OBJECTIVE:To establish a method for determining plasma concentrations of tanshinone ⅡA and use it for pharma-cokinetics study of tanshinone ⅡA in Beagle dogs. METHODS:HPLC was performed on the column of Phenomenex Luna C18, with the mobile phase of water(5 mmol/L of ammonium acetate+0.05% H3PO4)-acetonitrile at flow rate of 1.0 ml/min;the detec-tion wavelength was 270 nm,column temperature was 40 ℃ and the volume was 20 μl. 9 Beagle dogs were randomly divided into tanshinone ⅡA low,medium and high dose groups(2,4 and 8 mg/kg),blood was respectively taken from forelimb venous blood to determine the plasma concentration before and after 2,5,10,15,20,30,45,60,75,90 and 120 min of iv administration. DAS 2.0 software was used to calculate the pharmacokinetic parameters. RESULTS:The linear range of tanshinoneⅡA was 0.097 5-12.50 μg/ml (r=0.999 8);the RSDs of precision and stability tests were all less than 10%;the method recovery was 100.4%-107.2%,and extraction recovery was 90.2%-92.3%. The t1/2αin tanshinoneⅡA low,medium and high dose groups were respective-ly(1.01±0.27),(1.03±0.46)and(1.51±0.65)min;t1/2β were respectively(16.25±4.78),(22.42±9.32)and(24.45±12.02) min;AUC0-120 min were respectively(150.88±45.25),(305.44±92.55)and(643.67±178.27)μg·min/ml;and CL were respective-ly(12.01±4.36),(12.78±5.06)and(12.17±5.41)ml/(min·kg). CONCLUSIONS:The precision,stability and recovery of the method are all in line with the determination requirements of biological samples,and tanshinone ⅡA showed a good linear relation-ship with dose in Beagle dogs in AUC0-120 min.

BACKGROUND: The repair of periodontal tissue is dependent on the number and proliferation and differentiation abilities of periodontal ligament (PDL) cells. PDL cells have the potentiality of multi-directional differentiation such as cementoblast,osteoblast and fibroblast to fonn cement, alveolar bone and periodontal ligament and finally achieve periodontal tissue regeneration.OBJECTIVE:To observe the effects of Shuanghuangbu extract on the proliferation and differentiation of PDL cells.DESIGN:Observation trail.SETTING:Central Laboratory of Wuxi Third People's Hospital. MATERIALS: Periodontal tissue was provided voluntarily by the healthy young patients with deformity correction, and golden thread,skullcap,and rhizoma drynariae by the National Institute for the Control of Pharmaceutical and Biological Products.METHODS: The experiment was conducted in the Central Laboratory of Wuxi Third People's Hospital from July to October 2003.The crushed golden thread.skullcap,and rhizoma drynariae were mixed with distilled water at ratio of 1:10 (m:V),and refluxed in boiling water for 5 hours.The extract was collected,and after colation,the residue was refluxed in boiling water for another 3 hours. Both extract was blended, rotary evaporated and condensed, finally the liquid extract of 3 kg/L was obtained.There were 8 groups in the study including golden thread group, skullcap group,rhizoma drynariae group,golden thread plus skullcap group,golden thread plus rhizoma drynariae group, skullcap plus rhizoma drynariae group, Shuanghuangbu group and control group. PDL cells were cultured/n vitro assisted with Shuanghuangbu.The proliferation of cells was detected with MTT method and the ratio of collagen content in total protein was evaluated with hydroxyproline (HP).MAIN OUTCOME MEASURES:A value of proliferated PDL cells and the proportion of collagen protein in total protein.RESULTS:①Proliferation of PDL cells:Except golden thread group,all Chinese medicine promoted the proliferation of PDL cells significantly compared with control group (P＜0.05). The A value of Shuanghuangbu group was significantly increased.A value was increased with time and reached the peak on day 5.There were significant differences among each group at different time (P＜0.05).②Ratio of collagen content in total protein:Except golden thread group,the percentage was significantly increased by other Chinese medicines compared with control group (P＜0.05), especially Shuanghuangbu. The percentage was increased with time and reached the peak on day 5. There were significant differences among each group at different time (P＜0.05). CONCLUSION:The findings suggest that as a traditional Chinese herb,Shuanghuangbu can significantly stimulate the proliferation and differentiation of PDL cells.and increase the proportion of collagen content in total protein.It may act as an ideal Chinese medicine helper factor for the regeneration of PDL cells.

BACKGROUND: Multipotential mesenchymal stem cells (MSCs) can be differentiated into bone, fat, cartilage, muscle and endothelial cell at the specific conditions, so it draws great attentions of the related study.OBJ ECTTVE: To establish the method of in vitro culture and expansion of human umbilical blood-derived MSCs and investigate their influencing factors.DESTGN: Randomized and controlled trials.SETTING: Department of Tissue Engineering and Cell Biology in Wuxi Third People's Hospital.MATERTALS: Seventy cases of human umbilical cord blood (HUCB) of healthy neonates (selected from Parturient Room, Department of Gynecology and Obstetrics, Wuxi Third People's Hospital, with the informed consents of the puerpera and their relatives), 40 mL each; dulbecco's minimal essential medium (DMEM) of low glucose (LG) at the dose of 50 g/L, fetal bovine serum (FBS), mycillin and trypsin (all were from Gibco), FITC-labeled mice anti-human CD29, CD105 and CD106 (Ancell) monoclonal fluorescent antibody, PE-labeled mice anti-human CD34, CD44, CD45, CD19, HLA-DR (Immunotech)monoclonal fluorescent antibody, Ficoll separation medium (Pharmacia).METHODS: The experiment was carried out in Department of Tissue Engineering and Cell Biology in Wuxi Third neonatal cord blood were anticoagulated by 25 u/mL heparin, and 60 cases of isolated HUCB mononuclear cell were precipitated and suspended by cell culture fluid (50 g/L DMEM-LG + 50 g/L FBS + 10 g/L mycillin), while other 10 cases were purified with DMEM of high glucose (100 g/L) to produce adherent layer. Once the cells reached fluences of FBS coating on adhesive rats of MSCs: Some of the purified HUCB MSCs at logarithmic phase were growth curve of MSCs: The growth of cells were observed by phase contrast microscope (OLYMPUS CK40), and were detected by EPICS-ALTRA flow cytometry.FBS coating and without FBS coating at the different time points, and their adhesive rates.RESULTS: All 70 cases of HUCB MSCs were adopted in this study, however, 10 samples failed to obtain the ideal MSCs cord blood MSCs after culture. During 2 weeks of culture, primary cells began to double for 3-4 days and reached conand CD105, but did not express antigens CD34, CD45, CD106 and HLA-DR, which was identical to surface labeling of those without FBS coating (P＜0.01).CONCLUSrON: MSCs in HUCB can be cultured and expanded in vitro, and are regarded as an alternative source of MSCs for experimental and clinical applications. Moreover, high glucose can depress the growth and proliferation of MSCs in HUCB.

Objective To study role of MDC/CCI22-CCR4 axis in mouse milky spots with peritoneal carcinomatosis of gastric cancer. Methods We examined the expression of CCR4 in 615 Mouse gastric cancer cell (MFC) lines by RT-PCR and Western-blot; Peritoneal metastasis model on the 615mouse was established by intraperitoneal injection of 0.2 ml MFC cells(1×104 cells). Dil fluorescence was used to observe the transfer process and section of MFC. Immunohistochemistry was conducted to detect the expression of CCR4 and CCL22 in omental milky spot; the structure of Milky spot was observed by scanning electron microscopy. Mice were randomly divided into 2 groups, namely, the saline control group (received saline) and MFC group. The concentration of CCL22 in ascitic fluid was measured in the 615 mice injected MFC after 6,8,10 days and in the saline group. Results MFC first metastasizes to the milky spot on the omentum, the expression of CCR4 and CCL22 were observered in the milky spot. The surface layer cells in milky spot consisted of discontinuous mesothelial cells and mainly macrophages and lymphocytes. The average value of CCL22 was 43 pg/ml and 364 pg/ml respectively in saline control group and MFC group.Conclusions MDC/CCL22-CCR4 axis plays an important role in the development of peritoneal carcinomatosis in mouse gastric cancer.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Child, Preschool ; Female ; Humans ; Lymphoma, T-Cell ; classification ; diagnosis ; Lymphoma, T-Cell, Peripheral ; classification ; diagnosis ; Male ; Middle Aged ; Prognosis ; Young Adult

Hepatic fibrosis models were induced by CCl4 in rats. To explore vascular endothelial growth factor (VEGF), transforming growth factor-beta1 (TGFβ1) mRNA expression and bcl-2, Bax protein expression levels of intervention and explore the mechanism of the Aralia chinesis anti-hepatic fibrosis. Sixty male Sprague-Dawlley (SD) rats were randomly divided into six groups: nomal group, model group, high-dose (10 mL x kg(-1)), medium-dose (7.5 mL x kg(-1)), low-dose (5.0 mL x kg(-1)) of A. chinesis treated group and colchicine treated group. The change of liver histopathology was observed by HE and Masson staining. The mRNA of VEGF, TGF-β1 were detected by RT-PCR. The protein of Bcl-2 and Bax were detected by Western blot. In the model group liver cell obvious degeneration, necrosis, a large number of collagen fibers of the cable hyperplasia, part visible pseudolobule formation. A. chinesis large, medium, low-dose group and colchicine group liver cell degeneration and necrosis reduced A. chinesis small, medium, and high-dose group was gradually reduced trend and A. chinesis large, middle dose group degree of reduction is particularly significant. Compared with model group, A. chinesis of large, medium and small dose group and colchicine group VEGF mRNA expression, A. chinesis of large, medium-dose group TGF-β1 mRNA expression reduce (P < 0.05); compared with colchicine group, A. chinesis of large, middle dose group of VEGF mRNA expression decreased (P < 0.05); A. chinesis of large, middle dose group of TGF-β1 mRNA expression decreased (P < 0.01), and compared with colchicine group, large dose group of of TGF-β1 mRNA expression decreased (P < 0.05). Compared with model group, A. chinesis of large, medium and small dose group and colchicine group Bcl-2 protein expression reduce (all is P < 0.05). But A. chinesis of large, medium and small dose group and colchicine group of Bax protein expression were increased (P < 0.05). A. chinesis regulation of VEGF, TGF-β1 may prevent the activation of hepatic stellate cells, liver tissue by up regulating the anti-apoptotic protein Bax and down pro-apoptotic protein Bcl-2 expression, thereby to improve the degree of liver fibrosis.
Animals ; Apoptosis ; drug effects ; Aralia ; chemistry ; Drugs, Chinese Herbal ; administration & dosage ; Hepatic Stellate Cells ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; drug therapy ; genetics ; metabolism ; Male ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism

AIM: To determine the role of Fas antigen and caspase-8 in modulating apoptosis of osteosarcoma cells induced by bacterial redox protein azurin. METHODS: The human osteosarcoma cell line U2OS was treated with bacterial redox protein azurin (150 mg/L) for 6, 12, 24 and 48 h, respectively. Cell immunohistochemistry and quantitative image pattern analysis were applied for detecting the expression of Fas antigen. Caspase-8 activity was detected using caspase-8 fluorescent assay kit. The apoptotic rate was measured by FCM. RESULTS: Compared with the control group, the expression of Fas antigen and activity of caspase-8 significantly increased in U2OS cells treated with 150 mg/L azurin (P

Objective To preliminarily observe the clinical efficacy of hippocampal-sparing prophylactic cranial irradiation (HS-PCI) using helical tomotherapy (HT) in patients with limited-stage small-cell lung cancer (LS-SCLC) after chemoradiotherapy,and compare HT with intensity-modulated radiotherapy (IMRT) and volumetric modulated arc therapy (VMAT) in dose distribution.Methods From April to June,2014,six patients with LS-SCLC who had achieved a complete remission after chemoradiotherapy were assigned to HS-PCI using HT within a month after brain metastasis was ruled out using brain magnetic resonance imaging (MRI).After fusing CT images and MRI images,the hippocampus was contoured in the fusion images and hippocampal avoidance regions were created using a volumetric expansion of 3 mm around the hippocampus.A dose of 25 Gy in 10 fractions to 95％ of planning target volume (PTV) was prescribed in HT,IMRT,and VMAT.The clinical efficacy,adverse reactions,neurocognitive function,and brain metastasis were evaluated for HT.The dose distribution in PTV and hippocampus were compared between HT,IMRT,and VMAT.Results There were one patient with abdominal wall and abdominal lymph node metastases,one patient with local recurrence,and no patient with brain metastasis during the observation period.The numbers of patients with grade 1 and grade 2 headache,dizziness,and hair loss reactions were 3 and 1,3 and 1,and 4 and 2,respectively.There were no significant differences in the average score of the Mini-Mental State Examination before treatment and at 3 and 6 months after treatment (29.7,29.2,and 29.3 ; P =0.083,0.317,and 0.157).The mean dose to the hippocampus was 16.85 Gy for IMRT and 17.59 Gy for VMAT.For HT,the mean doses to the hippocampus and avoidance regions were reduced to 5.26 Gy and 6.21 Gy,respectively.The prescribed dose for HT was reduced by 79％ and 71％ compared with IMRT and VMAT,respectively.The average coverage rate of the prescribed dose was 94.48％ for HT.Conclusions HT achieves promising dose distribution and target coverage in sparing of the hippocampus.Moreover,HT dose not increase the incidence of adverse reactions.The change in neurocognitive function needs to be further studied with longterm observation and large-scale sampling.

Objective To explore the feasibility of evaluating cardiac structure, coronary artery,pulmonary artery and cardiac function in one single scan by 320-row CT ECG-gated double phase cardiac function scan mode. MethodsForty patients underwent the 320-detector row CT double phase cardiovascular angiography. The pulmonary phase and aortic phase were reconstructed in order to evaluate the pulmonary and coronary artery. MPR reconstructions of both pulmonary and aortic phase were used to analyze the function of the two ventricles. And the results of the cardiac function were compared with those of transthoracic echocardiography. Thirty-five cases could be analyzed and diagnosed, while the other 5 cases had to be given up because of the poor imaging quality. The mean heart rate was (71.2 ± 11.2) beat per min (bpm). No arrhythmia case included. Results ( 1 ) Pulmonary embolism were diagnosed in 11 cases,coronary artery disease (CAD) were found in 5 cases, while post-stent implantation were observed in 7 cases. Six cases of congenital heart disease were diagnosed with 3 ASD and 3 primary pulmonary hypertension. Another one was diagnosed with left atrial myxoma, and 5 cases were pulmonary embolism associated with CAD. All of above cases were verified by final clinical diagnosis. (2) The heart function parameters including LVEDd , RVEDd, LVESd, RVESd and LVEF were (36.7 ±3.3), (43.3 ± 3.4) mm,(31.6±5.1), (41.3 ±5.1) mm and (47.1 ±15.1) for CT, while those were (40.3 ±3.1), (47.3 ±4.2) mm,(37.3 ±5.6), (45.3 ±3.3) mm,and (46.0 ± 14.8) for ultrasound, respectively. The CT results were correlated with the ultrasound ( n = 35, r = 0.886-0.988, P ＜ 0.01 ). (3) The average radiation exposure was ( 5.4 ± 0.5 ) mSv. Conclusions 320-row CT ECG-gated double phase cardiac function scan mode is feasible for the "one-stop-shop" examination of the cardiovascular disease. This noninvasive method is recommended for the diagnosis, differential diagnosis, treatment and prognosis of cardiovascular disease.

Yeast is a widely used host for recombinant protein expression. However, glycoproteins derived from yeast contain N-glycan of high mannose type and are usually hyperglycosylated. alpha-1,6-mannosyltransferases gene (och1) encodes the enzyme that initiates the first step of out-chain elongation of high mannose type N-glycan in yeast, which is different from that in human. So, a high efficient method to knockout target gene by two-step recombination was established and was used to delete och1. In the first recombinant, a plasmid with och1::ADE1 and ura3 gene was linearized in the downstream of och1 and inserted to the och1 site of P. pastoris genome, where the upstream and downstream of och1 were duplicated. In the second recombinant, the duplicated fragments of och1 were exchanged and the och1 deletion strains were selected on the plates containing 5-FOA, but no adenine. Then the och1 deletion strain was applied to express an human serum albumin (HSA) granulocyte-macrophage colony-stimulating factor (GM-CSF) chimera. Different with the hyperglycosylated HSA/GM-CSF chimera expressed in wild type P. pastoris, the chimera expressed in the och1 deletion strain, contained smaller N-glycan. The results suggested that the och1 mutant yeast may be more suitable for production of recombinant glycoproteins. And the och 1 deletion strain could be used for further re-engineering to produce complex human glycoproteins.
Chimera ; Gene Deletion ; Gene Knockout Techniques ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Mannosyltransferases ; genetics ; Pichia ; enzymology ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Serum Albumin ; biosynthesis ; genetics