AIM In order to search inhibitors of protein kinase CK2, we observed the inhibitory effects of kaempferol on recombinant human protein kinase CK2 holoenzyme and its kinetics in vitro. METHODSCloning, prokaryotic expression and purification of human protein kinase CK2 α' and β subunits by gene engineering, the two subunits were mixed at equal molar ratio to reconstitute CK2 holoenzyme and identify its biological properties. The CK2 activity was assayed by detecting incorporation of 32P of [γ-32P]ATP into the substrate. The inhibitory effect of kaempferol on CK2 was assayed in the presence of different concentrations of kaempferol. Kinetic analysis of kaempferol-induced inhibition was carried out in the condition that casein concentration was fixed at 2 g·L-1 and ATP was changed at various concentrations(10, 20, 40, 80 μmol·L-1), or ATP was fixed at 10 μmol·L-1 and casein was changed at different concentrations (1, 2, 4, 8 g·L-1). RESULTS Kaempferol was shown to strongly inhibit the holoenzyme activity of recombinant human protein kinase CK2 with IC50 of 1.9 μmol·L-1, which was more effective than chrysin, morin and genistein which are both known as CK2 special inhibitors. Kinetic studies of kaempferol on recombinant human CK2 showed that kaempferol acted as a noncompetitive inhibitor with substrate ATP(Ki=1.1 μmol·L-1) and casein (Ki=3.1 μmol·L-1). CONCLUSIONKaempferol is a novel potent inhibitor of protein kinase CK2 in vitro. Discussions indicate that flavonoid inhibitors of CK2 may adopt different orientations in theactive site of CK2 and that these are determined by the number and position of their hydroxyl groups.

Objective To study the protection of taurine(TAU) against apoptosis of neurons induced by manganese(Mn) in vitro. Method Cortex neurons were separated from Wistar neonatal rats and cultured in vitro.The assays began when neurons grew under the best conditions. Cells were randomly divided into 7 groups: control group,Mn-added groups (Mn 0.2,0.6 and 1.0 mmol/L respectively),TAU-intervened groups (1.5mmol/L TAU with Mn). All treatments lasted 24h. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was used to observe the morphology of apoptosic neurons. Flow cytometry (FCM) was used to quantitate neuron apoptosic rates. Results (1) Typical morphologic charateristic was found in Mn-added groups. TAU intervention could protect against the effect of 1.5mmol/l Mn on neurons. (2) FCM indicated that TAU can protect against neurons apoptosis induced by Mn. Conclusion Taurine can protect neurons from apoptosis induced by Mn in vitro.

Objective To investigate the efficacy and mechanism of compound glycyrrhizin on patients with serious hepatitis. Methods Thirty patients who were hospitalized from August 2005 to June 2007 with diagnosed with serious hepatitis were enrolled into treatment group and treated by compound glycyrrhizin injection ( 80 to 100 ml per day,for 3 consecutive weeks) and common supporting medicines,while the other 30 patients in control group were treated only with same supporting medicines. Mortality,biochemical parameters, plasma levels of endotoxin and inflammatory factors in patients of both groups were observed during the treatment. Results By the end of three-week of treatment,8 patients in the treatment group died with the mortality of 26. 7% ( 8 /30) . Thirteen patients died in the control group and the mortality was 43. 3% ( 13 /30) . Serum ALT and AST levels in treatment group were significantly lower than those of control group during the treatment. The average level of serum total bilirubin and plasma prothrombin time in treatment group was lower than those of control group by end of the third treatment week. The level of TNF-alpha in treatment group was lower than that of control group during treatment. The levels of plasma endotoxin and interleukin-6 in treatment group were significantly lower than those of the control group at the second and third treatment week. Conclusion Compound glycyrrhizin improves the biochemical parameters of patients with serious hepatitis,and probably,improves the survival of patients with severe hepatitis. The implying mechanism might be that compound glycyrrhizin declines plasma endotoxin levels and lessen cytokine-induced secondary hepatic injuries.

Objective To investigate the X-ray and CT features of anterior mediastinal lymphoma.Methods 11 cases with lymphoma of anterior mediastinal confirmed by pathology were collected,the X-ray and CT features were reviewed.Results The tumors in all cases appeared as anterior mediastinal tumor,among them,the tumors in six cases grew at anterior mediastinum bilaterally and in five cases at one-side.There were soft tissue nodi around tumor in eleven cases,superior vena cava were involved in 6,neighborhood blood vessel embed by tumors in 5,pleural effusion could be seen in 3,combined with hydropericardium in one.Conclusion The anterior mediastinal lymphomas have certain imaging characteristics,most of them can be distinguish from other anterior mediastinal malignant tumors if one analysing their imaging features carefuly.

Objective To observe the effect of 7 flavonoids on recombinant human protein kinase CK2 holoenzyme activity and investigate their structure-activity relationship. Methods Recombinant hu-man protein kinase CK2 α' and β subunits were mixed at equal molar ratio to reconstitute CK2 holoen-zyme. The CK2 activity was assayed by detecting incorporation of 32p of [γ-32P] ATP into the substrate for the inhibitory effect by flavonoids and calculation of IC50 was performed according to probability unit (PROBIT) method. Results Myricetin, quercetin, morin, luteolin, kaempferol, apigenin, and chrysin were shown to obviously inhibit recombinant CK2 holoenzyme activity in a concentration-dependent man-ner with IC50 values of 1.18, 0.51, 16.16, 0.86, 1.88, 1.72, and 13.63 umol/L, respectively. Myricetin, quercetin, luteolin, kaempferol, and apigenin were more effective than DRB and A3, which were known as CK2 inhibitors in vitro. Whereas morin and chrysin displayed a similar effect to DRB. Structure-activity study indicated that the major structural requirements for the potent inhibition of CK2 by these flavonoids were hydroxyl group at position 6, 3' and 4'. Different from these requirements, absence of a hydroxyl group at position 3 did not modify their inhibitory potency, while addition of hydroxyl groups at positions 2' or 5' was detrimental to the inhibitory effect on CK2. Conclusion The inhibitory effect of flavonoid on protein kinase CK2 in vitro may be determined by the position of their hydroxyl groups.

Objective To clarify the expression of Tbx3 gene in breast cancer tissues and its clinical significance, and the expression of Tbx3 gene in different clinical pathological characteristic breast cancer. Methods Expression of Tbx3 gene in 53 specimens of breast cancer and 28 specimens of normal breast tissues were investigated by immunohistochemistry, analysing the clinical significance. Meanwhile the correlation of expression of Tbx3 gene in breast cancer classified was investigated by estrogen receptor ( ER),lymph node metastasis, Her-2 status. Results Positive rate of Tbx3 gene in breast cancer tissues was significantly higher than that in normal breast tissues [73.58% (39/53) vs 14.29% (4/28)] (P ＜ 0.05).Positive rate of Tbx3 gene in ER positive breast cancer tissues was higher than that in ER negative breast cancer tissues [ 84.38% (27/32) vs 57.14% ( 12/21 ) ] (P ＜ 0.05 ). Positive rate of Tbx3 gene in breast cancer tissues with lymph node metastasis was higher than that in breast cancer tissues without lymph node metastasis [86.21%(25/29) vs 58.33% (14/24) ] (P ＜ 0.05). Positive rate of Tbx3 gene in Her-2 positive breast cancer tissues was higher than that in Her-2 negative breast cancer tissues[ 93.75%(15/16) vs 64.86% (24/37) ] (P ＜ 0.05). Conclusions Tbx3 gene is over expressed in breast cancer tissues. It is related to ER, lymph node metastasis, Her-2 status.

BACKGROUND: Neural stem cells are a kind of special cells possessing self-renewal and multi-directional differentiation potential. They are ideal carriers for studying the occurence, development and development rule of nervous system and their inherent regulation mechanism of molecular biol ogy and cytology. They have wide applicative prospect in the treatment of nervous system disease and injury. Investigating a set of convenient, effective and useful culture system of neural stem cells cultured in vitro is the prerequisite and basis for application. OBJECTIVE: To observe the effect of serum culture medium containing basic fibroblast growth factor on the in vitro culture of neural stem cells of mice. DESIGN: Single-sample observation. SETTING: Department of Histology and Embryology, Youjiang Nationality Medical College. MATERIALS: Five 2-month-old Kunming white mice of either sex were used in this experiment. METHODS: This experiment was carried out at the Department of Histology and Embryology, Guangxi Medical University from June 2003 to May 2005. ① The cerebral cortical cells were isolated from cerebral cortex and cultured in vitro of the adult mouse cerebral cortex and made into cell suspension , then put in the DMEM/F12 (1:1)culture medium (containing fetal bovine serum of 0.15 volume fraction, basic fibroblast growth factor of 20 μg/L, penicillin of 100 u/mL and streptomycin of 100 u/mL ) .The attachment culture method was used to acquire the clone cells. ② Expression of nestin antigen of clone cells was detected with immunohistochemical technique. MAIN OUTCOME MEASURES: ① Morphological observation of cultured cells. ② Observation of cultured cells stained by haematoxylin and eosin . ③ Identification of neural stem cells. RESULTS: ① Morphological observation of cultured cells: After inoculation, most of the cells attached to the wall within 12 hours, cells were thin and flat. 3 days after inoculation, cells began to grow and proliferate. There were several scores to several hundreds of cell clones on the 5th day after inoculation and cells covered 80%-90% of the bottom of the bottle on the 7th to 8th days. After passage, the cells still grew adhesively with regular cellular morphology and clear borderline. ② Observation of cultured cells stained by haematoxylin and eosin: It was shown that the cells presented round or ellipse. There was little cytoplasm, presenting acidophily. Nucleus was big and round, single in the center, deep stained.③ Identification of neural stem cells: Immunofluorescent detection showed that cell nestin antigen positive. CONCLUSION: After cultured in serum culture medium containing basic fibroblast growth factor, cerebral cortex cells possess very strong reproductive activity and express nestin antigen, still possessing the characteristics of neural stem cells.

Objective To construct a novel tissue?engineered bone matrix gelatin (BMG)/poly[butylene succinate?co?tere?phthalate] (PBST) biphasic scaffold for annulus fibrosus. Methods The PBST spinning fibers were prepared by electrospinning and the porosity and water absorption rate were tested. Rabbit annulus fibrosus cells were isolated, cultured and identified through SafraninOstaining, and collagenⅡimmunohistochemical staining in vitro. And then annulus fibrosus cells were implanted on the PBST fiber, whose growth situation was observed by scanning electron microscope (SEM). Then the BMG/PBST biphasic scaf?fold was constructed by BMG as the outer annular fibrosus and PBST fiber as the inner annular fibrosus. The annulus fibrosus cells were implanted on the biphasic scafflod and cultured for 3, 7 and 21 days in vitro. The biomechanical and biological property was observed at the predetermined time point. Results The porosity of the fiber was 61.83%±7.33%and its water absorption rate was 297.34%± 57.13%. The identified result of annulus fibrosus cells were positive, suggesting that the cells have still kept their annulus fibrosus cells characteristics. The cells growth could be observed through SEM at 3rd and 7th day after implanted on the fi?bers. After cultured on the BMG/PBST scaffold, HE staining proved that the cells could ingress into the inner of fiber with time. SafraninOstaining and collagenⅡimmunohistochemical staining proved that the cells can secreted abundant proteoglycan and collagenⅡ, the special annulus fibrosus cell extracellular matrix. Compared with the BMG/PBST scaffold without cells, the elastic modulus of biphasic scaffold was increased from 14.83±1.02 MPa to 17.56±1.47 MPa after cultured with cells for 21 days in vitro. Conclusion The novel tissue?engineered biphasic scaffold for annulus fibrosus constructed with BMG and PBST fiber spinning has good cytocompatibility and biomechanical characteristics, which provide a basis for the complete tissue engineered interverte?bral disc.

BACKGROUND:At present,the most frequently agents used for neural induction of bone marrow stromal cells(BMSCs)in vitro are anti-oxidants,such as beta-mercaptoethanol and all trans-retinoids.The majorities of induction from BMSCs are neuron-like cells in these protocols;however,whether it has neuronal function or not should be further studied.OBJECTIVE:TO investigate the differentiated characteristics of inducing human BMSCs into neural cells in serum-free medium.DESIGN:Observational study.SETTING:Chaozhou Central Hospital.MATERIALS:The experiment was carried out in the Chaozhou Central Hospital from April 2004 to December 2005.Adult bone marrows were derived from femoral and tibial bone marrow of three patients with fracture.All patients provided the confirmed consent and were approved by the Ethics Committee of Chaozhou Central Hospital.DMEMIF12 medium(1:1),fetal bovine serum (FBS), glutamine, N2 supplements and B27 Supplements were from GIBCO/BRL Company;recombinant basic fibroblast growth factor(bFGF)and recombinant epidermal growth factor(EGF)from Sigma Company;monoclonal antibody for vimentin(1:100),monoclonal antibody for myelin basic protein(MBP) (1:100),monoclonal antibody for S1 00(1:1 00),monoclonal antibody for neuron specific enolase(NSE)(1:1 00),and monoclonal antibody for neurofilament 200(NF200)(1:1 00)from Beijing Zhongshan Company;monoclonal antibody for glial fibrillary acidic protein (GFAP)(1:200)and polyclonal antibody for nestin(1:100)from Boster Company(Wuhan);mouse monoclonal antibody for beta-tubulin 3(1:1 000)from Sigma Company;SP-9000 kits and quick AEC from Beijing Zhongshan Company; culture dishes and flasks from Coming Company.METHODS:BMSCs from human bone marrow were cultured in serum-containing medium.When the primary culture was established, BMSCs were transferred into serum-free medium containing N2 or B27 supplement with 20 μg/L basic fibroblast growth factor(bFGF)and epidermal growth factor(EGF),and cells were cultured in an incubator containing C02of 0.05 volume fraction at 37℃. Morphological changes of BMSCs in serum-free medium were observed under phase contrast microscope. And two days after culture. Expression of relative markers of BMSCs was detected withimmunocytochemistry.MAIN OUTCOME MEASURES:Morphological changes of BMSCs and expression of relative markers of nerve cells.RESULTS:A population of BMSCs could be isolated from adult human bone marrow,and they were processed to obtain a fibroblast-like population and were expanded as undifferentiated cells in culture for more than 10 passages.indicating their proliferative capacity.They could form spheroid state when they were sub-cultured in serum-free media supplemented with bFGF and EGF.these cells could express the markers for neural stem cells such as vimentin and nestin;they could expressed neuron specific enolase(NSE),beta-tubulin 3,TrkC and neurofilament 200(NF200)when they were plated on dishes with serum-containing medium; some cells exhibited the phenotypes for astrocytes.expressing gilal fibrillary acidic protein(GFAP)and S100 protein.CONCLUSION:The morphology,protein expression and differentiation ability of BMSCs in serum-free medium was similar to those of neural stem cells.The data support the hypothesis that adult bone marrow contains stem cells capable of differentiating into neural cells,the serum-free media make BMSCs overcome their mesenchymal commitment,showing the phenotypes for neural stem cells.

BACKGROUND: In many studies, rats were commonly used as models of retrorsine-induced hepatic injury. Some reports have confirmed that retrorsine cannot inhibit proliferation of mouse hepatic cells. Other reports have shown that retrorsine has inhibitory effects on proliferation of mouse hepatic cells. OBJECTIVE: To study the liver regeneration after hepatic injury by creating mouse models treated with partial hepatectomy combination with retrorsine. METHODS: A total of 40 C57BL/6J mice were equally and randomly assigned to 2 groups. In the partial hepatectomy combined with retrorsine group, intraperitoneal injection of retrorsine 70 mg/kg was conducted, twice, within an interval of 2 weeks. Four weeks later, 2/3 hepatectomy was performed. In the partial hepatectomy group, intraperitoneal injection of saline 70 mg/kg was performed, twice, with an interval of 2 weeks. Four weeks later, 2/3 hepatectomy was performed. At 14 days after partial hepatectomy, the restoration of the livers was observed. The liver cell injury was observed at 3, 7 days with hematoxylin-eosin staining. The hepatocyte proliferation was observed at 3 days with BrdU staining. Oval cell proliferation was observed at 3, 7and 14 days with CK19 and C-kit antibody immunohistochemistry.RESULTS AND CONCLUSION: In the partial hepatectomy group, the damaged liver nearly restored to normal at 14 days after partial hepatectomy, and the result was contrary to partial hepatectomy combined with retrorsine group. Hematoxylin-eosin staining demonstrated that significant degeneration changes in hepatic cells in the partial hepatectomy combined with retrorsine group. BrdU staining showed that hepatocyte proliferation at day 3 was significantly determined in the partial hepatectomy group, but few in the partial hepatectomy combined with retrorsine group. CK19 and C-kit immunohistochemistry demonstrated that visible oval cell proliferation was seen in mice of partial hepatectomy combined with retrorsine group. First of all, hepatic oval cells appeared in portal area and differentiated into hepatic cells and bile duct cells, and then grew into the hepatic lobule gradually. These indicated that retrorsine can obviously inhibit hepatocyte regeneration after liver injury in mice. The model of mice treated with retrorsine and partial hepatectomy could induce oval cell proliferation.