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Objective:To investigate the correlation of intratumoral fibrosis with the prognosis of clear cell renal cell carcinoma (ccRCC).Methods:The correlation of the transcriptional expression of the primary collagen with the prognosis in ccRCC was evaluated using the Cancer Genome Atlas (TCGA) database, including 530 ccRCC patients with complete information. Of them, 344 cases were male, 186 cases were female. The age of 264 cases was ≤ 60 years, and the age of 266 cases was > 60 years. The pathology grade of 241 patients was G 1-2 grade, and the pathology of 281 cases were G 3-4 grade, 8 cases were undetermined grade. There were 322 cases with AJCC stage Ⅰ-Ⅱ and 205 cases with AJCC stage Ⅲ-Ⅳ, and 3 cases with undetermined stage. There were 420 cases in M 0 and 78 cases in M 1, and 32 cases without distant metastases information. Furthermore, the paraffin sections of 158 non-cystic ccRCC patients confirmed by pathology from November 2005 to November 2017 were further used to evaluate the level of collagen of ccRCC and the status of the pseudocapsule by the Masson staining, Sirius red staining and multicolor immunofluorescence staining of collagen Ⅰ and collagen Ⅲ. Of them, 112 cases were male, 46 cases were female. There were 100 cases with age ≤ 60 years, and 58 cases with age > 60 years. The pathology grade of 111 cases were G 1-2, and the pathology grade of 47 cases were G 3-4. There were 144 cases with AJCC stage Ⅰ-Ⅱ, 14 cases with AJCC stage Ⅲ-Ⅳ. Kaplan-Meier survival curve were used to analyze the relationship between tumor collagen parameters and the overall survival prognosis of patients with ccRCC. Results:The transcriptome results of the TCGA database indicated that the expression level of COL1A1 in ccRCC tissues was significantly higher than that in adjacent normal tissues ( P<0.001). The high expression of collagen suggested a worse overall survival prognosis ( HR=1.165, P=0.002). In addition, the high ratio of COL1A1/COL3A1 indicated a worse overall survival prognosis ( HR=1.901, P<0.001) compared with the low ratio. We further confirmed that the abundance of collagen in tumor was significantly increased compared with the normal adjacent tissues by the Masson staining [41.0 (14.0-75.0) vs.15.0 (3.0-57.0), P<0.001] and the Sirius red staining [42.5 (10.0-90.0) vs.10.0 (2.5-60.0), P<0.001] on 30 ccRCC tissues and adjacent normal tissues. Based on the Masson staining, we found that high collagen abundance in tumor tissue was associated with more G 3-4 grade of tumor compared with low collagen abundance (38.5% vs.21.3%, OR=2.316, 95% CI 1.146-4.681, P=0.023). Kaplan-Meier survival curve showed that higher collagen abundance was associated with a worse overall survival prognosis in ccRCC ( HR=2.630, P=0.007). However, incomplete fibrous pseudocapsule was associated with a worse overall survival prognosis ( HR=11.140, P<0.001). Conclusions:In ccRCC, intratumoral collagen fiber level was overexpressed. High intratumoral collagen level and incomplete fibrous pseudocapsule may indicate a poor overall survival prognosis.
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Objective:To assess the association between warm ischemia time (WIT) and renal function in patients undergoing laparoscopic partial nephrectomy.Methods:A total of 344 patients treated with laparoscopic partial nephrectomy in Peking University People’s Hospital were included. There were 240 males (69.8%) and 104 females (30.2%) with a median age of 57 (23-89) years.The median BMI was 25.6 (16.7-36.0) kg/m 2.213 cases (61.9%) were associated with hypertension.There were 66 (19.2%) patients with diabetes mellitus. There were 92 cases (26.7%) with smoking history. The median preoperative creatinine was 73 (32-170) μmol/L. The median preoperative estimated glomerular filtration rate (eGFR) was 95 (33-142) ml/(min·1.73m 2). The maximum diameter of the tumor was 2.5 (7-9) cm.314 (91.3%) patients with renal cancer stage T 1. All patients underwent warm ischemia during the operation. The patients were divided into three groups for analysis. Restricted cubic spline regression analysis was used to assess the association between WIT as a continuous variable and percentage change of eGFR. Analysis of covariance was used to compare postoperative eGFR among the three groups, and to adjust for preoperative eGFR and tumor diameter. Results:There were statistically significant differences in the percentage change of postoperative eGFR ( P=0.009) and tumor diameter ( P<0.001) among the three groups. Restricted cubic spline regression analysis showed that with the prolongation of WIT, the percentage change of postoperative eGFR gradually decreased, and the curve began to stabilize after 30 minutes (R 2=0.044, P=0.015). The results of covariance analysis showed that after adjusting for baseline preoperative eGFR and tumor size, the effect of WIT on postoperative eGFR was significantly different among the three groups ( F=3.864, P=0.022). The postoperative eGFR in the WIT<20 min group was significantly higher than that in 20 min≤WIT<30 min group( P=0.009) and WIT≥30 min group( P=0.017). There was no significant difference in postoperative eGFR between the two groups with longer WIT( P=0.806). Conclusions:In partial nephrectomy, patients with WIT less than 20 minutes had higher postoperative eGFR levels than those with WIT greater than 20 minutes. However, when WIT exceeded 20 minutes, prolonged ischemia time did not lead to further decline in renal function.
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OBJECTIVE：To study the compositi on of the volatile oil from Compound chaihu guizhi decoction ，and to evaluate its in vitro anti-proliferative activity on human lung adenocarcinoma A 549 cells. METHODS ：The volatile oil from Chaihu guizhi decoction was extracted according to the steam distillation method of general rules 2004 in the 2015 edition of Chinese Pharmacopoeia（part Ⅳ）. The volatile oil components were analyzed by GC-MS combined with Kováts index ，and the relative content of each component was calculated by peak area normalization method. Using different concentrations of cisplatin （4，8， 16，32，64 mg/L）as positive control ，MTT assay was used to detect the inhibitory effects of different concentrations of volatile oil from Chaihu guizhi decoction （25，50，100，200，400 mg/L）on in vitro proliferation of A 549 cell after 48 h of treatment. Negative control group （with cells but without drugs ）was set up. RESULTS ：A total of 71 chemical components were isolated from the volatile oil ，among which there were 59 compounds identified ，sum of peak areas accounting for 84.99％ of the total peak area. The compounds with relatively high content included ar-curcumene （17.65％），β-bisabolene（9.57％），β-ocimene（7.05％）， α-curcumene（5.35％），2，5-dimethylbenzaldehyde（4.24％），linalyl isobutyrate （2.70％），α-cedrene（2.48％），δ-cadinene （2.07％）. Compared with negative control group ，the proliferation rate of cells were decreased significantly in 4-64 mg/L cisplatin groups and 25-400 mg/L volatile oil from Chaihu guizhi decoction groups （P＜0.05）. IC 50 of cisplatin and volatile oil from Chaihu guizhi decoction to in vitro proliferation of A 549 cells were 10.150 and 73.526 mg/L. CONCLUSIONS ：The volatile oil from Chaihu guizhi decoction mainly includes ar-curcumene ，β-bisabolene，β-ocimene，α-curcumene，which shows certain inhibitory effect on in vitro proliferation of A 549 cells.
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OBJECTIVE：To establish a method for simultaneous determination of calycosin glucoside ，ononin，calycosin， formononetin，astragaloside Ⅳ，isoastragaloside Ⅱ，cycloastragenol and isoastragaloside Ⅰ in Astragalus membranaceus before and after bidirectional solid fermentation with Cordyceps kyushuensis ，and to investigate the effects of fermentation on the contents of above 8 components in A. membranaceus . METHODS ：HPLC-DAD-ELSD was adopted. The determination was performed on Agilent 5 TC-C18 column with mobile phase consisted of 0.1％ formic acid aqueous solution-acetonitrile （gradient elution ）at the flow rate of 1 mL/min. The column temperature was set at 30 ℃. DAD detection wavelength was set at 260 nm，ELSD evaporation tube temperature was 100 ℃，atomizer temperature was 80 ℃，carrier gas flow rate was 1.6 L/min；injection volume was 15 μL. RESULTS：The eight components had a good linear relationship within their respective ranges of concentration （all R2＞0.999 0）； RSDs of precision ，stability and repeatability tests were all less than 3％（n＝3 or n＝6）；the recoveries was 97.88％-101.32％， and RSDs were 1.22％-2.39％（n＝6）. Setting the content of components in unfermented A. membranaceus as 100％，after bidirectional solid fermentation with C. kyushuensis ，the change rates of 8 components were －98.51％，－96.41％，－94.74％， －96.40％，289.20％，20.25％，－75.05％，562.46％，respectively. CONCLUSIONS ：After fermentation with C. kyushuensis ，the contents of active components as astragaloside Ⅳ，isoastragaloside Ⅰ and isoastragaloside Ⅱ can be increased significantly in A. membranaceus .
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Objective@#To observe the integration frequency of aadA2 resistance cassette at attI site of the integron under different concentration of streptomycin. @*Methods@#Class 1 integron with known gene sequence was cloned into plasmid pACYC184 to produce recombinant plasmid pACIDA, meanwhile the integrase gene was cloned into plasmid pET28a to construct recombinant plasmid pETINT. These two recombinant plasmids were consecutively transformed into E. coli BL(DE3). These transformed bacteria was cultured in the LB medium at 37 ℃ overnight with addition of different concentration of streptomycin. The copy number of total integrons and the copy number of integrated aadA2 at attI site of integrons were determined by using real-time PCR. and the integration frequency is the result of the former divided by the latter. @*Results@#The resulting frequencies were (1.97±0.24)×10-3, (3.23±1.77)×10-3, (3.27±0.67)×10-3, 0.45±0.13 and 1.32±0.11, with respective streptomycin concentrations of 0, 20, 30, 40 and 50 μg/ml. The background frequency of integration without integrase overexpression was less than (1.75±0.33)×10-7. @*Conclusion@#These findings indicate that antibiotic concentration significantly increase recombination frequency of aadA2 resistance cassette at attI site of the integron, catalyzed by integron integrase.(Chin J Lab Med, 2018, 41: 531-535)
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Objective To evaluate the health-related quality of life (HRQOL) for prostate cancer patients receiving androgen deprivation therapy (ADT).Methods We studied 200 patients with prostate cancer who were treated with primary ADT in Peking University People's Hospital from June 2014 to June 2015.The patients'average age was 73.9 years.The mean PSA level was 21.57 ng/ml when they were diagnosed with prostate cancer.Of these 200 patients,79％ (158/200) were localized and seclected ADT therapy due to age,body condition,basic diseases or individual will.21％ (42/200) were locally advanced or metastasic,which accord with the indication of ADT therapy.The scales,including the MOS item short from health survey (SF-36),Functional Assessment of Cancer Therapy-General module (FACT-G),Functional Assessment of Cancer Therapy-Prostate instrument (FACT-P),Self-Rating Depression Scale (SDS) and Self-Rating Anxiety Scale (SAS) were used to evaluate their HRQOL.Results According to results of SF36,the physical health component score and mental health component score was 67.41 ± 16.39 and 64.81 ± 17.51,respectively.They indicated that the overall quality of life of these patients was at an acceptable level.And the average score of all domains exceeded 60 except general health domain,which the score was only 40.03 ± 21.89.When it comes to FACT-P,the sum score,emotional well-being score and prostate cancer component score were 8.15 ± 3.72,12.30 ± 4.04 and 77.41 ± 9.95,respectively,which were less than half of their respective top score.However the physical well-being score was 20.41 ±4.29,which was a relatively satisfactory value.In addition,61％ (121/200) patient selected not at all in item I am able to have and keep an erection.The score of SDS and SAS was 46.76 ± 8.29 and 43.25 ±9.69,respectively.And there were 23.0％ (46/200) and 21.5％ (43/200) patiens exhibited depression and anxiety,respectively.Conclusion In the present study,the prostate cancer patients receiving ADT showed acceptable HRQOL,but some patients sufferd from depression,anxiety and erectile dysfunction.
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Objective:To investigate the association between metabolic factors,such as obesity,blood pressure,blood glucose,serum lipid profile,and the histopathological characteristics of renal cell carci-noma.Methods:The medical records of 382 consecutive renal cell carcinoma patients who underwent radical or partial nephrectomy at Peking University People’s Hospital from January 2009 to January 2015 were retrospectively reviewed.Metabolic factors were collected from the records,including weight,body mass index,waist circumstance,blood pressure,fasting blood glucose,serum total triglyceride,serum total cholesterol,serum low-density lipoprotein-cholesterol and serum high-density lipoprotein-cholesterol. The patients were divided into different groups according to tumor grade,stage and diameter.Statistics analysis,such as t test,Mann-Whitney U test and Logistic analysis,were performed to investigate the as-sociation between metabolic factors and grade,stage and tumor diameter of renal cell carcinoma.Re-sults:A total of 80 (20.94%)of the tumors were classified as high grade disease,63 (16.49%)were classified as advanced disease and 153 (40.05%)tumor diameter more than 4 cm.The patients in high grade group were found to have lower high-density lipoprotein-cholesterol level than in low grade group (P =0.015),body mass index,total cholesterol and high-density lipoprotein-cholesterol were found to be lower in advanced disease than in localized disease (P =0.022,P =0.005 and P =0.006,respec-tively),and low-density lipoprotein-cholesterol was found to be lower in larger tumors (P =0.030). Other factors were comparable between the different groups.The results of Logistic analyses showed that, body mass index (OR =0.906,95%CI:0.852 -0.986,P =0.023)and total cholesterol (OR =0.660,95%CI:0.492 -0.884,P =0.005)were associated with the tumor stage,high-density lipopro-tein-cholesterol level was significantly associated with tumor grade (OR =0.293,95% CI:0.108 -0.797,P =0.016)and stage (OR =0.204,95%CI:0.065 -0.635,P =0.006),and low-density lipoprotein-cholesterol level was significantly associated with tumor diameter (OR =0.756,95% CI:0.586 -0.975,P =0.031).Conclusion:The results of our study indicate that metabolic factors,espe-cially obesity and serum lipid profile,are closely related with the histopathological characteristics of renal cell carcinoma.
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Objective:To investigate the expression of MEK/ERK signaling pathways in renal cell car-cinoma with bone metastasis,and to analyze the differences of expressions of VEGFR-2,MEK,ERK on the primary and metastasis tissue and its mechanism.Methods:The tissue samples were obtained from 7 renal cell carcinoma patients kindly provided by Department of Urology,Peking University People’s Hos-pital from January 1,2009 to January 1,2010.The expression of MEK/ERK signaling pathways was de-tected in the 7 renal cell carcinoma patients`primary and matched metastatic tissues with ICH,The anti-body concentrations were 1 ∶200,1 ∶25,and 1 ∶250,respectively.The mutation of the twentieth exon of the PDGFRA gene,the second exon of the K-ras gene,the fifteenth exon of the Brafgene and the se-cond exon of the MEK1 gene were detected with PCR.Results:The expression intensities of VEGFR-2, MEK,and ERK were measured by H-score [intensity (1,2,3,or 4)multiplied by the distribution (%)].VEGFR-2,MEK,and ERK expressions were divided into 3 groups according to the positive dis-tribution of the tumor cells:1,0 -5%;2,6% -50%;and 3,>50%,To assess intratumor heteroge-neity,three distinct microscopic fields (×200)from each specimen were used to evaluate the expres-sions,Subsequently,the scores were averaged to obtain a single concatenated score for each tissue. VEGFR-2,MEK,and ERK expressions were assessed by 2 independent pathologists who were blinded to the clinicopathological data.The data were expressed as the mean value of the triplicate experiments.The expressions of MEK,and ERK were higher in the metastatic tissues than in the matched RCC tissues (6.10 ±4.10 vs.1.33 ±0.51,P =0.015;9.10 ±2.24 vs.4.43 ±2.84,P =0.021 )while the ex-pression of VEGFR-2 was not different between the primary and metastatic tissues (P =0.901).No mu-tation was detected on the twentieth exon of the PDGFRA gene,the second exon of the K-ras gene,the fifteenth exon of the Brafgene and the second exon of the MEK1 gene.Conclusion:MEK/ERK signa-ling pathways may play an important role in the metastasis and the resistance of sunitinib in RCC patients with bone metastasis.
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Objective:To investigate the change of biological characteristics after stable knockdown of CKLF-like MARVEL transmembrane domain containing 3 (CMTM3)expression in PC3 by lentivirus shRNA and to reveal new therapeutic targets.Methods:The research includes two groups:sh393 is the experimental group in which CMTM3 is knocked down in PC3 cell line;shN is the control group in which CMTM3 is negatively knocked down.The expression of CMTM3 was detected by Western blot.The mi-gration ability of PC3 after stable knockdown was detected by Transwell and Wound healing assay.The invasion ability of PC3 was detected by Matrigel assay.Results were obtained from at least three indivi-dual experiments.Results:The expression of CMTM3 in sh393 group is significant lower than shN group (0.004 0 ±0.000 4 vs.0.490 0 ±0.055 7,P <0.001)detected by Western blot.It also had statistical significance in Matrigel assays (248.6 ±4.5 vs.113.0 ±3.3),Transwell (203.6 ±1.9 vs.103.0 ± 1.2)and Wound healing assays (95.0 ±2.9 vs.33.0 ±1.5)that knockdown of CMTM3 promoted mi-gration,and invasion of PC3 cells in vitro (P <0.001).Conclusion:Negative correlation exists between the stable knockdown of CMTM3 and change of biological characteristics in PC3 cells,and knocking down CMTM3 affects migration,and invasion ability in PC3 cells.
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Objective:To establish a new approach to synthesis of diethyl N-[4-[(2,4-diaminopyrido [3,2-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glutamate.Methods:Target compound (5) was syn-thesized by the use of (2,4-dioxo-tetrahydropyridopyrimidin-6-yl) methyl acetate (1) as starting material via hydrolysis, chlorination, condensation with diethyl (p-aminobenzoyl)glutamate and aminolysis.Re-sults:A new approach to synthesis of diethyl N-[4-[(2,4-diaminopyrido[3,2-d]pyrimidin-6-yl)methyl-amino]benzoyl]-L-glutamatewas established .This synthetic route has hydrolysis reaction , chlorination, diethyl N-( p-aminobenzoyl )-L-glutamate condensation reaction and ammonolysis reaction .The total yield is 36.7%.The structures of those compounds have identified by 1 H nuclear magnetic resonance , 13 C nu-clear magnetic resonance and mass spectrometry .This synthetic route avoid the unstable brominated re-action product and improves the harsh condition of ammonolysis reaction .Conclusion:The new synthetic route has improved the reaction condition and the stability of the intermediate , and increased the extent of the derivative compounds , which has great significance to anti-folic acid of anti-tumor inhibitor synthesis .
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ObjectiveTo investigate the relation of learning and memory with the expression of postsynaptic density 95 (PSD-95) in traumatic brain injury (TBI) rats in Morris water maze.Methods Forty adult male Sprague-Dawley rats were randomly assigned to the TBI group and control group.The TBI group was produced using the impact acceleration injury model.Morris water maze memory paradigm was used to assess the learning and memory function in both groups one week after injury.Protein electro-phoresis was used to observe the expression of PSD-95 1,3,7 d after TBI.ResultsCompared with the control group,the TBI group showed a longer latency in the Morris water maze after one week,significantly longer than the latency in the first three days after TBI.The quantification of PSD-95 in the hippocampus was gradually reduced at one week after TBI ( P ＜ 0.01 ).ConclusionTBI may decrease expression of PSD-95 in the hippocampus of the rats,as may be one of the mechanisms for impairments of learning and memory of rats.