Objective To study isolation and identification and culture of rat type A spermatogonial cells in vitro.Methods Percoll discontinue density gradient centrifugation combined with different speeds of different cells adhering to dish was used to purify the type A spermatogonial cells.The c-kit and TERT special antibodies were used to identify the type A spermatogonial cells.The purified cells were cultured in vitro. Results 0.614?10~6 cells per testis finally were obtained and the percentage of viable cells was 92.1% by trypan blue dye exclusion test.The percentage of type A spermatogonial cells expressing c-kit and TERT were 91.7?1.2% and 90.8?1.0% respectively.Type A spermatogonial cells could proliferate and self-renew in the DMEM containing 10% NBS.Conclusion Percoll discontinue density gradient centrifugation combined with different speeds of different cells adhering to dish is an efficiency method for isolation of rat type A spermatogonial cells.The purified cells are type A spermatogonial cells by identification of the immunohistochemistry of c-kit and TERT antibodies.Type A spermatogonial cells can proliferate and self-renew in vitro.

Objective Aspirin is a non steroidal anti-inflammatory drugs, whether or not which does harm to renal function in the course of using, there is a big debate at home and abroad.In this paper, The aim of this study was to observe the effects of different doses of aspirin on renal function in elderly patients with cardiovascular disease at different ages.MethodsWe Selected a total of 288 patients with cardiovascular disease from January 2014 to March 2016 in Zhejiang Rongjun hospital.There were 288 cases of cardiovascular disease, including 144 cases of normal uric acid, 144 cases with hyperuricemia (including gout).The two groups according to the age of 60 to 70 years, 71 to 80 years old, over 80 years old respectively into three subgroups, each subgroup of 48 cases;Each subgroup was randomly selected without the use of aspirin (0mg/d) and the use of different doses of aspirin (50mg/d, 100mg/d, 150mg/d) for a total of four groups, each which is 12 patients.We detect patients routine blood tests, liver and kidney function, blood urea nitrogen (BUN), blood creatinine (SCR) levels and glomerular filtration rate (GFR) (MDRD) and blood and urine beta 2-microglobulin used aspirin before and used after 6 months and 12 months.ResultsBefore treatment, there were no significant differences in body mass index (BMI), blood pressure, hemoglobin, serum albumin and platelet count between various groups of the same age group.After treatment for 6 and 12 months,Uric acid normal patients and high uric acid patients with 0mg/d, 50mg/d, 100mg/d or 150mg/d dose of aspirin has no statistical difference in BUN, Scr and eGFR, 2-microglobulin Ccr, blood, urine beta 2-microglobulin.ConclusionRegardless of uric acid, different doses of aspirin had no significant effect on renal function in elderly patients with cardiovascular disease, and can be safely used.

OBJECTIVETo investigate the effects of c-jun on hCG-induced testosterone secretion in isolated rat Leydig cells by antisense oligodeoxynucleotides(ASODNs).

METHODSc-jun ASODNs were used to antagonise the effects of c-jun, hCG was used to induce the testosterone secretion of LC cultured in vitro and testosterone was measured by radioimmunoassay.

RESULTSThe testosterone secretion of LC in vitro could be induced by hCG, which was a good model for the functional study of LC. c-jun ASODNs decreased the hCG-induced testosterone secretion of LC in a dose-dependent manner(P < 0.05).

CONCLUSIONIt is suggested that c-jun proto-oncogene enhances the testosterone secretion of LC.

Animals ; Cells, Cultured ; Chorionic Gonadotropin ; pharmacology ; Dose-Response Relationship, Drug ; Leydig Cells ; secretion ; Male ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins c-jun ; physiology ; Rats ; Rats, Sprague-Dawley ; Testosterone ; secretion

Objective To investigate the mechanism of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair in the treatment of hypertension based on the network pharmacology method and animal experiment verification.Methods(1)TCMSP,BATMAN and TCMIP databases were used to screen the active components and targets of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair.The hypertension-related targets were obtained by searching the Drugbank,Genecard,TTD and Disgenet databases.The intersection(common target)of the active component target and the target related to hypertension disease was taken,and the obtained intersection target was the potential target of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair for the treatment of hypertension.The active ingredients and their targets of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair were imported into Cytoscape 3.9.1 software to construct a'Chinese medicines-active ingredients-targets'network and screen key active ingredients.The protein-protein interaction(PPI)network of potential targets was constructed to screen potential core targets.The Metascape platform was used to analyze the GO function and KEGG pathway enrichment of potential targets.The key active components and potential core targets were selected for molecular docking verification.(2)Thirty male spontaneously hypertensive rats(SHR)were randomly divided into model group,western medicine group(Candesartan Cilexetil,0.72 mg·kg-1)and low-,medium-and high-dose groups of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma(2.25,4.50,9.00 g·kg-1).Another male WKY rats were selected as blank group,with 6 rats in each group,once a day for 8 weeks.The systolic blood pressure of rat tail artery was detected before administration and 2,4,6 and 8 weeks after drug intervention.The pathological changes of thoracic aorta were observed by HE staining.The protein expression levels of GRP78,CHOP and Caspase-12 in aorta abdominalis were detected by Western Blot.Results(1)A total of 83 active components of Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma were obtained,and 158 potential targets(intersection targets)for the treatment of hypertension were screened out.Five key active ingredients:p-hydroxybenzoic acid,4-hydroxybenzylamine,tanshinone I,tanshinone,γ-sitosterol;6 potential core targets:IL6,TNF,CASP3,JUN,PTGS2,IL1B;GO functional enrichment analysis obtained 1 826 biological process items,89 cell component items,and 199 molecular function items.KEGG pathway enrichment analysis obtained 186 pathways,mainly involving neuroactive ligand-receptor interaction,calcium signaling pathway,inflammatory response(such as TNF and MAPK signaling pathway),vascular protection(such as HIF-1 and cAMP signaling pathway),oxidative stress(such as PI3K-Akt signaling pathway)and other signaling pathways.Tanshinone I and tanshinone had strong binding force to 6 potential core targets,and γ-sitosterol had strong binding force to IL6,CASP3,JUN,PTGS2 and IL1B.(2)Compared with the blank group,the systolic blood pressure of the model group was significantly increased(P＜0.01).The thoracic aortic endothelial injury was obvious,the endothelial cell morphology was abnormal,swelling and exfoliated cells could be seen,the intima of the tissue was disordered,the intima structure was incomplete,and the intima was thickened.The protein expressions of GRP78,CHOP and Caspase-12 in abdominal aorta were significantly increased(P＜0.01).Compared with the model group,the systolic blood pressure of the rats in the administration group was significantly decreased(P＜0.01);the injury of thoracic aorta was alleviated,and the morphology,intima structure and thickness of endothelial cells were improved to varying degrees.The protein expressions of GRP78,CHOP and Caspase-12 in abdominal aorta were significantly decreased(P＜0.01).Conclusion Gastrodiae Rhizoma-Salviae Miltiorrhizae Radix et Rhizoma drug pair may act on core targets such as IL6,TNF,CASP3,JUN,PTGS2,and IL1B through key active components such as p-hydroxybenzoic acid,tanshinone,and γ-sitosterol,and regulate key signaling pathways such as TNF signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway,and PERK signaling pathway to improve vascular endothelial dysfunction,inhibit endoplasmic reticulum stress,and lower blood pressure.

This article summarized the experience of Professor Xiong Jibai,a national TCM master,in treating pulmonary nodules based on the theory of"body fluids and blood stasis mixing"in Huang Di Nei Jing.Professor Xiong Jibai believes that the basic pathogenesis of pulmonary nodules is that"body fluids and blood stasis mixing"accumulate in lung collaterals,and the fundamental pathological factor is phlegm and blood stasis.Xiong's treatment is based on dissipating phlegm and activating qi,activating blood circulation and resolving masses,paying attention to syndrome differentiation and treatment,examining syndromes and seeking causes,flexibly selecting prescriptions and treating both symptoms and root causes;attaching importance to maintaining healthy qi,preventing both illness and change,and preventing recovery after illness.Clinical medical records were attached to prove the clinical thinking and medication characteristics.

Objective: To investigate the survival and prognostic factors of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) for patients with myeloid neoplasms and RUNX1 mutations. Methods: From July 2014 to April 2018, the clinical data of forty-two AML/MDS patients with RUNX1 mutations in the First Affiliated Hospital of Soochow University were retrospectively analyzed. The clinical characteristic features and distribution of the mutations frequently observed with RUNX1 mutations were summarized, the prognosis of allo-HSCT for these patients was also analyzed. Results: Among 42 AML/MDS patients with RUNX1 mutations, 27 were male, 15 were female. The median age was 43.5 (16-68) years old. There are 31 patients in allo-HSCT group and 11 patients in chemotherapy group. RUNX1 mutations co-occurred with many other gene mutations, the most frequent mutations were FLT3 (26.2%, 11/42) . Interestingly, FLT3 mutations only occurred in AML patients compared with MDS patients (P=0.014) . ASXL1 (25%, 3/12) mutations were observed as the most frequent co-mutations in MDS patients. One-year overall survival (OS) , disease-free survival (DFS) of allo-HSCT and chemotherapy patients were (70.6±9.0) %, (61.0±9.4) % and (34.4±16.7) %, (22.4±15.3) %, respectively. When OS and DFS between allo-HSCT and chemotherapy patients were compared, significant differences (χ²=4.843, 4.320, P<0.05) were showed. In univariate analysis, transplant age >45 years was a negative effect for OS [HR=4.819 (95% CI 1.145-20.283) , P=0.032] and DFS [HR=5.945 (95% CI 1.715-20.604) , P=0.005]. Also, complex chromosome karyotype abnormality was a negative effect for OS [HR=5.572 (95%CI 1.104-28.113) , P=0.038]. Conclusion Transplant age (>45 years) and complex chromosome karyotype abnormality were negative prognostic factors in allo-HSCT for myeloid neoplasms patients with RUNX1 mutations.

Objective To investigate the clinical diagnostic value of serum procalcitonin(PCT),D-dimer (DD),C-reactive protein(CRP)in acute-on-chronic liver failure(ACLF). Methods 124 ACLF patients, 63 chronic hepatitis B patients,32 chronic hepatitis C patients,24 chronic hepatitis E patients and 60 healthy controls from the second affiliated hospital of Nanchang University were enrolled in this study.PCT was detected by a sandwish immunodetection method. D-dimer was detected by Latex Turbidimetry. CRP was detected by rate nephenometry. The detection results were used for analyzing the clinical diagnostic value of ACLF with infection. Results(1)The level of PCT,DD and CRP in ACLF group were significantly higher than non-ACLF group and healthy controls(P<0.05).The levels of PCT,DD and CRP in the infection group were significantly higher than non-infection group(P<0.05).(2)The positive rates of PCT,DD and CRP in the infection group were 93.24%, 78.38%,89.19%,which were significantly higher than the non-infection group and healthy controls respectively (P < 0.05).(3)The sensitivity(93.24%)and specificity(90.00%)of PCT were the highest among all indexes. (4)The area under the ROC curve of PCT,DD,CRP were 0.892,0.715,0.755,respectively.PCT had the highest diagnostic value. Conclusion The levels of serum PCT,DD and CRP have a significant clinical value for the early diagnosis of ACLF with infection.

Objective: To investigate the prognosis of allogeneic hematopoietic stem-cell transplantation (allo-HSCT) for patients with acute myeloid leukemia and MLL rearrangement. Methods: From September 2009 to May 2016, the clinical data of 47 patients with MLL-rearranged AML undergoing allo-HSCT in the First Affiliated Hospital of Soochow University were retrospectively analyzed. Results: Among 47 MLL-rearranged AML patients, 24 were male and 23 female. The median age was 30 (15-58) years old. There are 36 (76%) patients were FAB-types M4/M5. Two-year overall survival (OS), disease-free survival (DFS), relapse incidence and transplant-related mortality (TRM) were (64.4±8.4)%, (47.3±9.3)%, 41.0% and 17.9%, respectively. Of them, 45 patients were detected with 11q23 translocations, and 2 patients with normal karyotype were MLL partial tandem duplication. According to different chromosome karyotype, 47 patients were divided into three groups: 16 cases of t (6; 11), 15 cases of t (9; 11) and 16 cases of other types. Overall survival was compared between the three groups, there was no significant difference (χ²=1.509, P=0.472). On multivariate analysis, independent risk factor on OS was transplant age >45 years [HR=4.454(95%CI 1.314-15.099), P=0.016]. The multivariate analysis also confirmed the higher TRM in patients at non-CR state when transplanted [HR=10.370(95%CI 1.043-103.110), P=0.046]. Positive minimal residual disease (MRD) before transplantation was a negative prognostic factor on DFS [HR=4.236(95%CI 1.238-14.495), P=0.021] and relapse incidence (RI) [HR=5.491(95%CI 1.371-21.995), P=0.016]. Conclusion Transplant age (>45 years), allo-HSCT in non-CR state adn positive MRD before transplantation were negative prognostic factors in allo-HSCT for MLL-rearranged AML patients.

ObjectiveThe type 2C protein phosphatases （PP2C） are involved in numerous plant signal transduction pathways. They mainly participate in plant stress response and regulate second metabolites biosynthesis via negatively regulating MAPK signaling pathway. Herein，we were to identify and analyze PP2C （CsPP2C） gene family from hemp genome，in hope of providing comprehensive insights for studying CsPP2C function during the development of hemp. MethodMolecular Evolutionary Genetics Analysis （MAGA）-X was used to construct phylogenetic tree. Expert Protein Analysis System （ExPASy），WoLF PSORT，Multiple EM for Motif Elicitation （MEME），Batch Conserved Domain Search （Batch-CD-Search），PlantCare，and TBtools were used，respectively，to predict CsPP2C physicochemical properties，subcellular localization，conserved motifs，protein structure，cis-element in promoter and collinearity with Arabidopsis PP2C. Cannabis sativa transcriptome and Real-time polymerase chain reaction（Real-time PCR） were used to analyze and verify gene expressions，respectively. ResultFifty-two CsPP2C with conserved domains were identified from the entire genome of hemp，encoding proteins ranging from 244 to 1 089 aa in length and with molecular weights ranging from 26.76 to 122.53 kDa. Those genes were mainly distributed in the nucleus，cytoplasm and chloroplast. The 47 CsPP2C were divided into 10 subfamilies，and the remaining 5 were not clustered. Seven pairs of homologous genes between hemp and Arabidopsis thaliana were identified according to collinear analysis. The light-responsive elements and abscisic acid elements are most abundant in the prediction. The gene expression heat map showed varied expression pattern of CsPP2C in different tissues. Real-time PCR results of three CsPP2C were consistent with transcriptome data. Moreover，alternative splicing analysis showed that some CsPP2C had alternative-splicing genes during evolution. ConclusionWe predicted and analyzed CsPP2C gene family in genomic scale and showed that CsPP2C are involved in many biological processes，whereby provides foundation for CsPP2C functional study.