ObjectiveTo construct DPC4 gene recombinant expression vector and to study the inhibitory effect of DPC4 on the growth of human pancreatic adenocarcinoma cell line (PC3) cells.MethodsDPC4 cDNA was amplified from K562 cell line using RTPCR, and was cloned into the pcDNA3.1 vector to construct a recombinant expression vector plasmid pcDNA3.1DPC4. The recombinant expression plasmid was transferred into PC3 cells by liposome method. After G418 selection, cell cycle and apoptosis were assessed by flow cytometry, then the cell growth rate was estimated by cell count. The cells being not transferred plasmid and transferred pcDNA3.1 plasmid were used as controls.ResultsThe DPC4 gene recombinant expression vector was constructed. Wildtype DPC4 gene attributed to the increase of G1 phase cells and the decrease of S phase cells in PC3 cells,and could inhibit the growth of PC3 cells, the cell growth rates was reduced to 34.3%-41.1% of that of the controls, but cell apoptosis was not observed on all groups. ConclusionWildtype DPC4 gene could inhibit the proliferation of human pancreatic adenocarcinoma cells and could become one of the target genes of pancreas adenocarcinoma gene therapy
For research the relationship between the expression of vascular endothelial growth factor (VEGF) and the cell proliferation. The expression of VEGF was evaluated while the cell cycle of hepatoma cell line Hep G2, which was synchronized at G0 phase with serum deprivation, and reinitiated with TPA and blocked with antisense oligonucleotides of c-jun. Results: Hep G2 cell did not express VEGF at G0 phase. TPA could induce the expression of VEGF as well as initiation of cell cycle. The antisense oligonucleotides of c-jun could prohibit the expression of VEGF and arrest the cell cycle at G0 phase. Conclusion: The fact that the expression of VEGF accompanies the initiation of cell cycle suggests that they be regulated by the same singnal pathway, the expression of VEGF may be a marker indicating the proliferation of hepatoma cells.
Objective To investigate the effect on proliferation and apoptosis of vascular endothelial cells exposed to high glucose. Methods The cultured human umbilical vein endothelial cells (HUVEC) were treated with high glucose at concentrations of 10,20,30,40,50 mmol/L for 2,4,6,8,10,12,14 days,cpm value was studied by using tritium-labelled thymidine deoxyribose(3H-TdR)incorporation assay.Flow cytometry was used to detect apoptosis index,proliferation index and cell cycle. Results Treated with high glucose,the proliferation index and cpm were significantly reduced in a dose and time dependent manner than the control groups(Plt;0.05).The apoptosis index of HUVEC were higher compared with the control groups(Plt;0.05).The percent of the cultured cells in G1 phase in all high glucose groups were increased,the percent in S phase were largely decreased(Plt;0.05). Conclusion Exposed to high glucose,the apoptosis of HUVEC was accelerated and the suppressive effect of high glucose on the proliferation of HUVEC was observed.Endothelial cells were possibly arrested in G1/S checkpoint. (Chin J Ocul Fundus Dis,2000,16:177-180)
ObjectiveTo investigate the effects of dipyridamole (DP), one of the human equilibrative nucleoside transporters (hENTs) blockers, on 5-fluorouracil (5-FU) induced cell apoptosis and cell cycle changes of the pancreatic cancer Panc-1 cell line. MethodsPancreatic cancer cell line Panc-1 was divided into hENTs blocked group and hENTs unblocked group. The hENTs blocked group was further divided into two subgroups according to the DP concentration, 5 μmol/L DP group and 10 μmol/L DP group. Each group was incubated in culture medium with or without 1.5×106 ng/L 5-FU for 24 h. Cell apoptosis and cell cycle were detected by flow cytometry. Results①The apoptosis results of each group： Incubated in culture medium with 1.5×106 ng/L 5-FU for 24 h, the apoptosis rates of 5 μmol/L DP group and 10 μmol/L DP group were higer than those of hENTs unblocked group （Plt;0.05）, and which of 10 μmol/L DP group was higer than that of 5 μmol/L DP group (Plt;0.05). Incubated in culture medium without 5-FU for 24 h, there were no significant differences of the apoptosis rates among three groups (Pgt;0.05). ②The cell cycle results of each group: Incubated in culture medium with 1.5×106 ng/L 5-FU for 24 h, the percentages of S phase cells in the 5 μmol/L DP group and 10 μmol/L DP group were less than those of hENTs unblocked group (Plt;0.05). The percentage of S phase cell of 5 μmol/L DP group reduced to 87.09% and that of 10 μmol/L DP group reduced to 74.06% as compared with hENTs unblocked group. 5-FU had little influence on G2 phase (Pgt;0.05) except for the percentage of G2 phase cells in the 5 μmol/L DP group increased significantly (Plt;0.05) as compared with the hENTs unblocked group. Incubated in culture medium without 5-FU for 24 h, there were no significant differences of the cell cycles among three groups (Pgt;0.05). ConclusionsIn pancreatic cancer Panc-1 cell, DP could enhance the cytotoxicity of 5-FU by blocking hENTs. The enhanced cytotoxicity is related to elevation of 5-FU concentration in cells, and unrelated to DP itself.
Objective To investigate the effect of the 8-bromum-cyclic adenosine monophosphate (8-Br-cAMP) on the telomerase activity and changes of cell cycle in retinoblastoma （RB） cells. Methods The cultured RB cells were divided into the experimental group (8-Br-cAMP) and control group. After cultured for 24, 48 and 72 hours in vitro, the telomerase activity of RB cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and the changes of cell cycle were detected by flow-cytometry. Results The difference of telomerase activity was significant between the experimental groups and control group (Plt;0.01). There was a negative correlation between the A value of absorbance and the time in the experimental groups (r=-0.778 9, F=33.936, Plt;0.01). The changes of the cell cycle were that the percentages increased in G1 phase and decreased in S phases. Conclusion 8-Br-cAMP may weaken telomerase activity, affect the cell cycle, and inhibit the proliferation of RB cells. (Chin J Ocul Fundus Dis，2004，20：358-360)
Objective To evaluate the role of the cell cycle related genes cyclinD1 and Bcl-2 protein expression in the pathogenesis and infilt ration of the uveal melanoma． Methods Using immunohis tochemistry to detect the cyclinD1 and bcl-2 protein expression in 96 cases of uveal melanoma． Results The expression content of bcl-2 was high in uveal melanoma, and there wasn't any relationship between bcl-2 cell positivity and tumor cell type and extrascleral extension． In contrast, cyclinD1 expression was higher in epithelial cell uveal melanoma than mix cell and spindle cell varieties． There was a positive correlation between cyclinD1 cell positivity and extrascleral extension． Conclusion The expression of bcl-2 protein is important for the survival of the uveal melanoma． CyclinD1 may serve as a sensitive index of its malignancy． (Chin J Ocul Fundus Dis, 2001,17:44-46)
Objective To investigate cell cycle as a new tool to evaluate the biocompatibility of biomaterials.Methods The cell cycle and the expression of related genes were analyzed by the methods of immunocytochemistry, protein blotting, RT PCR and flow cytometry. Results The physical properteis, chemical properties and topological properities of biomaterials could not only influence cell cycle of the cells attached onto biomaterials but also affect the expression of related genes of target cells. Conclusion As an important extension of routine proliferation epxeriments, the study of cell cycle control will be great help for us to to study the cell group as an organic society. It revealed the balance between cell proliferation, cell differentiation and apotosis. It is suggested that the study of cell cycle control will play a key role in the research of tissue engineering.
Objective To study the effects of L-arginine （L-Arg） on cell proliferation, inducible nitric oxide synthase (iNOS) expression and cell cycle in human colon carcinoma cell line LS174 through nitric oxide (NO) pathway. Methods LS174 cells were cultured in medium with L-Arg at different concentrations for different times. MTT method was employed to evaluate the level of the cell proliferation. The production of NO in culture supernatants of LS174 cell was detected with enzyme reduction of nitrate. The distribution of the cell cycle was detected with the flow cytometry (FCM). The expression level of iNOS in the cells was determined by Western blot and SP immunocytochemical staining method. Results The growth of LS174 was promoted by the L-Arg at low concentration (0.125 mmol/L) and inhibited at high concentrations (0.5， 2， 8 and 32 mmol/L). The level of NO was increased with the increasing concentration of L-Arg in culture medium. To compare with the control group, the ratio of cells at S phase was increased after 48 hours’ treatments with high concentrations (0.5, 2, 8 and 32 mmol/L) of L-Arg (P＜0.05, P＜0.01); while there was no obvious difference after treatments with low concentration (0.125 mmol/L) of L-Arg (Pgt;0.05). With the increase of the concentration of L-Arg, the expression of iNOS was increased as compared with control group. The higher the concentration of L-Arg was, the better the effect. Conclusion L-Arg can induce the expression of iNOS resulting in increase the production of nitric oxide (NO). Low concentration of L-Arg can promote the growth of LS174 cells, while high concentration ones can inhibit growth and proliferation. The high concentration of L-Arg could induce S phase arrestion in the cell cycle.
Objective To know the abnormal expression of the cell cycle-regulated proteins in pancreatic adenocarcinoma and their effect on tumor cell growth. Methods The expression of p16, p21, Rb and p53 protein in 47 cases were investigated by immunohistochemistry with wet autoclave pretreatment for antigen retriaval. Furthermore, tumor growth index were assessed by a novel anti-ki-67 antibody (ki-s5). Results All the expression of p53, p16, p21 and Rb protein were the nuclear stainning. The positive rates of p53, p16, p21 and Rb protein were 55%, 53%, 74% and 98% respectively. There was negative correlation between of p16, p21 or Rb protein expression and ki-67 growth index. No relation of p53 protein stainning and the expression of p21 protein was found. Conclusion In pancreatic adenocarcinoma, the negative expression of p16 protein and p21 protein may play an important role in tumor cell growth, but tumor proliferation caused by abnormality of Rb protein is rare. The expression of p21 protein was not associated with the expression of p53 protein.
Objective To observe apoptotic and proliferative characteristics of the retinal vascular end othelial cells (RVECs) of the 1～16 weeks diabetic rats and p53 and bcl-2 expressions of the rats,in order to probe the pathogenic mechanism of diabetic retinopathy(DR). Methods Models of diabetic Wistar rats were made by alloxan venous injection.The retinal blood vessels were filled by ink,the wholemounts and paraffin-embedded sections of the retinas were made,TUNEL staining and Immunohistochemical ABC staining were used,and light microscopy was taken,in succession. Results Apoptosis of the RVECs was not found.Compared with control group,the morphologic features of the RVECs and the structure of the retinal blood vessels remained unchanged.In the period from the 10th to the 16th week,the immunohistochemical stain of PCNA,BrdU,p53,and bcl-2 for RVECs revealed positive results,but there was no any sign of the RVECs stacking and proliferating or new blood vessels forming in the retinas.In control group,the reaction of immunological stain of the aforementioned parameters was negative. Conclusions No accelerated apoptosis and proliferation of the RVECs in the 1～16 week diabetic rats happen after alloxan injection.Almost all of the RVECs were stimulated to enter the cell cycle in the 10th week.Expression of p53 and bcl-2 might play an important role in stabilizing the RVECs in early stage of diabetes. (Chin J Ocul Fundus Dis, 1999, 15: 157-159)