Objective To search for the anticancer active substance from Caesalpinia sappan wood extractions. Methods Crude extracts were extracted from Caesalpinia sappan wood with different solvents.Liquid chromatography was applied to analyze the content of each essential component in the extraction fractions. Trypan blue exclusion test was performed to detect the growth suppression rate of human bladder carcinoma cell line T24 treated by the extraction fractions at different time course (20,40,60,80,100 min).The main component positively correlated with the cell suppression rate was separated out using repeated chromatography, thus the anticancer active monomer was obtained, with purity over 98 ％. The chemical constitution was determined using NMR (nuclear magnetic resonance), mass spectrum and infrared spectrum methods. T24 cell line, human ovarian cancer cell line SKOV3, mice sarcoma S180 and hepatic carcinoma H22 cell were chosen as target subjects, with mitomycin, hydroxycamptothecin as positive control drug, the inhibitory activity of the monomer was tested by trypan blue chromophobia method. Results Among the extraction fractions, R12 has a positive correlation with the cell suppression rate (r100 min=0.941, P＜0.001).Brazilin is the key component in R12.The inhibition rate of brazilin could reach 90.89 ％,98.65 ％,99.82 ％ and 100.00 ％ on T24,SKOV3,S180 and H22 respectively in 40 min at the concentration of 1.2 mg/ml,and its effect is much superior to that of the control drug mitomycin and hydroxycamptothecin. Conclusion Brazilin is one of the essential anticancer principles in Caesalpinia sappan wood.

Objective To observe caesalpinia sappan aqueous extract's growth inhibiting and apoptosis inducing effects on human ovarian cancer cell line SKOV3. Methods Cell growth inhibiting effect was detected by cytometry; Examined were by trypan blue staining under a light microscope; Electronic microscopy was used to observe ultrastructure of SKOV3 cell affected by caesalpinia sappan aqueous extract's;With different concentration of caesalpinia sappan aqueous extract's acting on cells, cells cycle and apoptosis rate were analyzed by flow cytometry (FCM). Results The growth of SKOV3 was inhibited effectively by caesalpinia sappan aqueous extract's; typical morphology of apoptosis (cell shrinkage, chromatin condensation and apoptotic body formation) were observed and in a concentration-dependent manner. Flow cytometry detection showed: the experiment in vitro revealed growth of SKOV3 after treatment by caesalpinia sappan aqueous extract's at the different concentration. Apoptosis rate was increased with the increasing of the concentration of caesalpinia sappan aqueous extract's. Conclusion Caesalpinia sappan aqueous extract's can obviously inhibit growth of human ovarian cancer cell line SKOV3 and induce its apoptosis.

Objective To observe hematoxylin's cytocidal and apoptosis-inducing effects on human urinary cancer cell-T24,and its cytocidal mechanism to the target cell.Methods Target cells were incubated in the medium 1640 for 24h,which contained hematoxylin in dosage of zero(blank),12.5,25,50,100,200μg/ml,respectively;under inverted microscopy to observe target cells'morphologic change,and then harvest them;by trypan blue tmpochrome method to determine hematoxylin's cytocidal activity to the target cells;by flow cytomelry to detect the effects of hematoxylin in its different levels on target cells'apoptosis.Results The control group(without hematoxylin)showed their target cells in a fusiform adherent growth,plump,close-arranged,and with a good transparence.With the addition and increment of hematoxylin,target cells turned round,not adherent,pyknotic,with a bad transparence,as well as chromatin condensation,the cells clumped.Cell death rate of control group was(2.63±0.29)%,with the increased dosage of hematoxylin the cell death rate of test groups was(10.00±4.82)%,(21.88±3.42)%,(76.41±4.82)%,(92.27±6.54)%,and(96.34±8.70)%respectively.Flow cytometry showed cell apoptosis rate in control group was 0.47%(occurred spontaneously),but hematoxylin in dose of 50μg/ml made the apoptosis rate increased markedly,to 43.1 8%,dead cell rate 48.47%,and survival cell rate 8.35%.With the increased hematoxylin dose,cell apoptosis rate decreased gradually,while dead cell rate increased.Conclusion Hematoxylin can inhibit the target cell by two routes:to induce apoptose or kill it.In a lower dose it is able to induce target cell to apeptose;hematoxylin in a dose over 100μg/ml can directly kill the target cell.Making this trial for checking the cell's morphologic changes benefits determining the optimal dosage level and optimal acting duration for the apoptosis induction.

Objective To establish a stable animal model of transplanted human colorectal cancer.Methods BALB/c nude mice were randomly divided into orthotopic colorectal model group and subcutaneous inoculation model group by random number table,and separately inoculated with 0.1 ml human colon cancer cell HCT116 under the density of 2×107/ml into the orthotopic colorectal (with the self-made inoculator) and right forelimb pit subcutaneously.The mice were observed for 60 days to compare the tumor formation and tumor growth in the two groups.Results The tumor formation rate of all 18 animals was 100 ％ (18/18) in the orthotopic colorectal group.The average tumor weight was (2.78±1.86) g and the average survival time was (45.00±11.99) d.The tumor formation rate was 27.78 ％ (5/18) in the subcutaneous inoculation group.The average tumor weight was (1.74±0.82) g,and the average survival time was (60.00±0.00) d.Conclusion 0.1 ml (2×107/ml) human colon cancer cell suspension HCT116 inoculated into BALB/c nude mice orthotopic colorectal with self-made inoculator could establish human colorectal cancer animal model successfully.

Objective To observe the effects of brazilin on proliferation and apoptosis in T24 cells.Methods Trypan blue exclusion test was performed to detect the inhibition of brazilin on the growth of T24 cell lines in vitro cultured within different time.After exposure to different concentrations of brazilin,homogeneous bioluminescence assay was used to detect the inhibitory action of brazilin,Caspase-3 and Caspase-9 activity on T24 cells.Cellular apoptosis was measured by flow cytometry (FCM) and observed by laser scanning confocal microscope.Results Brazilin could significantly inhibit the proliferation of T24 cells after 8 hours,the inhibitory rates of the brazilin at concentration of 25,50,100,200 μg/ml against T24 cells respectively were 43.19 ％,60.73 ％,86.38 ％ and 93.89 ％ (P ＜ 0.05).After exposured to 50 μg/ml of brazilin,the inhibition ration to T24 cells increased with time prolonging (52.72 ％ in 4 h,60.73 ％ in 8 h,91.77 ％ in 24 h,96.41％ in 48 h) (P ＜ 0.05).The activity of Caspase-3 and Caspase-9 increased slightly when brazilin was at 25 μg/ml,but there was no statistical differences compared with that in the control group (P ＞ 0.05).When cells were treated with an increase of the concentration of brazilin from range of 7.5-60 μg/ml for 16 hours,the apoptosis ratio in turn showed a upward trend of 0.15 ％,1.35 ％,2.91％,34.76 ％.It could be seen by laser scanning confocal microscope that the apoptosis occurred in the cells.Conclusion Brazilin can effectively inhibit the proliferation of T24 cells and induce apoptosis in a dose and time dependent manner.

AIM:To investigate the pathologic role of aldosterone and protective effect of aldosterone receptor antagonist on peritoneal fibrosis in peritoneal dialysis rats .METHODS:A peritoneal fibrosis rat model was established by intraperitoneal injection of lipopolysaccharide ( at 1 d, 3 d, 5 d and 7 d, 0.6 mg/kg) and dialysate ( daily intraperitoneal injection of 4.25%dialysate, 100 mL/kg).At the same time, spironolactone (an aldosterone receptor antagonist , 100 mg? kg -1? d-1 ) was given to the model rats .After 4 weeks, the expression of aldosterone synthase CYP 11B2, 11β-hydrox-ysteroid dehydrogenase type 2 (11β-HSD2), mineralocorticoid receptor (MCR), and inflammatory factors were detected by immunohistochemistry , real-time PCR and Western blotting .RESULTS:The rat model of peritoneal fibrosis was suc-cessfully established .At the same time, the injury of mesothelial cells , deposition of collagen fibers and thickness of perito-neal were increased .Moreover , the infiltration of macrophages in the peritoneum/dialysate was increased .The level of al-dosterone and the expression of MCR , 11β-HSD2 and CYP11B2 in fibrotic peritoneum were obviously up-regulated as com-pared with normal rats .The expression of NF-κB/MCP-1 was also increased .However , treatment with spironolactone alle-viated peritoneal fibrosis and reduced the expression of NF-κB/MCP-1.CONCLUSION:Local aldosterone is involved in the process of peritoneal fibrosis via NF-κB/MCP-1 pathway.Spironolactone alleviates peritoneal fibrosis of peritoneal dial-ysis.

Objective To compare the inhibitory effects to mice ascitic hepatoma (HepA) among hematoxylin,mitomycin,cisplatin,hydroxycamptothecine and other medicine in celiac chemotherapeutic route,probing into the credibility of hematoxylin in celiac chemotherapy.Methods Mice ascitic HepA model was set up by celiac inoculation of HepA strains.24 h after the inoculation,intraperitoneal administration was done,then raising the mice as routine.Their body weights were measured regularly,living quality observed,mean survival times compared among these groups.Having repeated the above experiment three tines,T/C values obtained in the three experiments were compared one another.Results The awerage survival time of mice of control group (inoculated but no medicine used) was 15.99-16.33 d; that of hematoxylin group was 36.35-39.81 d; that of mitomycin gro.up was 35.77-40.06 d; that of hydroxycamptothecine survived 20.79-38.47 d; and that of cisplatin group was 32.98-41.89 d.A comprehensive comparison showed that mitomycin group and hematoxylin group had better effects.Conclusion Hematoxylin in celiac chemotherapy has an outstanding effect to the mice's transplanted HepA,no significant difference with mitomycin.However,hydroxycamptothecine and cisplatin are not good enough.

Objective To observe the immunological effect of tumor cell vaccines to mouse hepatoma A(HepA) treated by Haematoxylin. Methods HepA cell was treated by 0.1% Haematoxylin and made into three vaccines: HepA vaccine with complete Freund' s adjuvant, HepA vaccine with incomplete Freund' s adjuvant; and HepA vaccine without any adjuvants. Five times of immunization were given the grouped mice with the above three vaccines, then active HepA cells (1×105 for each mouse) were inoculated by intraperitoneal injection to attack them; reckoning the mean survival time (MST) of the grouped mice, comparing the immunoprotective action of the three vaccines to the tmnor-bearing mice. Those mice only receiving normal saline (equal volume to the vaccine) were as a control group. Results MST of control group was (23.30±1.24) day; MST of mice receiving H22 vaccine with complete adjuvant was (43.90±15.20) day (P<0.02); MST of mice receiving H22 vaccine with incomplete adjuvant was (39.60±13.77) day (P<0.05); and MST of mice receiving HepA vaccine without any adjuvant was (38.40±12.54) day (P<0.05); As compared with the control group, the three treated groups showed a life-lengthening rate (LLR) 88.41%, 69.96 % and 64.81% respectively. Conclusion The vaccines treated by Haematoxylin give a marked immunoprotective action to those tumor-bearing mice. The HepA vaccine' s immunological effect is increased by the Freund' s adjuvant (complete or incomplete).

Objective To study the effects of sensitized dendritic cells in the treatment of bladder tumor and further discuss the mechanism of this immunotherapy. Methods 44 female F344 rats, which irrigated N-methyl-N-nitrosourea into bladders every other week for a total of five doses, were induced to bladder tumor. They were treated subcutaneously with either PBS, unsensitized DC, freeze thawing supernatant of tumor cells, or sensitized DC respectively every week for a total of four times. In the fifteenth week, their bladders were weighted and harvested for observation by naked eye and microscope, their blood was harvested for examination CTL by FCM. Results The weight of bladders in sensitized DC group was lower than those in PBS group, unsensitized DC group and freeze thawing supernatant of bladder tumor cells group (P<0.05). The stages of bladder tumor in sensitized DC group were statistically descended compared with those in PBS group (P <0.05). The CD+3 T cells in sensitized DC group was lower than those in PBS group, unsensitized DC group and freeze thawing supernatant of bladder tumor cells group (P <0.05). The CD+3 CD+8 CD+28 T cells in sensitized DC group was higher than those in PBS group, unsensitized DC group and freeze thawing supematant of bladder tumor cells group(P <0.001). Conclusion Sensitized DC injecting subcutaneously can reduce the stages of F344 rats' bladder tumor, Unsensitized DC injecting subcutaneously has not effect in the treatment of bladder tumor;, while the effect of freeze thawing supematant of tumor cells injecting subcutaneously is not well. The mechanism of sensitized DC in the treatment of blader tumor is that DC plays an immunol killing role by presenting antigen, stimulating CTL.

Objective To compare the effects between the impact of IL-7Rα,bacterial flagellin alone to the acute graft-versus-host disease and the combination of both,and to explore its possible mechanism.Methods The BALB/C (H-2d) female mice as recipients were divided into alone irradiation transplantation group (group A),IL-7Rα intervention group (group B),bacterial flagellin intervention group (group C),IL-7Rα combined with bacterial flagellin intervention group (group D),and 6 mice in each group.All mice were accepted 9 Gy 60Co total body irradiation,and 1×107 bone marrow cells and 2× 107 spleen cells of donors C57BL/6 (H-2b) mice were infused via the tail vein to recipient mice.The symptoms,signs,survival time and hematopoietic function recovery of the recipient mice were observed.Results Mice survival of group A was (22.5±2.30) d,30 d survival rate was 50.0 ％ (3/6),and aGVHD performances inculding the fatigue,anorexia,hair removal,arched,and so on appeared obviously.Survival of group B was (25.83±3.49) d,30 d survival rate was 33.3 ％ (2/6),aGVHD performances compared with group A was lighter.Survival of group C was (26.33±3.52) d,30 d survival rate was 33.3 ％ (2/6),also appeared aGVHD performance,which degree was same to the group B.survival of group D was (30.17±2.86) d,30 d survival rate was 66.7 ％ (4/6),aGVHD performances compared to the other three groups was lightest.The white blood cell count of four groups were reduced to minimum at +7 d,then the three intervention groups gradually recovered.The WBC recovery at 14,21,28,30 day after the transplant of group A compared with slowly was the intervention groups (P ＞ 0.05),WBC recovery of B was roughly equal to group C (P ＞ 0.05),while the WBC recovery of group D was faster than group B or C (P ＜ 0.05).At 2nd week after transplantation,CD3+ T cells was significantly decreased in 4 groups,and at 3rd week began gradually rised.Compared with group A,the proportion of CD3+ cells of other three groups were increased significantly,there was no statistical signifiance of CD3+ cell proportion between group B and group C at 2nd,3rd,4th week after transplantation (P ＞ 0.05),while the CD3+ T cell recovery in group D was faster than group B or C (P ＜ 0.05).Conclusions The stable aGVHD mouse model was established.Exogenous IL-7Rα and bacterial flagellin may reduce the incidence of aGVHD.There was no significant difference for aGVHD when they was used alone,but when combination of them,aGVHD is slighter and the hematopoietic recorery is faster.