BACKGROUND: Prostate cancer stem cell is an important reason for the invasion and recurrence of prostatic carcinoma. However, separation efficiency of prostate cancer stem cells is very low.OBJECTIVE: To explore the efficient method for isolating and identifying the prostate cancer stem cells from human prostatic carcinoma cell lines PC-3 and LNCap. METHODS: Prostate cancer cell lines PC-3 and LNCap were cultured in serum free medium (SFM) and serum supplemented medium (SSM), respectively. The percentage of prostate cancer stem cells from different medium was detected by flow cytometry through markers CD133 and CD44, and the properties of prostate cancer stem cells were preliminarily identified using inducing differentiation experiments.RESULTS AND CONCLUSION: PC-3 and LNCap formed sphere cells in SFM, which can be induced into adherent cells after culture in SSM. Higher percentage of CD44+/CD133+cells was obtained from LNCap cells (1.71%; 0.73%) than PC-3 cells (0.59%; 0.32%) in both SFM and SSM. The number of CD44+/CD133+ LNCap cells was more than PC-3 using both methods (P < 0.05), but the efficiency of SFM and SSM had no statistical significance (P > 0.05). However, the culture cycle was longer and number of obtained cells was less by SFM culture, directly influencing functional determination of prostate cancer stem cells. Compared with suspension culture method with SFM, SSM is more convenient and effective in isolating prostate cancer stem cells from LNCap cells.

Objective:To explore the role of matrix metalloproteinase 12 (MMP12) in airway macrophages and pulmonary neu-rogenic substance P ( SP ) in the pathogenesis of asthma by analyzing their relationship in different categories of asthmatic patients.Methods:Twenty patients of asthma remission phase ( remission asthma group ) , twenty ones of mild acute exacerbation asthma (mild asthma group) and twenty healthy adults (normal control group) were included,respectively.After lung function was measured,the numbers of macrophage in induced sputum were counted.The expression levels of MMP12 mRNA and protein in sputum macrophages were detected by quantitative reverse transcription polymerase chain reaction and Western blot.The concentration of sputum SP was assayed by enzyme immunometric assay.Results: ( 1 ) Compared with the subjects in normal control group, forced expiratory volume in 1 second%predicted ( FEV1 ) and forced expiratory flow rates at 50% of the forced vital capacity % predicted ( FEF50 ) were much lower and the numbers of sputum macrophages were much higher in the patients in different asthmatic groups.Compared with the patients in remission asthma group,FEV1 and FEF50 were much lower in the ones in mild asthma group.(2) MMP12 expressions in the macrophages and the concentrations of SP in sputum were significantly increased in the patients in different asthmatic groups compared with those in normal control group;Furthermore,MMP12 and SP in mild asthma group were much higher than in remission asthma.(3) In all patients from different asthmatic groups,mRNA expressions of MMP12 in the macrophages were positively correlated with the levels of sputum SP or the numbers of sputum macrophages,whereas negative correlations between mRNA expressions of MMP 12 and FEV 1 or FEF50 were observed.Conclusion: The regulatory imbalance of macrophages′MMP12 and pulmonary neurogic SP may participate in the pathogenesis of asthma and become the potential targets for asthma therapy.

Objective To assess the clinical diagnostic performance of liver stiffness measurement (LSM) and aspartate transaminase (AST)-to-platelet (PLT) ratio index (APRI) for liver fibrosis in chronic hepatitis B (CHB) patients with alanine aminotransferase (ALT) less than or equal to five times of the upper limit of normal (≤5×upper limit of normal [ULN]).Methods FibroScan,blood routine and liver function test were conducted at the day or one day before liver biopsy in 383 CHB patients with ALT≤5 × ULN.The Scheuer scoring system was used for liver histologic assessment.APRI was calculated.Based on the results of liver pathology,the areas under receiver operating characteristic curve (AUC) of LSM and APRI for diagnosis of liver fibrosis stage were compared.Results The median LSM were 5.10 kPa for S0 fibrosis stage,5.20 kPa for S1,6.60 kPa for S2,10.10 kPa for S3,and 18.80 kPa for S4.The median APRI values were 0.36,0.38,0.63,0.61 and 1.27,respectively.The AUC of LSM were 0.817 for ≥S2,0.891 for ≥S3 and 0.913 for ≥S4.And the AUC of APRI were 0.717 for ≥S2,0.711 for ≥S3 and 0.746 for ≥S4.The cut-offs of LSM values were 6.8 kPa for ≥S2,8.7 kPa for ≥S3,and 10.9 kPa for ≥S4.Conclusion LSM can accurately assess the degree of liver fibrosis in CHB patients with ALT ≤5 × ULN,which is superior to APRI in clinical utility.

Objective To introduce a practical high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)method for the detection of seven growth hormone-releasing peptides (GHRPs)including GHRP-1,GHRP-2,GHRP-4,GHRP-5,GHRP-6,Hexarelin and Alexamorelin and two growth hormone secretagogues(GHS)including anamorelin and ipamorelin,and study the stability of these nine substances in the human urine.Method The urine samples were purified and extracted by a solid phase extraction procedure using Oasis(R) WCX column.The urine was first centrifuged and taken out 1 mL into a small column,cleaned by 5％ NH4OH and 20％ CH3CN respectively,eluated using the mixture of water and acetomitrile(1/3)with 2％ formic acid,blow-dried in the nitrogen at 35℃ and finally redissolved to be injected into the LC-MS/MS.Result The limits of detection were between 0.01～0.5 ng/mL accordingly.The spiked recoveries at the low concentration(1 ng/mL),medium concentration(2 ng/mL)and high concentration(10 ng/mL)ranged between 40％ and 76％.The intra-and interday precisions of the target substances at these three concentrations were all less than 15％.The indoor temperature,refrigeration condition and multigelation were observed to have significant impact on the anamorelin,GHRP-2,GHRP-4 and GHRP-5.Conclusion The method established in this study is simple,and its specificity and sensitivity meets the international standard and technical documents for laboratories set up by the Wworld Anti-Doping Agency.It has been applied in our routine work.Multigelation should be avoided in the transport,detection and long-term laboratory storage of urine samples.

Objective To assess the diagnostic performance of liver stiffness measurement(LSM)and serum markers on hepatic fibrosis in chronic hepatitis B(CHB)patients with alanine aminotransferase(ALT)less than or equal to two times the upper limit of normal(≤2×ULN).Methods A total of 284 CHB patients with ALT≤2×ULN who were treated in Department of Hepatobiliary Medicine,Public Health Clinical Center,Shanghai from October 2015 to December 2017 were analyzed.FibroScan,routine blood tests and serum fibrosis markers were conducted on the day or one day before liver biopsy.The Scheuer scoring system was used for liver histologic assessment.Aspartate aminotransferase to platelet ration index(APRI)and FIB-4 were calculated.Based on the results of liver pathology,the area under receiver operating characteristic curve(AUROC)was used to evaluate the value of LSM and serum markers in the diagnosis of liver fibrosis stage.Non-normal distribution variables were expressed as M(QR)as appropriate,and compared by analysis of Kruskal-Wallis test as appropriate.The correlation between two variables was analyzed by Spearman correlation analysis.Results Of 284 CHB patients,175 were male and 109 were female.For inflammatory grading,175 cases were G1 grade,88 cases were G2,and 21 cases were G3.For fibrosis grading,153 cases were S1,53 cases were S2,34 cases were S3,and 44 cases were S4.Spearman correlation analysis showed that LSM,APRI and FIB-4 were positively correlated with hepatic fibrosis stage(r=0.650,0.484,and 0.317,respectively,all P<0.01).The AUC of LSM for predicting fibrosis≥S2,≥S3,and S4 were 0.840,0.902,and 0.942,respectively.The cut-off of LSM values were 6.10,8.40,and 10.10 kPa,respectively.The values of AUC of APRI and FIB-4 for predicting fibrosis≥S2 were 0.755 and 0.638,respectively,those for predicting fibrosis≥S3 were 0.737 and 0.657,respectively,and those for S4 were 0.804 and 0.694,respectively.The AUCs of LSM for predicting fibrosis≥S2 in patients with ALT≤1×ULN and those with ALT>1 -≤2×ULN were 0.857 and 0.813,respectively,those for fibrosis≥S3 were 0.890 and 0.892,respectively,and those for S4 were 0.925 and 0.908,respectively.The cut-off of LSM were 5.90 and 7.80 kPa,8.10 and 9.50 kPa,8.40 and 10.40 kPa,respectively.Conclusions LSM could accurately assess the degree of liver fibrosis in CHB patients with ALT≤2×ULN,which is superior to serum markers for predicting liver fibrosis stage.

ObjectiveTo investigate the value of aspartate aminotransferase-to-platelet ratio index (APRI), fibrosis-4 (FIB-4) score, and gamma-glutamyl transpeptidase-to-platelet ratio (GPR) in diagnosis of liver inflammation grade in patients with chronic hepatitis B (CHB). MethodsA total of 545 patients with CHB who underwent percutaneous liver biopsy and routine laboratory examinations during hospitalization in Shanghai Public Health Clinical Center Affiliated to Fudan University from October 2016 to October 2019 were enrolled. Inflammation grade (G) was determined according to the Scheuer scoring system, and APRI, FIB-4, and GPR were calculated based on related clinical indicators. The t-test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test was used for comparison of categorical data between two groups. A Spearman correlation analysis was used to investigate the correlation between two variables. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic performance of the three serum noninvasive diagnostic models in determining liver inflammation grade, and the Delong test was used for comparison of the area under the ROC curve (AUC). ResultsAmong the 545 patients, 224 had grade G0-1 liver inflammation, 209 had grade G2 liver inflammation, and 112 had grade G3 liver inflammation. The Spearman correlation analysis showed that APRI, FIB-4, and GPR were positively correlated with liver inflammation grade (r=0.611, 0.470, and 0.563, all P＜0.001). APRI, FIB-4, and GPR had an AUC of 0.820, 0.719, and 0782, respectively, in the diagnosis of G≥2 liver inflammation, with optimal cut-off values of 0.53, 1.48, and 0.20, respectively; for the diagnosis of G≥2 liver inflammation, GPR had a better performance than FIB-4 (P=0.01) and a slightly lower performance than APRI (P=0.048). The stratified analysis based on alanine aminotransferase (ALT) level showed that in the ≤1×upper limit of normal (ULN) group, the (1-2)×ULN group, and the (2-5)×ULN group, APRI had an AUC of 0.847, 0.786, and 0.724, respectively, in the diagnosis of G≥2 liver inflammation, FIB-4 had an AUC of 0.777, 0.729, and 0.626, respectively, and GPR had an AUC of 0.801, 0.781, and 0.607, respectively; the subgroup analysis showed that GPR had a similar diagnostic performance to APRI and FIB-4 in all ALT stratification groups except the (2-5)×ULN group, in which GPR had a lower diagnostic performance than APRI (P=0.042). APRI, FIB-4, and GPR had an AUC of 0.791, 0.725, and 0.801, respectively, in the diagnosis of G≥3 liver inflammation, with optimal cut-off values of 0.66, 1.49, and 0.25, respectively; in the diagnosis of G≥3 liver inflammation, GPR had a similar diagnostic performance to APRI and a better diagnostic performance than FIB-4 (P=0.006). The stratified analysis based on ALT level showed that in the ≤1×ULN group, the (1-2)×ULN group, and the (2-5)×ULN group, APRI had an AUC of 0.900, 0.742, and 0.693, respectively, in the diagnosis of G≥3 liver inflammation, FIB-4 had an AUC of 0.874, 0.683, and 0.644, respectively, and GPR had an AUC of 0.890, 0.805, and 0.668, respectively. The subgroup analysis showed that GPR had a similar diagnostic performance to APRI and FIB-4 in all ALT stratification groups except the (1-2)×ULN group, in which GPR had a better diagnostic performance than FIB-4(P=0.015). ConclusionAPRI, FIB-4, and GPR may accurately diagnose liver inflammation grade in CHB patients, which helps to monitor the progression of CHB and determine the timing of antiviral therapy.

Objective:To investigate the kinetic metrics of 68Ga-fibroblast activation protein inhibitor (FAPI)-04 in pancreatic cancers and normal organs by using total-body PET dynamic imaging. Methods:From December 2020 to December 2021, 68Ga-FAPI-04 total-body PET/CT dynamic imaging were performed on 6 pancreatic cancer patients (3 males, 3 females, median age 55.5 years) in Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. Images were respectively analyzed. Manual delineations of volume of interests (VOIs) on multiple normal organs and pathological lesions were performed and time-to-activity curves (TACs) were generated. A reversible two-tissue compartment model (2TCM) was fitted for each tissue TAC. Rate constants including K1, k2, k3 and k4, and the total volume of distribution ( Vt) were obtained and compared by tissue types. Wilcoxon rank sum test and Spearman correlation analysis were used for data analysis. Results:Kinetic metrics varied significantly among normal organs and pancreatic cancer lesions ( z values: 2.00-1 240.00, all P<0.05). The highest K1 among lesions was observed in primary tumor (0.30 min -1), which was observed in the spleen (1.42 min -1) among normal organs. The highest k2 among lesions was observed in peritoneal metastases (0.24 min -1), which was observed in the spleen (2.59 min -1) among normal organs. Primary tumor showed the highest k3 of 0.17 min -1 among lesions, and the pancreas had the highest k3 of 0.16 min -1 among normal organs. Primary tumor had the highest k4 of 0.03 min -1 among lesions, and the heart, lungs, parotid glands had high k4(0.06 min -1) among normal organs. Vt were higher in pathological lesions compared to normal organs, with the highest in primary tumor (13.78 ml/cm 3). There were correlations between Vt in lesions and SUV mean( rs=0.86, P<0.001) or SUV max ( rs=0.77, P<0.001). Conclusion:The rate constants including K1, k2, k3 and k4, and Vt of 68Ga-FAPI-04 vary among normal organs and lesions.

Background and purpose:For patients with human epidermal growth factor receptor 2(HER2)-positive metastatic breast cancer,trastuzumab treatment can prolong the overall survival and significantly improve the prognosis of patients.However,the reference original research trastuzumab(Herceptin?)is more expensive.Biosimilars have comparable efficacy and safety profiles while increasing patient access to treatment.This clinical trial aimed to evaluate the efficacy,pharmacokinetics,safety and immunogenicity of the trastuzumab biosimilar AK-HER2 compared to trastuzumab(Herceptin?)in patients with HER2-positive metastatic breast cancer.Methods:This multi-center,randomised,double-blind phase Ⅲ clinical trial was conducted in 43 subcenters in China.This study complied with the research protocol,the ethical principles stated in the Declaration of Helsinki and the quality management standards for drug clinical trials.It was approved by the hospital's medical ethics committee.The clinical trial registration agency is the State Food and Drug Administration(clinical trial approval number:2015L04224;clinical trial registration number:CTR20170516).Written informed consent was obtained from subjects before enrollment.Enrolled patients were randomly assigned to the AK-HER2 group and the control group,respectively receiving AK-HER2 or trastuzumab(initial loading dose 8 mg/kg,maintenance dose 6 mg/kg,every 3 weeks as a treatment cycle,total treatment time is 16 cycles)in combination with docetaxel(75 mg/m2,treatment duration is at least 9 cycles).The primary endpoint of this clinical trial was the objective response rate(ORR9)between the AK-HER2 group and the control group in the 9th cycle.Secondary efficacy endpoints included ORR16,disease control rate(DCR),clinical benefit rate(CBR),progression-free survival(PFS)and 1-year survival rate.In this study,100 subjects(AK-HER2 group to control group=1:1)were randomly selected for blood sample collection after the 6th cycle of medication,The collection time points were 45 minutes after infusion(the end of administration),4,8,24,72,120,168,336,and 504 hours after the end of administration.After collection,blood samples were analyzed by PK parameter set(PKPS).Other evaluation parameters included safety and immunogenicity assessment.Results:A total of 550 patients with HER2-positive metastatic breast cancer were enrolled in this clinical trial between Sep.2017 and Mar.2021.In the AK-HER2 group(n=237),129 subjects in the experimental group achieved complete response(CR)or partial response(PR),and the ORR9 was 54.4%.There were 134 subjects in the control group(n=241)who achieved CR or PR,and the ORR9 was 55.6%.The ORR9 ratio between the AK-HER2 group and the control group was 97.9%[90%confidence interval(CI):85.4%-112.2%,P=0.784],which was not statistically significant.In all secondary efficacy endpoints,no statistically significant differences were observed between the two groups.We conducted a mean ratio analysis of pharmacokinetics(PK)parameters between the AK-HER2 group and the control group,and the results suggested that the pharmacokinetic characteristics of the two drugs are similar.The incidence of treatment emergent adverse event(TEAE)leading to drug reduction or suspension during trastuzumab treatment was 3.6%(10 cases)in the AK-HER2 group and 8.1%(22 cases)in the control group.There was statistically significant difference between the two groups(P=0.027).The incidence rate was significantly lower in the AK-HER2 group than in the control group,and there was no statistically significant difference among the other groups.The differences in the positive rates of anti-drug antibodies(ADA)and neutralizing antibodies(NAB)between groups were of no statistical significance(P=0.385 and P=0.752).Conclusion:In patients with HER2-positive metastatic breast cancer,AK-HER2 was comparable to the trastuzumab(Herceptin?)in terms of drug efficacy,pharmacokinetics,safety and immunogenicity.

OBJECTIVE: To investigate changes in intestinal flora in patients with primary Sj?gren syndrome (pSS) and explore the relationship between pSS disease activity and intestinal flora structure. METHODS: Fecal samples were collected from 18 female pSS patients, including 9 patients with active disease (group A) and 9 with disease inactivity or low activity (group B), with 10 healthy subjects as the control group. The total bacterial DNA was extracted from the fecal samples for PCR amplification, and Illumina Hiseq 2500 high-throughput sequencing was performed for the v3-v4 region of 16Sr DNA gene to obtain the biological information of the intestinal flora. The intergroup OTU analysis, structural diversity analysis, significant difference analysis and LEFSE analysis were performed with information mining of the literature think tanks. RESULTS: The dilution curves generated based on the OTUshannon index for analysis of sample complexity showed that the measured data were relatively complete and could reflect the diversity of the microorganisms in the subjects. Analysis of the Alpha diversity index showed that the Shannon index differed significantly between group A and group B, and the Simpson index differed significantly between group A and group B and between group A and the control group ( < 0.05). Sequence analysis the 3 groups all consisted mainly of 4 phylum (, , , showed that the intestinal flora in and ) and 4 genera (, , , and ), all showing no significant differences among the 3 groups ( > 0.05) with the exception of genus, which differed significantly among the 3 groups ( < 0.05). The 16S v3-v4 region in the genus , , , , , , , , , , -, and differed significantly among the 3 groups ( < 0.05). The high-dimensional biometrics and genomic characteristics of the intestinal microorganisms differed significantly among the 3 groups ( < 0.05). According to the size of LDA SCORE (effect size), the core flora in group A included the genera , , -, , -, , , , and , as compared with the genera , , , , , -, , - and in the control group. CONCLUSIONS Patients with pSS have significant changes in the diversity of intestinal flora, especially in some specific bacteria in genus and in 16S v3-v4 region of the bacteria. The differences in the core bacteria in the intestinal flora of pSS patients suggest the role of flora structure changes in the pathogenesis of pSS.
Bacteria ; classification ; genetics ; Biodiversity ; DNA, Bacterial ; genetics ; Feces ; microbiology ; Gastrointestinal Microbiome ; Humans ; RNA, Ribosomal, 16S ; genetics ; Sjogren's Syndrome ; microbiology

OBJECTIVE: To investigate changes in intestinal flora in patients with primary Sj?gren syndrome (pSS) and explore the relationship between pSS disease activity and intestinal flora structure. METHODS: Fecal samples were collected from 18 female pSS patients, including 9 patients with active disease (group A) and 9 with disease inactivity or low activity (group B), with 10 healthy subjects as the control group. The total bacterial DNA was extracted from the fecal samples for PCR amplification, and Illumina Hiseq 2500 high-throughput sequencing was performed for the v3-v4 region of 16Sr DNA gene to obtain the biological information of the intestinal flora. The intergroup OTU analysis, structural diversity analysis, significant difference analysis and LEFSE analysis were performed with information mining of the literature think tanks. RESULTS: The dilution curves generated based on the OTUshannon index for analysis of sample complexity showed that the measured data were relatively complete and could reflect the diversity of the microorganisms in the subjects. Analysis of the Alpha diversity index showed that the Shannon index differed significantly between group A and group B, and the Simpson index differed significantly between group A and group B and between group A and the control group ( < 0.05). Sequence analysis the 3 groups all consisted mainly of 4 phylum (, , , showed that the intestinal flora in and ) and 4 genera (, , , and ), all showing no significant differences among the 3 groups ( > 0.05) with the exception of genus, which differed significantly among the 3 groups ( < 0.05). The 16S v3-v4 region in the genus , , , , , , , , , , -, and differed significantly among the 3 groups ( < 0.05). The high-dimensional biometrics and genomic characteristics of the intestinal microorganisms differed significantly among the 3 groups ( < 0.05). According to the size of LDA SCORE (effect size), the core flora in group A included the genera , , -, , -, , , , and , as compared with the genera , , , , , -, , - and in the control group. CONCLUSIONS Patients with pSS have significant changes in the diversity of intestinal flora, especially in some specific bacteria in genus and in 16S v3-v4 region of the bacteria. The differences in the core bacteria in the intestinal flora of pSS patients suggest the role of flora structure changes in the pathogenesis of pSS.
Bacteria ; DNA, Bacterial ; Feces ; Female ; Gastrointestinal Microbiome ; Humans ; RNA, Ribosomal, 16S ; Sjogren's Syndrome