Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent H...Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent HDFs showed several molecular and cytological changes such as large flat morphology, expression of senescence-associated β-galactosidase (SA β-gal) activity and altered gene expression. Small interfering RNA (siRNA) has been demonstrated to be a potential research tool to analyse gene function and pathway. Expression of an appropriate housekeeping or reference gene can be used as a measurement of transfection efficiency in siRNA. Therefore this study was designed to determine the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model. GAPDH knockdown with an appropriate transfection reagent was measured by quantitative real time RT-PCR while cellular senescence was characterized based on morphological changes, expression of SA β-gal and p16INK4a expression levels. Our findings showed that GAPDH knockdown represents silencing efficiency and down regulation of p16INK4a in senescent transfected HDFs caused morphological alterations which results in the formation of spindle shaped fibroblasts. This study demonstrated the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model.展开更多
OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of t...OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of the adherence factor CD44v6. METHODS PEI-PEG/siRNA nanoparticles, in different N/P ratios, were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the control group. The transfection efficiencies were observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay （mononuclear cell direct cytotoxicity assay）, and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. Based on the different N/P ratios, PEI-PEG/siRNA composites were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the controls. The transfection efficiency was observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. RESULTS After transfection, the transfection efficiency of the PEI-PEG/siRNA nanocomposites increased incrementally in N/P ratio value. The transfection efficiency improved with an increase in N/P ratio. When the N/P value was 15, fluorescence became more intense in the PEI-PEG/siRNA group than in the Lipo2000/siRNA group. At the same time, cell viability was （80.4 ± 5.6）% in the MTT reduction assay, which was similar to that in the Lipo2000/siRNA group. The results of Western blot analysis showed that the expression level of CD44v6 protein decreased to （59.7 ± 3.0）% after siRNA-CD44v6 was inhibited. CONCLUSION PEI-PEG could effectively form the nanocomposite in combination with siRNA, be transfected into the SGC7901 gastric cancer cell lines and inhibit CD44v6 protein expression. Moreover, as a genetic carrier, PEI-PEG copolymer has greater advantages, including high transfection e. ciency, less cytotoxicity and an easily alterable vector structure.展开更多
A biodegradable gene transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] has been developed by thermal polycondensation of aspartic acid and lysine, and branch poly(ethylenimine) (Mw less than ...A biodegradable gene transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] has been developed by thermal polycondensation of aspartic acid and lysine, and branch poly(ethylenimine) (Mw less than 600) was grafted to the backbone. The polymer was characterized by 1H NMR. It appeared lower cytotoxity compared to poly(ethylenimine) (25KDa), which was quantified by MTT assay. Electrophoresis indicated that the polymer could retardate DNA at N/P ratio 1.2-1.8 (w/w). Transfection efficiency of the complexes was studied in NT2 cell lines. It was 1.5 fold higher than molecular weight PEI (Mw = 25KDa).展开更多
Incorporating functional ligands and biodegradable bonds into biocompatible low-molecular-weight(LMW)polymers,such as 1.8 kDa poly(ethylenimine)(PEI1.8 k),is a common strategy to improve the properties of LMW polymers...Incorporating functional ligands and biodegradable bonds into biocompatible low-molecular-weight(LMW)polymers,such as 1.8 kDa poly(ethylenimine)(PEI1.8 k),is a common strategy to improve the properties of LMW polymers including biosafety and delivery efficacy.This study demonstrates the hypothesis that introducing different functional ligands and linked reductive disulfides in PEI 1.8k will achieve superior siRNA transfection efficiency.By incorporating PEI-X(X represents cholesterol(Ch),heptafluorobutyric anhydride(HFBA,F)and 4-carboxyphenylboronic acid(PBA))functional ligands into PEI 1.8k and subsequently crosslinking with each other via disulfide bond links,reductive-responsive PEI-X-SS-X-PEI copolymers were constructed to enhance the cellular transfection via the synergistic effect of the high affinity of Ch,F and PBA to cell membranes and the disulfide reduction triggered intracellular disassembly of micelles and subsequent siRNA release.Extraordinarily,ternary Ch-SS-F-SS-PBA micelles exhibited the strongest siRNA transfection efficiencies in in vitro cell experiments and in vivo animal experiments due to the coordination of enhanced serum stability,promoted cell uptake and endosomal escape,and cell targeting ability.This strategy of constructed multifunctional polymer here we called"building-block crosslinking"showed a simple and smart way to synthesize new materials.Also this strategy of constructing ligands-directed reduction-sensitive micelles improves the transfection efficiency of LMW PEI and provides a valuable insight to develop novel gene delivery systems.展开更多
文摘Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent HDFs showed several molecular and cytological changes such as large flat morphology, expression of senescence-associated β-galactosidase (SA β-gal) activity and altered gene expression. Small interfering RNA (siRNA) has been demonstrated to be a potential research tool to analyse gene function and pathway. Expression of an appropriate housekeeping or reference gene can be used as a measurement of transfection efficiency in siRNA. Therefore this study was designed to determine the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model. GAPDH knockdown with an appropriate transfection reagent was measured by quantitative real time RT-PCR while cellular senescence was characterized based on morphological changes, expression of SA β-gal and p16INK4a expression levels. Our findings showed that GAPDH knockdown represents silencing efficiency and down regulation of p16INK4a in senescent transfected HDFs caused morphological alterations which results in the formation of spindle shaped fibroblasts. This study demonstrated the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model.
基金This work was supported by the following funds： National Natural Science Foundation of China （No.30670951） Guangdong Provincial+5 种基金 Natural Science Foundation （No.06021322） Fund of Guangzhou Municipal Scientific Problem-Solving Program （No. 2003 Z 3-E0381） Fund of Guangdong Provincial Scientific Problem-Solving Program （No.2005 B31211002） Guangdong Provincial Government and Ministry of Education Project com- bining project initiation, study and research （No.2009B090300277）.
文摘OBJECTIVE To investigate the effect of polyethylene imine glycol （PEI-PEG）/siRNA nanocomposites in the in vitro transfection of human gastric cancer SGC7901 cell lines and the down-regulation of gene expression of the adherence factor CD44v6. METHODS PEI-PEG/siRNA nanoparticles, in different N/P ratios, were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the control group. The transfection efficiencies were observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay （mononuclear cell direct cytotoxicity assay）, and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. Based on the different N/P ratios, PEI-PEG/siRNA composites were synthesized and transfected into gastric cancer cells. Lipo2000/siRNA was used in the controls. The transfection efficiency was observed under fluorescence microscope. The cytotoxicity of the nanoparticles was measured using the MTT assay and the down-regulation effect of siRNA on CD44v6 gene was evaluated by Western blot. RESULTS After transfection, the transfection efficiency of the PEI-PEG/siRNA nanocomposites increased incrementally in N/P ratio value. The transfection efficiency improved with an increase in N/P ratio. When the N/P value was 15, fluorescence became more intense in the PEI-PEG/siRNA group than in the Lipo2000/siRNA group. At the same time, cell viability was （80.4 ± 5.6）% in the MTT reduction assay, which was similar to that in the Lipo2000/siRNA group. The results of Western blot analysis showed that the expression level of CD44v6 protein decreased to （59.7 ± 3.0）% after siRNA-CD44v6 was inhibited. CONCLUSION PEI-PEG could effectively form the nanocomposite in combination with siRNA, be transfected into the SGC7901 gastric cancer cell lines and inhibit CD44v6 protein expression. Moreover, as a genetic carrier, PEI-PEG copolymer has greater advantages, including high transfection e. ciency, less cytotoxicity and an easily alterable vector structure.
文摘目的比较三种转染试剂对大鼠施万细胞(Schwann cell,SC)mi RNA的转染效率和细胞毒性,为更好地研究SC创造条件。方法以大鼠SC株(RSC96)为研究对象,采用Lipofectamine 2000、Fu GENE6 Transfection Reagent和Super Fectin II Reagent分别转染绿色荧光素FAM标记的mi RNA阴性对照产品,比较和分析三者的转染效率以找出转染效率最高的转染试剂,并以CCK-8法细胞活性检测和TUNEL凋亡染色对这三种转染试剂的细胞毒性进行观察。结果 Fu GENE6 Transfection Reagent不适合RSC96转染mi RNA,lipofectamine 2000转染效率偏低,Super Fectin II Reagent转染效率能够满足实验要求,但其细胞毒性较大,应注意换液。结论 Super Fectin II Reagent可用于RSC96转染mi RNA,转染后宜进行换液以减少毒性。
文摘A biodegradable gene transfer vector, poly(ethylenimine)-grafted-poly[(aspartic acid)-co-lysine] has been developed by thermal polycondensation of aspartic acid and lysine, and branch poly(ethylenimine) (Mw less than 600) was grafted to the backbone. The polymer was characterized by 1H NMR. It appeared lower cytotoxity compared to poly(ethylenimine) (25KDa), which was quantified by MTT assay. Electrophoresis indicated that the polymer could retardate DNA at N/P ratio 1.2-1.8 (w/w). Transfection efficiency of the complexes was studied in NT2 cell lines. It was 1.5 fold higher than molecular weight PEI (Mw = 25KDa).
基金supported by the National Natural Science Foundation of China(81903556)the Natural Science Fund for Colleges and Universities in Jiangsu Province(19KJB350004)supported by the National Health and Medical Research Council(NHMRC)Early Career Fellowship(1112258)of Australia。
文摘Incorporating functional ligands and biodegradable bonds into biocompatible low-molecular-weight(LMW)polymers,such as 1.8 kDa poly(ethylenimine)(PEI1.8 k),is a common strategy to improve the properties of LMW polymers including biosafety and delivery efficacy.This study demonstrates the hypothesis that introducing different functional ligands and linked reductive disulfides in PEI 1.8k will achieve superior siRNA transfection efficiency.By incorporating PEI-X(X represents cholesterol(Ch),heptafluorobutyric anhydride(HFBA,F)and 4-carboxyphenylboronic acid(PBA))functional ligands into PEI 1.8k and subsequently crosslinking with each other via disulfide bond links,reductive-responsive PEI-X-SS-X-PEI copolymers were constructed to enhance the cellular transfection via the synergistic effect of the high affinity of Ch,F and PBA to cell membranes and the disulfide reduction triggered intracellular disassembly of micelles and subsequent siRNA release.Extraordinarily,ternary Ch-SS-F-SS-PBA micelles exhibited the strongest siRNA transfection efficiencies in in vitro cell experiments and in vivo animal experiments due to the coordination of enhanced serum stability,promoted cell uptake and endosomal escape,and cell targeting ability.This strategy of constructed multifunctional polymer here we called"building-block crosslinking"showed a simple and smart way to synthesize new materials.Also this strategy of constructing ligands-directed reduction-sensitive micelles improves the transfection efficiency of LMW PEI and provides a valuable insight to develop novel gene delivery systems.
