【Abstract】ObjectiveTo compare the effects of newcastle disease virus (NDV) and adriamycin (ADM) on surface structure and actin of hepatocellular carcinoma cell lines SMMC-7721. Methods SMMC-7721 carcinoma cell lines were divided into 2 groups. NDV was added into one group, while ADM was added into the other group. The cells were then cultured at 5 time phases (8, 16, 24, 36 and 48 h). Intracellular actin and Ca2+ were examined by using immunofluorescence method. CD44 and intercellular adhesion molecule-1 (ICAM-1) were detected by using immunochemical method and flow cytometry, respectively. The change of cellular surface structure was observed by scan electron microscope. Results Cells gradually contracted and turned round over time. It was observed that actin was segmented and cells alignment became disordered. The mean fluorescence intensity of actin decreased in both groups, but it was obvious in NDV group. There were significant differences of fluorescence intensity between 2 groups at the phases of 16 h (P＜0.05), 24 h (P＜0.05), 36 h (P＜0.01) and 48 h (P＜0.05), except the one after 8 h. Intracellular Ca2+ concentration increased gradually in both groups, and the amplifications in NDV group were significantly higher at the phases of 24 h, 36 h and 48 h than those in ADM group (P＜0.01， P＜0.05 and P＜0.01, respectively). There were also differences at 8 and 16 h, but there were no statistical significance. The expression of CD44 in cells decreased. The mean fluorescence intensity of ICAM-1 raised gradually, and then came to peaking at 36 h, but there was no significant difference between two groups. All the above indices between different phases in the same group showed significant differences (P＜0.05). Conclusion Both NDV and ADM could make tumor cells degenerate and rupture, but the effect of NDV is more intensive. It could increase the fragility of cells and hasten the process of cell rupture. Disintegrated cancer cell and changes of adhesion molecule could lead cancer cells be identified, encapsulated, and killed by immune cells under static condition.
ObjectiveTo study the relationship of the expression of CD44v6 and bcl2 protein with histological type,pathological grading and metastasis.MethodsImmunohistochemical technique was used to investigate the expression of CD44v6 and bcl2 in 50 primary gallbladder carcinoma,20 gallbladder adenoma and 10 chronic cholecystitis.ResultsThe positive rate of CD44v6 and bcl2 was 82.0% and 60.0%，which was positively correlated with the histological type,pathological grading and metastasis of gallbladder carcinoma(P＜0.05) and was higher than that in gallbladder adenoma (CD44v6 45.0% and bcl2 30.0% respectively).Expression of CD44v6 was significantly correlated with the expression of bcl2(r=0.36,P＜0.05).ConclusionCD44v6 and bcl2 might be an important biologic marker to evaluate the malignancy and prognosis of gallbladder carcinoma.There might be some extent of coordinated regulation between them.
ObjectiveTo approach the role of CD4+CD25+ regulatory T cells in the maintenance of immunotolerance in mouse liver allograft. MethodsThe mouse orthotopic liver transplantation was performed. After the liver transplantation immunotolerance induction, antiCD25 monoclonal antibody (PC61) was injected into the recipients with a delayed timing to remove the CD4+CD25+ T cells. The percentage of CD4+CD25+ T cells and the expression of forkhead/winged helix transcription factor (Foxp3) in the recipients were examined. Furthermore, the survival time of the recipient was observed. ResultsC3H/HeJ recipients receiving DBA/2 hepatic allografts survived over 70 d as in the syngeneic liver transplantation (C3H/HeJ recipients receiving C3H/HeJ hepatic grafts). With various protocols of the delayed PC61 treatment, the CD4+CD25+ T cell was completely disappeared as observed. However, the removal of CD4+CD25+ regulatory T cells after the induction of transplantation immunotolerance did not affect the survival of hepatic allografts. ConclusionCD4+CD25+ regulatory T cells are not essential for the maintenance of spontaneous mouse liver transplantation immunotolerance.
ObjectiveTo investigate the role of preoperative peripheral blood CD4/CD8 ratio in predicting the prognosis of patients with coronary atherosclerotic heart disease (CAD) after off-pump coronary artery bypass grafting (OPCABG).MethodsA total of 118 patients with CAD who underwent OPCABG in our hospital from September 2016 to April 2017 were included in the study, including 82 males and 36 females aged 62.74±4.50 years. The primary end point was the incidence of major adverse cardiovascular events (MACE). Patients were divided into a high CD4/CD8 group (≥1.40, 62 patients) and a low CD4/CD8 group (<1.40, 56 patients) according to the results of flow cytometry. The correlation between CD4/CD8 ratio and prognosis of patients after OPCABG and the value of CD4/CD8 ratio for predicting postoperative MACE were evaluated.ResultsMedian duration of follow-up was 23.25 (20.91, 24.70) months, during which 21 patients (17.80%) experienced MACE and 4 patients (3.39%) were lost to follow-up. Kaplan-Meier analysis revealed that high CD4/CD8 group had a significantly higher MACE rate than the low CD4/CD8 group did (log-rank χ2=5.797, P=0.02). The results of adjusted Cox proportional hazards model showed that CD4/CD8 ratio (HR=3.103, 95%CI 1.557-6.187, P<0.01) was an independent risk factor of MACE in patients with CAD after OPCABG. The receiver operating characteristic curve showed that area under curve was 0.778 (95%CI 0.661-0.894, P<0.01), the optimal cut off value was 2.24, the sensitivity was 57.1%, and the specificity was 87.6%.ConclusionPreoperative peripheral blood CD4/CD8 ratio is an independent predictor of MACE after OPCABG in patients with CAD.
【Abstract】ObjectiveTo study the relationship between expression of CD44v6 in gastric carcinoma and neoplasm metastasis and prognosis. MethodsExpression of CD44v6 in 52 cases of gastric carcinoma was assayed by flow immunocytometry, and its relation with clinical pathology and prognosis was analyzed. ResultsIn 52 cases of gastric carcinoma tissue, the positive rate of CD44v6 expression was 67.31%(35/52); but the positive rate of CD44v6 expression in normal gastric tissue was 25.00% (13/52). The positive rate of expression was significantly different (P＜0.01). The positive rate of CD44v6 expression in gastric carcinoma tissues was related to the depth of carcinoma infiltration, lymph node metastasis and pTNM stage （P＜0.05）. ConclusionExpression of CD44v6 plays an important role in invasion, metastasis and pTNM stage of gastric carcinoma. It may be used as a new indicator to predict metastatic potential and prognosis of gastric carcinoma.
Objective To explore the effect and mechanism of glutamine to the aberrant crypt foci (ACF) in rat injured by acetic acid. Methods Thirty Wistar rats were averagely divided into three groups: control group, acetic acid group and glutamine group. The colon of the rat was infused with 1% acetic acid. Started to gavage with glutamate two days after modeling glutamine group. The injured colons were studied after fourteen days with light and scanning electronic microscope. Paraffin sections of specimens were prepared and stained with HE. The colon crypts were isolated by HCl digestion method. The expressions of CD44 and ICAM-1 in the epithelial cell of the large intestine mucosa were detected by immunohistochemistry method. Results On the days of 14， the number of ACF in the glutamine group were remarkably decreased as compared with that of the acetic acid group and a branch-like. The expressions of ICAM-1 and CD44 (every 1 000 cells) were 302.1±30.1 and 298.6±28.3 in glutamine group, 223.6±23.5 and 221.5±28.6 in control group, 198.5±19.5 and 215.3±17.8 in acetic acid group, respectively. While the expressions of CD44 and ICAM-1 in intestine were increased remarkably in the glutamine group compared with the control group and acetic acid group (P＜0.05). Conclusion Glutamine could decrease the formation of the ACF injured by acetic acid. Increasing the expressions of CD44 and ICAM-1 may be one of the important factors to decrease the ACF.
ObjectiveTo study the relationship between the expression of sialyl Lewisx (SLeX) antigen and CD44v6 products and biological behaviors in cholangiocarcinomas. MethodsThe expression of SLeX and CD44v6 in 43 cases of cholangiocarcinoma tissue was respectively investigated by catalyzed signal amplification immunohistochemical technique.The relationship between expression of SLeX and CD44v6 and the clinicopathological factors of cholangiocarcinoma was analyzed.ResultsThe positive expression rate of SLeX and CD44v6 in cholangiocarcinoma was 67.4% and 62.8% respectively,which was significantly higher than that in control group (20.0%,P＜0.05).The high level expression of SLeX and CD44v6 were correlated with the TNM phase, differentiation degree,metastasis to lymph nodes and viscera in cholangiocarcinoma (P＜0.05). Moreover,there was a positive correlation between the SLeX and CD44v6 expression in cholangiocarcinoma (r=0.49,P＜0.001).Conclusion Expression of SLeX and CD44v6 could be helpful in predicting the biological behavior and prognosis of cholangiocarcinoma.
Objective To study the effect of allogeneic canine cord blood mesenchymal stem cells（cbMSC）transplantation on the distribution of CD4+T and CD8+T in infracted area of hearts. Methods Mononuclear cells of cord blood were isolated by density gradient centrifugation and amplified by adherent culture. 36 adult male dogs were divided into experimental group and control group. Animal models of acute myocardial infarction were established by ligating anterior descending coronary artery. The fourth generations of mesenchymal stem cells (MSC) were transplanted into infarcted area of hearts by left anterior descending coronary artery after 72h induced by 5-aza and transfected by LacZ. The survival of transplanted cells in hearts can be confirmed by βgal expression. CD4+T and CD8+T cells distributed in infarcted area were detected by immunohistochemical staining method. The ImagePlus 5.1 software was used to analyze the images. Results Cells transplanted into infarcted area could survive for a long time. 2, 4, 8 weeks after transplantation, the IOD of CD4+T in experimental group were 44.35±7.03, 19.29±4.11 and 20.27±3.51 respectively, and the CD4+T/CD8+T ratios were 0.63±0.12, 0.51±0.15 and 0.66±0.08. In control group, the IOD of CD4+T at 2, 4, 8 weeks after transplantation were 65.78±10.27, 28.02±2.59, 29.79±6.83, and the CD4+T/CD8+T ratios were 1.28±0.20, 1.34±0.09 and 1.50±0.16. The IOD of CD4+T and CD4+T/CD8+T ratio in experimental group were significantly lower than that in control group. In experimental group the IOD of CD8+T at 2, 4, 8 weeks after transplantation were 69.88±7.84 , 37.80±8.83 and 30.81±7.42, higher than that in control group which were 51.28±10.01, 20.87±4.50 and 19.91±2.87. Conclusion The preliminary results indicated that allogeneic cbMSC transplanted in infarcted area can escape from immune rejection, its mechanism may be associated withdecreasing the amount of CD4+T cells infiltrated in periphery of infarcted area and maintaining CD4+T/CD8+T ratios at a lower level.
To diminish the specific lymphocytes that responsive to the rejection of allograft. Anti-rat CD4,CD8 monoclonal antibodies and trichosnthin (TCS) was conjugated to immunotoxin by heterobifunctional reagent SPDP, 2-IT. The free TCS was removed from conjugates mixture by a column of Sephacryl S-200. The SDS-PAGE and cytotoxic assay was used to measure the biological activity of immunotoxin. SDS-PAGE showed the immunotoxin, free McAb and TCS were in the mixture of conjugation, and the free TCS can be separated by Sephacryl S-200. In Vitro, the lymphocytes of rat can be killed by antiCD4,antiCD8 immunotoxin. The kill capability was relay to the amount of immunotoxin. The authors consider that the immunology unresponsiveness can be induced by antiCD4,antiCD8 immunotoxin. That was useful in induced transplantation tolerance.
【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.