtained by reverse transcriptase polymerase chain reaction (RT-PCR).The Cyclin D1 cDNA was sequenced, and sub-cloned to the PET32a+.The prokaryotic expressed was used to obtain the Cyclin D1.Results The 483 bp Cyclin D1 cDNA was obtained.The sequence of Cyclin D1 was corrected.The 36 KD CyclinD1 was obtained by prokaryotic expression .Conclusions The Cyclin D1 cDNA was obtained.Cyclin D1 was expressed in BL21.

Objective To investigate the value of urinary trehalase (T) in patients with renal proximal tubular damage.Methods 134 patients with kidney disease (male:66 female:68 age:18-59 ; 31cases with acute renal failure,30 cases with chronic renal failure,20 cases with drug-induced renal impairment,21 cases with renal transplantation and 32 cases with nephritic syndrome) and 101 healthy controls (58 males and 43 females) were selected.Urinary T,N-acetyl-D-glucosaminidase (NAG),β2-MG,gamma-glutamyl transferase (GGT) were detected.Data were analyzed by SPSS 11.5,including nonparametric rank test,ROC analysis.Results The level of urinary trehalase in control group was normally distributed (7.1 ± 4.1) μmol/h · g Cr (0-25 μmol/h · g Cr).There was no significant difference between men and women (t =0.63,P =0.53).Urinary T levels were significant higher in all kidney disease groups than in control group (Z =6.80,5.90,5.23,6.00,8.04,P ＜0.01).According to ROC curve,the area of urinary T under the ROC curve (AUC) in 134 patients was 0.9,significantly different with NAG,β2-MG,GGT area (P ＜ 0.01),the AUCs of T were 0.94,0.85,0.90,0.90,0.91 in acute and chronic renal failure group,drug-induced renal impairment group,renal transplantation group and nephritic syndrome group,respectively; Youden index were 0.85,0.65,0.77,0.66,0.72 respectively.Corresponding to the Youden index,sensitivity and specificity were 90.3％ and 95.1％,73.3％ and 92.1％,85.0％ and 92.1％,81.0％ and 85.2％,87.5％ and 84.2％ respectively.Conclusions The Urinary trehalase is better than other markers in the diagnosis of the proximal renal tubular damage.It was better to evaluate the proximal tubular function early in time.The diagnostic value of urinary trehalase played a key role in diagnosis,treatment and prognosis of kidney diseases.

BACKGROUND:Bone marrow stem cells are defined by their multi-potential ability, and can be differentiated into intrinsic cells in the kidney.
OBJECTIVE:To study the effects of mobilizing autologous bone marrow stem cells by granulocyte colony-stimulating factor plus stem cellfactor on cellapoptosis and proliferation of rats with renal ischemia-reperfusion injury.
METHODS:Total y 160 male Sprague-Dawley rats were randomly divided into four groups:control group, model group, cytokine treatment group, cytokine control group. Rat models of unilateral renal ischemia-reperfusion injury were established in the model and cytokine treatment groups. Rats in the cytokine treatment group and cytokine control group received subcutaneous injection of granulocyte colony-stimulating factor (50μg/kg) and stem cellfactor (200μg/kg), once a day, for 5 continuous days. Rats in the model and control groups had no treatment. Apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method, and the expression of CD34-positive cells, Caspase-3, Bcl-2, proliferating cellnuclear antigen in the kidney were measured using immunohistochemistry staining.
RESULTS AND CONCLUSION:The number of CD34-positive cells in renal tissue of the cytokine treatment group was significantly higher than that of the control group and model group (P<0.05). The apoptotic index and expression of Capase-3 in the model group and cytokine treatment group were higher than those in the control group and cytokine control group (P<0.05). The apoptotic index and expression of Capase-3 in the cytokine treatment group were lower than that in the model group (P<0.05). The expression of Bcl-2 in the model group and cytokine treatment group was higher than that in the control group and cytokine control group (P<0.05). The expression of Bcl-2 in the cytokine treatment group was higher than that in the model group (P<0.05);however, as time went on, Bcl-2 expression was obviously decreased. Proliferating cellnuclear antigen expressed both in the model group and in the cytokine treatment group. Additional y, the proliferative index reached peak at 24 days in the model group, and then decreased gradual y;while in the cytokine treatment group, it reached the peak at 10 days, maintained a high level until the 17th day, and then decreased gradual y. Mobilization of autologous bone marrow stem cells by combination of granulocyte colony-stimulating factor and stem cellfactor can increase proliferation and decrease apoptosis of renal tubular epithelial cells after renal ischemia-reperfusion injury, and thus, promote the recovery from renal tubular injury.

Objective:To understand whether there are drug resistant and disinfectant resistant Yersinia pestis strains in China, and to provide accurate information for clinical treatment of plague. Methods:A total of 2 753 Yersinia pestis strains isolated from 10 natural plague foci in China from 1943 to 2016 were collected. According to National Center for Biotechnology Information (NCBI) released sequences of aminoglycoside streptomycin resistant genes strA, strB, β-lactam antibiotics resistant genes TEM, SHV and CTX-M, sulfamilamide resistant genes sul1, sul2 and sul3, and disinfectant resistant gene qacE△1-sul1, a pair of primers of each gene was designed for above-mentioned genes. Genomic DNA of 2 753 strains of Yersinia pestis was extracted, and the 9 target genes of all DNA samples were amplified by PCR. Results:Negative and positive controls of PCR detection were established. No corresponding target bands of aminoglycoside streptomycin resistant genes strA, strB, β-lactam antibiotics resistant genes TEM, SHV and CTX-M, sulfamilamide resistant genes sul1, sul2 and sul3, and disinfectant resistant gene qacE△1-sul1 were found in the DNA samples of 2 753 strains of Yersinia pestis.Conclusion:The above-mentioned genes of drug resistance and disinfectant resistance have not been detected in Yersinia pestis of China, but the monitoring of drug resistance of Yersinia pestis still needs to be carried out continuously.

Objective:To investigate the drug resistance of Yersinia pestis to 11 kinds of antibiotics in the natural foci of plague in Inner Mongolia Autonomous Region, and to provide a theoretical basis for scientifically and effectively selecting antibiotics for treatment of the plague. Methods:A total of 137 strains of Yersinia pestis isolated from the natural foci of plague in Inner Mongolia Autonomous Region at different times, regions, hosts and vectors were collected. According to Clinical and Laboratory Standard Institute (CLSI), the agar plate dilution method was used to determine the minimum inhibitory concentration (MIC) of the 11 kinds of antibiotics against 137 strains of Yersinia pestis, including ofloxacin, ciprofloxacin, kanamycin, streptomycin, ceftriaxone, ampicillin, chloramphenicol, spectinomycin, cefuroxime, tetracycline and sulfamethoxazole-trimethoprim. The MIC 50 and MIC 90 (the minimum concentration of drug which could inhibit 50% and 90% of bacterial growth) were calculated, and their sensitivity was determined according to CLSI standards. Results:Among 137 strains of Yersinia pestis tested, no strains of Yersinia pestis had single or multiple resistance to ofloxacin, ciprofloxacin, kanamycin, streptomycin, ceftriaxone, ampicillin, chloramphenicol, spectinomycin, cefuroxime, tetracycline and sulfamethoxazole-trimethoprim. According to CLSI standards, 137 strains of Yersinia pestis were all sensitive to the 11 kinds of antibiotics; among them, ofloxacin, ciprofloxacin, ceftriaxone and sulfamethoxazole-trimethoprim had higher antibacterial activity, with MIC 90 < 0.250 μg/ ml; the antibacterial activity of spectinomycin was the lowest, with MIC 90 of 16.000 μg/ml. Conclusions:The Yersinia pestis isolated from the natural foci of plague in Inner Mongolia Autonomous Region is not found to have single or multiple resistance to the 11 kinds of antibiotics. Continuous drug resistance monitoring of Yersinia pestis should be carried out to provide a basis for clinical medication.

Abstract: Objective To understand the phenotypic and genetic characteristics of Yersinia pestis strains isolated from Himalayan marmot natural focus area and domestic rat plague focus area in southern China, and provide reference for mastering the pathogenic characteristics of Yersinia pestis of two plague foci. Methods A total of 412 of Yersinia pestis strains isolated from Himalayan marmot plague focus and domestic rat plague focus of southern China were subjected to to sorbitol fermentation assays, virulence factor, different region (DFR) typing, and clustered regularly interspaced palindromic repeats (CRISPR) typing. Results The biochemical types of Y. pestis from the two plague foci showed distinct regional distribution features. Five biochemical phenotypes were identified in Yersinia pestis isolated from Himalayan marmot natural focus area, while only one biochemical phenotype was identified in strains isolated from the domestic rat plague focus of Southern China. Most of the Yersinia pestis isolated from the two plague foci were capable of producing the virulence factors of Fl and PstI. Among the strains from Himalayan marmot focus, 70.53% (201/285) were VW-positive, 75.09% (214/285) were Pgm-positive, 20.00% (57/285) of the strains were Pgm-negative, and 5.26% (15/285) were Pgm mixed-type strains. Among strains from domestic rat plague focus of southern China, 37.80% (48/127) were VW-positive, 29.13% (37/127) were Pgm-positive, 58.27% (74/127) were Pgm-negative, and 12.60% (16/127) were Pgm mixed-type strains. DFR typing revealed 22 genotypes of Y. pestis from the Himalayan marmot plague focus, with the main genotypes being type 5, 7, 8, 10, 19, 32 and 49. All strains from domestic rat plague focus area in southern China belonged to type 9. CRISPR typing revealed that all strains from the Himalayan marmot natural focus were classified into 7 CRISPR gene clusters and 14 CRISPR genotypes, with the main genotypes being G7, G22, G26-a1'and G22-A1'. All strains from domestic rat plague focus area in southern China belonged to CRISPR genotype G30, with the gene cluster being Ca8. Conclusions The phenotypes and genotypes of the Yersinia pestis of Himalayan marmot plague focus are diverse, with an obvious characteristics of geographical distribution. The phenotype and genotype of the Yersinia pestis of domestic rat plague focus of Southern China are single. DFR and CRISPR genotyping methods with phenotypic characteristics can effectively identify the Yersinia pestis isolated from the two plague foci, thereby meeting the needs of identification and traceability research.

Objective: To investigate the surgical complications in the treatment of stage Ⅰ endometrial cancer by robotic-assisted laparoscopy, the risk degree of Clavein-Dindo complications and the main risk factors affecting the occurrence of surgical complications. Methods: A retrospective case-control study was conducted in the First Affiliated Hospital of Zhengzhou University from October 2014 to June 2019. The patients were divided into robotic-assisted laparoscopy group and traditional laparoscopy group according to the operation mode, including 131 cases in robot group and 290 cases in traditional laparoscopy group. To compare the complications during and after operation and the risk degree of complications between the two groups by Clavein-Dindo classification standard, the age, body mass index (BMI), comorbidities, past history of pelvic surgery, American Society of Anesthesiologists (ASA) grade, preoperative anemia, number of pelvic lymph node resection, number of abdominal aortic lymph node resection, the total number of lymph node resection, operation time, surgical methods (robot surgery or traditional laparoscopic surgery) and other clinicopathological data were analyzed by logistic regression analysis. Results: (1) Complications of operation: the incidence of operative complications (including intraoperative and postoperative complications) in robot group was significantly lower than that in traditional laparoscopy group [(20.6%, 27/131) vs (34.8%, 101/290); χ²=8.620, P=0.003)]. The incidence of intraoperative complications in robot group was lower than that in traditional laparoscopy group [1.5% (2/131) vs 6.2% (18/290); χ²=4.368, P=0.037]. The incidence of intraoperative vascular injury in robot group was significantly lower than that in traditional laparoscopy group [0.8% (1/131) vs 5.2% (15/290); χ²=4.798, P=0.022]. The incidence of postoperative complications in robot group was also lower than that in traditional laparoscopy group [19.1% (25/131) vs 28.6% (83/290); χ²=4.303, P=0.038], but the incidence of postoperative lymphatic leakage in robot group was higher than that in traditional laparoscopy group [10.7% (14/131) vs 5.2% (15/290); χ²=4.279, P=0.039]. (2) Clavein-Dindo classification: the incidence of Clavein-Dindo Ⅰ, Ⅲ, Ⅲ, Ⅳ and Ⅴ grade between two groups were respectively 3.8% (5/131) vs 11.0% (32/290), 13.7% (18/131) vs 14.5% (42/290), 3.1% (4/131) vs 8.6% (25/290), 0 (0/131) vs 0.3% (1/290), 0 (0/131) vs 0.3% (1/290), and the incidence of grade Ⅰ (χ²=5.684, P=0.015) and Ⅲ (χ²=4.361, P=0.037) complications were statistically significant. The incidence of severe complications in robot group (grade Ⅲ and above) was lower than that in traditional laparoscopy group [3.1% (4/131) vs 9.3% (27/290); χ²=5.179, P=0.023]. (3) Analysis of influencing factors of surgical complications: univariate analysis showed that BMI (χ²=15.801, P=0.000), preoperative anemia (χ²=14.299, P=0.000), total number of lymph node resection (χ²=10.425, P=0.001), surgical methods (χ²=8.620, P=0.003) were related to the occurrence of surgical complications of endometrial carcinoma. Multivariate analysis showed that BMI (OR=0.289, 95%CI: 0.097-0.864, P=0.026), preoperative anemia (OR=0.309, 95%CI: 0.129-0.740, P=0.008), the total number of lymph node resection (OR=0.624, 95%CI: 0.403-0.966, P=0.034) and surgical methods (OR=3.491, 95%CI: 1.030-11.840, P=0.045) were independent risk factors for surgical complications of endometrial carcinoma. Conclusions Compared with traditional laparoscopic surgery, robot-assisted laparoscopic surgery has fewer complications and lower incidence of severe complications. BMI, preoperative anemia, the total number of lymph node resection and surgical methods are independent risk factors for the occurrence of surgical complications of stage Ⅰ endometrial cancer.