Metallothionein gene (MT) has been transferred into mushroom protoplasts by electroporation. It is a low molecular weight, cysteine-rich and metal-binding protein. MT can bind metals. Its synthesis is induced by Zn io...Metallothionein gene (MT) has been transferred into mushroom protoplasts by electroporation. It is a low molecular weight, cysteine-rich and metal-binding protein. MT can bind metals. Its synthesis is induced by Zn ion. Thus the expression of MT gene in mushroom can improve the accumulation of Zn in this fungus. This transgenic mushroom, consumed as a kind of vegetable, can supply the necessary Zn to people who are short of the element. When protoplasts were prepared, the concentration (C) of protoplasts is 6.745 x 10(6) /mL. After protoplast electroporation, the transformation rate of protoplasts is 0.01 %. Polymerase chain reaction (PCR) analysis showed that the gene had been integrated into the mushroom chromosome, SDS-PAGE, Western blot analysis indicated that the MT gene had been expressed in the transgenic mushroom. The expressing level, detected by ELISA, is 0.6 % - 0.8 %. Tested for metal resistance, the wild-type mushroom growth was inhibited on die medium containing 1.0 - 1.2 mmoL/L ZnSO4. While the transgenic mushroom was inhibited on the medium containing 1. 5 - 2.0 mmol/L ZnSO4. The mycelium can develop into hymenophore in the medium of rice bran: sawdust = 1: 3, and not in the medium of rice bran: sawdust = 1: 4.展开更多
AIM:To investigate the inhibitory and anti-metastatic effect of mutant p27 gene(p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism.METHODS:Inhibitory effect of p27mt gene on...AIM:To investigate the inhibitory and anti-metastatic effect of mutant p27 gene(p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism.METHODS:Inhibitory effect of p27mt gene on the growth of colorectal cancer xenografts was determined by measurement of tumor size before and after direct intratumoral injection of Ad-p27mt in a preestablished transplantation model of human colorectal cancer in nude mice.Cell cycle and apoptosis were detected by flow cytometry performed on single-cell suspension from an isolated tumor.Expression of MMP-9 in tumor tissue was detected by immunohistochemistry.RESULTS:The average sizes of transplantation tumors were 1.94 ± 0.67 cm3,2.75 ± 0.83 cm3 and 3.01 ± 0.76 cm3 in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.05).The average proliferation rates were 37.34% ± 1.45%,53.16% ± 3.27% and 54.48% ± 2.43%,in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.05).The average apoptosis rates were 19.79% ± 3.32%,6.38% ± 4.91% and 7.25% ± 5.20% in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.01).The average MMP-9 expression rates were 20%,75% and 66.7% in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.01).CONCLUSION:p27mt inhibits the growth of transplanted tumor by blocking the proliferation of cancer xenografts and by promoting apoptosis of transplantated tumor cells,as well as decrease transpl-anted tumor metastasis.展开更多
目的:探讨突变型p27基因(p27mutant type gene;p27mt)对恶性黑素瘤A375细胞凋亡的调节作用。方法:利用重组腺病毒Ad-p27mt转染培养的人恶性黑素瘤A375细胞,用3H-TdR掺入法检测细胞增殖;流式细胞术、DNA片段分析法、TUNEL法检测细胞凋亡...目的:探讨突变型p27基因(p27mutant type gene;p27mt)对恶性黑素瘤A375细胞凋亡的调节作用。方法:利用重组腺病毒Ad-p27mt转染培养的人恶性黑素瘤A375细胞,用3H-TdR掺入法检测细胞增殖;流式细胞术、DNA片段分析法、TUNEL法检测细胞凋亡。结果:重组腺病毒Ad-p27mt在MOI≥50时,可达到100%的转导效率。Ad-p27mt转染恶性黑素瘤细胞A375后,3H-TdR掺入法检测发现细胞增殖抑制;流式细胞术检测在G1期前出现亚二倍体凋亡峰;细胞DNA抽提电泳后发现凋亡特征性梯带。TUNEL法检测凋亡指数分别为48.5±3.6(Ad-p27mt组)及4.2±0.8(空白对照组),差异有显著性(P<0.01)。结论:重组腺病毒介导的p27mt基因转移可诱导恶性黑素瘤A375细胞在Gl期阻滞并导致细胞凋亡。展开更多
文摘Metallothionein gene (MT) has been transferred into mushroom protoplasts by electroporation. It is a low molecular weight, cysteine-rich and metal-binding protein. MT can bind metals. Its synthesis is induced by Zn ion. Thus the expression of MT gene in mushroom can improve the accumulation of Zn in this fungus. This transgenic mushroom, consumed as a kind of vegetable, can supply the necessary Zn to people who are short of the element. When protoplasts were prepared, the concentration (C) of protoplasts is 6.745 x 10(6) /mL. After protoplast electroporation, the transformation rate of protoplasts is 0.01 %. Polymerase chain reaction (PCR) analysis showed that the gene had been integrated into the mushroom chromosome, SDS-PAGE, Western blot analysis indicated that the MT gene had been expressed in the transgenic mushroom. The expressing level, detected by ELISA, is 0.6 % - 0.8 %. Tested for metal resistance, the wild-type mushroom growth was inhibited on die medium containing 1.0 - 1.2 mmoL/L ZnSO4. While the transgenic mushroom was inhibited on the medium containing 1. 5 - 2.0 mmol/L ZnSO4. The mycelium can develop into hymenophore in the medium of rice bran: sawdust = 1: 3, and not in the medium of rice bran: sawdust = 1: 4.
基金Supported by The Natural Science Foundation of Hubei Province,No.2003ABA193Bureau of Science and Technology of Shiyan City,No.2005ZD036
文摘AIM:To investigate the inhibitory and anti-metastatic effect of mutant p27 gene(p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism.METHODS:Inhibitory effect of p27mt gene on the growth of colorectal cancer xenografts was determined by measurement of tumor size before and after direct intratumoral injection of Ad-p27mt in a preestablished transplantation model of human colorectal cancer in nude mice.Cell cycle and apoptosis were detected by flow cytometry performed on single-cell suspension from an isolated tumor.Expression of MMP-9 in tumor tissue was detected by immunohistochemistry.RESULTS:The average sizes of transplantation tumors were 1.94 ± 0.67 cm3,2.75 ± 0.83 cm3 and 3.01 ± 0.76 cm3 in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.05).The average proliferation rates were 37.34% ± 1.45%,53.16% ± 3.27% and 54.48% ± 2.43%,in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.05).The average apoptosis rates were 19.79% ± 3.32%,6.38% ± 4.91% and 7.25% ± 5.20% in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.01).The average MMP-9 expression rates were 20%,75% and 66.7% in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.01).CONCLUSION:p27mt inhibits the growth of transplanted tumor by blocking the proliferation of cancer xenografts and by promoting apoptosis of transplantated tumor cells,as well as decrease transpl-anted tumor metastasis.
文摘目的:探讨突变型p27基因(p27mutant type gene;p27mt)对恶性黑素瘤A375细胞凋亡的调节作用。方法:利用重组腺病毒Ad-p27mt转染培养的人恶性黑素瘤A375细胞,用3H-TdR掺入法检测细胞增殖;流式细胞术、DNA片段分析法、TUNEL法检测细胞凋亡。结果:重组腺病毒Ad-p27mt在MOI≥50时,可达到100%的转导效率。Ad-p27mt转染恶性黑素瘤细胞A375后,3H-TdR掺入法检测发现细胞增殖抑制;流式细胞术检测在G1期前出现亚二倍体凋亡峰;细胞DNA抽提电泳后发现凋亡特征性梯带。TUNEL法检测凋亡指数分别为48.5±3.6(Ad-p27mt组)及4.2±0.8(空白对照组),差异有显著性(P<0.01)。结论:重组腺病毒介导的p27mt基因转移可诱导恶性黑素瘤A375细胞在Gl期阻滞并导致细胞凋亡。
