To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly ...To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.展开更多
Objective:To explore the suppressing effect ofγ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.Methods:The SHG-44 cell was treated by DAPT with different concent...Objective:To explore the suppressing effect ofγ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.Methods:The SHG-44 cell was treated by DAPT with different concentration.The proliferation of cells was detected by MTT assay;cell cycle and TSC of CD133^+were determined by flow cytometry analysis technique;the key factor in Notch signaling pathway(Notch-1,Delta-1,Hes-1)was measured by reverse transcrip tase-polymerase chain reaction and western blotting.Results:DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05).And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner.DAPT increased the rate of cells in G_0/G_1 phase of SHG-44 cells,while it decreased the rate of cells in S phase.TSC of CD133^+was significantly reduced after DAPT treated SHC-44 cells.The expression of protein and mRNA of Notch-1,Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.Conclusions:DAPT can downregulate these key factor in Notch signaling pathway,reduce the TSC of CD133+and inhibit the proliferation of SHC-44 cells.展开更多
The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the go...The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.展开更多
BACKGROUND: Progenitors of the immortalized human glioma cell line, SHG-44, are significantly less sensitive to irradiation. Two hypotheses regarding the mechanism of this effect exist: several studies have suggeste...BACKGROUND: Progenitors of the immortalized human glioma cell line, SHG-44, are significantly less sensitive to irradiation. Two hypotheses regarding the mechanism of this effect exist: several studies have suggested that there is a subgroup with different radiosensitivities in identical cell group, and the progenitors of irradiate is a adaptive response subgroup, so its radiosensitivity is descend. A second hypothesis suggests that irradiated glioma progeny have a stronger ability to repair DNA damage. This would suggest that when progeny are continuously irradiated, resistance to irradiation-induced DNA increases, and radiosensitivity decreases. OBJECTIVE: To investigate radiosensitivity and growth features after irradiation to progeny of the human glioma cell line SHG-44. DESIGN, TIME AND SETTING: A randomized, controlled experiment, which was performed at the Department of Radiology Laboratory, the First Hospital Affiliated to Soochow University, between September 2004 and January 2006. MATERIALS: The glioma cell line SHG-44 was provided by the Institute of Neuroscience, First Affiliated Hospital of Suzhou University. Propidium iodide reagent was provided by Coulter Corporation. A linear accelerator, KD-2 type, was provided by Siemens, Germany. The flow cytometer EPICS-XL was provided by Coulter Corporation. METHODS: Brain glioma SHG-44 cells were divided into four groups: SHG-44, SHG-44-2, SHG-44-6, and SHG-44-10 . The SHG-44-2, SHG-44-6, and SHG-44-10 cells were vertically irradiated with varying doses of 2, 6 and 10 Gy by a linear accelerator (6 MVX). The cells were passaged for 15 generations and cultured in RPMI-1640 culture media. MAIN OUTCOME MEASURES: Community re-double time, mean lethal dose (D0), extrapolation number (N), fraction surviving fraction irradiated by 2 Gy dose (SF2), quasi-threshold dose (Dq), and cell cycle. RESULTS: The Population doubling time (PDT) of SHG-44-2, SHG-44-6, and SHG-44-10 cell groups was not significant (P = 0.052). Compared to these three groups, the PDT of the SHG-44 cell group was significantly difference (F = 7.878, P 〈 0.002). SHG-44 cell clone ratewas 26.5%, and SHG-44-10 cell group was 15.5%. The SHG-44-10 cell group also exhibited radiosensitivity, but was less than the radiosensitivity of the SHG-44 cell group. Compared to the SHG-44 cell group, the ratio of the G2/M phase was decreased in the SHG-44-10 cell group, and the radio of S phase was increased. The SHG-44 and SHG-44-10 cell groups were irradiated with 8 Gy. After 12 hours, the G2/M ratio was compared to pre-irradiation times, indicating a significantly higher ratio in the pre-irradiated groups (P 〈 0.01). The cells between S HG-44 and SHG-44-10 groups were harvested 12 hours after irradiation: G2 phase of SHG-44-10 cells was arrested and the G2/M ratio was increased, which was intensified with increasing irradiation doses. CONCLUSION: In the present study, the proliferation delay and decreased radiosensitivity were confirmed in progeny of irradiated human glioma cells, and radiosensitivity was dose-dependent.展开更多
目的构建人MCHR2真核表达载体,转染CHO细胞,建立稳定转染的CHO细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR2基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和测序鉴定后,用脂...目的构建人MCHR2真核表达载体,转染CHO细胞,建立稳定转染的CHO细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR2基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和测序鉴定后,用脂质体转染法转染CHO细胞,通过G418筛选,建立稳定转染的CHO细胞系,用RT-PCR、W estern b lot及免疫荧光法检测MCHR2的表达。结果成功构建了pcDNA3.1(+)/MCHR2真核表达载体,并建立了稳定转染的CHO细胞系,成功地表达目的基因。结论真核表达载体成功构建和稳定转染CHO细胞系的建立为进一步研究MCHR2的功能奠定良好的实验基础。展开更多
目的:建立一种新的可动态观察肿瘤细胞生长的大鼠神经胶质瘤原位移植瘤模型。方法:利用脂质体将含有强化绿色荧光蛋白(enhanced green fluorescence protein,EGFP)基因的真核表达质粒pEGFP-N1转染大鼠神经胶质瘤C6细胞,经G418筛选及亚克...目的:建立一种新的可动态观察肿瘤细胞生长的大鼠神经胶质瘤原位移植瘤模型。方法:利用脂质体将含有强化绿色荧光蛋白(enhanced green fluorescence protein,EGFP)基因的真核表达质粒pEGFP-N1转染大鼠神经胶质瘤C6细胞,经G418筛选及亚克隆,获取稳定表达EGFP的细胞系C6-gfp。采用CCK-8试剂盒检测C6及C6-gfp细胞增殖情况;将C6-gfp细胞接种于Wistar大鼠颅内,通过B超、大体标本、荧光体视镜、H-E染色和免疫组化观察颅内成瘤情况及瘤组织中绿色荧光蛋白的表达。结果:流式细胞术分析体外培养的C6-gfp细胞97.7%产生绿色荧光;C6-gfp细胞与亲代C6细胞相比,生长曲线无明显不同(P>0.05)。C6-gfp接种于颅内3周后成瘤率达70%;利用B超可动态观察肿瘤生长,利用荧光体视镜可见肿瘤组织发出绿色荧光,可与周围正常组织区别。H-E染色可见肿瘤细胞核大,染色深,核分裂明显,瘤内有很多新生血管。免疫组化可见GFP阳性细胞染色深浅不一。结论:成功构建了能稳定高水平表达EGFP的大鼠神经胶质瘤C6-gfp细胞株,其生物学行为未发生改变,能在同系大鼠颅内成瘤。该模型为神经胶质瘤防治研究提供了良好的基础。展开更多
目的建立稳定高效表达正反义A lu-Sx的细胞系。方法根据A lu亚家族Sx序列合成两对两端带有限制性酶切位点的引物,提取HEK293细胞的总DNA后PCR扩增,产物连接到真核表达载体pcDNA3.1/myc-H is A中构建成重组体。经酶切及测序鉴定后,阳离...目的建立稳定高效表达正反义A lu-Sx的细胞系。方法根据A lu亚家族Sx序列合成两对两端带有限制性酶切位点的引物,提取HEK293细胞的总DNA后PCR扩增,产物连接到真核表达载体pcDNA3.1/myc-H is A中构建成重组体。经酶切及测序鉴定后,阳离子脂质体转染法转染HEK293细胞后G418筛选稳定表达的细胞克隆,Northern杂交检测并挑选出表达最强的亚克隆。结果成功构建A lu亚家族Sx的正反义真核表达载体并获得高效稳定表达的HEK293细胞亚克隆。结论高效稳定表达正反义A lu Sx的HEK293细胞亚克隆可用于下一步研究。展开更多
文摘To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.
基金supported by Shaanxi Province Health Department Key Funds(sx201227273)
文摘Objective:To explore the suppressing effect ofγ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.Methods:The SHG-44 cell was treated by DAPT with different concentration.The proliferation of cells was detected by MTT assay;cell cycle and TSC of CD133^+were determined by flow cytometry analysis technique;the key factor in Notch signaling pathway(Notch-1,Delta-1,Hes-1)was measured by reverse transcrip tase-polymerase chain reaction and western blotting.Results:DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05).And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner.DAPT increased the rate of cells in G_0/G_1 phase of SHG-44 cells,while it decreased the rate of cells in S phase.TSC of CD133^+was significantly reduced after DAPT treated SHC-44 cells.The expression of protein and mRNA of Notch-1,Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.Conclusions:DAPT can downregulate these key factor in Notch signaling pathway,reduce the TSC of CD133+and inhibit the proliferation of SHC-44 cells.
基金Supported by Key Teachers Foundation of Education Office of Heilongjiang Province, 2005 (1055G005)
文摘The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.
文摘BACKGROUND: Progenitors of the immortalized human glioma cell line, SHG-44, are significantly less sensitive to irradiation. Two hypotheses regarding the mechanism of this effect exist: several studies have suggested that there is a subgroup with different radiosensitivities in identical cell group, and the progenitors of irradiate is a adaptive response subgroup, so its radiosensitivity is descend. A second hypothesis suggests that irradiated glioma progeny have a stronger ability to repair DNA damage. This would suggest that when progeny are continuously irradiated, resistance to irradiation-induced DNA increases, and radiosensitivity decreases. OBJECTIVE: To investigate radiosensitivity and growth features after irradiation to progeny of the human glioma cell line SHG-44. DESIGN, TIME AND SETTING: A randomized, controlled experiment, which was performed at the Department of Radiology Laboratory, the First Hospital Affiliated to Soochow University, between September 2004 and January 2006. MATERIALS: The glioma cell line SHG-44 was provided by the Institute of Neuroscience, First Affiliated Hospital of Suzhou University. Propidium iodide reagent was provided by Coulter Corporation. A linear accelerator, KD-2 type, was provided by Siemens, Germany. The flow cytometer EPICS-XL was provided by Coulter Corporation. METHODS: Brain glioma SHG-44 cells were divided into four groups: SHG-44, SHG-44-2, SHG-44-6, and SHG-44-10 . The SHG-44-2, SHG-44-6, and SHG-44-10 cells were vertically irradiated with varying doses of 2, 6 and 10 Gy by a linear accelerator (6 MVX). The cells were passaged for 15 generations and cultured in RPMI-1640 culture media. MAIN OUTCOME MEASURES: Community re-double time, mean lethal dose (D0), extrapolation number (N), fraction surviving fraction irradiated by 2 Gy dose (SF2), quasi-threshold dose (Dq), and cell cycle. RESULTS: The Population doubling time (PDT) of SHG-44-2, SHG-44-6, and SHG-44-10 cell groups was not significant (P = 0.052). Compared to these three groups, the PDT of the SHG-44 cell group was significantly difference (F = 7.878, P 〈 0.002). SHG-44 cell clone ratewas 26.5%, and SHG-44-10 cell group was 15.5%. The SHG-44-10 cell group also exhibited radiosensitivity, but was less than the radiosensitivity of the SHG-44 cell group. Compared to the SHG-44 cell group, the ratio of the G2/M phase was decreased in the SHG-44-10 cell group, and the radio of S phase was increased. The SHG-44 and SHG-44-10 cell groups were irradiated with 8 Gy. After 12 hours, the G2/M ratio was compared to pre-irradiation times, indicating a significantly higher ratio in the pre-irradiated groups (P 〈 0.01). The cells between S HG-44 and SHG-44-10 groups were harvested 12 hours after irradiation: G2 phase of SHG-44-10 cells was arrested and the G2/M ratio was increased, which was intensified with increasing irradiation doses. CONCLUSION: In the present study, the proliferation delay and decreased radiosensitivity were confirmed in progeny of irradiated human glioma cells, and radiosensitivity was dose-dependent.
文摘目的构建人MCHR2真核表达载体,转染CHO细胞,建立稳定转染的CHO细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR2基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和测序鉴定后,用脂质体转染法转染CHO细胞,通过G418筛选,建立稳定转染的CHO细胞系,用RT-PCR、W estern b lot及免疫荧光法检测MCHR2的表达。结果成功构建了pcDNA3.1(+)/MCHR2真核表达载体,并建立了稳定转染的CHO细胞系,成功地表达目的基因。结论真核表达载体成功构建和稳定转染CHO细胞系的建立为进一步研究MCHR2的功能奠定良好的实验基础。
文摘目的:建立一种新的可动态观察肿瘤细胞生长的大鼠神经胶质瘤原位移植瘤模型。方法:利用脂质体将含有强化绿色荧光蛋白(enhanced green fluorescence protein,EGFP)基因的真核表达质粒pEGFP-N1转染大鼠神经胶质瘤C6细胞,经G418筛选及亚克隆,获取稳定表达EGFP的细胞系C6-gfp。采用CCK-8试剂盒检测C6及C6-gfp细胞增殖情况;将C6-gfp细胞接种于Wistar大鼠颅内,通过B超、大体标本、荧光体视镜、H-E染色和免疫组化观察颅内成瘤情况及瘤组织中绿色荧光蛋白的表达。结果:流式细胞术分析体外培养的C6-gfp细胞97.7%产生绿色荧光;C6-gfp细胞与亲代C6细胞相比,生长曲线无明显不同(P>0.05)。C6-gfp接种于颅内3周后成瘤率达70%;利用B超可动态观察肿瘤生长,利用荧光体视镜可见肿瘤组织发出绿色荧光,可与周围正常组织区别。H-E染色可见肿瘤细胞核大,染色深,核分裂明显,瘤内有很多新生血管。免疫组化可见GFP阳性细胞染色深浅不一。结论:成功构建了能稳定高水平表达EGFP的大鼠神经胶质瘤C6-gfp细胞株,其生物学行为未发生改变,能在同系大鼠颅内成瘤。该模型为神经胶质瘤防治研究提供了良好的基础。
文摘目的建立稳定高效表达正反义A lu-Sx的细胞系。方法根据A lu亚家族Sx序列合成两对两端带有限制性酶切位点的引物,提取HEK293细胞的总DNA后PCR扩增,产物连接到真核表达载体pcDNA3.1/myc-H is A中构建成重组体。经酶切及测序鉴定后,阳离子脂质体转染法转染HEK293细胞后G418筛选稳定表达的细胞克隆,Northern杂交检测并挑选出表达最强的亚克隆。结果成功构建A lu亚家族Sx的正反义真核表达载体并获得高效稳定表达的HEK293细胞亚克隆。结论高效稳定表达正反义A lu Sx的HEK293细胞亚克隆可用于下一步研究。