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.
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
Purpose To study changes of cell cycle of vascular endothelial cell in non-proliferative diabetic retinopathy. Methods Alloxan induced Wistar-rats were employed and immunohistochemistry,Western blotting methods were used. Results The vascular endothelial cells of retinas of 8～20 weeks diabetic rats were observe to be cyclinD1,cyclinD3,cyclinB1,p21 and p27 positive stained with light and electronmicroscopies.CyclinE immuno-stained vascular endothelial cells was observed occasionally.Meanwhile,the evidences of morphologic changes of the vascular en dothelial cells were proved:less plasma,thinner cell,more bubble organelles than those of controls.But,the ultra-structures of pericytes and other type of retinal cells did not change and they also immunostain negative.Komas blue and Western blotting methods also proved that the vascular endothelial cells of retina of 20th week diabetic rats expressed cyclinD1,cyclinB1,p21 and p27 protein. Conclusion Glucose induced retinal vascular endothelial cells of 8～20th weeks diabetic rats enter cell cycle and were arrested at G1/S restriction point.This study also suggested that retinal vascular endothelial cells may possess the ability to resist glucose damage and mechanism of selfstability during very early stage of diabetes. (Chin J Ocul Fundus Dis,2000,16:173-176)
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 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.
In order to investigate the effect of insulin-like growth factor-1 (IGF-1 on the cyclic change of tendon cell, the 6th generation of cultured tendon cell were selected, and 20 ng/ml IGF-1 was added to the medium. After 48 hours, the cells were determined by flow cytometer, as well as the control cells. The results showed that the time of G1 phase, DNA synthesis phase and G2M phase in IGF-1 group were 11.8 hours, 21.4 hours and 6.8 hours respectively, while those were 25.6 hours 22.6 hours and 21.8 hours respectively in the control group. It was showed that the time needed for G1 phase and G2M phase was shortened by IGF-1.
Objective To investigate the effect of quercetin on human breast cancer cells and related mechanism. MethodsHuman breast cancer cell line MDA-MB-435S was treated by different concentrations of quercetin (12.5, 25, 50, 100 and 200 μmol/L). The inhibition ratio of human breast cancer cell was measured by trypan blue dye exclusion test, the proliferation cycle of human breast cancer cell was analyzed by flow cytometry and the expression of caspase-3 mRNA was determined by RT-PCR. ResultsQuercetin could inhibit the proliferation of breast cancer cell in the dose- and time-dependent manner. Quercetin could change the cell cycle of breast cancer cell, with the concentrations of quercetin increasing, the percentages of breast cancer cell at G0/G1 phase decreased, which increased at S phase, and increased at G2/M phase followed decrease. Quercetin could activate the expression of caspase-3 mRNA in breast cancer cell. ConclusionQuercetin can inhibit proliferation and induce apoptosis of human breast cancer cell, the mechanism may be associated with the change of cell cycle and the upregulation of caspase-3 expression.
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 study the effects of recombinant human growth hormone (rhGH) on proliferation of human rectal cancer cell in vitro. MethodsThe experiment was divided into control group,rhGH group,Oxaliplatin (LOHP) group and rhGH+LOHP group. The double proliferation time of cells,cell inhibition rate,cell cycle, proliferation index (PI) and DNA inhibition rate of human rectal cancer line,HR8348,were studied by cell culture, MTT assay and flow cytometry on different concentration of rhGH. ResultsIn vitro the markedly accelerated effects of rhGH on multiplication of HR8348 cell line were not found: there was no statistical significance as compared rhGH group with control group or compared rhGH+LOHP group and LOHP group （Pgt;0.05）. The double proliferation time of cells was markedly lengthened, cell inhibition rate and the cells arrested in G0-G1 phase were obviously increased, meanwhile, the cells in S phase （P＜0.05） and G2-M phase and PI were markedly decreased and DNA inhibition rate was obviously risen as compared rhGH+LOHP group with control group or rhGH+LOHP group and rhGH group （P＜0.01）.ConclusionIn vitro rhGH does not accelerate the multiplication of human rectal cancer cells.
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.