OBJECTIVE:To develop an HPLC method for the determination of Tanshinone Ⅰ,Tanshinone ⅡA,Cryptotanshinone,and Dihydrotanshinone in Shandantong retard tablets.METHODS:The samples were separated on Lichrospher C18 column(250 mm?4.6 mm,5 ?m) with the mobile phase consisted of methanol-water(80∶20).The detection wavelength was set at 270 nm.RESULTS:The linear ranges of Tanshinone Ⅰ,Tanshinone ⅡA,Cryptotanshinone,and Dihydrotanshinone were 0.20～2.00 ?g,0.30～3.00 ?g,0.20～2.00 ?g,and 0.20～2.00 ?g,respectively.The average recoveries were 99.54%,98.89%,100.10%,and 99.44%,respectively with RSD at 0.40%,0.92%,1.30% and 1.53%(n=6),respectively.CONCLUSION:This method is simple,rapid and accurate,and suitable for the quality control of Shandantong retard tablets.

Background Intracorneal macrophages play a critical role in corneal neovascularization (CNV)by secreting relative chemokines.But macrophages are characteristic by heterogeneity which has different biologic functions under different induction or stimulation from microenvironment.Objective This study was to detect the effects of chemokine (C-X3-C motif) ligand 1 (CX3CL1) and chemokine (C-C motif) ligand 2 (CCL2) on macrophages in vitro.Methods CNV was induced by corneal alkali burn in the left eyes of 20 male BALB/c mice aged 7-8 weeks.The CNV was evaluated under the slit lamp microscope 4 days after alkali burn,and then the corneal sections were prepared after mice were sacrificed.The expressions of CCR2 and CX3CR1 in the corneal specimens were detected by histo-fluorescence staining.Human peripheral blood mononuclear cells were separated using density gradient centrifugation and incubated in RPMI-1640 medium containing 10％ fetal bovine seruml(FBS) with 30 μg/L granulocyte-macrophage colony-stimulating factor (GM-CSF).The cells were divided into CD68 +CCR2 group and CD68+CX3CR1 group,and the percentage of the CX3CR1 and CCR2 expressions in the infiltrated macrophages of corneal specimens and human monocyte-derived macrophages was assayed by flow cytometry.The cultured cells were stimulated using human recombinant CX3CL1 and CCL2 proteins,and real-time PCR was used to detect the relative expressions of angiogenesis-related factors in macrophages.Results CNV was found in corneas 4 days after alkali burn and the CNV onsets from corneal limbus to central zone observed by a slit lamp.CCR2 and CX3CR1 were expressed in the F4/80-positive macrophages in alikali burned corneas.The macrophages grew for two weeks and appeared more dead cells in without GM-CSF group,but in GM-CSF induced group,the number of macrophages was increased.The percentage of CX3CR1-positive cells was 75％ and that of CCR2-positive cells was 45％.Real-time PCR showed that expression level of vascular endothelial growth factor (VEGF) mRNA increased and that ADAMTS-1 mRNA or TSP-1 mRNA decreased on macrophages after CCL2 stimulation,with significant differences in the 150 mg/L CCL2 group compared with the control group (t =-5.09,P =0.03 ; t =3.01,P =0.04 ; t =4.27,P =0.02).However,the VEGF mRNA expression decreased and ADAMTS-1 mRNA and TSP-1 mRNA increased after CX3CL1 stimulation,showing significant differences between the 150 mg/L CX3CL1 group and the control group (t=6.35,P=0.O2;t=-2.92,P=0.04; t=-3.81,P=0.03).Conclusions These results suggest that the macrophages have high heterogeneity.CCL2-and CX3CL1-expressing macrophages can regulate the expressions of angiogenesis-related factors.Macrophage chemokine signal may be a good target for treatment of neovascular ocular disease.

Objective To analyze the clinical efficacy,toxicity and survival prognosis of patients diagnosed with Siewert type Ⅱ and Ⅲ locally advanced adenocarcinoma of esophagogastric junction (AEG) undergoing preoperative involved-field irradiation with concurrent chemotherapy. Methods A total of 45 cases were recruited in this prospective clinical trial. Prior to surgery, patients received 2 cycles of chemotherapy with XELOX and concurrent radiotherapy ( a total of 45 Gy in 25 fractions,5 times weekly). After 6-8 weeks,they underwent surgical resection. After the surgery,patients received 6 cycles of adjuvant chemotherapy. The completion of preoperative neoadjuvant chemoradiotherapy, postoperative pathological status,TNM down-staging effect and adverse reactions were observed. Kaplan-Meier method was applied to estimate survival analysis. Results All 45 patients completed preoperative neoadjuvant chemoradiotherapy. Among them, 39 patients completed 2 cycles of chemotherapy, and 6 patients completed 1 cycle of chemotherapy. The median time of surgical interval was 6 weeks. The R0resection rate was 96%.The pathological complete response (pCR) rate was 22%. The TNM down-staging rate was 69%.The incidence of acute radiation-induced esophagitis or gastritis was 44% and the incidence of radiation-induced pneumonitis was 7%. The incidence of grade 1-3 leukocytopenia,thrombocytopenia and neutropenia was 78%,47% and 44%,respectively. In terms of gastrointestinal reactions,the incidence of nausea,vomiting and loss of appetite was 62%,24% and 71%,respectively. No hematologic or nonhematologic adverse effects was observed at grade 4 or 5.The median follow-up time was 30 months. 11 patients died of cancer,1 patient was treatment-related death in the perioperative period and 1 patient died of pneumonia. The 1-,2-and 3-year progression-free survival (PFS) rates were 90%,70% and 67%,respectively. The 1-,2-and 3-year overall survival rates were 95%,80% and 75%,respectively. The 1-,2-and 3-year local control rates were 95%,84% and 84%, respectively. The 1-, 2-and 3-year distant metastasis rates were 7%, 25% and 25%, respectively. Conclusions Preoperative involved-field irradiation with concurrent chemotherapy yields relatively high clinical efficacy and is well tolerated by patients with Siewert typeⅡandⅢlocally advanced AEG.Patients are recommended to receive 4 cycles of adjuvant chemotherapy following neoadjuvant chemoradiotherapy and surgery.

Objce tive To investigate the effect and significance of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells.Methods Breast cancer cell line MDA-MB-231 cells were used in this study.Breast cancer stem cells were isolated and enriched by serum-free culture.The obtained stem cells were identified through calculating the percentages of CD44 and CD24 stem cells by FACS and evaluating the paclitaxel resistance in vitro and tumorigenicity in mice.RT-PCR, real-time PCR (qPCR) and Western blot were used to detect Oct4 expression.RNA interference was applied to induce Oct4 down-regulation.The interference experiment set up a control group ( no siRNA transfection) , negative control group ( negative siRNA group,transfection of siRNA sequences without any interfering effect on the cells) and Oct4 siRNA group ( transfection of siRNA with interfering effect on the Oct4 gene) .Methyl thiazolyl tetrazolium ( MTT ) and Transwell chamber tests were conducted to detect the proliferation and invasion ability of MDA-MB-231 breast cancer stem cells after Oct4 knock-down, and paclitaxel inhibition test was applied to evaluate drug resistance of MDA-MB-231 breast cancer stem cells after Oct4 knock-down. Resulst MDA-MB-231 breast cancer stem cells grew as spheres cultured in serum-free suspension.MDA-MB-231 breast cancer stem cells showed a higher percentage of CD44+C/D24 -/low cells (97.2%) than that in MDA-MB-231 breast cancer cells ( 76.6%) ( P<0.05) .The tumor size in mice inoculated with MDA-MB-231 breast cancer stem cells was (124.60±13.65)mm3, significantly larger than that of mice inoculated with breast cancer cells (68.20±9.99 mm3) (P=0.0007).MDA-MB-231 breast cancer stem cells were less sensitive to paclitaxel inhibition than MDA-MB-231 breast cancer cells showing by 50% inhibitory concentration (IC50) [(4.40±0.48) μg/ml vs.(8.20±0.34) μg/ml, P<0.05].However, the expression of transcriptional factors Oct4 was higher in MDA-MB-231 breast cancer stem cells than that in breast cancer cells (P<0.05).The proliferation potential of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was significantly lower than that in the negative siRNA and control groups ( P<0.05) from the third day.The invasion ability of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was obviously reduced than that in the control and negative siRNA groups shown by number of penetrated cells [(46.52±2.58) vs.(79.67±3.85) and (77.29±2.13), P<0.05 for both].As for resistance to paclitaxel, IC50 of MDA-MB-231 breast cancer stem cells with Oct4siRNA interference was significantly decreased [(4.48±0.22) μg/ml] compared with that in the control [(7.99±0.59) μg/ml] and negative siRNA group [(8.10±0.68) μg/ml] (P<0.05 for both).Conclusions MDA-MB-231 breast cancer cells are successfully obtained by serum-free culture. The proliferation potential, invasion ability and drug resistance of breast cancer stem cells were down-regulated by Oct4 gene knock-down.

Objce tive To investigate the effect and significance of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells.Methods Breast cancer cell line MDA-MB-231 cells were used in this study.Breast cancer stem cells were isolated and enriched by serum-free culture.The obtained stem cells were identified through calculating the percentages of CD44 and CD24 stem cells by FACS and evaluating the paclitaxel resistance in vitro and tumorigenicity in mice.RT-PCR, real-time PCR (qPCR) and Western blot were used to detect Oct4 expression.RNA interference was applied to induce Oct4 down-regulation.The interference experiment set up a control group ( no siRNA transfection) , negative control group ( negative siRNA group,transfection of siRNA sequences without any interfering effect on the cells) and Oct4 siRNA group ( transfection of siRNA with interfering effect on the Oct4 gene) .Methyl thiazolyl tetrazolium ( MTT ) and Transwell chamber tests were conducted to detect the proliferation and invasion ability of MDA-MB-231 breast cancer stem cells after Oct4 knock-down, and paclitaxel inhibition test was applied to evaluate drug resistance of MDA-MB-231 breast cancer stem cells after Oct4 knock-down. Resulst MDA-MB-231 breast cancer stem cells grew as spheres cultured in serum-free suspension.MDA-MB-231 breast cancer stem cells showed a higher percentage of CD44+C/D24 -/low cells (97.2%) than that in MDA-MB-231 breast cancer cells ( 76.6%) ( P<0.05) .The tumor size in mice inoculated with MDA-MB-231 breast cancer stem cells was (124.60±13.65)mm3, significantly larger than that of mice inoculated with breast cancer cells (68.20±9.99 mm3) (P=0.0007).MDA-MB-231 breast cancer stem cells were less sensitive to paclitaxel inhibition than MDA-MB-231 breast cancer cells showing by 50% inhibitory concentration (IC50) [(4.40±0.48) μg/ml vs.(8.20±0.34) μg/ml, P<0.05].However, the expression of transcriptional factors Oct4 was higher in MDA-MB-231 breast cancer stem cells than that in breast cancer cells (P<0.05).The proliferation potential of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was significantly lower than that in the negative siRNA and control groups ( P<0.05) from the third day.The invasion ability of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was obviously reduced than that in the control and negative siRNA groups shown by number of penetrated cells [(46.52±2.58) vs.(79.67±3.85) and (77.29±2.13), P<0.05 for both].As for resistance to paclitaxel, IC50 of MDA-MB-231 breast cancer stem cells with Oct4siRNA interference was significantly decreased [(4.48±0.22) μg/ml] compared with that in the control [(7.99±0.59) μg/ml] and negative siRNA group [(8.10±0.68) μg/ml] (P<0.05 for both).Conclusions MDA-MB-231 breast cancer cells are successfully obtained by serum-free culture. The proliferation potential, invasion ability and drug resistance of breast cancer stem cells were down-regulated by Oct4 gene knock-down.

Objective To investigate the prevalence of high-risk HPV subtypes in different pathological types of cervical cancer, and analyze the attribution of carcinogenic HPV subtypes in different pathological types. Methods A total of 1 541 patients with cervical cancer were treated between February 2009 and October 2016 in Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. The median age at diagnosis was 49 years (ranged 20-82 years old). The numbers of patients with cervical cancer from North China, Northeast China, East China, Central China and other regions (including Northwest, Southwest and South China) were 961, 244, 175, 87 and 74 cases, respectively. Pathological types: 1 337 cases of squamous cell carcinoma (SCC), 87 usual adenocarcinoma (ADC), 23 adenosquamous carcinoma (ASC), 20 mucinous carcinoma (MC), 19 clear cell carcinoma (CCC), 12 endometrioid carcinoma (EC), 25 neuroendocrine carcinoma (NEC), 9 serous carcinoma (SC), 5 villous adenocarcinoma (VADC) and 4 minimal deviation adenocarcinoma (MDAC). The prevalence of high-risk HPV in different regions, age groups at diagnosis and pathological types in cervical cancer were analyzed. The attribution of 13 high-risk HPV subtypes in different pathological types of cervical cancer based on proportional attribution method, and the attribution of high-risk HPV subtypes prevented by 9-valent HPV vaccine in SCC and ADC were calculated. Results (1) The prevalence of high-risk HPV in 1 541 patients with cervical cancer was 86.6% (1 335/1 541). The multiple high-risk HPV infection rate in patients with SCC ≥60 years old (23.0%, 37/161) was significantly higher than those in patients aged 45-59 years old and≤44 years old [11.4% (85/747) vs 11.7% (50/429), P<0.01], and the high-risk HPV infection rates of patients with cervical cancer in North China, Northeast China, East China, Central China and other regions were respectively 86.8% (834/961), 87.7% (214/244), 83.4% (146/175), 83.9% (73/87) and 91.9% (68/74). SCC (86.8%, 1 337/1 541) and ADC (5.6%, 87/1 541) were the most common pathological types in cervical cancer. The high-risk HPV prevalence of SCC, ADC, ASC, MC, NEC and VADC were 90.1% (1 205/1 337), 74.7% (65/87), 87.0% (20/23), 65.0% (13/20), 72.0% (18/25) and 5/5 respectively. The high-risk HPV infection rates of SC, EC, CCC and MDAC were 4/9, 3/12, 2/19 and 0/4 respectively. (2) According to proportional attribution, HPV 16 (69.5%), HPV 18 (5.6%), HPV 58 (2.2%), HPV 31 (1.9%), HPV 52 (1.4%) and HPV 33 (1.3%) were the six common high-risk HPV subtypes in SCC. While, HPV 18 (44.1%), HPV 16 (20.5%), HPV 52 (2.3%), HPV 58 (1.2%) and HPV 51 (1.2%) were the main carcinogenic subtypes in ADC. The main carcinogenic high-risk HPV subtypes of ASC, NEC and MC were HPV 18 and HPV 16. The total attribution of HPV 16, 18, 31, 33, 45, 52 and 58 prevented by 9-valent HPV vaccine in SCC and ADC were 82.6% and 68.1% respectively; the attribution of HPV 45 in SCC and ADC were only 0.8% and 0. Conclusions SCC and ADC are the main pathological types in cervical cancer. SCC, ADC, ASC, MC, NEC and VADC are closely related to high-risk HPV infection. HPV 16 is the main carcinogenic genotypes of SCC. HPV 18 maybe play an important role in the pathogenesis of ADC.

Objective:To explore the serum cyclic polypeptide biomarkers for ovarian cancer diagnosis.Methods:A total of 54 patients with epithelial ovarian cancer confirmed by pathology in Cancer Hospital, Chinese Academy of Medical Sciences from March 2018 to September 2018 were selected as the study subjects, and 40 healthy women with normal examination results in the cancer screening center were selected as the control. All of the samples were randomly divided into training set and validation set at the ratio of 1∶1 with a random number. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with magnetic bead technology was used for detecting peptide profiling in serum samples to screen significantly differently expressed peptides between ovarian cancer group and control group of the training set (score>5). Receiver operating characteristic (ROC) curve analysis was used to screen differential peptide peaks with area under curve (AUC) ≥0.8, sensitivity and specificity>90% in the training set and validation set. Liquid chromatography-mass spectrometry (LC-MS/MS) was further used to determine the composition of differentially expressed peptides.Results:By comparing the peptide profiles of the two groups, 102 differential peptide peaks were initially detected in the mass-to-charge ratio range of 1 000 to 10 000. ROC curve analysis showed that there were 42 differential peptide peaks with AUC ≥0.8 in both training set and validation set, 19 of which were highly expressed in ovarian cancer group, and 23 were lowly expressed. There were 15 different peptide peaks in highly expressed ovarian cancer group with sensitivity and specificity over 90%. The mass-to-charge ratios were 7 744.27, 5 913.41, 5 329.87, 4 634.21, 4 202.02, 3 879.26, 3 273.35, 3 253.79, 3 234.34, 2 950.33, 2 664.51, 2 018.38, 1 893.37, 1 498.69 and 1 287.55. There were 15 different peptide peaks in lowly expressed ovarian cancer group with sensitivity and specificity over 90%, the mass-to-charge ratios were 9 288.46, 7 759.77, 5 925.24, 4 652.77, 4 210.42, 3 887.02, 3 279.90, 3 240.82, 2 962.15, 2 932.70, 2 022.42, 1 897.16, 1 501.69, 1 337.38 and 1 290.13. No protein composition was identified in 15 different peptide peaks in lowly expressed ovarian cancer group. The two protein compositions identified in 15 different peptide peaks in highly expressed ovarian cancer group were recombinant serglycin (SRGN) and fibinogen alpha chain (FGA), the mass-to-charge ratios of which were 1 498.696 and 5 913.417, respectively. The sensitivity and specificity of the two proteins for ovarian cancer diagnosis were 100%, 100% and 90.9%, 100%, respectively.Conclusion:SRGN and FGA are highly expressed in the serum of ovarian cancer patients, which may be potential diagnostic markers for ovarian cancer.

Objective:To explore the serum cyclic polypeptide biomarkers for ovarian cancer diagnosis.Methods:A total of 54 patients with epithelial ovarian cancer confirmed by pathology in Cancer Hospital, Chinese Academy of Medical Sciences from March 2018 to September 2018 were selected as the study subjects, and 40 healthy women with normal examination results in the cancer screening center were selected as the control. All of the samples were randomly divided into training set and validation set at the ratio of 1∶1 with a random number. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) combined with magnetic bead technology was used for detecting peptide profiling in serum samples to screen significantly differently expressed peptides between ovarian cancer group and control group of the training set (score>5). Receiver operating characteristic (ROC) curve analysis was used to screen differential peptide peaks with area under curve (AUC) ≥0.8, sensitivity and specificity>90% in the training set and validation set. Liquid chromatography-mass spectrometry (LC-MS/MS) was further used to determine the composition of differentially expressed peptides.Results:By comparing the peptide profiles of the two groups, 102 differential peptide peaks were initially detected in the mass-to-charge ratio range of 1 000 to 10 000. ROC curve analysis showed that there were 42 differential peptide peaks with AUC ≥0.8 in both training set and validation set, 19 of which were highly expressed in ovarian cancer group, and 23 were lowly expressed. There were 15 different peptide peaks in highly expressed ovarian cancer group with sensitivity and specificity over 90%. The mass-to-charge ratios were 7 744.27, 5 913.41, 5 329.87, 4 634.21, 4 202.02, 3 879.26, 3 273.35, 3 253.79, 3 234.34, 2 950.33, 2 664.51, 2 018.38, 1 893.37, 1 498.69 and 1 287.55. There were 15 different peptide peaks in lowly expressed ovarian cancer group with sensitivity and specificity over 90%, the mass-to-charge ratios were 9 288.46, 7 759.77, 5 925.24, 4 652.77, 4 210.42, 3 887.02, 3 279.90, 3 240.82, 2 962.15, 2 932.70, 2 022.42, 1 897.16, 1 501.69, 1 337.38 and 1 290.13. No protein composition was identified in 15 different peptide peaks in lowly expressed ovarian cancer group. The two protein compositions identified in 15 different peptide peaks in highly expressed ovarian cancer group were recombinant serglycin (SRGN) and fibinogen alpha chain (FGA), the mass-to-charge ratios of which were 1 498.696 and 5 913.417, respectively. The sensitivity and specificity of the two proteins for ovarian cancer diagnosis were 100%, 100% and 90.9%, 100%, respectively.Conclusion:SRGN and FGA are highly expressed in the serum of ovarian cancer patients, which may be potential diagnostic markers for ovarian cancer.

Clostridioides difficile is a key pathogen of antibiotic related diarrhea and hospital associated infection, causing several outbreaks in Europe and North Americans and resulting in severe disease burden. However, the standardized diagnostic principle and detection specifications in C. difficile infection (CDI) survey are limited in China, and the infection rate and disease burden of CDI in China are unclear. Therefore, National Institute for Communicable Disease Control and Prevention,National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, together with another 11 institutions, draft the group standard entitled "Diagnosis of Clostridium difficile infection (T/CPMA 008-2020)" of Chinese Preventive Medicine Association. Based on the principle of "legality, scientificity, advancement, and feasibility", this standard clarifies risk factors, diagnosis principles, diagnoses and differential diagnoses in order to improve the accuracy of CDI diagnosis in clinical practice, guide the surveillance for CDI, and understand the infection rate and disease burden of CDI in China .

ObjectiveTo examine the clinical features of fractures and related risk factors in patients with rheumatoid arthritis(RA) in China.MethodsSix hundred and eighty-one RA patients were randomly selected from department of rheumatology of 18 hospitals of China.Data were obtained from the questionnaire,including age,sex,disease duration,the involvement of joints,treatment regimen,features of fractures etc.The possible risk factors of fracture in patients with RA were analyzed with a multi-variate Logistic regression analysis.Results① In 681 RA patients of the survey,48 patients had 54 fractures,and the incidence of fractures was about 8％.② Fractures occurred at various sites.Foot/ankle,femur,spine and wrist were the mostfrequent sites.③ The Logistic regression analysis showed that several factors increased the risk of fracture in RA patients,including long disease duration (OR:1.245,95％CI:0.987-1.570,P=0.065),male gender(OR:0.433,95％CI:0.199-0.942,P=0.035),more deformed joints(OR:1.042,95％CI:1.006-1.079,P=0.023),family history of RA (OR:2.201,95％CI:0.984-4.923,P=0.055),and high scores of SF-36(OR:1.017,95％CI:1.002-1.033,P=0.028).④ According to the degree of correlation from strong to weak,the risk factors of fracture were disease duration,SF-36,sex,number of deformed joints and family history of rheumatoid arthritis.ConclusionThe incidence of fracture is high in patients with rheumatoid arthritis.Several factors could increase the risk of fractures in RA patients,including long disease duration,male gender,more deformed joints,and family history of RA.In order to prevent the occurrence of fractures,cautions should be taken to prevent the development of fractures and treat the disease aggressively to suppress the disease activity of RA.