Effects of miR-369-3p on the proliferation and apoptosis of bladder cancer cells by regulating the expression of VEGFC gene / 中国医师杂志

Objective To observe the effect of miR-369-3p on vascular endothelial growth factor C (VEGFC) gene in bladder cancer cells EJ and 5637 and observe its effect on cell proliferation and apoptosis.Methods Bladder cancer cell lines EJ and 5637 were transfected with miR-NC (control group) or transfected with miR-369-3p (experimental group).The target gene of miR-369-3p was predicted by Targets can target gene prediction software.Fluorescence real-time quantitative polymerase chain reaction (qPCR) was used to detect the changes of VEGFC at mRNA level.The changes of VEGFC,p-Raf-1 and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) at the protein level were detected by Western blotting.The effect of miR-369-3p on the proliferation of bladder cancer cells was detected by cell counting kit (CCK-8) and clone formation experiment.The effect of miR-369-3p on apoptosis was detected by flow cytometry.Results After transfection with miR-369-3p,the expression of VEGFC at mRNA and protein levels was significantly decreased (P ＜ 0.05),the expression of p-Raf-1 and p-ERK1/2 protein was significantly decreased,the cell proliferation ability was significantly decreased (P ＜ 0.05),the number of clones formed was significantly decreased (P ＜ 0.05) and the apoptosis was significantly increased (P ＜ 0.01).Conclusions miR-369-3p can inhibit the proliferation and promote the apoptosis of bladder cancer cells by interfering with the expression of VEGFC gene,which may provide a new target for the biological therapy of bladder cancer.

Experimental study on apoptosis of ovarian cancer cell line SKOV-3 induced by triptolide / 中国医师杂志

Objective To investigate the effect of triptolide (TP) on the apoptosis of ovarian cancer cell line SKOV-3 and its molecular mechanism.Methods Ovarian cancer cell line SKOV-3 was cultured and treated with different doses of TP (1 × 10-6 mg/ml,1 × 10-5 mg/ml,1 × 10-4 mg/ml,1 × 10-3 mg/ml),Iscoves modification of DMEM (IMEM) medium without TP was negative control.At the 12 h,24 h and 48 h after treatment,apoptosis rate was determined by terminal dexynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) kit.At the 48 h after treatment,mRNA expression of apoptosis molecules and invasion molecules were determined by fluorescence quantitative polymerase chain reaction (PCR) kit.Results (1) At the 12 h,24 h,and 48 h after treatment with different doses of TP,at the same treatment time,the higher the TP dose was,the higher the apoptosis rate of SKOV-3 cells was;at the same treatment dose,the longer the treatment time was,the higher the apoptosis rate of SKOV-3 cells was;and TP increased the apoptosis rate on dose dependent and time dependent manner;(2) After 48 h treatment with different doses of TP,the higher the TP dose was,the higher the mRNA expression of cytochrome C (CytC),Bax,and Caspase-3 was,the lower the mRNA expression of Bcl-2,matrix metalloproteinases (MMP)2,MMP7,MMP9,N-cadherin,and vimentin was;TP increased mRNA expression of CytC,Bax,and Caspase-3 and decreased mRNA expression of Bcl-2,MMP2,MMP7,MMP9,N-cadherin,and vimentin on dose dependent manner.Conclusions TP can induce the apoptosis of ovarian cancer cells,activation of mitochondrial apoptosis pathway and inhibition of cell invasion are molecular mechanism of TP promoting apoptosis.

The effect of cyclooxygenase-2 on proliferation in gastric cancer cell line SGC7901 / 中国医师杂志

Objective To investigate the influence of cyclooxygenase-2 (COX-2) knock-down on cell proliferation and apoptosis in gastric cancer cell line SGC7901.Methods The mRNA levels of COX-2 in SGC7901 and gastric epithelium cells (GES) were determined by quantitative real-time polymerase chain reaction (qRT-PCR) assays.The influence of COX-2 on the proliferation and apoptosis of cell line SGC7901 was evaluated with methyl thiazolyl tetrazolium (MTT) and fluorescence-activated cell sorter (FACS) assay.Results qRT-PCR assay indicated that the mRNA level of COX-2 in SGC7901 was significantly higher than that in GES (P ＜ 0.01).MTT and FACS assays showed that the proliferation was reversed by COX-2 knock-down in SGC7901 cells.Up-regulated Bax and down-regulated Bcl-2 can inhibit the expression of COX-2.Conclusions COX-2 could contribute to the proliferation of gastric cancer cell line SGC7901.

Influence of brain-derived neutrophic factor on tissue of oxidative stress and cell apoptosis of secondary brain injury after traumatic brain injury in rats / 中国医师杂志

Objective To investigate the changes of the level of oxidative stress and cell apoptosis of secondary brain injury after traumatic brain injury,and the influence of brain-derived neutrophic factor on these parameters in rats,as well as its potential mechanisms.Methods A total 84 adult and healthy male rats was divided randomly into 2 groups:control (n =42) and traumatic brain injury (TBI) groups (n =42).The brain-derived neurotrophic factor (BDNF) group was induced using improved Feeney method and was received abdominal injections of BDNF (0.5 μg/μl) immediately after injury,the control group were received abdominal injections with the same dose sodium chloride injection immediately after injury and repeat one time everyday until the rats was killed.Each group was divided into seven subgroups by sacrificed time after injury,those are 1 h,3 h,6 h,12 h,24 h,3 d,and7 d,each subgroup got6 rats.Each subgroup was randomly selected three rats after being killed.The water content,superoxide dismutase (SOD),malonic aldehyde (MDA),and glutathione (GSH) of rats were measured contusion peri tissues brain tissue.Specimens were taken from left three rats of subgroup,which was part of the brain tissue.The expression of NF-κB p65,around the brain tissue with immunohistochemical methods were detected.TdT-mediated dUTP nick-end labeling (TUNEL) method was used to observe the peri cell apoptosis after brain contusion.Results NF-κB p65 was expressed obviously around the lesion in 1h group,and strongly expressed in TBI-3 h-12 h,and reached a peak in 24 h after the injury,while NF-κB p65 expression reduced in TBI-3 d-7 d,and still in high expression.NF-κB p65 expression strongly correlated with the degree of cerebral edema (r =0.651,P ＜0.05).For two groups,NF-κB p65 expression strongly correlated with the level of MDA (r1 =0.947,P ＜0.01;r2 =0.961,P ＜0.01).Conclusions Changes of NF-κB protein expression after brain injury were involved in a series of pathological processes of secondary brain injury,such as oxidative stress,and apoptosis,brain-derived neutrophic factor is probably through inhibit oxidative stress levels,control apoptosis,prevent the development of vasogenic cerebral edema,and reduce or mitigate secondary brain injury.

Effect of TRPM8 on proliferation and apoptosis of human bladder transitional cell carcinoma cell T24 / 中国医师杂志

Objective To investigate the effect of down-regulation of transient receptor potential melastatin 8 (TRPM8) on the proliferation and apoptosis of human bladder transitional cell carcinoma cells T24.Methods shRNA targeting TRPM8 was designed and synthesized, and then transfected into the T24 cells via lipofectamine 2000 mediation.The proliferation and apoptosis of T24 cells were detected with methyl thiazolyl tetrazolium (MTT) assay and flow cytometry.Expression of extracellular regulated kinase (ERK), cyclin D1, and Bcl-2 were detected with Western blot.Results TRPM8-targeted shRNA downregulated TRPM8 expression of T24 cells.MTT assay showed a significant acceleration of the proliferation of shRNA interference group compared to blank and control groups (P ＜0.01).Compared to control group, cell apoptosis rate was significantly higher in shRNA interference group (P ＜ 0.01).In addition, the expressions of PI3K, cyclin D1, and Bcl-2 were decreased in shRNA interference group.Conclusions Down-regulation of TRPM8 can induce inhibition of proliferation and promotion of cell apoptosis in human bladder transitional cell carcinoma cells T24 via regulating PI3K.It might be regarded as a novel target for clinical diagnosis and gene therapy of bladder cancer.

Study on the mechanism of apoptosis of hepatic stellate cells induced by nerve growth factor / 中国医师杂志

ObjectiveTo investigate the effect of NGF on apoptosis of HSC in vitro and explore the possible mechanism.MethodsHSC was incubated with different concentrations of NGF.HSC apoptosis was identified by FCM.The expressions of apoptosis-regulating proteins Caspase-3,p53 and Bcl-2 of HSC after apoptosis induced by NGF were examined by immunohistochemical staining.Expressions of NGF and p75NTR were detected by immunofluorescence.ResultsApoptosis index of HSC was higher than that of control group [(22.36±9.51)％ vs (5.88±1.36)％] after treated with NGF (100 ng/ml) (P＜0.05).After incubating with 100 ng/ml NGF for 24 h,the positive expression rates of p53 and Caspase-3 of HSC increased significantly than those of control group [(78.41±4.00)％ vs (34.96±3.84)％,(39.26±1.57)％ vs (9.27±1.01)％,P ＜0.05].The positive expression rate of Bcl-2 protein of HSC significantly decreased compared with that of control group (18.12±1.38)％ vs (91.53±2.98)％ (P＜0.05).When HSC was stimulated with 100 ng/ml NGF for 24 h,the average optical density of NGF increased significantly than control group (6.53±1.40 vs 1.77±0.17) (P＜0.05),while the expression of p75NTR was not significantly changed (3.52±0.36 vs 4.24±0.38) (P＞0.05).ConclusionsThe mechanism of NGF to induce HSC apoptosis may be associated with the up-regulating expression of Caspase3,P53 and down-regulating expression of Bcl-2 on HSC.NGF could be used as an initiating factor and effect factor to increase the expression of NGF on HSC,but it had no significant effect on p75NTR expression.

The role of Survivin in the apoptosis of PC-3 cells induced by Oridonin / 中国医师杂志

Objective To study the apoptosis-inducing effect of Oridonin on PC-3 cells line and the role of Survivin in the process.Methods After PC-3 cells were incubated with different concentrations of Oridonin,cell viability was analyzed with MTT assay.The percentage of earlier apoptosis cell was analyzed by flow cytometry.The protein expression of Survivin in PC-3 cells were detected by Western blot and fluorescent quantitative PCR.Results Oridonin effectively inhibited the proliferation of PC-3 cells in a concentration-time dependent way.After PC-3 cells were treated with Oridonin ( 2.5,5,10,20,40 μmol/L)for 48 hours,the cytotoxicity index were 9.2％,25.3％,39.3％,77.2％,92.5％ and the IC50 of PC-3 cells was 10.29 μmol/L,respectively.Flow cytometry was used to detect the effect of different concentration of Oridonin (0,10,20,40 μmol/L) for 48 hours,the apoptotic rates of PC-3 cells were 4.8％,15.4％,19.5％,27.4％ ( P ＜ 0.05).Oridonin down-regulated Survivin protein in a concentration-dependent way in PC-3 cells.Conclusions Oridonin can induce the apoptosis of PC-3 cells by a concentration-dependent manner.Oridonin can induce the apoptosis of PC-3 cells by down-regulated Survivin protein.

Mechanism of apoptosis effect induced by triptolide in PC3 cells / 中国医师杂志

Objective To detect the treatment effect of PC3 cells with triptolide on altering the expression of genes.Methods MTT assay was used to detec the inhibition of proliferation.Apoptosis was detected by Annexin-V/PI staining.RT-PCR was used to analyze the mRNA expressions of BCL-2,BAX,PIG3,P21,FAS,CASPASE3.Results Triptolide caused a time - and dose - dependent inhibition of cell proliferation,and IC50 of 24 h and 48 h were 18.3 ng/ml and 13.5 ng/ml,respectively.Compared with the 24 h group,the low concentration of triptolide(5 ng/ml,t =1.47,P ＞0.05)and the high concentration of triptolide ( 160 ng/ml,t =0.91,P ＞0.05)had no statistical significance in 48 h group,while 10 ng/ml( t =3.26,P ＜0.05),20 ng/ml( t =4.21,P ＜0.05),40 ng/ml( t =4.09,P ＜0.05),80 ng/ml( t =2.91,P ＜ 0.05 )had statistical significance.At the concentration of 18.3 ng/ml,triptolide induced PC3 cells apoptosis in a time - dependent manner.Compared with the control group,Anexin-V( + )/PI(-)was(5.42±2.21)％(t =3.52,P ＜0.05)in 6h,(13.51±3.37)％(t =6.53,P ＜0.01) 12h,(29.3 ±4.53)％ ( t =8.74,P ＜0.01) 24 h group separately,and it had statistical significance.RTPCR showed that 18.3 ng/ml triptolide up-regulated the mRNA expression of BAX and PIG3,down-regulated P21 and BCL-2.FAS and CASPASE3 did not show obvious changes.Conclusions Triptolide inhibits the proliferation of PC3 and induces apoptosis,and the changes of BCL-2,BAX,PIG3 and P21 may play an important role in the apoptosis of PC-3 cells.

Effects of As2 O3 in combination with cinobufacini on proliferation and apoptosis of the K562 cells / 中国医师杂志

Objective To investigate the effect of As2 O3 in combination with cinobufacini on proliferation and apoptosis of the K562 cells and provide theoretical basis for clinical application.Methods Cell proliferation was assayed by analyzing the growth and viability of the cells.Apoptosis was assayed by performing cell morphology,Annexin-V/PI staining,DNA-PI staining,and DNA gel electrophoresis.Results After exposure to As2O3 and cinobufacini,the growth of K562 cells was inhibited and the viability of K562 cells was decreased. After treated with 1.0μmol/L As2O3,0.125μg/ml cinobufacini,0.25μg/ml cinobufacini,1.0μmol/L As2O3 + 0.125 μg/ml cinobufacini,1.0μmol/L As2O3 + 0.25μg/ml cinobufacini for 24 and 48 hours,the proliferation inhibition rate were(24 ± 1.3)%,(21 ± 1.5)%,(38 ± 3.1)%,(57 ±2.7)%,(66 ±3.3)% and(49 ±2.9)%,(48 ±2.7)%,(61 ±2.1)%,(77 ±3.8)%,(82 ±4.2)%,the apoptosis rate of K562 cells were(4.8 ± 0.5)%,(5.6 ± 0.7)%,(9.8 ± 0.6)%,(11.9 ± 1.2)%,(15.2±1.5)% and(11.0 ±0.9)%,(12.9 ±1.1)%,(18.4 ±1.5)%,(21.0 ±2.0)%,(28.0 ±1.9)%.The percentage of apoptotic cells was a time- and dose-dependent manner.Typical DNA ladder was shown by DNA gel electrophoresis.Conclusions As2O3 combined with cinobufacini inhibited the proliferation of K562 cells and induced apoptosis of the K562 cells,the combination of the two drugs had better effect.

Research on apoptosis of human colon carcinoma cell induced by NDGA combined Celecoxib / 中国医师杂志

Objective To study the effect of 5-lipoxygenase(5-LOX) inhibitor nordihyroguaiaretic acid (NDGA) combined the selective cyclooxygenase-2 (COX-2) inhibitor Celecoxib on the apoptosis of human colon carcinoma cell line HT-29. Methods Different concentration of NDGA and Celecoxib combinations were used to process cancer cell, and thiazolyl blue tetrazlium bromide (MTT) and phase contrast microscope and Annexin V/PI fluorescence staining and reverse transcription polymerase chain reaction (RT-PCR) were used to study the proliferation inhibited effect and apoptosis induced effect caused by combination of NDGA combined Celecoxib. Results MTT results showed that the viability of NDGA group, Celecoxib group and the group of NDGA combined Celecoxib (0.432±0.024,0.425±0.013,0.303±0.014 vs 0.693±0.018,t＝18.79,25.75,37.64,P＜0.01) was obviously lower than control group. The group of NDGA combined Celecoxib was significantly lower than NDGA group or Celecoxib group (t＝10.21, 14.14,P＜0.01). Under inverted phase contrast microscope, cell morphology significantly changed, and the group of NDGA combined Celecoxib changed most obviously. Apoptosis was observed by laser scanning confocal microscope (LSM) after NDGA and Celecoxib were used to process the HT-29. RT-PCR showed that up-regulation of Caspase-3 after treatment, and the combination of two drugs increased the most. Conclusions NDGA combined Celecoxib inhibited proliferation and induced apoptosis in human colon carcinoma cell line HT-29, and combined therapy had better effect than that of any drug used separate-ly. The mechanism may be associated with up-regulation of Caspase-3.

Research on the apoptosis of malignant melanoma cell induced by transmembrane form of human blood group A mimotope vaccine / 中国医师杂志

Objective To investigate the apoptotic effect of the transmembrane form vaccine of human blood group A mimotope on malignant melanoma cell line B16. Methods B16 cells were transfected with different recombinant plasmid through Lipofectamine 2000 and incubated with different concentration of monoclonal anti-A antibody at 2.5 μg/ml, 5 μg/ml,10 μg/ml and 20 μg/ml. Apoptosis rate of cells was determined with Annexin Ⅴ/PI double staining by flow cytometry. Results Apoptosis rate to P/F-M-pIRES group B16 cells was 74.74% when anti-A monoclonal antibody concentration was 10 μg/ml; apoptosis rate of plasmids carrying peptide/Fas fusion gene such as P/F-M-pIRES group and P/F-pIRES group were significantly higher than M-pIRES group and pIRES group. The apoptosis rate was statistically significantly different between different recombinated plasmid groups (F＝669.707,P＜0.01). The apoptosis rate was statistically significantly different between different antibody groups (F＝106.596,P＜0.01). The interaction between recombinated plasmid groups and antibody groups was statistically significant (F＝34.806,P＜0.01). Conclusions The transmembrane form vaccine of human blood group A mimotope could induce B16 cell apoptosis in vitro. This vaccine may be a promising candidate for potential malignant melanoma therapy.

Eicosapentaenoate inhibits palmitic acid induced apoptosis in INS-1 / 中国医师杂志

Objective To study whether eicosapentaenoate can decrease the apoptosis effects in-duced by palmitic acid in INS-1 or not. Methods Based on different condition, there were four groups in this study, including control group , EPA group, palmitic acid group, combination of EPA and palmitic acid group. The grow curve was detected by MTT, and the expressions of bax were detected by Western blot.Cell apoptosis was detected by caspase-3. ROS was detected by CM2H2DCFDA kit after 48h. Result The grow curve of combination of EPA and palmitic acid group was higher than that in plamitic group[24 h :(37.33±1.15)OD vs (30. 79 ± 1.55 )OD, P < 0. 01 ;48 h:(31.50 ± 1.56)OD vs (23.94 ± 1.10)OD, P<0. 01] . Caspase-3 activity and ROS and the expression of bax and SREBP1c in combination of palmitic acid and T0901317 group were lower than those in palmitic acid group[(3566. 67 ± 305.51 )OD vs (4233. 33 ±416. 33)OD]. Conclusion EPA can inhibit apoptosis in INS-1 induced by palmitic acid.

Rosiglitazone and ATRA on gastric cancer SGC7901 cell line proliferation in vitro / 中国医师杂志

Objective To investigate the influence of PPARγ excitomotor RSG and ATRA on gastric cancer SGC7901 cell line proliferation in vitro and its potential mechanism study.Methods Human gastric cancer SGC7901 cell line was cultured in vitro.Experiment samples were divided to blank group,10μmol/L ATRA group, 12.5μmol/L RSG group, 25μmol/L RSG group, 10μmol/L ATRA + 25μmol/L RSG group.Proliferation inhibitory effect was determined by MTI assay.Flow cytometry was used to detect cell cycle, H.E stain was used to observed micrography alteration.Expression of PPARγ protein in gastric cancer cells were measured by immunohistochemistry.PPARγ mRNA in gastric cancer cells were measured by RT-PCR.Results ATAR at concentration 10μmol/L, RSG at 12.5 μmol/L and RSG at 25 μmol/L could inhibit the proliferation of SGC7901 cells in a dose-and time-dependent, and when both agents were combined for 72h, growth inhibition ratio was (29.73 ± 0.69) %.Flow cytometry analysis revealed a cell cycle arrest at G1 and S phase, and when both agents combined, S% was (12.87 ± 0.35 )%, cell micrography tended to be normal when both agents combined.Up-regulation of PPARγ protein and PPARγ mRNA expressions were also observed, those effects were enhanced when both agents combined, and grey scale ratio was 0.646.Conclusion The ATRA and RSG could significantly induced growth inhibition of human gastric cancer SGC7901 cell, which may be associated with cell cycle arrest and inducing differentiation, activation of PPARγ protein and PPARγ mRNA expression.Synergistic effect could be caused by the combined use of the two agents.

Effects of Ang-( 1-7 ) on the apoptosis of cultured endothelial cells induced by Ang Ⅱ / 中国医师杂志

Objective To investigate the effect of Ang-(1-7) on the apoptosis in human umbilical vein endothelial cells (HUVECs) induced by Ang Ⅱ.Methods HUVECs were isolated and cultured.Cultured HUVECs were incubated for 24 h with Ang-(1-7), Ang Ⅱ, Ang-(1-7) A-779, Ang-(1-7) + Ang Ⅱ, A-779 + Ang Ⅱ + Ang-( 1-7), respectively.Cultured HUVECs without incubating stimulator were chosen as controls.The apoptosis of endothelial cells were detected by flow cytometry.Results The apoptosis of endothelial cells in HUVECs were upregulated by AngⅡ ( 10-6 mol/L) (25.60% ±3.17% vs 2.32% ±0.24%, P ＜0.005).Compared with the AngⅡ group, Ang-(1-7) dose-dependently inhibited the apoptosis of endothelial cells in HUVECs ( (20.04% ± 2.21% ,16.04% ± 1.32 %, 10.04% ±2.05,7.79% ±1.50% vs AngⅡ group 25.60% ±3.17%, P ＜0.05 , P ＜0.05).The effects of Ang-(1-7) could be blocked by A-779 (23.37% ±0.75% vs 20.04% ± 2.21%, 16.04% ± 1.32,10.04% ± 2.05% ,7.79%± 1.50%, P ＜ 0.05 ).Conclusion Ang-(1-7) can attenuate the apoptosis of endothelial cells induced by Ang Ⅱ in HUVECs in a dose-dependent manner.The effects of Ang-(1-7) could be blocked by A-779( P＜0.05).

Effects of glucose on apoptosis of endothelial progenitor cells from human peripheral blood / 中国医师杂志

Objectives To investigate the effects of glucose on apoptosis rate of cultured endothelial progenitor cells (cEPCs). Methods The peripheral blood of healthy adults was isolated by density gradient centrifugation, and mononuclear cells (MNCs) were inducted to differentiate at cultured conditions.EPCs were identified by Dil-acLDL and FITC-UEA-1 as double fluorescent-positive cells. The effectsof glucose at different concentrations on apoptosis rate of the harvested EPCs were measured by fluorescent microscope and fluorescence-activated cell sorting (FACS) after staining with Annexin V-FITC and PI. Results No significant differences were observed in apoptosis rate between samples treatedwith 5.6mmol/l glucose and 11. 1 mmol/l. P ＞0. 05). 25.5 mmol/L glucose enhanced the EPCsapoptosis rate in a time-dependent manner( P ＜0. 05). Conclusion High concentration glucose can accelerate apoptosis rate of EPCs in a time-dependent manner.

The influence of VEGF expression on the apoptosis of pancreas subjected to ischemia/reperfusion injury / 中国医师杂志

Objective To investigate the influence of VEGF expression on the apoptosis of pancreas subjected to ischemia/reperfu-sion injury. Methods Thirty male SD rats were randomly divided into three groups (rt = 10). Group A was served as sham-operation group. Groups B were subjected to 30 min of ischemia by clamping of celiac artery and superior mesenterie artery then releasing for 6 hours to produce ischemia/reperfusion injury model. Groups C were treated with VEGF antisense oligodeexynueleotide after isehemia. Rats were sacrificed, the pancreas was obtained to detect the VEGF expression by immunohistochemical method, and apoptosis was determined by TUNEL staining. Results Apoptosis appeared and VEGF expression up regulated in pancreas after Isehemia/reperfusion injury. Comparison between Groups C and Groups B showed a significant down regulation of VEGF expression (P <0. 05) and a notable increase of apoptotic index (P < 0. 05). Conclusion VEGF expression suppresses apeptosis of pancreas during the course of ischemia/reperfusion injury and may play an important role in protection of pancreas against the ischemia/repeffusion injury.