Objective To investigate the effects of recombinant adenovirus-mediated co-transfection of carcinoembryonic antigen (CEA) gene and erythropoietin (EPO) gene on promoting hematopoietic stem cells directly producing erythrocyte vaccine against colon cancer. Methods The expression adenovirus vectors carrying CEA and EPO or green fluorescent protein (GFP) gene were constructed respectively, and recombinant adenovirus carrying CEA, EPO or GFP were packaged and produced respectively. The bone marrow-derived mesenchymal stem cells (MSCs) of mice were isolated and cultured in vitro by anti-CD117 magnetic bead separation, and were transfected with CEA (CEA group), EPO (EPO group) or GFP (blank vector group), co-transfected with CEA and EPO (CEA-EPO group). The expressionsof CEA and EPO gene and its protein after transfection in supernatant fluid of culture were detected by realtime-PCR and Western blot method in each group. We had checked and obtained the vaccine with co-transfection of CEA gene and EPO gene by cell red line marker antibody CD71 and GPA, then we carried on experiments with the vaccine in vitro and in vivo. There were 4 groups in our trail: blank vector group, CEA group, EPO group, and CEA-EPO group. Results We had successfully gathered the hematopoietic stem cells, flow cytometry analysis result showed that there were significant differences before and after purification for positive selected samples (P<0.05). The expressions of double genes (CEA-EPO gene) and protein showed CEA-EPO gene were successfully transfected into the hematopoietic stem cells. We had confirmed erythrocyte vaccine with co-transfection of CEA and EPO gene by antibody CD71 and GPA with flow cytometry. The monocytes cytotoxicity on colon cancer cell line CT26 showed that lysis of target cells of CEA-EPO group were higher than those of other 3 groups when in proportion of 40∶1 (P<0.05). In the experimentation of neoplasma format, the volume of tumor and mortality were smaller or lower, but survival time was longer of CEA-EPO group in2 weeks after treatment (P<0.05). Conclusions The erythrocyte vaccine with co-transfection of CEA gene and EPO gene has efficient anti-tumor effects on colon cancer. Not only can promote hematopoietic stem cell directly producing erythrocyte vaccine, but also can produce tumor antigen vaccine against colon cancer.
Objective To summarize the research status of echinococcosis- specific vaccine antigens, analyze their sources and application prospects, and to provide new ideas for the development of echinococcosis vaccine antigens and drug treatment. Method Research on echinococcosis-specific vaccine antigens at home and abroad was searched and reviewed. Results Natural hydatid antigens, such as cystic fluid crude antigen, protoscolex segment, germinal layer, etc. often appear due to the difficulty of material acquisition and cumbersome preparation, resulting in unstable evaluation indicators such as sensitivity and specificity. The gene or protein sequences of a new recombinant hydatid antigen was accessible, the reproducibility and specificity were better, and it was more suitable for batch production testing, which was the main direction of current research, such as rAgB8/1, rEm18, rEm2, etc. Conclusions Vaccine development is one of the main directions for the elimination of hydatidosis. In the interaction between echinococcus and human or animal hosts, the natural structural proteins or excretion/secretion proteins of echinococcus stimulate the host to produce anti-parasites immunity and immune clearance, and the search for these specific protein antigens is of great significance for vaccine development, and new drug treatment.
Objective To analyze inducing factors and clinical characteristics of deep venous thrombosis (DVT) and to explore clinical value of soluble cell surface differentiation antigen 40 ligand (sCD40L) in early diagnosis of DVT. Methods The patients with the DVT of lower extremity who had not received the anticoagulant and thrombolytic therapy in the Nanchong Central Hospital from January 2012 to January 2017 were collected, these patients were divided into an early-acute stage, mid-acute stage, late-acute stage, and subacute stage according to the clinical course of DVT. The sCD40L expression in the peripheral blood of DVT patients were detected by the enzyme linked immunosorbent assay. Results There were 100 patients with the DVT were included, including 31 cases of early-acute stage, 26 cases of mid-acute stage, 21 cases of late-acute stage, and 22 cases of subacute stage; 66 patients with the peripheral type, 28 patients with the central type, and 6 patients with the mixed type. ① The fracture, malignant tumor, long time in the bed following the thoracic or abdominal operation, joint replacement, and caesarean section were the successively main risk factors of the DVT. ② The early-acute stage of DVT was more common in the fracture patients, the mid- and late-acute stage of DVT often occurred in the joint replacement sufferer, and the subacute stage of DVT was usually found in the malignant tumor patients. ③ The sCD40L expression in the patients with the different stage DVT was signifiantly higher than that in the control group (20 healthy people in the physical examination, P<0.05). Furthermore, there was a significant difference in the different stage DVT patients (F=26.57, P=0.02), that is, the expression of sCD40L was the highest in the early-acute stage of DVT, and then gradually reduced (P<0.05). ④ The sCD40L expression had a significant difference among the central type DVT, mixed type DVT, and peripheral type DVT (F=12.51, P=0.02), which in the peripheral type DVT was significantly higher than that of the central type DVT (P<0.05) and mixed type DVT (P<0.05), but had no difference between the central type DVT and the mixed type DVT (P>0.05). ConclusionsCD40L might act as a blood index of early diagnosis and judgement of extent of DVT, especially be helpful in early-acute stage of DVT.
Objective To evaluate the clinical relationship between serum carcinoembryonic antigen (CEA) and mortality of anti-melanoma differentiation associated gene 5 (MDA5) antibody positive dermatomyositis with interstitial lung disease (ILD). MethodsThe consecutive clinical data of 214 patients with anti MDA5 antibody positive dermatomyositis from West China Hospital of Sichuan University from February 2017 to September 2019 were collected retrospectively, including demographic, laboratory examination and imaging examination data. Patients were divided into CEA elevated group (CEA≥4.63 ng/mL) and CEA normal group (CEA<4.63 ng/mL) according to CEA level. R4.1.2 software was used for statistical analysis of all data, and Kaplan Meier method was used to draw the survival curve. Cox proportional hazard model was used to analyze the survival of patients with ILD, and to explore the risk factors associated with the survival of patients with anti-MDA5 antibody positive dermatomyositis with ILD. Results There were 180 patients with ILD who met the inclusion and exclusion criteria, 57 patients with rapidly progressive pulmonary interstitial fibrosis (RPILD), and 123 patients without RPILD; 121 women and 59 men, with an average age of 50.2±10.7 years; The average follow-up was 23.5 months, and 52 patients died. Univariable analysis suggested that CEA≥4.63 ng/mL, smoking, RPILD, lactate dehydrogenase (LDH) ≥321 IU/L, albumin<30 g/L and dyspnea were risk factors associated with death in patients with anti MDA5 dermatomyositis combined with ILD. Multivariable Cox regression analysis showed that CEA≥4.63 ng/mL [hazard ratio (HR) =3.01, 95% confidence interval (CI) 1.23 - 7.32, P=0.015], RPILD (HR=3.87, 95%CI 2.09 - 7.19, P<0.001), smoking (HR=2.37, 95%CI 1.25 - 4.47, P=0.008), LDH≥321 IU/L (HR=2.47, 95%CI 1.23 - 4.96, P=0.011), albumin<30 g/L (HR=2.57, 95%CI 1.38 - 4.78, P=0.003) were independent predictors for mortality. ConclusionsSerum CEA level can be used as a clinical prognostic predictor in patients with anti-MDA5 positive dermatomyositis and ILD. RPILD, smoking, LDH≥321 IU/L, and albumin<30 g/L are independent predictors for mortality.
Purpose To observe the expression of proliferating cell nuclear antigen（PCNA）and bcl-2 of cultured human retinal pigment epithelial cells（RPE）． Methods SABC techniques were applied for immunocytochemical staining of cultured RPE with mouse anti-human PCNA monoclonal antibody and rabbit antihuman bcl-2 antibodies． Results 31.2% and 50.6% cultured cells were positive to anti-human PCNA at 24h and 48h after seeding，respectively．The positive staining was mottled in the nucleus．positive staining for bcl was seen in 76%to 90% cells as fine granules scattered within the cytoplasm. Conclusion One half of cultured RPE expressed PCNA，indicating that the cells were in phase S of the cell cycle．Positive staining for bcl-2 appeared in much more RPE cells．These biological markers may be associated with the growth activity of cultured RPE． (Chin J Ocul Fundus Dis,1998,14:26-28)
ObjectiveTo assess the inhibitory ability of sarpogrelate on neointimal hyperplasia of carotid artery in rat balloon-injuried model, and to compare the proliferation of vascular smooth muscle cell (VSMC) by monitoring the expression of proliferative cell nuclear antigen (PCNA). MethodsTwenty-four male SD rats (SPF, 8 weeks) were allocated prospectively and randomly into 3 groups: blank group, sarpogrelate group, and clopidogrel group. Each group included 8 rats. All the rats were fed high-fat diet for 1 week before the operation. No drug was fed in the blank group, and sarpogrelate 100 mg/(kg·d) or clopidogrel 20 mg/(kg·d) was fed in the sarpogrelate group or clopidogrel group respectively. The carotid artery of rat was dilated by the Forgarty balloon catheter. The rats were killed 2 weeks later and the samples were got in the balloon-injuried carotid arteries. Histomorphological analysis and immunohistochemical analysis were proceeded. The thickness ratio and area ratio of intima and media, the ratio of PCNA positive cells and PCNA absorbance were calculated among the three groups. ResultsCompared with the blank group, the average intimal thickness, average intimal area, thickness ratio of intima and media, area ratio of intima and media, PCNA absorbance, and ratio of PCNA positive cells were significant decreased in the sarpogrelate group (P < 0.001) and the clopidogrel group (P < 0.001), but which had no significant differences between the sarpogrelate group and the clopidogrel group (P > 0.05). There was no significant difference in the average media thickness or area among the 3 groups (P > 0.05). ConclusionSarpogrelate and clopidogrel could significantly reduce the thickness or area of intima, the absorbance of PCNA and the ratio of PCNA positive cells.
Objective To study whether the porcine endothelial cells (PECs) lines transfected by HLA-G1 can alter the lysis mediated by human peripheral blood mononuclear cell (PBMC) and natural killer cell 92(NK-92). Methods By use of liposomes pack, the pcDNA3.0 eukaryotic expression vector carrying HLA-G1 was transfected into PECs. Using indirect immunofluorescence and RT-PCR assays, the HLA-G1 expression in PECs was detected. The alteration of the lysis mediated by PBMC and NK-92 was detected by51Cr-release assays. Results HLA-G1 expression could be detected in PECs after transfection of HLA-G1 at the levels of protein andRNA. It also could be found that the survival rate of transfected PECs was muchhigher than that of non-transfected PECs, when both of them faced the lysismediated by human PBMC and NK-92.After transfecting the expression of HLA-G1 could be found in the transfected PECs and the lysis mediated by PBMC and NK-92 to PECs decreased obviously (Plt;0.05). Conclusion The PECs- transfected by HLAG1 can decrease the NK lysis, so that it may provide us a new thought to inhibit the xeno-cell-rejection.
【Abstract】Objective To investigate the expression of PCNA in gastric cancer and its relationship with telomerase activity of peritoneal washings and peritoneal dissemination, and to compare the efficacy of telomerase activity and cytology of peritoneal washings for prediction of peritoneal metastasis of gastric cancer. MethodsTelomeric repeated amplification protocol (TRAP)enzymelinked immunosorbent assay (ELISA) was performed to measure the telomerase activity of peritoneal washings collected from 60 patients with gastric cancer. Exfoliate cytologic analysis of the corresponding samples was used for comparison.Expression of PCNA was measured with immunohistochemical staining.Their relationship with clinicopathologic features were evaluated. ResultsThe positive rate of telomerase activity in peritoneal washing collected from patients with gastric cancer was 41.7%,which well related to serosal invasion, histology types, depth of infiltration and peritoneal metastasis of gastric cancer. The positive rate of telomerase activity increased with the increased depth of infiltration and serosal involvement areas (P＜0.05).The positive rate of exfoliative cytology was 25.0%, which was obviously high in the group with macroscopic peritoneal metastasis (the group of P1-3). The positive rate of exfoliative cytology also increased with the increased depth of infiltration and serosal involvement areas (P＜0.05). Although the positive rate of telomerase activity in peritoneal washing collected from patients with gastric cancer was not significantly higher than that of exfoliative cytology in general, it was significantly higher than that of exfoliative cytology in the group of pT4, P1-3 and undifferentiated type.The PCNA proliferation index (PI) of positive telomerase activity group was significantly higher than that of negative. The PCNA PI was significantly higher in the group of P1-3 and serosal invasion thanthat of P0 and without serosal invasion. ConclusionTo detect telomerase activity in peritoneal washings and to detect tumor cells by cytologic method are useful to predict subclinical metastasis to the peritoneum in patients with gastric cancer,but telomerase activity is more sensitive than the other one.Telomerase activity is well related to proliferating activity of gastric cancer,which was the very important reason of peritoneal metastasis and serosal invasion.
ObjectiveTo investigate the expression and distribution of CD15s antigen in breast cancer and its relationship with carcinogenesis, progression and metastatic proclivity. MethodsCatalyzed signal amplification(CSA) immunohistochemical technique was used to detect the expression of CD15s antigen in breast cancer and in adjacent normal mucosa. Immunoelectromicroscopic ultrastructural localization of CD15s antigen labelled by colloidal gold was also bserved.ResultsThe positive rate of CD15s antigen expression in primary breast cancer was 79.8%(75/94). In adjacent normal mucosa (n=10) CD15s antigen showed weaker staining. The positive rate of CD15s antigen expression in grade Ⅱ-Ⅲ (87.3%) was notably higher than that in grade Ⅰ (69.2%, P＜0.05). In patients with lymph node metastasis, the positive rate of CD15s antigen expression was 90.2%, which was significantly higher than 67.4% in nodes with no metastasis (P＜0.05). CD15s antigen immunoreactivity was mainly localized in the border membrane of cytoplasm, endoplasmic reticulum, golgi complex and surrounding nuclear membrane in tumor tissue, and in the border membrane of cytoplasm in adjacent normal tissue. Conclusion CD15s antigen is a practical parameter for evaluating the degree of malignancy and lymphatic metastatic proclivity of breast cancer. It can provide a new pathway to investigate the carcinogenesis and progression of breast cancer.
ObjectiveTo investigate the expression changes and the repair effect of mitogen and stress- activated protein kinase 1 (MSK1) on spinal cord injury (SCI) in rats.MethodsOne hundred and twenty male Sprague Dawley (SD) rats (weighing 220-250 g) were used for the study, 70 of them were randomly divided into sham-operation group and SCI group (n=35), the rats in SCI group were given SCI according to Allen’s method, and the sham-operation group only opened the lamina without injuring the spinal cord; spinal cord tissue was collected at 8 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after invasive treatment, each group of 5 rats was used to detect the expression of MSK1 and proliferating cell nuclear antigen (PCNA) by Western blot assay. Another 20 SD rats were grouped by the same method as above (n=10). In these rats, a negative control lentiviral LV3NC dilution was injected at a depth of approximately 0.8 mm at the spinal cord T10 level. The results of transfection at 1, 3, 5, 7, and 14 days after injection were observed under an inverted fluorescence microscope to determine the optimal transfection time of the virus. The other 30 SD rats were randomly divided into group A with only SCI, group B with a negative control lentiviral LV3NC injected after SCI, and group C with MSK1 small interfering RNA (siRNA) lentivirus injected after SCI, with 10 rats each group. The Basso, Beatlie, Bresnahan (BBB) score of hind limbs was measured at 1, 3, 5, 7, and 14 days after treatment; spinal cord tissue collected at the optimal time point for lentivirus transfection was detected the expression changes of MSK1 and PCNA by Western blot and the localization by immunofluorescence staining of MSK1 and PCNA proteins.ResultsWestern blot assay showed that there was no significant changes in the expression of MSK1 and PCNA at each time points in the sham-operation group. In the SCI group, the expression of MSK1 protein was gradually decreased from 8 hours after injury to the lowest level at 3 days after injury, and then gradually increased; the expression change of PCNA protein was opposite to MSK1. The expression of MSK1 in SCI group was significantly lower than that in the sham-operation group at 1, 2, 3, and 5 days after injury (P<0.05), and the expression of PCNA protein of SCI group was significantly higher than that of the sham-operation group at 8 hours and 1, 2, 3, 5, and 7 days after injury (P<0.05). The fluorescence expression of both the SCI group and the sham-operation group has be found and peaked at 7 days. There was a positive correlation between fluorescence intensity and time in 7 days after transfection. With the prolongation of postoperative time, the BBB scores of groups A, B, and C showed a gradually increasing trend. The BBB score of group C was significantly lower than those of groups A and B at 5, 7, and 14 days after treatment (P<0.05). After transfection for 7 days, Western blot results showed that the relative expression of MSK1 protein in group C was significantly lower than that in groups A and B (P<0.05); and the relative expression of PCNA protein was significantly higher than that in groups A and B (P<0.05). Immunofluorescence staining showed that MSK1 was expressed in the nuclei of the spinal cord and colocalized with green fluorescent protein, neuronal nuclei, and glial fibrillary acidic protein (GFAP). The relative expression area of MSK1 positive cells in group C was significantly higher than that in group B (P<0.05), and the relative expression areas of PCNA and GFAP positive cells were significantly lower than those in group B (P<0.05).ConclusionLentivirus-mediated MSK1 siRNA can effectively silence the expression of MSK1 in rat spinal cord tissue. MSK1 may play a critical role in the repair of SCI in rats by regulating the proliferation of glial cells.