反义周期蛋白B1基因抑制HT29细胞的增殖

用ＤＮＡ重组技术将人类ｃｙｃｌｉｎＢ１基因的全长ｃＤＮＡ，反向插入到真核表达载体ｐＸＪ４１－ｎｅｏ的ＢａｍＨ１位点，得到ｃｙｃｌｉｎＢ１基因的反义ＲＮＡ表达载体ｐＡＳ－Ｂ１。利用ＤＮＡ磷酸钙共沉淀方法，将ｐＡＳ－Ｂ１转染进ＨＴ２９细胞。在含有Ｇ４１８的培养基中，挑选出独立的细胞克隆。利用ｃｙｃｌｉｎＢ１ｃＤＮＡ作探针，Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交鉴定出一株内源性ｃｙｃｌｉｎＢ１ｍＲＮＡ受到抑制的细胞株ＨＴＢ１。比较ＨＴＢ１与对照细胞ＨＴ２９发现，ｃｙｃｌｉｎＢ１的反义ＲＮＡ明显抑制ＨＴ２９细胞的增殖，细胞内３Ｈ－ＴｄＲ参入量减少。流式细胞光度术分析，处于Ｇ１期细胞比率的升高与Ｓ期、Ｇ２＋Ｍ期细胞数量的下降都十分显著。

ANTISENSE CYCLIN B1 GENE INHIBITS THE PROLIFERATION OF HT29 CELLS

The full length cDNA of human cyclin B1 gene was inserted into the eukaryotic expression vector pXJ41 neo in its antisense orientation at the BamH I site to produce the plasmid pAS B1 which expressed antisense RNA of cyclin B1 gene by DNA recombinant technique. The plasmid pAS B1 was transfected into HT29 cells by DNA Ca 3(PO 4) 2 co precipitation. Individual cell clones were selected in the medium supplemented with G418. The expresison of endogenous cyclin B1 mRNA was inhibited and was measured by Northern blot with cyclin B1 cDNA probe. Compared with HT29 cells, we found that antisense RNA of cyclin B1 could obviously inhibit the proliferation and 3H thymidine incorporation of HTB1 cells. By flow cytometry analysis we found that the cell number of G1 phase increased obviously and the cell number of S phase and G2 M phases decreased markedly in HTB1 cells.