Objective To investigate the detection of peritoneal free cancer cells and its clinical significance. Methods The peritoneal free cancer cells, the positive rates of CK20 protein and CK20 mRNA expressions of peritoneal lavage fluid were detected by peritoneal lavage cytology (PLC), flow cytometry (FCM) and real-time fluorescent quantitative RT-PCR in 50 cases of gastric cancer patients, respectively. The sensitivity of three kinds of detection method to peritoneal free cancer cells was compared. Results The positive rates of peritoneal free cancer cells, CK20 protein and mRNA expression of peritoneal lavage fluid were 20.0% (10/50), 36.0% (18/50) and 58.0% (29/50), respectively. The positive rate of CK20 mRNA expression detected by real-time fluorescencequantitative RT-PCR in peritoneal lavage fluid was significantly higher than those of the CK20 protein expression detected by FCM and peritoneal free cancer cells detected by PLC (Plt;0.05 or Plt;0.001). The difference of positive rate of CK20 protein expression and peritoneal free cancer cells was not significant (Pgt;0.05). The positive rate of CK20 mRNA expression of peritoneal lavage fluid was related to the tumor invasion depth, differentiation degree, TNM stage, and lymph node metastasis (Plt;0.05). Conclusion Real-time fluorescence quantitative RT-PCR is an effective method for the detection of peritoneal free cancer cells.
PURPOSE:To evaluate the value of the apoptosis-suppressing oncogene bcl-2 protein expression in the development and progression of uveal and conjunctival melanomas. METHODS:Using flow cytometry and immunofluorescence methods to detect the bcl-2 protein expression in 40 cases of uveal malignant melanomas (UMM), 5 cases of conjunctival nevi (CN) and 7 cases of conjunctival malignant melanomas (CMM). RESULTS :The expression content of bcl-2 protein in CMM was significantly higher than that in CN (P＜0.05);the bcl-2 protein positive expression percentages in CMM and UMM were 85.71% and 72.50% respectively. The expression content of bcl-2 protein in UMM was not related to pathological classfication, scleral invasion,ciliary body involvement,and tumor dimensions (P＞0.05). CONCLUSIONS: The over-expression of bcl-2 protein and apoptosis suppressing might be related to the pathogenesis of CMM and UMM;bcl-2 protein expression might be helpful in discriminating CN from CMM, but unavailable in evaluating the patholgical malignancy of UMM. （Chin J Ocul Fundus Dis,1997,13: 73-74 ）
Objective To explore the clinical significance of the platelet glycoproteins CD62p and CD63 in septic patients.Methods The expressions of CD62p and CD63 in peripheral platelet were measured in 40 septic patients within 24 hours after onset by flow cytometry.The expression levels of CD62p and CD63 in mild and severe sepsis and normal subjects were compared.Meanwhile the correlation of CD62p and CD63 with APACHE Ⅱ score was analyzed.Results Significant differences in the CD62p and CD63 levels were found in the septic patients when compared with the normal subjects [CD62p：（2.56±1.51）% vs （1.48±0.40）%；CD63：（2.15±0.50）% vs （1.29±0.35）%；all Plt;0.01].The expressions of CD62p and CD63 in the severe septic patients were significantly higher than those in the mild septic patients [CD62p：（3.31±1.94）% vs （2.05±0.87）%；CD63：（2.37±0.36）% vs （2.00±0.53）%；all Plt;0.05].The positive correlations of CD62p and CD63 with APACHE Ⅱ score were also found（CD62p：r=0.377，P=0.016；CD63：r=0.452，P=0.003）.Conclusion Platelets were significantly activated in septic patients at early stage which was correlated with the severity of sepsis.
ObjectiveTo establish a methodology for alveolar macrophages (AMs) phagocytosis of AlexaFluor 488 (AF488) labeled bacteria by flow cytometry.MethodsStaphylococcus aureus and Streptococcus pneumoniae were labeled with different concentrations of AF488. A flow cytometric assay was used to quantify in vivo bacterial uptake by AMs. AMs and different ratio of fluorescent-labeled bacteria were incubated at 37 ℃ for 2 hours, 4 hours, 6 hours and 8 hours, respectively. AMs were washed with DPBS and extracellular fluorescence was quenched with 1% (w/v) trypan blue. Trypan blue was aspirated and phagocytosis of fluorescent-labeled bacteria by AMs was measured using a flow cytometry. Confocal microscopy was performed to ensure that bacterial in positive AM had been internalized rather than bound to the cell surface.ResultsWhen the concentration of AF488 was more than 50 μg/mL, the labeling rates of Staphylococcus aureus and Streptococcus pneumoniae were higher than 92% (P<0.05), and has quickly reached the upper limit. With the prolongation of incubation time, the phagocytic rate of AMs increased from 20.4% at 2 hours to 76.5% at 8 hours. With the increase in the number of bacteria, the phagocytic rate of AMs increased from 7.7% by ratio of 1∶10 to 85.1% by ratio of 1∶300.ConclusionDetection of AMs phagocytosis of AF488 labeled bacteria by flow cytometry is an effective method, but the dye concentration, incubation time and the proportion of bacteria will influence the results.
Objective To investigate the effect of peritoneal exudative cells as feeder cells on growth state of primary culture of adult rat retinal Muuml;ller cells. Methods Peritoneal exudative cells were gained from adult rats, which were identified with specifically biological marker of macrophage (CD68). The phagocytosis was evaluated by the ink particles experiment. Retinal Muuml;ller cells of adult rats were cultured by enzyme digestion method, and identified by GFAP and vimentin immunocytochemically. As the feeder cells, peritoneal exudative cells were cocultured with Muuml;ller cells. The proliferation cycle of Muuml;ller cells was assayed by flow cytometry. One-step TUNEL staining was employed to detect the apoptotic Muuml;ller cells. Results Over ninety-five percent of rat peritoneal exudative cells were macrophage, which have a favourable phagocytic ability for the ink particles. The primary cultured Muuml;ller cells adhered to the wall of flask and grew fast, with large applanate cell bodies. The third-generation cells grew slowly. After cocultured with feeder cells, the Muuml;ller cells showed more rapid growth rate with more cells in S and G2/M phase(S phase, t=4.172, Plt;0.001; G2/M phase, t=3.562, Plt;0.01） and less apoptotic rate (t=3.804, Plt;0.01). The growing cycle was cut down from 25-30 days to 1822 days for the firstgeneration cells, from 10-15 days to 7-10 days for the second-generation cells. Conclusion It is an effective method to use the peritoneal exudative cells as feeder cells cocultured with primary culture of retinal Muuml;ller cells, which can shorten the culture period of Muuml;ller cells in adult rats.
Objective To examine the influence of retinal pigment epithelium(RPE) cells on antigen-specific activatedlymphocytes in vitro,and to explore the role of RPE cells in the immune privilege of the eye. Methods Co-culture systems of RPE cells with antigen-specific T lymphocyte lines and resting T lymphocytes were established in vitro．Induction of apoptosis was detected by genomic DNA electrophoresis,DNA in situ end-labelling and flow cytometry. Results RPE cells induced apoptosis in antigen-specific activated T lymphocytes. 24 hours after culture,the signs of apoptosis appeared in lymphocytes co-incubated with RPE cells．As time of co-culture went on,the number of apoptosic cells increased．Quantitative analysis of apoptosic cells showed that apoptosic cells accounted for 5.95% after 24 hours, 9.38% after 48 hours,and 17.95% after 72 hours．In contrast,RPE cells induced few apoptosis in resting T lymphocytes. Conclusions These results suggest that RPE cells possess the ability to induce the apoptosis of invading lymphocytes. This phenomenon serves as a restrain mechanism of immune response and may be of vital importance in the maintenance of immune privilege in posterior segment of eye and in the protection of eye from the damage of immunogenic inflammation. (Chin J Ocul Fundus Dis, 1999, 15: 241-244)
ObjectiveTo investigate the diagnostic value of products triggered by endotoxin including cytokines and procalcitonin for differentiating bacterial pneumonia from pulmonary tuberculosis. MethodsFifty patients diagnosed to have hospital-acquired pneumonia and another 50 patients diagnosed with tuberculosis admitted into West China Hospital between January and August 2015 were recruited in this study. The frequencies of CD4+ interferon (IFN)-γ+, CD4+ tumor necrosis factor (TNF)-α+, CD4+ interleukin (IL)-2+, CD4+ IL-10+ as well as CD8+IFN-γ+, CD8+TNF-α+, CD8+IL-2+, CD8+IL-10+ populations in peripheral blood were detected by flow cytometry after endotoxin stimulation. Meanwhile, the levels of procalcitonin, IL-6 and C reactive protein were measured by immunofluorescence staining. ResultsThe frequencies of CD4+ IFN-γ+, CD4+ TNF-α+, CD4+ IL-2+, CD4+ IL-10+ as well as CD8+ IFN-γ+, CD8+ TNF-α+, CD8+ IL-2+, CD8+ IL-10+ populations in the pneumonia group increased significantly compared with those in the tuberculosis group (P < 0.05). The levels of procalcitonin, IL-6 and C-reactive protein in the pneumonia group increased statistically compared with the counterparts in the tuberculosis group (P < 0.05). The positive rates of procalcitonin, IL-6 and C-reactive protein in the pneumonia group were significantly higher than those in the tuberculosis group (P < 0.05). ConclusionMeasurement of products triggered by endotoxin is beneficial for differential diagnosis of pneumonia from tuberculosis.
【Abstract】ObjectiveTo find a stable and efficient method for ex vivo concentration of CD4+CD25+ regulatory T cells in rats. MethodsCD4+CD25+ regulatory T cells were separated from the rat splenic cells in two steps by magnetic cell sorting (MACS) system. CD4+ T cells were first negatively sorted by cocktail antibodies and antiIgG magnetic microbeads. And then CD4+CD 25+T cells were positively sorted by antiCD25 PE and antiPE magnetic microbeads. The purity and the cell survival rate of the sorted cells were measured by flow cytometry and trypan blue dyeing respectively, and the immunosuppressive action of CD4+CD25+ T cells on the proliferation of CD4+CD25- T cells was also assessed by in vitro cell proliferation assay. ResultsThe purity of negatively sorted CD4+ T cells were (83.6±2.5)% (79%~87%), and the purity of positively sorted CD4+CD25+ T cells was (90.2±1.8)% (86%~93%) with the survival rate of (92.8±3.4)% (92%~95%). These concentrated cells significantly suppressed the proliferation of CD4+CD25- T cells in mixed lymphocyte culture （CD25+/CD25- versus CD25-, P＜0.01）. ConclusionWe created a twostep procedure of magnetic cell sorting system for CD4+CD25+ regulatory T cells sorting, which insures the cells to be satisfactorily purified and well functioned.
Objective To observe the proapoptotic effect ofthe homogenate of different parts of pig’s full thickness dermal wounds on cultured fibroblasts. Methods The tissues were dissected from the wound center and subneoepithelium separately 15 days after homogenization and sterilization, the specimens stored at -70℃. The forth passage of the fibroblasts were cultured for 16 hours in different culture solutions and were grouped into 7 groups: DMEM containing 5% fetal bovine serum as Group Ⅰ, DMEM containing 5% homogenate of tissue from wound center as GroupⅡ, DMEM containing 5% homogenate of tissue from subneoepithelium as Group Ⅲ, the culture solution of Group Ⅱmixed with 10 μg/ml GM6001 in Group Ⅳ, with the culturing medium of Group Ⅲplus 10 μg/ml GM6001 as Group Ⅴ, the culture solution of Group Ⅱ mixed with 10 ng/ml aFGF as Group Ⅵ, and the culture solution of Group Ⅲ mixed with 10 ng/ml aFGF as Group Ⅶ. In all groups except Group Ⅰ, the fibroblasts of the 6 pigs were treated with the homogenate derived from the same animal respectively. After being incubated in Annexin Ⅴ-FITC and PI, cells were analyzed by Flow Cytometry and the rate of apoptotic cells was acquired. The data were analyzed by SPSS 11.0 using Leastsignificant Difference test(LSD). Results The apoptotic rate of the 7 groups were as follows:4.39%±0.41% in Group Ⅰ，10.98%±1.42% in Group Ⅱ，13.47%±1.44% in Group Ⅲ，7.2%±0.46% in Group Ⅳ，12.1%±0.85% in Group Ⅴ，3.9%±0.63% in Group Ⅵ，9.8%±0.50% in Group Ⅶ； there were significant differences between every two groups except Group Ⅰand Group Ⅵ. Conclusion Homogenate of the tissue derived from the subneoepithelium has greater proapoptotic effect than that from the wound center; the proapoptotic effect of homogenate of the tissue both under neoepithelium and in wound center can be significantly alleviated by acid fibroblast growth factor, partly because of MMPs.
Objective To investigate the role of expression of T cell costimulatory molecule CD28 and variance of T cell subpopulations in the development and prognosis of gastric cancer and colorectal cancer. Methods The peripheral blood lymphocytes were tested for T cell subpopulations and T cell costimulatory molecule CD28 by flow cytometry in 38 patients with gastric cancer, 42 patient s with colorectal cancer , and 21 healthy peoples as control group . Results Expressions of T cell costimulatory molecule CD28 in patients with gastric cancer and colorectal cancer were (25. 80 ±10. 56) % and (28. 95 ±9. 29) % , and significantly higher than that of control group 〔(0. 82 ±0. 98) % , Plt; 0. 01〕. Expression percentage of total T cell (CD3 + ) in patient s with gastric cancer and colorectal cancer were significantly lower than that of control group 〔(53. 61 ±13. 84) % and (55. 96 ±10. 68) % vs (72. 07 ±7. 83) % , Plt; 0. 01〕. Expression percentage of CD4 + T cell (CD4 + CD3 + ) in patients with gastric cancer and colorectal cancer were significantly lower than that of control group 〔( 29. 84 ±9. 71) % and ( 33. 75 ±9. 04) % vs (38. 79 ±5. 08) %; Plt; 0. 01 , Plt; 0. 05〕; Expression percentage of CTL cell (CD8 + CD28 + CD3 + ) in patient s with gastric cancer and colorectal cancer were significantly higher than that of control group 〔( 1. 57 ±1. 99) % and (1. 93 ±2. 61) % vs (0. 02 ±0. 04) %; P lt; 0. 01〕; Expression percentage of CD8 + inhibitory T cell (CD8 + CD28 -CD 3 + ) and CD4 / CD8 ratio in patient s with gastric cancer were significantly lower than that of control group 〔(16. 06 ±6. 94) % vs (20. 56 ±6. 54) % , Plt; 0. 05 ; (1. 10 ±0. 51) % vs (1. 36 ±0. 31) % , P lt; 0. 05〕; Expression of regulatory T cell (CD4 + CD25 + CD3 + ) of patients with colorectal cancer was (19. 74 ±6. 89) % , which was significantly higher than that of control group 〔(13. 72 ±3. 08) % , Plt; 0. 01〕. No difference of expression was found in peripheral T cell subpopulations of postoperative patients with gastric cancer and colorectal cancer after one week ( Pgt; 0. 05) . Conclusion T cell number is fall ,T cell costimulatory molecule CD28 useless expression is increase in patient s with gastric cancer and colorectal cancer. CD4 + T cell subpopulation is significantly decreased in patient s with gast ric cancer. The regulatory T cell of patient s with colorectal cancer is significantly increased.