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
[Objectives]This study aimed to optimize the siRNA transfection method for ARID4B gene and lay a foundation for further study on the effect of ARID4B low expression on the biological function of liver cancer cells Hep...[Objectives]This study aimed to optimize the siRNA transfection method for ARID4B gene and lay a foundation for further study on the effect of ARID4B low expression on the biological function of liver cancer cells HepG2.[Methods]HepG2 cells were cultured for 0,4 and 24 h,respectively and then transfected with ARID4B targeting siRNA.The mRNA expression of ARID4B gene was detected by RT-qPCR,and the protein expression was detected by Western blot.[Results]When HepG2 cells were transfected at 0 h after seeding,the expression of ARID4B was the lowest in both mRNA and protein levels,that is,the inhibition effect was the best.[Conclusions]Transfection at different time points after seeding of HepG2 cells had different inhibition effect on the expression of ARID4B.Transfection at 0 h after seeding showed the best inhibition effect.展开更多
【Objective】To assess the impact of siRNA transfection on the tumor development under experimental pathology of thyroid gland.【Methods】Experiments were performed on rats weighing(180±20)g,which were divided in...【Objective】To assess the impact of siRNA transfection on the tumor development under experimental pathology of thyroid gland.【Methods】Experiments were performed on rats weighing(180±20)g,which were divided into five groups:IA and IB-animals with simulated hypo and hyperthyroid states and transplanted Guérin's carcinoma;ⅡA andⅡB-animals with simulated hypo and hyperthyroid states and transplanted Guérin's carcinoma in combination with siRNA transfection;Ⅲ-control group with transplanted Guérin's carcinoma in combination with siRNA transfection.Orthogonal dimensions of the tumor was measured.Histological and immunohistochemical researches of tumor samples were performed.【Results】It has been shown that the inhibitory effect of short interfering RNA realized in a greater extent in hypothyroid state,indicating an important role of thyroid hormones in the regulation of expression of genes that control the cell cycle.【Conclusion】siRNA transfection leads to inhibition of tumor growth in animals with both hypo and hyperthyroidism,but this process is more obvious in hypothyroidism status.展开更多
Successful gene silencing by small interfering RNA (siRNA) requires efficient uptake of siRNA into targeted cells. For in vitro transfection of siRNA using cationic liposomes, two types of transfection method are curr...Successful gene silencing by small interfering RNA (siRNA) requires efficient uptake of siRNA into targeted cells. For in vitro transfection of siRNA using cationic liposomes, two types of transfection method are currently being used: conventional (forward;Fw) and reverse (Rev) transfections. Here, to investigate an efficient siRNA transfection method using cationic liposomes, we compared the transfection efficiency of siRNA between Fw-transfection and Rev-transfection methods with various types of cationic liposomes. In Fw-transfection, siRNA/cationic liposomes complex (siRNA lipoplexes) was added to pre-plated cells. In contrast, Rev-transfection was performed by co-incubation of cells with siRNA lipoplexes in suspension. As a result, Rev-transfection with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based or cationic cholesterol derivative-based liposomes could deliver siRNA into the cells via efficient cellular association, and induce an improved gene silencing effect by siRNA compared with Fw-transfection. Furthermore, Rev-transfection did not show increased cytotoxicity compared with Fw-transfection. These findings suggested that Rev-transfection in suspension has better potential for efficient transfection of siRNA into cells with minimal toxicity.展开更多
目的:研究磷脂酶C-ε(Phospholipase C epsilon,PLCε)siRNA表达质粒的转染对人膀胱癌细胞系T24侵袭转移能力的影响。方法:分别将携有PLCεsiRNA基因的真核表达质粒两对p11-PLCε和p12-PLCε载体体外转染T24细胞,筛选稳定转染的细胞并...目的:研究磷脂酶C-ε(Phospholipase C epsilon,PLCε)siRNA表达质粒的转染对人膀胱癌细胞系T24侵袭转移能力的影响。方法:分别将携有PLCεsiRNA基因的真核表达质粒两对p11-PLCε和p12-PLCε载体体外转染T24细胞,筛选稳定转染的细胞并扩增培养,用逆转录聚合酶链反应法(RT-PCR)检测PLCεmRNA的表达情况,应用Transwell小室侵袭试验、明胶酶谱分析分别研究转染前、后膀胱癌细胞侵袭转移能力的变化,并以空载质粒HK-A转染和未转染细胞为对照。结果:RT-PCR检测显示pll-PLCε,p12-PLCε转染成功后T24细胞PLCεmRNA表达明显比空质粒HK-A转染和未转染细胞减弱;p12-PLCεsiRNA转染细胞穿透侵袭小室滤膜的数目(26.8±5.8)和pll-PLCεsiRNA转染细胞穿透侵袭小室滤膜的数目(25.8±6.2)明显少于未转染细胞(34.8±6.9)和HK-A转染细胞(33.8±5.7)(P<0.01);明胶酶谱分析显示转染pll-PLCε、p12-PLCε均使细胞分泌MMP-2、MMP-9明显低于未转染细胞和HK-A转染细胞。结论:推测转染外源性PLCεsiRNA基因能下调PLCε基因的表达并能抑制膀胱癌细胞侵袭转移。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Scientific Research Foundation of Chengde Medical University for High-level Talents(10204030004).
文摘[Objectives]This study aimed to optimize the siRNA transfection method for ARID4B gene and lay a foundation for further study on the effect of ARID4B low expression on the biological function of liver cancer cells HepG2.[Methods]HepG2 cells were cultured for 0,4 and 24 h,respectively and then transfected with ARID4B targeting siRNA.The mRNA expression of ARID4B gene was detected by RT-qPCR,and the protein expression was detected by Western blot.[Results]When HepG2 cells were transfected at 0 h after seeding,the expression of ARID4B was the lowest in both mRNA and protein levels,that is,the inhibition effect was the best.[Conclusions]Transfection at different time points after seeding of HepG2 cells had different inhibition effect on the expression of ARID4B.Transfection at 0 h after seeding showed the best inhibition effect.
文摘【Objective】To assess the impact of siRNA transfection on the tumor development under experimental pathology of thyroid gland.【Methods】Experiments were performed on rats weighing(180±20)g,which were divided into five groups:IA and IB-animals with simulated hypo and hyperthyroid states and transplanted Guérin's carcinoma;ⅡA andⅡB-animals with simulated hypo and hyperthyroid states and transplanted Guérin's carcinoma in combination with siRNA transfection;Ⅲ-control group with transplanted Guérin's carcinoma in combination with siRNA transfection.Orthogonal dimensions of the tumor was measured.Histological and immunohistochemical researches of tumor samples were performed.【Results】It has been shown that the inhibitory effect of short interfering RNA realized in a greater extent in hypothyroid state,indicating an important role of thyroid hormones in the regulation of expression of genes that control the cell cycle.【Conclusion】siRNA transfection leads to inhibition of tumor growth in animals with both hypo and hyperthyroidism,but this process is more obvious in hypothyroidism status.
文摘Successful gene silencing by small interfering RNA (siRNA) requires efficient uptake of siRNA into targeted cells. For in vitro transfection of siRNA using cationic liposomes, two types of transfection method are currently being used: conventional (forward;Fw) and reverse (Rev) transfections. Here, to investigate an efficient siRNA transfection method using cationic liposomes, we compared the transfection efficiency of siRNA between Fw-transfection and Rev-transfection methods with various types of cationic liposomes. In Fw-transfection, siRNA/cationic liposomes complex (siRNA lipoplexes) was added to pre-plated cells. In contrast, Rev-transfection was performed by co-incubation of cells with siRNA lipoplexes in suspension. As a result, Rev-transfection with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based or cationic cholesterol derivative-based liposomes could deliver siRNA into the cells via efficient cellular association, and induce an improved gene silencing effect by siRNA compared with Fw-transfection. Furthermore, Rev-transfection did not show increased cytotoxicity compared with Fw-transfection. These findings suggested that Rev-transfection in suspension has better potential for efficient transfection of siRNA into cells with minimal toxicity.
文摘目的:研究磷脂酶C-ε(Phospholipase C epsilon,PLCε)siRNA表达质粒的转染对人膀胱癌细胞系T24侵袭转移能力的影响。方法:分别将携有PLCεsiRNA基因的真核表达质粒两对p11-PLCε和p12-PLCε载体体外转染T24细胞,筛选稳定转染的细胞并扩增培养,用逆转录聚合酶链反应法(RT-PCR)检测PLCεmRNA的表达情况,应用Transwell小室侵袭试验、明胶酶谱分析分别研究转染前、后膀胱癌细胞侵袭转移能力的变化,并以空载质粒HK-A转染和未转染细胞为对照。结果:RT-PCR检测显示pll-PLCε,p12-PLCε转染成功后T24细胞PLCεmRNA表达明显比空质粒HK-A转染和未转染细胞减弱;p12-PLCεsiRNA转染细胞穿透侵袭小室滤膜的数目(26.8±5.8)和pll-PLCεsiRNA转染细胞穿透侵袭小室滤膜的数目(25.8±6.2)明显少于未转染细胞(34.8±6.9)和HK-A转染细胞(33.8±5.7)(P<0.01);明胶酶谱分析显示转染pll-PLCε、p12-PLCε均使细胞分泌MMP-2、MMP-9明显低于未转染细胞和HK-A转染细胞。结论:推测转染外源性PLCεsiRNA基因能下调PLCε基因的表达并能抑制膀胱癌细胞侵袭转移。
