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 investigate the effect of up-regulation of microRNA-31(miR-31) on the biological behaviour in AGS cell of gastric cancer and on the expression of liver receptor homolog-1(LRH-1), and to analyze the possible mechanisms of miR-31 on initiation and development of gastric cancer. MethodsAGS cells were divided into 3 groups, receiving miR-31 transfection(MT group), empty liposomes transfection(NC group), and treatment of PBS (BC group). Then the cells' proliferation was determined by cell counting kit-8(CCK-8), the apoptosis situation was determined by flow cytometer, the migration was determined by Transwell test, the expression of LRH-1 protein was tested by Western blot method, and the target of miR-31 was tested by luciferase reporter assay. ResultsThe cell's proliferation results showed that the mean of A450 value in MT, NC, and BC groups were 1.31, 2.26, and 2.14 respectively on the 4 days after transfection, which lower in MT group(P<0.01).Results of flow cytometer experiment showed that the mean of apoptosis ratio of MT, NC, and BC groups were 39.5%, 9.3%, and 10.0% respectively, the mean of proportion of cell in G1+S stage were 92.54%, 73.23%, and 74.58% respectively, which both lower in MT group (P<0.05).Results of Transwell experiment showed that the mean of number of migrated cells in MT group was lower (P<0.05).Results of Western blot experiment demonstrated that the expression level of LRH-1 protein in MT group was lower than those of BC group and NC group(P<0.01). ConclusionsUp-regulation of miR-31 can obviously inhibit the proliferation of AGS cell, promoting its apoptosis and depressing its migration ability. On the other side, the up-regulation of miR-31 can also inhibit the expression level of LRH-1 protein, which indirectly induces the inhibition of proliferation of AGS cell. So miR-31 may be an important regulator in the initiation and development of gastric cancer through regulating LRH-1 gene.
【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 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.
Objective To investigate the effect on expression of c-myc and proliferating cell nuclear antigen （PCNA） of vein grafts transferred by c-myc antisense oligodeoxynucleotides（ODN） of soluble stent. Methods A rabbit model of common carotid arteries grafted by external jugular veins was constructed in 50 New Zealand rabbits and were randomly divided into five groups, 10 rabbits each group. Control group： no stents ; group 1： soluble stent ; group 2： soluble stent with sense-ODN; group 3： soluble stent with antisense-ODN; group 4.. soluble stent with mismatch-ODN. At 7 d, 28 d and 90 d after surgery, vein grafts were harvested. The expression of c-myc and PCNA were identified by immunochemistry methods. Results At 7d, 28d, 90d after surgery, the expression of c-myc and PCNA of the intima and media of vein grafts in control group, group 1, group 2, group 4 were higher significantly than that in group 3 （P〈0. 01）. At 28d, 90d after surgery, the expression of c-myc in five groups were higher than that in the same group at 7d after surgery （P〈0. 01）. Conclusion Soluble stent can transfer ODN effectively. C- myc antisense-ODN transferred by soluble stent can inhibit significantly the expression of c-myc and PCNA in the intima and media of vein grafts.
Objective To investigate the effect of methylprednisolone on the expression of glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) in Müller cells of rats’ retinae injured by laser. Methods Forty SD rats were randomly divided into two groups and inflicted with laser photocoagulation.The rats in treatment group were given methylprednisolone by intraperitoneal injection with a dose of 30 mg/kg for 3 days.At the 3rd,7th,14th,and 28th day after photocoagulation respectively, the eyes were enucleated,fixed and cut into sections.Immunohistochemical examination was used to detect the expression of PCNA and GFAP. Results After photocoagulation the Müller cells expressed PCNA both in the treatment and control group,and the expression of PCNA decreased sharply after 3 days. The expression of PCNA in treatment group was less than that in control group. After photocoagulation the Müller cells also expressed GFAP and the expression of GFAP lasted for at least 28 days ,and the expression of GFAP expression in the treatment group was less than that in the control group. Conclusion Methylprednisolone can reduce the expression of GFAP and PCNA in Müller cells of rats’ retinae injured by laser. (Chin J Ocul Fundus Dis, 2002, 18: 299-301)
Objective To study the expression of proliferating cell nuclear antigen (PCNA) in the occurrence and development of hepatocellular carcinoma. Methods Sixty SD rats were randomly divided into control group and experimental group. 3′MeDAB was administrated into rats to establish the experimental model of hepatocarcinoma. The expressions of PCNA of different phases were detected by immunohistochemistry and the liver pathologic changes were observed by optical microscope. Results The process of canceration was divided into three stages: inflammation, proliferative fibrosis and hepatic carcinoma. The expression of PCNA firstly presented in the oval cells that located in the portal area at the stage of inflammation, and a part of PCNA were hyperexpressed in the portal area. The expression rate of PCNA in the middle phase of inflammatory stage was higher than that of any other phases but declined later. Yet, when it came to the stage of hepatic carcinoma, the rate increased again. Conclusion Under the experimental circumstance when liver cancer is caused by the carcinogenic agent, PCNA may be firstly expressed in the oval cells, and the dynamic expression of PCNA may be an indicator for the early diagnosis of hepatocarcinogenesis.
PURPOSE:To investigate the relationship between the proliferative activity of refinoblastoma (RB)cell and the RB differentiation degree and the infiltration capability. METHOD:The proliferating cell nuclear antigen (PCNA)expression in RB tissues of 48 cases was analysed by using LSAB immunohistochemical method. RESULTS :The mean PCNA labelling index(LI)in differentiated RB tissues of 12 cases was markedly lower than that in non-differentiated of 36 cases(P＜0.05). The mean PCNA LI in RB tissues of the optic nerve infiltrated group(22 cases)was significantly higher than that of the optic nerve non-infiltrated group(26 cases)(P＜0.05). The results indicate that the PCNA LI is significantly related with the differentiation degree of RB and the infiltration capability. CONCLUSION :The determination of PCNA LI is of significance for evaluating the histologic characteristics and biological behavior of RB.
ObjectiveTo investigate the succession model for hyperthyroidism and thyroid carcinoma secondary to nodular goiter in iodine deficiency area. MethodsA total of 216 specimens of goiter patients from iodine deficiency area were collected in the former 3rd hospital of Norman Bethune Medical College from January 1980 to December 1994. Twentyfour heteroploid samples were selected by the method of Hedley with Flow cytometry (FCM) analysis. Paraffin-embedded tissues from the same position were used to perform immunohistochemical staining for proliferating cell nuclear antigen (PCNA), laminin (LN), factor Ⅷ related antigen (FⅧ-RAg), and p53. The proliferative activity, stroma change, and angiogenesis were observed. ResultsPCNA label index (PCNA-LI) and proliferation index (PI) consistent in 24 heteroploid samples with PCNA staining were significantly higher value. PCNA positive cells were mainly distributed over nonfollicular parenchymatous structures, small follicles, and multilayered structures with large bubbly follicles. Destroyed basement membrane and necrosis were found by LN staining in PCNA positive position with vigorous reproductive capacity. Combining FⅧ-RAg staining with LN staining, interstitial proliferation and angiogenesis were obvious in follicular epithelial cells with vigorous reproductive capacity, providing nutrition and superior environment for them. ConclusionsThe reproduction of thyroid follicular epithelial cells, interstitial proliferation, and angiogenesis are all involved in tuberosis and hyperthyroidism, forming precancerous lesion, which suggest the succession model of goiter in iodine deficiency area.