聚酰胺-氨纳米微粒介导5-氟尿嘧啶联合小RNA-21抑制乳腺癌细胞生长的研究

Objective To study the effect of PAMAM-mediated 5-fluorouracil combined with miR-21 inhibitor gene therapy to suppress MCF-7 human breast cancer cell growth in vitro. Methods 5-Fu/PAMAM complex was prepared by dialysis method and then incubated with miR-21 inhibitor at room temperature. Transmission electronic microscopy (TEM) was performed to observe the morphology of the nanoparticles. The drug loading efficiency and encapsulation efficiency was determined by ultraviolet spectroscopy (UV). The transfection of PAMAM dendrimer was detected by flow cytometry. MTT assay was carried out to determine MCF-7 cell growth survival rate. Cell apoptosis was analyzed by flow-cytometry. Transwell assay was performed to detect invasion ability after MCF-7 cells treated with 5-Fu chemotherapy combined with miR-21 inhibitor gene therapy. Results The morphology of the complex was sphere observed under TEM. Encapsulation efficiency and loading efficiency of drug were (66. 21±4. 11)% and (31.77±0. 73)% , respectively. Flow cytometry revealed that 5-Fu/PAMAM transfection efficiency was (60.54 ±6. 97)%. 5-Fu combined with miR-21 inhibitor treatment significantly suppressed cell growth, and the survival rate was only (55. 85±3. 71)% on the 6th day of the observation period. The apoptosis rate in combined treatment group was (18. 32±2.42)% , dramatically higher than in control group (F=58. 326,P<0. 01). In combined treatment group, the number of invasion cells was only 18. 96 ±3. 14, suggesting the greatly decreased invasion ability of MCF-7 cells (F=16. 409,P < 0. 01). Conclusion PAMAM could effectively deliver miR-21 inhibitor and 5-Fu simultaneously, and combined therapy can suppress growth of MCF-7 cells effectively in vitro.     

绿脓杆菌制剂对人肝癌MHCC97L细胞增殖和侵袭能力的影响

Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.     

绿脓杆菌制剂对人肝癌MHCC97L细胞增殖和侵袭能力的影响

Objective To investigate the effects of Pseudomonas aeruginosa vaccine (PA) on proliferation and invasiveness of the hepatocellular carcinoma cell line MHCC97L with metastatic potential. Methods Proliferation, growth curve, plate efficiency, flow cytometry, transwell invasion assay, cell motility assay, scarification test, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2) protein activity were evaluated after cells were treated with PA at various concentrations. Results PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose-dependent manner. The IC50 of cells treated with PA for 48 h and 72 h was 3.1 ×108/ml and 1.9 × 108/ml, respectively. The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5 × 108/ml, 1 × 108/ml and 2 × 108/ml PA (P＜0.01). PA could induce cell cycle arrest at the G1 phase in a dose-dependent manner by flow cytometric analysis. The average amount of invading cell per field in cell invasion assay and motility assay were 4. 8 ± 1.3 and 8. 8±2.2 when cells treated with 1× 108/ml PA, which was significantly lower than that of control group (8. 6±2. 1 and 15. 6±1.2 ) (P＜0.01) Scarification test showed that the metastatic ability of cells treated with 1 × 108/ml PA significantly lower than that in the control group. Comparison between cells treated with 1 × 108/ml PA and control group, no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of cell culture. Conclusion PA can inhibit proliferation and plate efficiency of HCC cell line MHCC97L, which is in part mediated by the cell cycle arrest at the G1 phase. PA could inhibit invasiveness of HCC cell line MHCC97L, which is unrelated to the VEGF and MMP2 protein activity.     

神经生长因子基因修饰的骨髓间充质干细胞在糖尿病大鼠膀胱中的生长及表达

Objective To confirm that rat bone marrow mesenchymal stem cells (MSC) transfected with nerve growth factor (NGF) gene in the bladder tissue of diabetic rats bladder tissues can survive and stably express NGF. Methods A diabetic rat model was constructed. The BrdU-labelled MSC transfected with NGF gene were transplanted into the diabetic rats bladder tissues. BrdUlabelled immunohistochemistry was used to observe the growth of MSC transfected with NGF gene in the diabetic rats bladder tissues. The expression of NGF mRNA and protein were checked by RT-PCR and ELISA. Results A diabetic rat model was successfully built by a single intraperitoneal injectionof STZ. The blood glucose was still high after 8 weeks. NGF gene modified MSC could be detected in the bladder of diabetic rats by BrdU-labelled immunohistochemistry. The concentration of NGF in the control group, disease group and treatment group were ( 114 ± 3), ( 70 ± 2), ( 110 ± 2) pg/ml by ELISA and mRNA quantity by RT-PCR were 0. 183±0. 004, 0. 032±0. 139, 0. 130±0. 165, respectively. Compared with the control group, the expression of NGF gene was decreased (P＜0. 05) in the incidence group. The expression of NGF gene was increased (P＜0. 05) in the treatment group compared with the disease group. Conclusions The NGF gene-modified MSC could survive in diabetic rats bladder tissues. The NGF gene in MSC could stably express in diabetic rats bladder tissues.     

神经生长因子基因修饰的骨髓间充质干细胞在糖尿病大鼠膀胱中的生长及表达

Objective To confirm that rat bone marrow mesenchymal stem cells (MSC) transfected with nerve growth factor (NGF) gene in the bladder tissue of diabetic rats bladder tissues can survive and stably express NGF. Methods A diabetic rat model was constructed. The BrdU-labelled MSC transfected with NGF gene were transplanted into the diabetic rats bladder tissues. BrdUlabelled immunohistochemistry was used to observe the growth of MSC transfected with NGF gene in the diabetic rats bladder tissues. The expression of NGF mRNA and protein were checked by RT-PCR and ELISA. Results A diabetic rat model was successfully built by a single intraperitoneal injectionof STZ. The blood glucose was still high after 8 weeks. NGF gene modified MSC could be detected in the bladder of diabetic rats by BrdU-labelled immunohistochemistry. The concentration of NGF in the control group, disease group and treatment group were ( 114 ± 3), ( 70 ± 2), ( 110 ± 2) pg/ml by ELISA and mRNA quantity by RT-PCR were 0. 183±0. 004, 0. 032±0. 139, 0. 130±0. 165, respectively. Compared with the control group, the expression of NGF gene was decreased (P＜0. 05) in the incidence group. The expression of NGF gene was increased (P＜0. 05) in the treatment group compared with the disease group. Conclusions The NGF gene-modified MSC could survive in diabetic rats bladder tissues. The NGF gene in MSC could stably express in diabetic rats bladder tissues.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.     

靶向Survivin的siRNA联合吉西他滨抑制胰腺癌细胞增殖

Objective To construct the siRNA eukaryotic expression vector targeting Survivin gene, and investigate the chemotherapy sensitivity of pancreatic cancer line Panc-1 treated by gemcitabine. Methods The siRNA eukaryotic expression vector targeting Survivin gene was constructed. Panc-1 cells were transfected with negative control vector or siRNA vector and selected by G418, and the cell growth curve was drawn. The expression of Survivin mRNA and protein was detected by RT-PCR and Western blot respectively. Panc-1 cells or transfected cells were treated with gemcitabine for 24 h,and then the growth inhibition rate was measured by MTT assay, and cell apeptosis rate was measured by flow cytometry. Results The result of endonuclease digestion and DNA sequencing revealed that the recombinant plasmid psiRNA-Survivin was constructed successfully. Survivin mRNA and protein levels were reduced by 79.2% and 83.6% respectively in stably transfected Panc-1 cells as compared with control group, and the cell growth curve was much smoother, and the growth inhibition rate [ ( 24.6±4.5 ) % / (38.7±5.2 ) % ] and apoptosis rate of these cells [ ( 16.7±2.5 ) %/( 26.8±3.4 ) % ] were significantly increased after treatment by gemcitabine (P < 0.05 ). Conclusion The constructed siRNA eukaryotie expression vector targeting Survivin could decrease the Survivin expression,inhibit the growth of Panc-1 cells significantly,and increase the chemotherapy sensitivity to gemcitabine.