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)
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.
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.
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)
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.
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)
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.
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)
【Abstract】 Objective To investigate the effect of IGF-1 on the growth of primary human embryonicmyoblasts. Methods The method of incorporation of 3H-TdR was used to evaluate the abil ity of prol iferation of myoblasts.The count per minute (CPM) values of myoblasts at different concentrations(1, 2, 4, 8, 16 and 32 ng/mL) of IGF-1 were measured,and dose-effect curves were drawn to choose the optional concentration of IGF-1 to promote the prol iferation. Then theexperimental group of myoblasts received the addition of the optional concentration of IGF-1 in the growth medium, the controlgroup just received the growth medium. The flow cytometry was used to detect the cell cycle . The method of incorporation of3H-TdR was used to measure the peak-CPM. The myotube fusion rate was measured in myoblasts with different concentrations(0, 5,10, 15, 20, 25 and 30 ng/ mL) of IGF-1 in fusion medium, the dose-effect curves were also drawn, so as to decided the optional concentrationof IGF-1 in stimulating differentiation. Fusion medium with optional concentration of IGF-1 was used in experimentalgroup, and the control group just with fusion medium. The fusion rate of myotube and the synthesis of creatine kinase(CK) weredetected in both groups. Results The optional concentration of 5 ng/mL IGF-1 was chosen for stimulating prol iferation . It was shown that the time of cell cycle of control was 96 hours, but that of the experimental group was reduced to 60 hours. The results of flow cytometry showed that the time of G1 phase, S phase and G2M phase was 70.03, 25.01 and 0.96 hoursrespectively in control group, and were 22.66, 16.47 and 20.87 hours respectively in experimental group. The time-CPM value curves showed that the peak-CPM emerged at 96 hours in control group and 48 hours in experimental group, which was in agreementwith the results of the flow cytometry. The optional concentration stimulating prol iferation was 20 ng/mL IGF-1. Compared with control, the quantity of CK was increased by 2 000 mU/mL and the fusion rate was elevated by 30% in experimental group. Conclusion The concentrations of 20 ng/mL IGF-1 can elevat obviously the fusion rate and the quantity of CK. IGF-1 can enhance the prol iferation and differentiation of myoblasts via inducing the number of myoblasts at G1 phase and increasing the number of myoblasts at S and G2M phases.