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 clarify the relationship between inhibition of proliferation and cxpression of Ki-67 in cultured human retinal pigment epithelial(RPE) cells. Methods The cultured human RPE cells were treated with daunoblastina at a dose of 180 mu;g/L for 12h.Twenty-four hours later,DNA inhibiting rate was studied by using tritium-labelled thymidine deoxyribose(3H-TdR)incorporation assay.The expression of Ki-67 was evaluated by immunocytochemical staining technique and image analysis system.Flow cytometry was used to analyse cell cycle. Results DNA inhibiting rate was directly proportional to the dosage of daunoblastina.The proportion of the cells positive staining to Ki-67 in the control and the daunoblastina-treated group were 89.3% and 45.6%(Plt;0. 01),and the integral optical density values for expression of Ki-67 in the two groups were 68.1plusmn;6.2 and 27.3plusmn;5.5(Plt;0.01),respectively.The percen tage of cells in G2 phase of cell cycle increased from 8.9% to 29.5%. Conclusion G2 block was induced and poliferation was inhibited by daunoblastina in cultured human RPE cells.There is a relatively good correlation between Ki-67 immunostaining and inhibition of RPE cell proliferation. (Chin J Ocul Fundus Dis，2000，16：1-70)
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 evaluate the prognostic and pathobiologic significance of DNA content. Methods DNA content was conducted on 140 hepatocellular carcinoma patients by flow cytometry. Cancer recurrence was followed up after the patients were discharged. The statistical software used was SPSS. Results DNA ploidy did not correlate with clinicobiologic features, except with the age of the patients (Ｐ＜0.05), tumor size and AFP level (Ｐ＜0.01). The mean following up time of the patients with diploid was 31.2 months. The recurrence rate was 23.1%. In aneuploid group the mean following up time was 22.6 months. The recurrence rate was 50.0%. Ploidy correlated significantly with recurrence rate, the recurrence rate for patients with aneuploid were significantly higher than for those of diploid (Ｐ=0.013), also the recurrence rate of aneuploid within one year (37.9%) was much higher than that of diploid (4.3%) Ｐ=0.002. In a Logistic multivariate analysis of DNA content, the grade of cirrhosis severity and the tumor size were considered to be independent factors that related with recurrence. Conclusion FCM DNA analysis of radically resected HCC is a simple and valid method to predict the recurrence.
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.
The process of multi-parametric flow cytometry data analysis is complicate and time-consuming, which requires well-trained professionals to operate on. To overcome this limitation, a method for multi-parameter flow cytometry data processing based on kernel principal component analysis (KPCA) was proposed in this paper. The dimensionality of the data was reduced by nonlinear transform. After the new characteristic variables were obtained, automatical clustering can be achieved using improvedK-means algorithm. Experimental data of peripheral blood lymphocyte were processed using the principal component analysis (PCA)-based method and KPCA-based method and then the influence of different feature parameter selections was explored. The results indicate that the KPCA can be successfully applied in the multi-parameter flow cytometry data analysis for efficient and accurate cell clustering, which can improve the efficiency of flow cytometry in clinical diagnosis analysis.
Human SW480 colonic cancer cell line was evaluated for its growth response to pentagastrin, gastrin receptor antagonist proglumide (PGL) in vitro by MTT assay and flow cytometry. The results showed that gastrin possessed a proliferative effect on SW480 cell, PGL alone had no obvious effect on SW480 cell, but it inhibited gastrin-induced growth of SW480 cell with dosage dependent when it was used with gastrin, its inhibitive effect did not steadly increase at a dose＞32μg/ml. This suggests that effect of gastrin is achieved via gastrin receptor. Gastrin promoted the sythesis of DNA, protein and triggered the cancer cell shifting from phase G0/G1 to phase S, G2M. PLG inhibited the effect of gastrin, it suggests that gastrin possessed a proliferation on SW480 cell at post receptor is achieved by the effect of gastrin on cell cycle.
Objective To investigate the value of a 4-color and 10-antibody flow cytometry immunophenotyping panel using 10 antibodies including CD45, CD38, CD19, CD56, CD20, CD5, CD10, human leukocyte antigen-DR (HLA-DR), κ antibody and λ antibody marked by four kinds of fluorescein including R-phycoerythrin (PE), fluorescein isothiocyanate (FITC), peridinin chlorophy Ⅱ protein (PerCP) and allophycocyanin (APC) in the diagnosis of multiple myeloma (MM). Methods A 4-color and 10-antibody flow cytometry immunophenotyping panel which used CD45dim/-/CD38high as gating strategy supplemented by CD19, CD56, CD20, CD10, CD5, HLA-DR, κ antibody and λ antibody was used to test the bone marrow (BM) specimens of 45 MM patients treated between December 2013 and March 2015. Then by morphological examination, we analyzed the quantitative results and characteristics of myeloma cells. Results In all the 45 MM patients, the myeloma cell detection rate was 100% by flow cytometry. The proportion range of myeloma cells in BM was between 1.17% and 72.31%, which showed a good consistency with the results of 7.5%-90.0% detected by morphological examination. The positive expression rates of antigen on myeloma cells were: 100.00% for CD38, 11.11% for CD45, 2.22% for CD19, 73.33% for CD56, 17.78% for CD20, 42.22% for HLA-DR, and 0% for CD10 and CD5. About 64.44% of the MM patients were restricted cytoplasmic λ light chain typing, and 35.56% were restricted cytoplasmic κ light chain typing. There was no obvious phenotype difference among the 3 Durie-Salmon stages of MM (P>0.05). The expression of CD56 was different among different immunoglobulin types of MM, and the types of immunoglobulin with an expression from high to low were non-secretory, IgA, IgG, and light chain (P<0.05). Conclusion The 4-color and 10-antibody flow cytometry immunophenotyping panel using 10 antibodies including CD45, CD38, CD19, CD56, CD20, CD5, CD10, HLA-DR, κ antibody and λ antibody marked by four kinds of fluorescein including PE, FITC, PerCP and APC has a good diagnostic value for MM.
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)