Objective To Clone,express,purify 38 ku protein of mycobacterium tuberculosis,to study its immunological characteristics,and to evaluate its potenial value for serodiagnosis of tuberculosis.Methods Extract DNA from standard strain of H37Rv as the template,amplify gene of 38 ku protein by PCR,insert to PET-30a(+)and construct the recombinant plasmid,express 38 ku protein in E.coli BL21(DE3),purify by Nickel affinity chromatography,at last get target proteins of higher purity,analyze its immol/Lunological characteristics by Western blotting and ELISA technology from Feb.2003 to Mar.2004. Results The clone was analyzed at the nucleotide lever and showed the same DNA sequence coding for natural 38 ku protein. The recombinant protein expressed in inclusion body in E.coli BL21(DE3). The purity of terget protein was 92.7% by Nickel affinity chromatography,Western blotting assays showed that the recombinant protein had satisfactory antigenicity . 38 ku protein detected TB postive and negative refference serum based on the mechanism of indirect ELISA,results showed that the sensitivity was 80.5%(33/41)and the specificity reached to 96%(25/26).Conclusion The recombinant protein expressed in inclusion body in E.coli BL21(DE3)and had satisfactory antigenicity,and might be selected as one of serodiagnostic antigen of tuberculosis.

Objective To investigate the efficacy and accuracy of quantitative computed tomography (QCT) in assessment of fracture healing. Methods Twenty-four healthy New Zealand rabbits were enrolled in the study and randomly divided into two groups, ie, Croup A (union model group, transverse fracture open created on mid-shaft of tibia and inter-fixed by kirschner wire) and Group B (non-union model group, transverse fracture with 5 mm defect on mid-shaft of tibia open created, then sealed with bone wax on fracture gap and medulla cavity, inter-fixed by kirschner wire). At 2, 4, 8 and 12 weeks, Kirschner wire was withdrawn and plain X-ray and QCT scanning were performed on the bilateral tibia. Then, rabbits were scarified and its bilateral tibia were desected and histologically examined. The result of X-ray and histological analysis was used as the "golden standard" for evaluation of fracture healing. Receiver operating characteristic curve (ROC) was used to analyze the evaluation performance of QCT. The corresponding segments of the contralateral healthy tibia were used as control to investigate the change of QCT parameters. Results In Group A, X-ray and histological analysis verified clear fracture line filled with irregular callus at 2 and 4 weeks but proved vague or vanishing fracture line and continuous and intact cortex of irregular callus at 8 and 16 weeks. In Group B, X-ray and histology analysis found clear fracture line with inactive ossification at 2 and 4 weeks but vague fracture line with scarce osteocyte and bone trabecula at 8 and 16 weeks. ROC analysis of QCT results showed the following results; (1) the areas under curve (accuracy) of material parameters including bone mineral density (BMD) and bone mineral content (BMC) were 0.781 and 0.750 respectively; (2) structure parameter-cross-sectional area (CSA) and the area under curve of cross-sectional moment of inertia (CSMI) were 0.781 and 0.469 respectively (P <0.05); (3) the areas under curve of the extending parameters bone strength indices (BSICSA) and CSMI bone strength indices ( BSICSMI) were 0. 913 and 0. 813 respectively (P < 0.05); (4) the area under curve (accuracy) of BSICSA, CSA and BMD were 0.905, 0.921 and 0.905 respectively (P<0.05). Conclusions QCT has potential in distinguishing fracture union and nonunion models in measurement of local fracture pattern. The screening parameters with more accuracy are BSICSA, CSA, BMD, which have advantages of accuracy and specialty in assessing fracture healing.

Numbness is a common symptom in many diseases.In terms of cognition about numbness in past dynasties,and based on clinic observation and practice,the research of Guizhi Huangqi Wuwu Decoction is necessary.As a classic prescription,it is important for doctors to concern about combination of disease and syndrome,but also attaches importance to correspondence of prescription and syndrome.

Objective To explore the inhibitory effects of Corilagin on the production of proinflammatory cytokines in microglia induced by radiation. Methods The cytotoxicity of Corilagin was measured by MTT assay. Microglia BV-2 cells were irradiated 0 or 32 Gy after pretreated with Corilagin for 12 hours. Realtime-PCR was used to detect the mRNA levels of inflammatory cytokines, such as IL-1β,TNF-α on several time-points. The content of nitric oxide (NO) was determined with nitrate reductase method. The translocation of NF-κB was measured by Western blot and immunocytochemical stain.Confocal microscopy was used to observe the expression of Iba-1 and Nemo. Results No cytotoxicity was detected on BV-2 cells with 1-10 μg/ml Corilagin. Iba-1 expression in microglia cells was activated by irradiation, the expression levels of inflammatory cytokines, such as IL-1β, TNF-α and NO were also elevated. Whereas, the production of IL-1 β, TNF-α in activated microglia cells was significantly inhibited with 5 μg/mL corilagin ( tIL-1β = 6. 341, tTNF-α = 3.41 1, tNO = 3. 134, P ＜ 0. 05 ). Corilagin significantly inhibited the expression of Nemo and the translocation of NF-κB p65. Conclusion Corilagin could inhibit the activation of irradiated microglia cells and down-regulate the expression of inflammatory cytokines, via inhibition of the NF-κB signaling pathway.

ObjectiveTo investigate the expression and significance of Thl7 cells in peripheral blood of patients with Rheumatoid Arthritis (RA).MethodsThirty-three active RA and 27 stable RA patients and 30 healthy individuals were enrolled in the study.Intracellular flow cytometry detection of TH17 cells in peripheral blood was established.The correlations between Th17 cells and RA disease activity (DAS28 scale) as well as bone erosion were analyzed.ResultsThe expressions of Th17 cells in the patients with RA were significantly higher than those of healthy controls [ ( 1.60 ± 0.51 ) ％ vs (0.81 ± 0.21 ) ％,t =10.30,P ＜ 0.001 ].The Th17 levels in active RA patients were also higher than that of stable RA patients and healthy subjects [ ( 1.94 ±0.38 ) ％ vs ( 1.19 ± 0.27) ％,t =8.85,14.60,P ＜ 0.01 ].And the Th1 7 levels in stable RA patients were also higher than that of healthy subjects (t =5.88,P ＜0.01 ).Active RA patients with bone erosion ( 19 cases) also had higher levels of Th17 cells than those without bone erosion ( 14 cases) [ ( 2.09 ± 0.39 ) ％ vs.( 1.74 ±0.29 ) ％,t =2.83,P ＜ 0.01 ].The expression of Th17 cells was positively correlated with the DAS28 score ( r =0.86,P ＜ 0.01 ).ConclusionThere is a high expression of Th17 cells in peripheral blood of RA patients,which is related to disease activity and bone erosion.Th17 cells might play an important role in the pathogenesis of RA.

Objective To assess the therapeutic effect of human adipose-derived mesenchymal stem cells on radiation-induced vascular injury in the small intestine of rat. Methods A total of 34 male Sprague-Dawley rats were enrolled in this study. To establish a model of radiation-induced intestinal injury, each rat was irradiated with 15 Gy in whole abdomen. 17 rats were randomly selected and infused intraperitoneally with passage 6 ( P6 ) Ad-MSCs, and the other 17 rats that received PBS were set as control. 10 days post-irradiation, the number of CD31+ endothelial cells in the small intestine villus was measured by flow-cytometry, the expressions of CD31, CD105 and isolectin-B4 in the na?ve endothelial cells with detected by IHC-staining, and the vascular integrity was evaluated by measuring VE-Cadherin. The origination of na?ve endothelial cells within injured intestine was also analyzed. In addition, total mRNA were extracted from irradiated small intestine to assay the expressions of VEGF, bFGF, Flk-1 and SDF-1 using quantitative Real-time PCR. Results Compared to the control, the amount of CD31-postive endothelial cells within irradiated intestine was significantly increased after Ad-MSCs infusion ( t=12?15, P<0?05). The microvascular density in the injured sites was also significantly increased by the infusion of Ad-MSCs (20 d:t=10?33, P<0. 05;30 d:t=32?85, P<0?05). Moreover, the expressions of VEGF, bFGF, Flk-1 and SDF-1 were significantly up-regulated after delivery of Ad-MSCs ( VEGF:t =10?34, bFGF:t=11?25,Flk-1:t=6?73, SDF-1:t=6?73, all P<0?05), which was beneficial in maintaining the integrity of intra-villus blood-vessels as well as promoting neovascularization in the injured sites. Conclusion Ad-MSCs had potentials in healing radiation-induced vascular injury in rat small intestine.

Objective To study the efficacy and toxicity of gensenoside-Rg3 (Rg3) combined with radiotherapy on non-small cell lung cancer ( NSCLC ) at advanced stages (Ⅲ and Ⅴ ).Methods Sixty-three patients with stage Ⅲ or Ⅳ NSCLC were divided randomly into two groups:treatment group ( n =35 ) treated with Rg3 combined with radiotherapy and control group ( n =28 ) treated with radiotherapy alone.The efficacy and side effects were compared after the treatment.Results The response rate ( CR + PR) of the treatment group was 57.14％,significantly higher than that of the control group (32.14％,x2 =3.91,P ＜ 0.05).The median survival time of the treatment group was 14.2 months,significantly longer than that of the control group ( 11.2 months,x2 =2.07,P ＜ 0.05 ).The one-year survival rate of the treatment group was 62.86％,significantly higher than that of the control group (39.29％,x2 =4.40,P ＜0.05).The incidence rates of side effects of the treatment group were all lower than those of the control group,but there were not significant difference. Conclusions Gensenoside-Rg3 combined with radiotherapy is effective for advanced stage NSCLC,with attenuation and synergistic effects.

Purpose To investigate the application value in diagnosis of pleural effusion by cell block combined with immunohisto-chemistry. Methods 60 cases of pleural effusion were collected, and paraffin-embedded cell block was prepared and immunohisto-chemistry was used to detect the expression of CK7, TTF-1, E-cadherin, CEA and Calretinin. Results By use of cell block combined with immunohistochemistry, malignant detected rate was higher than that of the conventional centrifugal smear. There was statistical significance in the expression of CK7, TTF-1, E-cadherin, CEA and Calretinin between pleural effusion lung adenocarcinoma and reac-tive hyperplasia mesothelial cells (P<0. 05). CK7, TTF-1, E-cadherin and CEA were highly expressed in pleural effusion of lung ad-enocarcinoma cell. Calretinin was highly expressed in hyperplastic mesothelial cells. Conclusion Cell block and immunohistochemi-cal technique combination in the differential diagnosis of difficult pleural effusion has important clinical significance. It is worthy of popularization and further clinical application.

Objective To detect the efficiency and function of NK cell differentiation from human umbilical cord he?matopoietic stem cells (HSCs) in vitro. Methods CD34+hematopoietic stem cells were isolated from human umbilical cord blood, and inoculated into SCGM medium containing 20 μg/L FMS like tyrosine kinase 3 ligand (Flt-3L), stem cell fac?tor (SCF), interleukin (IL)-7, IL-15 and IL-21. And CD34+HSCs were differentiated into NK cells in directional inducing. The growth state of cells was observed. The expressions of CD56, NKG2D, NKp46, CD3, CD19 and CD34 were detected by flow cytometry in the differentiation of 7, 14, 21 and 28 d. In the differentiation of 21 d and 28 d, the differentiation cells were used as effector cells, and K562 cells as target cells. The ratios of effector cells and target cells were 8∶1, 4∶1, 2∶1 and 1∶1. The killing activity of the differentiated cells was detected by lactate dehydrogenase (LDH) cell toxicity assay and 7AAD/CFSE labeling method. Results CD34+HSCs derived from human umbilical cord blood can proliferate in vitro under appropriate condition. There were no significant differences in the expression of CD3 and CD19 between different differentia?tion stages (7, 14, 21 and 28 d, P>0.05). The expressions of CD56, NKG2D and NKp46 were significantly different (P<0.05), and the ultimate expression amount was (72.57±1.60)%, (32.83±1.29)%and (29.53±2.40)%. The expression of CD34 decreased gradually, and the lowest was (12.13 ± 2.01)%. The maximum killing activity detected by LDH cell toxicity assay and 7AAD/CFSE labeling method reached(49.91±2.76)%and (40.87±1.12)%.The killing activity of NK cells was decreased in the order of 8∶1, 4∶1, 2∶1 and 1∶1 groups (P<0.05). There was no significant difference in the killing activity between NK cells of 28 d and 21 d. Conclusion Human umbilical cord hematopoietic stem cells can differentiate into NK cells un? der appropriate conditions in vitro, and the NK cells induced from differentiation are with killing activity.

Objective To evaluate the therapeutic effect of adipose-derived mesenchymal stem cells on radiation enteritis.Methods A total of 52 male Sprague-Dawley rats were used in the present study.Herein,46 rats were randomly selected and irradiated with a dose of 15 Gy at their abdomens.Two hours post-irradiation,23 rats were randomly selected and infused intraperitoneally with adipose-derived mesenchymal stem cells in passage 6 from young-female donor.The other 23 rats were intraperitoneally infused with PBS.The rest 6 rats were set as normal control.During the first 10 days post-irradiation,peripheral blood-samples from irradiated rats were harvested for testing the levels of IL-10 in serum using ELISA assay.Additionally,after isolating the thymic cells and peripheral blood mononuclear cells,the percentages of CD4/CD25/Foxp(3)-positive regulatory T cells in thymus and peripheral blood were tested by flow-cytometry.Finally,infiltration of inflammatory cells and deposition of collagens within irradiated small intestine were analyzed by H&E staining and Masson Trichrome staining,respectively.Based on the MPO-immunohistochemistry staining,the type of infiltrated cells was identified.The Kaplan-Meier method was used for analyzing the survival rate of irradiated rats.Results During a period of 30 days post-irradiation,the irradiated rats receiving adipose-derived mesenchymal stem cells survived longer than those receiving PBS (t =4.53,P ＜ 0.05).Compared to the irradiated rats with PBS-treatment,adipose-derived mesenchymal stem cells could elevate the level of IL-10 in serum (7 d:t =13.93,P ＜ 0.05) and increase the percentages of CD4/CD25/Foxp(3)-positive regulatory T cells in both peripheral blood (3.5 d:t =7.72,7 d:t=11.11,10 d:t =6.99,P ＜0.05) and thymus (7 d:t =16.17,10 d:t =12.12,P＜ 0.05).Moreover,infiltration of inflammatory cells and deposition of collagens within irradiated small intestine were mitigated by adipose-derived mesenchymal stem cells.Conclusions Adipose-derived mesenchymal stem cells were capable of curing radiation enteritis.