We prepared bovine epidermal keratin (EK) antibody, which can be used as a specific marker of cholangiocarcinoma(CC) cells to be distinguished from hepatocellular carcinoma (HCC) cells. The intrahepatically implanted solid tumors of FSK 7901 and FSK 7902 have the histological structures of CC and HCC, respectively. Using immunofluorescence and PAP techniques, we demonstrated that, in the solid tumors, FSK 7901 cells are definitely EK-positive, while most of the cells of FSK 7902 are EK-negative. Therefore, we considered FSK 7901 and FSK 7902 as CC and HCC, respectively. In the solid tumors of FSK 7902, few cells which are EK-positive may be those in which there are Mallory bodies or the structures are forming.

Objective To clone and construct eukaryotic expressing vector of bcr-abl fusion gene and to express the gene in the mammal COS-7 cell lines. Methods bcr-abl fusion gene was amplified from human chronic myeloid leukemia (CML) K562 cell lines by RT-PCR and the fragment of cDNA was retrieved,purified and cloned into the pEGFP-N3 eukaryotic expressing vector. After the selection of the positive clone and by restriction enzyme analysis and DNA sequencing, the correct plasmid was transfected into COS-7 cell lines and observed the transient expression. Results A 874 bp DNA fragment was amplified by RT-PCR. The sequence analysis showed it was consistent with bcr-abl gene of GeneBank. RT-PCR, Western blotting analysis provided strong evidences that bcr-abl gene was expressed successfully in transfected COS-7 cells.Conclusion The eukaryotic expressing vector of bcr-abl fusion gene was constructed, it will lay the foundation for further study of bcr-abl gene in the diagnosis and treatment of CML.

Objective To construct an eukaryotic expression vector of the hepatitis C virus(HCV)NS5A gene.and obtain a stable transfected Huh-7 cell line which provides a basis for further investigation of the hepatitis virus C NS5A protein.Methods The HCV NS5A gene from the pcDNA3.1(+)/HCV NS345 plasmid with HCV NS5A gene was amplified by PCR,and cloned to pGEM-T vector,and transformed into E.coli JM109.The positive colonies were first confirmed by restriction enzyme digestion and sequencing and then were inserted to eukaryotic expression vector pCI-neo,and verified by enzyme digestion and sequencing.Results After TA colon of the HCV NS5A was amplified by PCR,the positive colonies were finally verified by sequencing,which were totally in line with the designed coding sequence of HCV NS5A gene.Conclusion Eukaryotic expression vector of HCV NS5A gene has been successfully constructed.

ObjectTo develop the reliable RP-HPLC methods for the determination of salidroside, tyrosol, rosavin, rosin, and rosarin in the plants of Rhodiola L. and to evaluate their species from different habitats. Methods Method Ⅰ: methanol-water (0.5 mmol/L SDS in 1% acetic acid aqueous solution) system for the analysis of salidroside; method Ⅱ: acetonitrile-water system for rosavin; method Ⅲ: aqueous acetonitrile-phosphoric gradient system for salidroside, tyrosol, rosavin, rosin, and rosarin. Results The contents of salidroside in different species range from 0.021% to 1.420%, and those of rosavin in all species are very limited or undetected except in Rhodiola rosea L. and R. sachalinensis. The contents of the five marker ingredients are significantly species- and habitat-dependent. Conclusion Three RP-HPLC methods are established for quantitative analysis of the above five marker ingredients in the meantime, respectively. Evaluation of the quality of varied species of Rhodiola L. shows that R. rosea growing in Xinjiang Uygur Autonomous Region and R. sachalinensis growing in Jilin province are the two better species contained with abundant above-mentioned ingredients in China.

Objective To determine the contents of scutellarin and chlorogenic acid in Qingkailing extract by HPLC.Methods Kromasil C18 column was adopted and the mobile phase consisted of methnol-water-phosphoric acid (20 ∶80 ∶0.2) with the flow rate being 1.0 mL?min-1 and column temperature being 25 ℃.The detection wavelength was 327 nm.Results The linearity of scutellarin was good in the range of 0.090～0.540 ?g,r=0.999 9,and its average recovery rate was 100.3 %(RSD=0.99 %);the linearity of chlorogenic acid was good in the range of 0.196～1.176 ?g,r=0.999 9,and its average recovery rate was 99.9 %(RSD=1.06 %).Conclusion This method is sensitive,simple,accurate,and can be used for the quality control of Qingkailing extract.

Objective:To clone the RC-RNase gene and prepare its recombinant prokaryotic construct, and then to express RC-RNase protein using Escherichia coli system. Methods: RC-RNase cDNA was obtained by RT-PCR from liver of Rana catesbeiana, and cloned into pUCm-T plasmid for nucleotide sequencing. Its expression construct was prepared using the 6?His vector pRSET-A, and induced to express by IPTG in Escherichia coli BL21(DE3). Western blotting identified the expression product. Results: A 380 bp long cDNA was obtained from liver of Rana catesbeiana, restriction sites and sequence being consistent to those reported for RC-RNase. After introducing the gene into Escherichia coli and through the induction by IPTG, it was observed a new peptide at the expected position (Mr 16000) on SDS-PAGE gel. This product was proved to be the target protein via Western blotting. It existed in a form of inclusion body and its efficiency reached 12.5% of total bacterial proteins. Conclusion: RC-RNase gene was cloned and expressed in Escherichia coli. The protein could be used for characterizing the biological activities and function of RC-RNase.

Objective To investigate the prevalence of adult epileptic patients with interictal depression symptoms(IDs) and identify early predictors of IDs. Methods Adult patients with epilepsy were recruited ( n =110,45 females and 65 males) ,age between 16 and 67 years ( median 24 years). The sociodemographic and clinical factors of patients were recorded. Hamilton Depression Scale ( HAMD ) were applied to evaluate interictal symptoms of depression ( at least 72 hours after the last epileptic seizure). According to HAMD score,the epileptic patients were divided into IDs ( ≥8 ) and non-IDs(＜8) groups. The sociodemographic and clinical factors were compared between the two groups to identify the prevalence and early predictors of IDs in adult epileptic patients.Results The prevalence of IDs in adult patients with epilepsy was 38.2% ,49.0% in active epilepsy and 12.1 %in seizure freedom. 30.0% ,5.5% ,and 2.7% were experiencing mild-to-moderate (HAMD score≥8),moderateto-severe ( ≥ 18 ) and severe ( ≥25 ) depression. 42 patients who met the HAMD score≥8 were classified as IDs group,and the remaining 68 patients were classified as non-IDs group. With multiple stepwise backward logistic regreasion, independent predictors of IDs were epileptic seizures ( OR = 8. 845, P = 0. 003 ); symptomatic or cryprogenic epilepsy ( OR = 3.132, P = 0. 045 ); prolonged duration of illness ( OR = 1. 106, P = 0.004 ) and employment status (OR =0. 154, P=0.001 ). There were no relationship between seizure frequency and severity of IDs ( Kruskal-Wallis test, x2 = 4.5, P = 0. 104). Conclusion IDs is a frequent psychiatric comorbidity in adult patients with epilepsy. The prevalence of IDs is higher in those with active epilepsy compared with those in seizure freedom and most of them are mild-to-moderate. Epileptic seizure, symptomatic or cryprogenic epilepsy, prolonged duration of illness and employment status are independent predictors of IDs, but seizure frequency has nothing to do with the IDs severity of patients.

Objective To assess the neuropsychological characteristics in active epileptic patients and investigate itsrisk factors. Methods Ninety adult epileptic patients included 60 active epileptic patients (two or more unprovoked seizures within 12 months) and 30 age-, sex-, education-, course of disease- and seizure type-matched seizure-free subjects (without epileptic seizure for at least 1 year) . The neuropsychological tests including trail making test,digit symbol test, verbal fluency test,digit span test and hamilton depression scale( HAMD) ,were used to detect mental and motor speed, attention, language, working memory and depression symptoms respectively. The neuropsychological tests were compared between active and seizure-free epileptic patients and identified the risk factors of neuropsychological deficits in active epileptic patients. Results Compared to seizure-free subjects, active epileptic patients had significantly worse scores in digit symbol test, verbal fluency test, digit span test ((47.45 ±18. 812) vs(56.40 ±13. 631), (25. 25 ±8. 163) vs(30.40 ±8. 414), (10. 39 ±2. 228) vs( 11. 80 ± 2.074) respectively) ; more time to accomplish the trail making test A and B((64. 35 ±31.710) vs( 45. 47 ± 16. 309) , ( 133. 18 ± 47. 331 ) vs ( 98. 00 ± 35. 003 ) respectively) ; and higher scores in depressive symptoms ((9.12 ±6.219)vs(3.77 ±3.997) ,all P<0.05). Within active epileptic group,significant predictors of neuropsychological deficits were identified in a stepwise linear regression analysis: advancing age was significantly negatively correlated with digit symbol test(β = -0. 468, P = 0. 000) , digit span test (β = -0. 439, P = 0. 000), trail making test A (β =0.365, P = 0.003) and B(β = 0.346, P=0.002) ; higher scores on depressive symptoms was significantly negatively correlated with digit symbol test (β = -0.244, P = 0.015) ; mental work,high-education level and monotherapy were positively correlated with some of the cognitive function subscales. Conclusion This study suggests that active epilepsy can have a direct adverse effect on cognition and depression symptoms. Multi-drug therapy, severity of depression symptoms, advancing age, low-education level and non-mental work are the predictors of neuropsychological impairment in active epilepsy. In addition, good seizure control even after 1 year can have a beneficial impact on cognitive and depression prognosis.

Objective:To investigate the prognostic value of estrogen receptor (ER), progesterone receptor (PR), and Ki-67 in breast can-cer patients receiving neoadjuvant chemotherapy (NAC) and explore the association of chemotherapy regimens and cycles with the outcome of NAC. Methods:Clinical data of cancer patients receiving NAC were retrospectively analyzed. All the patients were admit-ted in Tianjin Medical University Cancer Institute and Hospital from January 2015 to December 2015. All statistical analyses were per-formed using SPSS version 19.0. The relationship among the outcome of NAC, molecular subtype, expression levels of ER, PR, and Ki-67, and chemotherapy regimens and cycles was investigated. Results:Only five HER-2(+) patients accepted the addition of trastuzum-ab in treatment, and few cases were excluded from the statistical analysis based on the effect of chemotherapy regimens. The effec-tiveness of NAC was positively correlated with the expression of Ki-67 whereas negatively correlated with the expression levels of ER and PR (P<0.05). In patients receiving NAC, the patients with Luminal subtype had worse outcome than those with non-Luminal sub-type (P=0.033). The invalid efficacy of pathologic evaluations of Luminal and non-Luminal NAC were 10.1%and 1.3%, respectively. No significant difference was found in the outcome among patients receiving TE, TEC, or EC-T;however, patients who received more than four cycles of NAC had better outcome than others (P=0.016). The outcome was statistically significant when the cut-off value of Ki-67 was 25%. Conclusion:Ki-67 proliferative index could be used as a prognostic marker to NAC in breast cancer patients. The cut-off value of Ki-67 should be determined on the basis of the data of each cancer patient. The curative effect of NAC was poor, and Luminal pa-tients with chemotherapy were insensitive and could be considered for surgical treatment. Patients who received less than four cycles of NAC had worse outcome than others, and prompt NAC foot treatment could improve the efficiency.