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Abstract The aim of this study was to investigate the effect of TiO2/N-succinyl-chitosan composite (TiO2/ NSCS) photodynamic therapy (PDT), while considering the effects of various light sources on the activation of photosensitizer. The methyl thiazolyl tetrazolium assay was used to examine the cell survival rate of the cells. The results showed that glioma cell strain (U251) was the most sensitive cancer cell strain to TiO2/NSCS. When the concentration of TiO2/NSCS was between 0 and 800 μg·mL-1, there was no obvious cytotoxicity to normal liver cells (HL-7702) and U251 cells. During the PDT process, the photokilling effect of TiO2/NSCS on U251 cells under ultraviolet-A (UVA) light irradiation was stronger than that of pure TiO2, and its killing effects were positively correlated with concentration and irradiation time. In addition, both UVA and visible light could excite TiO2/ NSCS, which had significant killing effect on U251 cells. The results of acridine orange/ethidium bromide fluorescent double staining and Annexin V/propidium iodide double staining indicated that TiO2/NSCS under UVA and visible light irradiation could kill U251 cells by inducing apoptosis, and the apoptosis rate of TiO2/NSCS treatment groups was higher than that of TiO2 treatment groups. Therefore, TiO2/NSCS might be used as a potential photosensitizer in PDT.
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Objective:To investigate the effect of apatinib on radiosensitivity of glioma cells U87MG and its potential mechanism.Methods:U87MG cells were divided into control group, apatinib group, radiation group and combination group treated with apatinib and radiation. The effect of different concentrations of apatinib (5, 10, 20, 40, 80 μmol/L) on cell proliferation was detected by CCK8 assay. The effect of apatinib on cell migration and invasion was detected by wound-healing assay and transwell assay, respectively. The effect of apatinib on cell radiosensitivity was detected by plate cloning assay, the cell apoptosis rate was detected by flow cytometry, and the protein expressions of Bax and Bcl-2 were detected by Western blot.Results:Apatinib significantly inhibited the proliferation of U87MG cells in a manner depended on the drug treatment time and radiation. Compared with the radiation group, the cell proliferation, migration and invasion in the combination group were inhibited much significantly ( t=9.857, 18.704, 4.197, P<0.05), so that the value of D0, Dq and SF2 of the combination group was lower, resulting in a radiosensitivity enhancement ratio (SER D0 ) of 1.3. Moreover, compared with the radiation group, the apoptosis rate of the combination group was increased, the expression of Bcl-2 protein was decreased, and the expression of Bax protein was increased ( t=16.187, 8.890, 5.222, P< 0.05). Conclusions:Apatinib inhibits cell proliferation, invasion and migration, induces apoptosis and increases radiosensitivity of glioma cells.
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Aim To investigate the effects of oridonin on proliferation, migration and apoptosis of U87 glioma cells and to explore the involvement of the mechanism in the inhibition of Yes-associated protein (YAP)-c - Myc signaling pathway. Methods The effect of oridonin on U87 viability was measured by MTT assay; the migration and invasiveness of cells were measured by transwell assays; the apoptotic rates of cells were assessed by flow cytometry; the caspase-3, B c l - 2, Bax, YAP, c - MycmRNA expression in U87 glioma cells was detected by real-time quantitative P C R; the caspase-3, Bcl-2, Bax, YAP, p-YAP (Seri 27), c-Myc protein expressions were detected by Western blot. Results The proliferation of U87 cells was significantly inhibited by oridonin in a dose-dependent manner (P < 0. 05), and the ability of cell migration and invasion was weakened (P <0. 01), cell apoptosis rate in flow cytometry analysis increased significantly (P <0. 01), the protein and mRNA expression of caspase-3 increased (P < 0. 05), the mRNA and protein expression of Bcl-2/Bax decreased (P < 0. 05), the mRNA and protein expression of Y A P and c-Myc decreased (P < 0. 05), and the protein expression of p - Y A P increased (P < 0. 05). Conclusions Oridonin can significantly inhibit the proliferation and migration of U87 glioma cells and promote the transformation apoptosis of glioma cells; the mechanism may be related to the inhibition of YAP-c-Myc signaling pathway.
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Aim To investigate the mechanism of heparin-binding epidermal growth factor (HBEGF) in upregulating the expression of programmed death ligand 1 (PD-L1) in glioma cells by regulating leukemia inhibitory factor (LIF). Methods Pearson analysis was used to analyze the correlation between HBEGF, LIF and PD-L1 mRNA in primary gliomas, U251 and U87 cells were treated with HBEGF neutralizing antibody and HBEGF was added after HBEGF knockout, the mRNA and protein levels of LIF and PD-L1 were detected by real-time fluorescence quantitative PCR (RT-qPCR) and Western blot, HBEGF knockout cells were added with LIF neutralizing antibody on the basis of adding HBEGF, and the levels of PD-L1 mRNA and protein were detected. Results Pearson analysis showed that HBEGF, LIF, and PD-L1 mRNA were positively correlated (P < 0.05). After adding HBEGF neutralizing antibody to U251 and U87 cells, the mRNA and protein levels of LIF and PD-L1 gradually decreased with cell passage (P < 0.05). After HBEGF knockout, the mRNA and protein levels of LIF and PD-L1 decreased (P < 0.05), and the mRNA and protein levels of LIF and PD-L1 increased after HBEGF was added (P < 0.05). In HBEGF knockout cell lines, the levels of PD-L1 mRNA and protein were up-regulated after HBEGF was added (P<0.05), and the levels of PD-L1 mRNA and protein was down-regulated by LIF neutralizing antibody (P < 0.05). Conclusion The partial induction of LIF by HBEGF in glioblastoma is an intermediate process for HBEGF to maintain PD-L1 expression.
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OBJECTIVE：To study the anti-tumor effect of artemether （ARM）self-microemulsifying drug delivery system （SMEDDS） on human glioma subcutaneously transplanted model mice. METHODS ：Human glioma cell line SHG 44 was inoculated and passed on to establish subcutaneous transplanted tumor model of nude mice. At the 5th，10th，15th，20th and 25th day after inoculation ，the tumor tissue volume was measured and the growth curve was drawn to confirm the initial stage of rapid tumor proliferation. Thirty nude mice was collected to establish subeutaneously transplanted tumor nude model ，and then divided into control group （normal saline ），ARM suspension group [ 60 mg/（kg·d）]，ARM-SMEDDS low-dose ，medium-dose and high-dose groups [ 10，20，30 mg/（kg·d）] at the initial stage of rapid tumor proliferation. They were given normal saline and relevant solution intragastrically once a day ，for consecutive 30 d. The weight change and general sibuation of mice were recorded. The change of tumor volume was determined and relative tumor proliferation rate was calculated. RESULTS ：The subcutaneously transplanted tumor tissue entered the initial stage of rapid tumor proliferation from the 10th day after transplantation. The general situation was normal ，and there was no obvious abnormal reaction in mice of each group during treatment. Since 10th day of administration，tumor tissue volume of mice in ARM-SMEDDS groups were shortened significantly than control group （P＜0.05）. At 15th day of administration ，tumor volume of mice in ARM-SMEDDS groups were shortened significantly than ARM suspension group（P＜0.05）. After last administration ，relative tumor proliferation rates of mice in ARM-SMEDDS groups were decreased significantly，compared with ARM suspension group （P＜0.05）. CONCLUSIONS ：ARM-SMEDDS show significant inhibitory effect on the proliferation of human glioma ，and are better than suspension with higher dosage.
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Objective To investigate the expression of miR-124 in glioblastoma (GBM) cell lines LN229 and LN229R,as well as the regulatory mechanism of miR-124 on radiosensitivity of LN229R cells.Methods miR-124 mimic (miR-124) and negative control (miR-NC),STAT3 overexpression plasmid (STAT3) and pcDNA3.1 vector (pcDNA) were transfected or co-transfected into radioresistant glioma cells LN229R.qRT-PCR was employed to analyze the expression of miR-124 in LN229 and LN229R cells.The survival rate and sensitivity-related parameters of LN229R cells at different doses were analyzed by cloning formation assay.Cell apoptosis of LN229R was evaluated by flow cytometry.Targeting gene of miR-124 was predicted using Targetscan software and verified by the double-luciferase reporter assay.Western blot assay was performed to detect STAT3 protein expression.Results The expression of miR-124 in LN229R cells (0.32 ± 0.03) was significantly lower than that in LN229 cells (1.02 ± 0.09) (t =12.780,P＜0.05).Transfection of miR-124 mimics promoted the expression of miR-124 in LN229R cells (4.02±0.39) compared with miR-NC group (0.95±0.06) (t=13.476,P＜0.05).After 8 Gy irradiation,the survival rate of LN229R cells transfected with miR-124 mimics (0.003 ± 0.000 4) was significantly lower than that in miR-NC group (0.033±0.005 0) (t=5.655,P＜0.05),and the apoptosis rate (22.34±2.42) ％ was significantly higher than that in miR-NC group (4.69 ± 0.51) ％ (t =12.361,P＜0.05).STAT3 was identified to be a target gene of miR-124.Exogenous restoration of STAT3 reversed the inhibitory effect of miR-124 on LN229R cell survival.Conclusion miR-124 increases the radiosensitivity of LN229R cells by targeting STAT3.
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Objective To investigate the effects of titanium dioxide ( TiO2 ) nanoparticles coupled with nuclear localization sequence ( NLS ) on the radiosensitivity of U251 glioma cells. Methods Synthesis and characterization of the TiO2-NLS nanoparticles with nuclear targeting property. U251 cells were treated with nanoparticles and/or ionizing radiation. Flow cytometry analysis was performed to measure the ROS content and the cell apoptotic percentage. The DNA damage was detected by using γ-H2AX foci staining. Clonogenic survival assay was used to evaluate the radiosensitivity of U251 cells. Results NLS modification promoted nuclear translocation of TiO2 nanoparticles. Compared with the control nanoparticles, TiO2-NLS treatment increased radiation-induced cell apoptosis (t=8. 96, P<0. 05). Clonogenic survival assay showed the radiosensitization ratios of TiO2 nanoparticle-treated group and TiO2-NLSnanoparticle-treated group were 1. 18 and was 1. 29, respectively. These two ratios had statistically difference ( t =14. 72, P< 0. 05). Conclusions Nuclear targeting TiO2 nanoparticles enhances the radiosensitivity of U251 glioma cells.
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Objective To investigate the inhibitory effect of rosuvastatin on the migration and invasion of U87 glioma cells and its related mechanisms. Methods Cultured U87 cells were treated with 0, 5, 10 and 20 μmol/L rosuvastatin for 24,48 and 72 h. Cell viability was measured by CCK-8 assay. Migration ability of the cells was detected by scratch assay,and invasion ability of the cells was detected by Transwell assay. The expression of matrix metalloproteinase 2(MMP2),MMP9 and the Wnt/β-catenin signaling pathway-related proteins was detected by Western blot assay. Results Compared with the control group,the viability of U87 cells in the 5,10 and 20 μmol/L rosuvastatin groups was decreased (P < 0.01)in a time- and dose-dependent manner. Migration and invasion abilities of the cells were decreased(P <0.01). In addition,the expression levels of MMP2,MMP9,Wnt1,Wnt3a,Wnt7a,and β-catenin were decreased as well (P < 0.01). Conclusions Rosuvastatin can significantly inhibit the migration and invasion abilities of U87 glioma cells, probably related to the blocking of Wnt/β-catenin signaling pathway.
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Objective To investigate the mRNA-based anticancer gene transfer in MSCs-mediated cytotox-icity of glioma cells. Methods TRAIL mRNA and PTEN mRNA were synthesized in vitro. Immunoblotting assay was used to detect the expression of TRAIL and PTEN in the transfected MSCs.Transwell co-culture was perform to detect the migration ability of MSCs after gene transfection. The bioluminescence,live/dead staining and real time cell analyzer were used to analyze the viability of DBTRG cells. Results Compared with non-transfected MSCs, an enhanced migration rate was observed in MSCs with two kind of mRNA transfection.TRAIL-and PTEN-mRNA-induced cytotoxicity in DBTRG glioma cell was correlated with the ratio of the conditioned medium of the transfect-ed MSCs. A synergistic action was observed in TRAIL and PTEN in the transwell co-culture model. Conclusion The present study reveals the effect of synthesized mRNA-based gene transfer on mesenchymal stem cell-mediated cytotoxicity of glioma cells(DBTRG).
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Aim To explore the effect of Cx43 over-expression on proliferation of C6 cells and its mechanisms by transfecting pCMV-Cx43cDNA plasmid into C6 cells.Methods pCMV-Cx43cDNA plasmid was transfected into C6 cells by liposome to up-regulate the expression of Cx43, and C6 cells with over-expression of Cx43 was stably cloned by using G418.Determination of cell doubling time and soft agar colony formation assay to detect the degree of cell proliferation.The cells were treated with ERK1/2 specific blocker PD98059(30 μmol·L-1) and p38MAPK specific blocker SB202190(10 μmol·L-1)respectively, the expression of Cx43, p-Cx43, p-ERK1/2 and p-p38MAPK of each group were detected by Western blot, and the activity of each group was detected by MTT Assay.Results pCMV-Cx43cDNA plasmid was transfected into C6 cells successfully.Cell lines with over-expression Cx43(C6-Cx43) or empty vector (C6-pCMV) were stably selected by using G418.Determination of cell doubling time and soft agar colony formation experiments showed that the proliferative rate and the colony number of C6-Cx43 group were significantly decreased, compared with that of C6 group and C6-pCMV group(P<0.01);ERK1/2, p38MAPK specific blockers were treated with each group,Western blot showed that the expression of Cx43 protein was increased(P<0.01), while p-Cx43 protein was decreased (P<0.05) in C6-Cx43+PD98059 group and C6-Cx43+SB202190 group,compared with that of C6-Cx43 group.Conclusion Cx43 may decrease the proliferation of glioma cells through ERK1/2, p38MAPK pathways.
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Objective:To investigate the effect of oxaliplatin inhibit the growth in glioma U 87 cells by regulating PI3K/Akt sig-nal pathway.Methods:The glioma U87 cells were cultivated in vitro ,using 0,20,40,80 μg/ml oxaliplatin treated U87 cells 24,36, 48,72 h respectively,MTT was used to detect cell proliferation.Using 40 μg/ml oxaliplatin treated U87 cells 48 h,flow cytometry was used to detect cell cycle and apoptosis .Cells apoptosis protein and PI 3K/Akt pathway protein expression after 40 μg/ml oxaliplatin treated U87 cells 48 h was detected by Western blot .Results:MTT assay showed that compared with the 0μg/ml treatment ,oxaliplatin treatment could significantly inhibited U 87 cell survival ( P0.05).Conclusion:Oxaliplatin may suppress U87 cell proliferation,ar-rest cell cycle,and promote apoptosis by inhibit PI3K/Akt signaling pathway.
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Objective The derivative of Gefitinib was used to treat glioma cells in vitro to explore a more effective new drug for the clinical treatment of astrocytoma. Methods (1) Fifteen kinds of gefitinib derivatives, gefitinib and temozolomide were used to treat glioma cells, and the effect of 0, 10, 15, 20, 25 and 30 μmol/L of each kind of drug on cell proliferation was detected by by MTT assay , respectively. (2) To calculate the concentration of IC50 , then select lower IC50 of derivativs combinate gefitinib and temozolomide with 10, 20 and 30 μmol/L to treat cells, then the apoptosis of cells were detected by flow cytometry. Expression of p-EGFR was detected by western–blot assay. Results (1) NO.LPY-5,9,11, but not other derivatives of Gefitinib could effectively inhibit the growth of cells. (2) IC50 of NO.LPY-9 was less than that of the 5th drug, and both of them were lower than those of gefitinib and temozolomide; NO. LPY-11 was excluded. (3) The cell apoptosis of No. LPY-9 was higher than that of gefitinib and temozolomide , respectively. However, No.LPY-9-induced cell apoptosis was significantly higher than that of No. LPY-5-induced cell. (4) Levels of p-EGFR expression in No.LPY-9 and gefitini-induced cells were significantly lower than that in the negative control group. Conclusion No.LPY-9 has asignificant inhibitory effect on glioma cells in vitro , resulting from the inhibition of the ERFR-mediated signaling pathways and induction of cell apoptosis.
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Objective To investigate the inhibitory effect of a disintegrin and metalloprotease 12 silenced by shR?NA on self-renewal capacity of CD133 positive giloma cells. Methods The shRNA recombinant lentivirus aimed at si?lencing ADAM12 was prepared. Human glioma cells U87 were employed in this study and assigned into three groups:shRNA-ADAM12, shRNA-NCandshRNA-C. ADAM12 expression was detected at mRNA and protein level using Re?al-time quantitative-PCR and western bloting, respectively. U87 cells were cultured with stem cell culture medium, to obtain cell sphere formation in which CD133 positive glioma cells were enriched. Immunofluorescence was employed to detect the expression of ADAM12 and CD133 in cell spheres and U87 cells; Self-renewal was tested by using tumor sphere formation assay. Molecular markers for differentiated or undifferentiated cells (CD133,GFAP and Tuj1) were de?tected at protein using western blotting. Western blotting was employed to test protein expression of HES1. Results AD?AM12 shRNA significantly down-regulated the mRNA and protein expression levels of ADAM12. Compared with shRNA–C group, the relative expression levels of mRNA in shRNA-ADAM12 group and shRNA-NC group were 0.22 ± 0.03 and 0.98 ± 0.06 (F=425.37,P<0.01). The relative expression levels of protein in shRNA-ADAM12 group, shRNA-NC group and shRNA-C group were 28.72%±2.36%, 69.21%±3.92%and 69.04%±3.57%, respectively (F=145.42,P<0.01). Immunofluorescence staining showed that expression levels of ADAM12 and CD133 in cell spheres were significantly higher than those in normal cells. The number of spheres in three groups were 45.5±2.3、104.2±5.8 and 109.6±6.2, tumor sphere formation ability of shRNA-ADAM12 group was lower than that of shRNA-NC group and shRNA-C group (F=147.03,P<0.01). Compared with the shRNA-NC group and shRNA-C group, the protain expression of GFAP and Tuj1 were increased up to 166% and 146% (P<0.01) whereas the protein expression levels of CD133 and HES1 were down-regulated by 54% and 50% (P<0.01). Conclusion Knockdown of ADAM12 may suppress self-renewal ability of CD133 positive glioma cells by inhibiting the Notch pathway activity.
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Objective To investigate the effect of ganoderic acid A( GA-A) on apoptosis, invasion and KDR expression of human U251 cells.Methods Ganoderic acid A( GA-A) was prepared, human U251 cells were treated with 0.1, and 0.5 mmol/L GA-A, and the experiment was divided into blank control, low concentration and high concentration group.The expressions of KDR mRNA and KDR protein was assayed by RT-PCR and Western blot.The effect of GA-A on the proliferation and invasion capability of U251 cells was determined by CCK-8 and transwell assay in vitro, respectively.Flow cytometry was used to detect the influence of GA-A on the cell cycle and apoptosis of U251 cells, and TUNEL staining was detected the cell apoptosis too.Results Compared with the control group, KDR mRNA and protein expression of high concentration and low concentration group were significantly decreased(P <0.05), GA-A can significantly reduce the cell growth rate, reduce the proportion of cells in G1 phase and increase the proportion of S phase and G2 /M phase,cells apoptosis was significantly increased in the high concentration and low concentration group ( P <0.01), and cells proliferation and invasion was significantly decreased (P <0.05).Compared with low concentration group, the high concentration group induce cell apoptosis and inhibit the expression of KDR more significant (P <0.05). Conclusions Ganoderma acid A can induce apoptosis in U251 cells, inhibit proliferation and invasion, and can inhibit the expression of KDR mRNA and protein, which may be one of the mechanisms of anti-tumor.
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Objective:To study the inhibitory effect of deoxyschizandrin on the growth of brain glioma C6 cells, and to explore its mechanism.Methods:The rat glioma C6 cells were cultured and divided into control group,50, 100,and 200 mg·L-1 deoxyschizandrin groups.The proliferation rates of C6 cells were examined by MTT assay;the changes of cell cycles were examined by flow cytometry;the expression levels of CyclinD1,Bax,Bcl-2 and Caspase-3 proteins in supernant were detected by ELISA assay. Results:Compared with control group, the proliferation rates at 24 and 48 h in 50,100,and 200 mg·L-1 deoxyschizandrin groups were significantly decreased (P <0.01),and the proliferation rates at 72 h in 100 and 200 mg·L-1 deoxyschizandrin groups were significantly decreased (P < 0.05 or P < 0.01 ). Compared with control group, the percentage of cells at SubG1 phase in 200 mg·L-1 deoxyschizandrin group was increased (P < 0.05 ), and the percentage of cells at S phase was decreased (P <0.05).Compared with control group,the expression levels of CyclinD1 in 100 and 200 mg· L-1 deoxyschizandrin groups were decreased (P < 0.01 );the expression levels of Bax protein in deoxyschizandrin groups were increased (P < 0.05 or P < 0.01 ), and the expression level of Bcl-2 protein in 200 mg · L-1 deoxyschizandrin group was decreased (P < 0.01 ), and the Bax/Bcl-2 value in deoxyschizandrin groups were increased (P < 0.01 ); the expression level of Caspase-3 protein in 200 mg · L-1 deoxyschizandrin group was increased (P < 0.01 ).Conclusion:Deoxyschizandrin could inhibit the growth of glioma cells through down-regulating the expression levels of CyclinD1 protein and up-regulating the expression levels apoptotic factors Bax and Bcl-2.
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Objective To study the temozolomide combined with curcumin on the inhibitory effect and apoptosis of the C6 glioma cells. Methods The C6 glioma cells were treated with temozomide in combination with curcumin. The anti proliferation effect of liposomes on the C6 glioma cells was investigated by using the method of sulforhodamine B (SRB). Flow cytometry was used to detect apoptosis of the C6 glioma cells. Confocal laser scan-ning microscope was used to observe apoptosis and location in the C6 glioma cells. Results The results of SRB as-say showed that temozolomide in combination with curcumin inhibition rate were (91.22 ± 0.51)%in 48 h of the C6 glioma cells; Flow cytometry showed that the apoptosis rate were (33.15 ± 0.79)% with temozolomide (5 μmol/L) in combination with curcumin (10 μmol/L). Laser scanning confocal scanning microscope indicated that the apop-tosis of in the C6 glioma cells treated with temozolomide in combination with curcumin was more than that of free drug. Conclusion The temozolomide in combination of curcumin can inhibit the growth and induce apoptosis of the C6 glioma cells.
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Objective:To observe the expression of IL-12 family subunit genes by real-time quantitative PCR in mice C6 glioma cells,construct the basis of the brain glioma research on IL-12 family in the future.Methods:Mice C6 glioma RNA was abstracted and reversed transcription cDNA.The mice C6 glioma cells mRNA expression influence of IL-12 family subunit genes was compared and analyzed by real-time quantitative PCR.Results: In mice C6 glioma cells, high expression abundances in IL-23a, IL-12a, midlde expression abundances in EBI3, IL-27, low expression abundance in IL-12b.Conclusion: IL-12 families are closely related to the occurrence and development of glioma,IL-12,IL-23 are regarded as the most potential anti-glioma cytokines among them,research de-velopments will bring a new way of brain glioma immune therapy.
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OBJECTIVE: To study the effects of Vaccinium vitis procyanidin on glioma SHG-44 cells and explore the mechanism of Vaccinium vitis procyanidin induced apoptosis. METHODS: SHG-44 cells were cultured. The rates of inhibition were examined by MTT assay; and the density of SHG-44 cells was observed by inverted microscope; the expression of bax protein was detected by cyto-histoimmunochemistry, bax, bcl-2 and caspase 3 proteins were examined by Western blot assay. RESULTS: The rates of inhibition of SHG-44 cells were increased in 8, 40 and 400 μg · L Vaccinium vitis procyanidin groups, and the density was decreased; the apop-totic rates were increased with dose increased; the expression of bax and caspase 3 proteins was increased in treated groups, but the expression of bcl-2 protein was decreased in treated groups. CONCLUSION: Vaccinium vitis procyanidin could induce the apoptosis in glioma SHG-44 cells through up-regulating the expression of bax protein and down-regulating the expression of bcl-2 protein, and then activated the mitochondrial pathway.
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Objective To study the influence of Rhizoma typhonii extract on the growth of glioma SHG-44 cells cultured invitro,and to explore the mechanism of the inhibitory effect of Rhizoma typhonii extract on the growth of glioma cells.Methods The SHG-44 cells were cultured and divided into blank control group and 8, 40, 200, 1 000μg·L-1 Rhizoma typhonii extract groups.The inhibitory effect of Rhizoma typhonii extract on the growth of glioma SHG-44 cells was measured by MTT assay. The secretion levels of Bax and caspase-3 proteins were examined by ELISA assay. The expression level of caspase-3 protein was examined by Western blotting method. Results Compared with blank control group, the inhibitory rates of the growth of SHG-44 cells in 200, 1 000μg·L-1 Rhizoma typhonii extract groups at 24 h,and 8,40,200,and 1 000μg·L-1 Rhizoma typhonii extract groups at 48 h were significantly increased (P<0.05 or P<0.01).The secretion levels of Bax and caspase-3 proteins in 40,200,and 1 000μg· L-1 Rhizoma typhonii extract groups at 48 h were increased compared with blank control group (P<0.05 or P<0.01 ). Compared with blank control group, the expression levels of caspase-3 protein in different doses of Rhizoma typhonii extract groups were increased significantly (P<0.01). Conclusion Rhizoma Typhonii extract can inhibit the growth of cells through up-regulating the expression of Bax protein,increasing the expression level of caspase-3 protein and activating apoptosis pathway.
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Objective:To construct a recombinant eukaryotic expressive vector of pEGFP-N3-M-IL-2(88 Arg,125 Ala),and to study the expression of this gene in the Glioma cell line U 87,and to detect its antitumor activities of the fusion protein M-IL-2(88 Arg,125 Ala).Methods:The target fusion gene M-IL-2 (88 Arg,125 Ala) was amplified by PCR from pPICZαA/M-IL-2 (88 Arg,125 Ala) and cloned into pEGFP-N3 vector after digestion to construct recombinant eukaryotic expressive vector pEGFP -N3-M-IL-2( 88 Arg,125 Ala).And then recombinant plasmid pEGFP-N3-M-IL-2(88Arg,125Ala) was transfected into Glioma cell U87 by LipofectamineTM2000 immediately after it was confirmed by restrictive enzyme analysis and sequencing .RT-PCR and Western blot were used to confirm expression of the fusion gene in the U87.Prohibitory effect of recombinant M-IL-2(88Arg,125Ala) protein on U87 was assessed by CCK-8 assay.Results:Restrictive analysis and sequence analysis revealed that M-IL-2(88Arg,125Ala) fusion gene was cloned into the vector pEGFP-N3 suc-cessfully,fusion gene M-IL-2(88Arg,125Ala) could express in U87 cells and could inhibit the growth of U87 cells.Conclusion:The eu-karyotic expression plasmid pEGFP-N3-M-IL-2( 88 Arg,125 Ala) was constructed and expressed in U 87 cells successfully ,the fusion protein could inhibit the growth of U87 cells.We laid a foundation for further research of gene M-IL-2(88 Arg,125 Ala).