Objective: To establish an HPLC fingerprint method of Verbenae officinalis L. and determine the contents of its main components. Methods: Many batches of Verbenae officinalis fingerprint and the contents of main components were determined by HPLC, and the similarity and cluster analysis of the fingerprints of each batch were compared and the contents of the main components were compared. Results: The results showed that the difference of the main content of medicinal materials and fingerprints were between each batch of Verbenae officinalis. Conclusion: The fingerprint and its main components determination method have been established to provide the scientific basis for the quality evaluation of Verbenae officinalis.

Objective: To establish the HPLC fingerprint of herbs of Patriniae Herba. Methods: The fingerprints of Patriniae Herba were built using Orca C18 column and acetonitrile-0.05% phosphoric acid as mobile phase. The flow rate was 1.0 mL/min. The detecting wavelength was set at 230 nm. The temperature of column was at 35℃. Results: Under the selected spectrum conditions, HPLC fingerprint of herbs of Patriniae Herba was established, and eight public peaks were shown in the HPLC fingerprint. The methodological study met the required standards. Cluster analysis showed that class I medical BJ1, BJ2, BJ3, BJ4, BJ5, BJ6, BJ7, BJ8, BJ9, both, BJ13, BJ14 each batch Patriniae Herba and control the similarity between fingerprints was 0.987-0.907, Indicating that there are good consistency between batches of medicinal materials; class II medical BJ11, BJ12, and BJ15 group of Patriniae Herba and control fingerprint differences larger. Conclusion: The method is accurate and reliable, simple and efficient, and can be used as the evaluation of Patriniae Herba from different origins and with different varieties.

Aim To investigate the effects of diallyl di-sulfide( DADS) on G2/M arrest in Chk1/MGC803 and Chk2/MGC803 cells so as to establish stable human gastric cancer MGC803 cells with overexpression of Chk1/2 gene. Methods The colony formation, flow cytometry, RT-PCR and Western blot were used to de-tect the proliferation, cell cycle, and expression of Chk1/2 mRNA and protein, p-Chk1/2, CDC25C and cyclinB1, respectively. Results The colony formation showed that the colony forming efficiency in Chk1/MGC803 and Chk2/MGC803 cells treated by 30 mg· L-1 DADS was lower than in control group and vector group ( P <0. 05 ) . Flow cytometry demonstrated that 41. 3%, 57. 4%, 68. 9% and 42. 9% of G2/M cells in Chk1/MGC803 were increased than in MGC803 and Chk2/MGC803 , respectively after treated by DADS in 12,24, 36 and 48 h(P <0. 05). At the same time, RT-PCR disclosed that expression of Chk1 and Chk2 mRNA had no marked change. Western blot showed that total proteins of Chk1 and Chk2 and p-Chk2 had invisible change, but expression of p-Chk1 was up-reg-ulated, and CDC25C and cyclinB1 were down-regula-ted time-dependently in Chk1/MGC803 cells ( P <0. 05 ) . Conclusion DADS arrests MGC803 cells at G2/M by increasing p-Chk1 expression to cause down-regulation of CDC25C and cyclinB1 simultaneously.

Objective To optimize purification technology of total flavonoids in Callicarpa nudiflora Hook. et Arm. by macroporous adsorption resin. Methods Macroporous resin models including AB-8, D-101, HP-20, HP2MG, were optimized by static adsorption and desorption experiments regarding to adsorption rate and desorption rate of total flavonoids. Purification technology parameters of total flavonoids were optimized by single factor test. Results HP-20 macroporous resin presented the best purification efficiency,the optimum purification conditions were that taking 4. 46 mg·mL-1 of total flavonoidsat pH 3. 0, loading at 3 BV·h-1, washed with 3BV of water at 3 BV·h-1,then eluted with 4 BV 75% ethanol at 2 BV·h-1, finally obtaining the total flavonoids from the dry extract of Callicarpa nudiflora Hook. et Arn. with the purity of 47. 4%. Conclusion HP-20 macroporous resin is suitable for preliminary purification of total flavonoids in Callicarpa nudiflora Hook. et Arn.

To observe BAG-1, EGFR, and PARP-1 expressions in invasive breast cancer and its correlation with clini-cal pathological indicators, as well as to evaluate their clinical significance. Methods:The BAG-l, EGFR, and PARP-1 expressions in a tissue microarray of invasive breast cancer and peritumoral tissues were detected through immunohistochemical staining. The clinical and pathological significance of BAG-1, EGFR, and PARP-1 were evaluated. Results:The BAG-1, EGFR, and PARP-1 expression lev-els are higher in invasive breast cancer tissues than in peritumoral tissues (P<0.05). BAG-1 expression in invasive cancer tissues is not related to age, tumor site, lymph node metastases, and clinical TNM staging of patients, but is related to size, grade, ER, PR, and HER-2 expressions and molecular subtype (P<0.05). EGFR expression is related to size, clinical TNM staging, and molecular subtype (P<0.05). PARP-1 expression is related to grade, lymph node metastases, ER, and molecular subtype (P<0.05). BAG-1 expression is not significantly correlated with EGFR and PARP-1 in all cases, but BAG-1 and PARP-1 expressions are positively correlated in tri-ple-negative breast cancer tissues (P<0.05). Results of the univariate analysis revealed that the BAG-1 and PARP-1 expressions and the molecular subtypes are associated with the prognoses of breast cancer patients. Multivariate analysis revealed that BAG-1 and PARP-1 expressions are factors that are independent of the prognosis. Conclusion: BAG-1, EGFR, and PARP-1 overexpressions in human breast tissues suggest that BAG-1, EGFR, and PARP-1 are related to breast cancer development. BAG-1, EGFR, and PARP-1 are poten-tial biomarkers of breast cancer diagnosis and prognosis.

AIM:To research the effects of lithium chloride on transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) in cultured human Tenon capsule fibroblasts (HTFs) and explore its mechanism.METHODS:HTFs were cultured and identified by vimentin staining with immunofluorescence and the morphological characteristics.The experimental group was processed 48h with LiCl in concentration of 80mmol/L, the control group without LiCl.The mRNA expression of TGF-β and CTGF in two groups were analyzed with real-time fluorescent quantitative polymerase chain reaction (real time-qPCR) and the protein expression was detected with Western blot.RESULTS:The cultured HTFs expressed TGF-β and CTGF.The mRNA expression of TGF-β and CTGF significantly decreased compared with the control group(t=20.042, 14.995, P<0.05).the protein expression of TGF-β and CTGF also decreased significantly compared with the control group(t=46.058、12.452, P<0.05)CONCLUSION:The cultured HTFs can express TGF-β and CTGF in mRNA and proteins' level.LiCl can reduce the expression of TGF-β and CTGF both in gene and proteins' level.LiCl has the potential to modulate wound healing for glaucoma filtration surgery.

Objective To study the role of glucagon-like peptide-1 (GLP-1) analogue liraglutide played in the proliferation of CD4+CD25 T cells in normal people and newly-onset type 1 diabetic patients,and to evaluate the possible immune regulatory role of liraglutide in the therapy of type 1 diabetes.Methods CD4+ CD25-T cells of 10 normal people and 10 newly-onset type 1 diabetic patients were separated from peripheral blood by MACS immunomagnetic beads and stimulated by Human T-Activator CD3/CD28 Dynabeads to proliferate.CFSE labeling technique was used to evaluate the proliferation of CD4+ CD25-T cells by flow cytometry.Liraglutide of different concentrations(0,25,50,and 100 nmol/ml) was added to the proliferation system,then the proliferation of CD4+CD25-T cell was measured.Results (1) Liraglutide suppressed the proliferation of CD4+ CD25-T cells from either normal people or type 1 diahetic patients with dose-dependent manner (P ＜ 0.05).(2) Under the different concentrationsofliraglutide,the proliferation ofCD4+CD25 T cells from diabetic patients was mueh more robust than that of normal people (P＜0.01).(3) The inhibitory effects of liraglutide on CD4+ CD25-T cells proliferation in normal people and diabetic patients were similar (P＞0.05).Conclusion The proliferation of CD4+ CD25 T cells in type 1 diabetic patients was more robust than normal people,which indicated cellular immune dysfunction in type 1diabetes.Liraglutide inhibits the proliferation of CD4+ CD25-T cells of type 1 diabetic patients in vitro.The immunosuppression effect of liraglutide may have potential value in the treatment of type 1 diabetes.

Objective To decrease the p53 gene expression at cellular and animal levels in marmoset using RNA interference technique.Methods The shRNA interference sequences were designed and inserted into the adeno-associated virus vector plasmid after bioinformatics analysis.The plasmids were transfected into African green monkey kidney cos-7 cells.The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western bolt.The adeno-associated virus-8 was injected through the hind leg vein.The changes of p53 protein expression in the liver tissue was detected by Western blot and immunohistochemistry.Results We screened two RNA interference effective arget sequences.The expression of p53 mRNA was suppressed ( 82.7 ±8.1 )% and ( 80.7 ± 7.5)%, respectively (P<0.05), and the expression of p53 protein was decreased (77.3 ±11.5)% and (73.7 ± 10.7)%, respectively (P<0.05).The two marmosets after virus infection showed that there were virus distributions in the liver, testes, and neck detected by in vivo fluorescence imaging.The expression of p53 in the marmoset liver was detected by western blot, immunohistochemistry analysis showing no obvious changes.Conclusions In the present study, the decrease of P53 gene expression at cellular level is achieved, however, the liver P53 protein in the marmoset liver is not significantly changes.Further optimization of the way of infection is needed in the future.

A preliminary exploration of the application and effects of digital orthopaedic technique combined with the clinical practice in our clinical teaching of orthopaedics and trauma for 8-year program medical students has been made. Construction of 3D-model of bone and joint with the digital reconstruction technology, combination of 3D-display with detailed clinical cases, individual studying of knowledge, and fracture models performed by three dimensional printing, all have enhanced the students' understanding of the basics knowledge of orthopaedic trauma and accelerated the process of its joining into clinical practice. Related software has been used to realize the virtual design of fracture reduction and fixation and based on it, students have been guided to personally involve in the development and realization of surgical simulation program on a personal computer, thus improving students' capabilities of analysis, diagnosis and treatment of illness. Besides, students have been actively guided to use digital technology to conduct the finite ele-ment analysis of posterior malleolar fractures and calcaneus fractures , and to produce APP software for mobile devices such as ankle fracture classification and function scoring , which has aroused students' in-terest of clinical research and innovation.

Objective To investigate the clinical diagnostic value and pathogenesis of serum protein identification in Keshan disease (KD).Methods A total of 65 chronic KD patients were selected as the patient group in KD endemic areas,while 29 cases of dilated cardiomyopathy (the DCM group),62 healthy cases from KD endemic areas (control 1 group) and 28 healthy cases from non-endemic areas (control 2 group) were selected as controls.Liquid chip time of flight mass spectrometry (ClinProtTM MALDI-TOF-MS) was used to determine the expression of proteins/peptide peaks.ClinProTools 2.2 software was used to analyze the protein profiles to determine differentially expressed proteins/peptide peaks.The Genetic Algorithm (GA),QuickClassifer Algorithm (QC) and Supervised Neural Network Algorithm (SNN) methods were used to screen marker proteins.Matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometry technique (MALDI-TOF/TOF) was also used as a secondary mass spectrometry to identify differentially expressed peptides.Results Between the KD and control 1 groups,34 differentially expressed proteins/peptides and 5 marker proteins were identified,while 52 differentially expressed proteins/peptides and 5 marker proteins were identified between the KD and control 2 groups,and there were 67 differentially expressed proteins/peptides and 5 marker proteins between the KD and DCM groups.During secondary mass spectrometry,two peptides for mass-to-charge ratio (m/z) 2 079 and 1 465 were obtained,peptide of matching β-globin showed low expression while peptide of matching fibrinogen showed high expression in the KD patients.Conclusions Serum marker proteins can be used as biomarkers for diagnosis and differentiation of KD.β-globin and fibrinogen play an important role in the development of KD myocardial injury.