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.
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 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 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 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.
Objective To investigate the changes of CD4 + CD25 + Foxp3 + regulatory T cells( Treg) in peripheral blood of patients with acute exacerbation of COPD( AECOPD) , and analyze the relationship of CD4 + CD25 + Foxp3 + Treg with insulin resistance. Methods A total of 79 patients with AECOPD were divided into four groups according to disease severity( 11 cases in stage Ⅰ,31 cases in stage Ⅱ,28 cases in stage Ⅲ, an 9 cases in stage Ⅳ) .42 healthy volunteers were recruited as control. Fast blood glucose( FBG) and fast insulin( FINS) were measured for calculating the insulin resistance index. The CD4 + CD25 + Foxp3 + Treg were detected by flow cytometry. The relationship between the proportion and number of CD4 + CD25 + Foxp3 + Treg with insulin resistance was statistically analyzed. Results Compared with the healthy control group, the levels of FBG, FINS, and insulin resistance index in the AECOPD patients were significantly higher ( P lt; 0. 01, P lt; 0. 05) . The proportion and number of CD4 + CD25 + Foxp3 + Treg in peripheral blood decreased significantly( P lt; 0. 01, P lt; 0. 05) . The insulin resistance index increased with the severity of AECOPD while the proportion and number of CD4 + CD25 + Foxp3 + Treg in peripheral blood decreased. The insulin resistance index in the AECOPD patients of stage Ⅲ and Ⅳ were higher than those of stage Ⅰ and Ⅱ. The proportion and number of CD4 + CD25 + Foxp3 + Treg in the AECOPD patients of stage Ⅲ and Ⅳ were significantly lower than those of stage Ⅰ and Ⅱ. Both the proportion and number of CD4 + CD25 + Foxp3 + Treg were negatively correlated with insulin resistance ( r = - 0. 633, - 0. 871, P lt; 0. 01) . Conclusions CD4 + CD25 + Foxp3 + Treg cells might may play important role in modulating insulin resistance in AECOPD. The more serious the disease, the lower the CD4 + CD25 + Foxp3 + Treg and the worse insulin resistance.
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 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.
ObjectiveTo investigate the expression of costimulatory molecules( B7，CD28, and CTLA-4) of peripheral blood lymphocytes in patients with Behcet′s disease(BD).MethodsLymphocytes were obtained in 24 patients with BD and 20 healthy individuals, and the expression of CD80(B7-1), CD86(B7-2), CD28 and CTLA-4 on T and B cells were detected by direct three-color immunofluorescence flow cytometry.ResultsSignificantly increased expression of CTLA-4 on CD 4+ T cells[(3.18±1.18)%]was found in BD patients compared with that in controls[(1.73±0.66) %](t=－3.722,P＜0.01). The expression of CD86 on CD19+B cells was also significantly increased in BD patients[(4.49±1.73)%]compared with that in controls[(2.40±1.49) %] (t=-2.071,P＜0.05). No significant difference in the expression of the other costimulatory molecules was found.ConclusionsInteraction of B7 and CD28 on peripheral lymphocytes promote the occurrence of uveitis in BD patients. Intervention with these costimulatory signals may lead to a new prevention or treatment for uveitis patients.(Chin J Ocul Fundus Dis, 2003,19:357-359)
The human wild-type Rb cDNA has been inserted into a retrovirus vector DOL and introduced into the human breast cancer cell MDAMB468,which has a large deletion of exons 3-27 of Rb genes,by electroporation transfection techniques.The exogenous Rb gene expresses the 110kd Rb protein.The morphology of the transfected cells is similar to that of the parent MDAMB468 cells.With the expression of Rb protein,the growth rate of MDAMB468 cells decreases by about 50%,and the colony formation ability in soft agaris repressed completely.After injection of 3times;106Rb+ cells and Rb-MDAMB468 cells into nude mice,the tumors formed from 106Rb+ cells are smaller than those from Rb-cells.The cell population of G1 and S phase of Rb+ MDAMB468 cells increases and the proliferation quotient decreases by about 50%.This result supports the former report the Rb protein. （Chin J Ocul Fundus Dis,1993,9:135-140）