ABSTRACT
Objective To investigate the role of CD4 ~+ CD25~+ regulatory T lymphocytes (Treg)in modulating the cellular immune response and pathogenesis of murine pulmonary tuberculosis.Methods Inactivation of Treg was achieved by intraperitoneal injection anti-CD25 (clone PC61,50 μ/mouse) in PC61 group, and rat-IgG (50 μ/mouse) was injected intraperitoneally in control group. All the mice were inoculated intravenously with H37Rv 0. 1 mL (1 × 10~6 CFU) 3 days after Treg inactivation. The effects of Treg inactivation in different tissues were analyzed by flow cytometry. The cellular immune response, pulmonary histopathology and bacterial load were determined in vitro at different time points. The data were compared using homogeneity of variance F test and non-paired t test. Results In spleen, the percentages of Treg/CD4 T lymphocytes in PC61 group and control group were (21. 13± 3. 58)% and (30. 42± 4. 20)%, respectively at day 10 of inoculation (t = 2. 38, P < 0. 05), and those were (16. 12 ± 1. 26)% and ( 17. 34± 1. 62)%,respectively at day 30 of inoculation (t = 0. 84,P>0. 05). The percentages of Foxp3~+/CD4~+ T lymphocytes in PC61 group and control group were (32. 07 ± 3. 95)% and (60. 55 ± 5. 48)%,respectively at day 10 of inoculation (t = 5. 96, P<0. 05). Similar results were achieved in the peripheral blood. Bacillus calmette-guerin (BCG)-specific 1L-17 (ng/L) secreted by murine spleen cells in PC61 group and control group at day 10, 30 and 60 of inoculation were 5. 1± 0.9 vs 0, 43. 1± 10.0 vs5. 9± 2. 8 and 124.8 ± 5.8 vs 102. 5±8. 1, respectively (t = 7. 90, t=5. 10,t = 3. 19; all P<0.05); those of BCG-specific IFN-γ (ng/L) were 28. 4 ± 8. 2 vs 4. 0±1. 3, 685. 9± 128. 6 vs418. 7±20.4 and 310.9±119. 7 vs 32. 8±7. 5, respectively(tO = 4. 21,t = 8. 43, t = 3. 27; all P<0.05);those of TNF-a (ng/L) were 38. 6±5.0 vs 16. 3±4. 0, 112. 9 ±12. 3 vs 71. 5±12. 6 and 86. 2±8. 2vs0, respectively(t = 4. 95, t=3. 33,t/=14.8; all P<0. 05). The lung bacterial load at day 10 of inoculation in PC61 group was lower than that in control group (t = 4. 63, P < 0. 01), but the differences were not significant thereafter. The changes of lung histopathology at late stage of infection (day 120) in PC61 group were less severe than those in control group. Conclusions Murine Tregs increase dramatically after Mycobacterium tuberculosis infection. Treg could inhibit the specific cellular immunity against Mycobacterium tuberculosis, and therefore, may facilitate the persistent infection of Mycobacterium tuberculosis and development of tuberculosis.
ABSTRACT
BACKGROUND: An ideal marked molecule has not been found to detarmine bone marrow mesenchymal stem cells (MSCs) so as to make sure the homogenicity.OBJECTIVE: To verify the in vitro differentiation from BrdU-treced MSCs into osteoblasts.DESIGN, TIME AND SETTING: A cytological observation/n vitro was performed in Shanxi Medical University from January to October 2008.MATERIALS: Wistar rats aging 4 weeks old were provided by Experimental Animal Center of Shanxi Medical University.METHODS: MSCs were isolated and cultured by using density gradient cantrifugation combined with attachment culture method.At about 80% confluence, trypsin was used for passage and amplification. MSCs at density of 5×1010/L were inoculated in a 25-mm culture dish with L-DMDM culture medium containing dexamethasone, β -phosphoglycarol, vitamin C, and 10% fetal bovine serum. The third-passaged MSCs were labeled in vitro with 10 μmol/L BrdU tracer. Thereafter, 10 visual fields were randomly selected to calculate numbers of positive and negative ceils so as to obtain BrdU tracing rate under a fluorescence microscope (×200).MAIN OUTCOME MEASURES: Inverted microscope was used to observe cell morphology; flow cytometry was used to detect cell surface antigen, differentiation into osteoblasts, and BrdU tracing rate in vitro.RESULTS: The purified MSCs which were like fibroblasts were adherent and fusiform. The third-passaged cells were changed equidirectionally and whirlpool-arranged, and the survival rate was more than 95%. The seventh-passaged cells still grew rapidly.CD44, CD71, and CD105 expressions were positive, but CD45 expression was negative. Black particles were visualized in MSCs after Von kossa staining. BrdU tracing rate was more than 90%.CONCLUSION: Density gradient centrifugation combined with attachment culture method can effectively isolate and purify rat MSCs which are cultured in vitro for a long period and differentiated into osteoblasts. BrdU tracer is safe, effective, and convenient to successfully label MSCs.