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)
【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 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.
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 investigate bax expression and induction of apoptosis in normal cultured retinal pigment epithelium (RPE) cells ． Methods Cultured human RPE cells were transfected by PMDNA3-hbax，which incoded the whole bax gene and may be induced by Zn2+ under the MTII promoter， through lepofectin mediated protocol．The tested RPE cells were divided into three groups of A，PMDNA3-hbax transfected ;B，PMDNA3 (nude vector) transfected and C ，normal RPE cells．After transfection， DNA gel electrophoreses were perform ed ，the tested RPE cell cycles were analyzed with flow cytometry (FCM)． Results The gel electrophoretogram showed DNA ladder phenomenon，FCM confirmed the apoptosis of RPE cells PMDNA3-hbaxtransfected ， consisting of significant apoptotic peak sited before the G 1 phase and the apoptotic rate was 36%． Conclusion The foreign bax gene can be effectively conducted in to the RPE cell through lepofectinmediated protocol and induced expression ． The foreign bax overexpression may induce the cultured human RPE cell susceptibi lity to apoptosis． (Chin J Ocul Fundus Dis, 2001,17:132-134)
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.
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.
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)
Objective To study the research method of cell survival rate at the procedure of cryopreservation of tissue engineered tendons.Methods In the 4thgeneration of human fibroblasts, the dead cells were stained with propidium iodine (PI), while the living cells with Hoechst 33342(Ho). The living cells and dead cells emitted fluorescence of red and blue respectively after they were stimulated by suitable ultra-violet, then flow cytometry was applied to distinguishthem. The seeding cells were collected to make them to be the cell suspension of suitable concentration(6.0×105 cell/ml) before they were divided into two parts. We cryopreserved and defrosted one part three times to kill the cells and didnot cryopreserve the other part, then we made cell suspension at different ratios of cryopreserved cell to noncryopreserved cells. The fluorescence staining and flow cytometry were used to study the correlation between cell ratios of cryopreservedcell to non-cryopreserved cell and the cell survival rates. We compared the cll survival rates between immediate flow cytometry and that 2 hours after fluorescence staining. Results The results of flow cytometry showed that correlation between the ratio of cryopreservation and the cell survival rate was significant (r=0.970，Plt;0.05), image analysis study also showed the correlation was significant (r=0.982，Plt;0.05).The cell survival rate decreased by use of flow cytometry twohours after fluorescence staining, but there was no significant difference when compared with that of immediate flow cytometry (Pgt;0.05). We could also observe the cells on the tissue engineered tendons by fluorescence image directly.Conclusion Flow cytometry and fluorescence image afterPI and Ho staining is a good way in study cell survival rate at the procedure of cryopreservationof tissue engineered tendons.