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 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 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)
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 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 observe the proportion changes of CD4+CD25+FOXP3+ T cells in peripheral blood of patients with VogtKoyanagiHarada disease (VKH) before and after one month of treatment. Methods he peripheral blood samples from 15 patients with VKH disease before and after one month of treatment by glucocorticoid, and from 15 healthy volunteers were collected，and lymphocytes were separated from them. CD4+CD25+ regulatory T cells were labeled by antibodies of cell surface marker CD4、CD25 and transcription factor FOXP3. The proportion of CD4+CD25+FOXP3+ T cells were detected by flow cytometry. Results Before the treatment, the percentage of CD4+CD25+FOXP3+ T cells in periphery blood was（0.30plusmn;0.19）% of CD4+ cell in VKH patients, and（1.41plusmn;0.52）% in control group, the difference was statistically significant（t=7.665,Plt;0.01）; after one month of treatment, the VKH patients group was（1.28plusmn;0.54）% which close to the control group. However there were two patients whose CD4+CD25+ T cells increased extraordinarily after one month of treatment. Conclusions The proportion of CD4+CD25+ FOCP3+ T cells in periphery blood in VKH patients were lower than control group obviously before treatment, but were close to control group after treatment. Those results indicated that VKH diseases may be associated with the decreased proportion of CD4+CD25+ regulatory T cells.
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 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.
Objective The aim is to sort CD90+ subpopulation cells in human liver cancer cell lines and investigate efficiency of magnetic cell sorting (MACS) on sorting the liver cancer stem cells. Methods ①Expressions of CD90. Immunohistochemical method was used to determine the expressions of CD90 in normal liver tissues in 8 cases, liver cancer and adjacent liver cancer tissues in 58 cases. ②Screened the cell lines. Huh-7, MHCC97-H, Bel-7402, and SMMC-7721 cell lines were divided into blank control group and experimental group (5.5×105 cells per hole, 1 hole), cells of the experimental group were added with 5 μL CD90–PE while cells of the blank control group were treated with 5 μL CD90–PE non fluorescent antibody. Determined the proportion of CD90+ cells in the 2 groups by flow cytometry (FCM). ③MACS. Huh-7 and MHCC97-H cell lines were labeled with magnetic beads respectively and sorted by MACS, 1 mL cell suspensionsorted by magnetic sorting (MS) was collected as CD90– group, and 1 mL PBS after MS wash was collected as CD90+ group, as well as blank control group and experimental group. Determined the proportion of CD90+ cells in 4 groups by FCM. Two times of MACS were performed in Huh-7 cells. ④Serum free culture and serum culture. Huh-7 cells were divided into serum-free culture group and serum culture group (1 hole), and proportions of CD90+ cells were determined by FCM at 1 week after culture. Results ①The positive rate of CD90 was 0 (0/8), 65.5% (38/58), and 20.7% (12/58) in normal liver tissues, liver cancer tissues, and adjacent liver cancer tissues respectively, and the positive rate of CD90 was higher in liver cancer tissues than those of normal liver tissues (χ2=6.78, P<0.05) and adjacent liver cancer tissues (χ2=20.83, P<0.05). ②For Huh-7, MHCC97-H, SMMC-7721, and Bel7402 cell lines, the proportions of CD90+ cells in the experimental group was 0.851%, 1.090%, 2.710%, and 4.050% respectively, the proportions of CD90+ cells in the blank control group was 0.241%, 0.688%, 1.890%, and 2.080% respectively, so we chose Huh-7 and MHCC97-H cell lines to perform MACS. ③Results of MACS for Huh-7 cell line. For the first MACS, the proportions of CD90+ cells in the blank control group, experimental group, CD90– group, and CD90+ group was 0.241%, 0.851%, 0.574%, and 1.100% respectively. For the second MACS, the proportions of CD90+ cells in the blank control group, experimental group, CD90– group, and CD90+ group was 0.032%, 0.961%, 0.426%, and 9.700% respectively. Conclusions The normal liver tissues do not express the CD90, but the liver cancer tissues express CD90 highly. There is a few CD90+ cells in Huh-7 and MHCC97-H liver cancer cell lines. The MACS has a certain effect on improving the proportion of CD90+ cells in the cell lines. The serum-free suspension culture has no effect on enriching CD90+ cells.