目的:观察PTEN(phosphatase and tensin hom o logy deleted on chrom osom e ten,第10号染色体上磷酸酶和张力蛋白同源缺失的基因)对人乳腺癌细胞ZR-75-1细胞增殖和细胞周期的影响。方法:利用脂质体介导法将携带有野生型和突变型PTEN c...目的:观察PTEN(phosphatase and tensin hom o logy deleted on chrom osom e ten,第10号染色体上磷酸酶和张力蛋白同源缺失的基因)对人乳腺癌细胞ZR-75-1细胞增殖和细胞周期的影响。方法:利用脂质体介导法将携带有野生型和突变型PTEN cDNA的真核表达载体pBP-w t-PTEN和pBP-G 129R-PTEN导入人乳腺癌ZR-75-1细胞(质粒转染成功后实验分为C-W T-PTEN组、C-G 129R-PTEN组和未转染质粒组即对照组)后,以RT-PCR、W estern b lot分析目的基因的表达,并采用M TT法和流式细胞术检测细胞增殖和细胞周期。结果:C-W T-PTEN组、C-G 129R-PTEN组细胞PTEN mRNA及PTEN蛋白出现明显的高表达。C-W T-PTEN组细胞生长的抑制率可高达42.7%,与对照组比较,差异有显著性(P<0.05)。但C-G 129R-PTEN组细胞生长的抑制率与对照组比较,差异无显著性(P>0.05)。流式细胞术显示C-W T-PTEN组细胞周期从G1期到S期已发生抑制。结论:野生型PTEN可依赖其磷酸酶活性抑制肿瘤细胞的增殖,并最终诱导细胞凋亡。展开更多
Objective To establish a method cloning the exon 3 of eck gene from normal tissue and ZR 75 1 cell line (a human breast cancer cell line)and study whether these genes exist mutant. Methods Designed a pair of s...Objective To establish a method cloning the exon 3 of eck gene from normal tissue and ZR 75 1 cell line (a human breast cancer cell line)and study whether these genes exist mutant. Methods Designed a pair of specific primers and amplified the exon 3 of eck gene fragment from the extracted genomic DNA derived from normal epithelial cells from skin tissue and ZR 75 1 cell line respectively by PCR technique. Transformed the E.coil. JM109 with recombinant plamids constructed by inserting the amplified fragments into medium vector pUCm T and sequenced these amplified fragments after primary screening of endonuclease restriction digestion and PCR amplification. Results ① Obtained the genomic DNA of human normal epithelial cells and ZR 75 1 cell line respectively. ② Obtained the amplified fragments of human exon 3 of eck gene through PCR technique. ③ Obtained the cloning vectors of exon 3 of eck gene of human normal epithelial cells and ZR 75 1 cell line respectively. ④ ZR 75 1 cell line exists mutation of nucleotides. Conclusion Successfully established the method of cloning the human exon 3 of eck gene and found some mutations in the detected samples. This study lays a foundation for further studying the function of eck gene in tumorgenesis.展开更多
文摘目的:观察PTEN(phosphatase and tensin hom o logy deleted on chrom osom e ten,第10号染色体上磷酸酶和张力蛋白同源缺失的基因)对人乳腺癌细胞ZR-75-1细胞增殖和细胞周期的影响。方法:利用脂质体介导法将携带有野生型和突变型PTEN cDNA的真核表达载体pBP-w t-PTEN和pBP-G 129R-PTEN导入人乳腺癌ZR-75-1细胞(质粒转染成功后实验分为C-W T-PTEN组、C-G 129R-PTEN组和未转染质粒组即对照组)后,以RT-PCR、W estern b lot分析目的基因的表达,并采用M TT法和流式细胞术检测细胞增殖和细胞周期。结果:C-W T-PTEN组、C-G 129R-PTEN组细胞PTEN mRNA及PTEN蛋白出现明显的高表达。C-W T-PTEN组细胞生长的抑制率可高达42.7%,与对照组比较,差异有显著性(P<0.05)。但C-G 129R-PTEN组细胞生长的抑制率与对照组比较,差异无显著性(P>0.05)。流式细胞术显示C-W T-PTEN组细胞周期从G1期到S期已发生抑制。结论:野生型PTEN可依赖其磷酸酶活性抑制肿瘤细胞的增殖,并最终诱导细胞凋亡。
文摘Objective To establish a method cloning the exon 3 of eck gene from normal tissue and ZR 75 1 cell line (a human breast cancer cell line)and study whether these genes exist mutant. Methods Designed a pair of specific primers and amplified the exon 3 of eck gene fragment from the extracted genomic DNA derived from normal epithelial cells from skin tissue and ZR 75 1 cell line respectively by PCR technique. Transformed the E.coil. JM109 with recombinant plamids constructed by inserting the amplified fragments into medium vector pUCm T and sequenced these amplified fragments after primary screening of endonuclease restriction digestion and PCR amplification. Results ① Obtained the genomic DNA of human normal epithelial cells and ZR 75 1 cell line respectively. ② Obtained the amplified fragments of human exon 3 of eck gene through PCR technique. ③ Obtained the cloning vectors of exon 3 of eck gene of human normal epithelial cells and ZR 75 1 cell line respectively. ④ ZR 75 1 cell line exists mutation of nucleotides. Conclusion Successfully established the method of cloning the human exon 3 of eck gene and found some mutations in the detected samples. This study lays a foundation for further studying the function of eck gene in tumorgenesis.
