【作者】 王志红；

【导师】 朱月永；

【作者基本信息】 福建医科大学 ， 内科学， 2007， 硕士

【摘要】 目的观察p53和血管内皮生成抑素基因(AS)共转染对HL60细胞生长和VEGF、bax、bcl-2表达水平的影响，探讨联合基因治疗急性白血病的可能性。方法选用脂质体将p53、AS共转染于HL60细胞，以RT-PCR法检测转染后HL60细胞目的基因的表达。通过细胞集落形成实验、MTT生长曲线及流式细胞仪观察p53、AS共转染后对HL60细胞生长和凋亡的影响。采用RT-PCR及免疫组化检测p53、AS共转染后HL60细胞的VEGF、bax、bcl-2表达水平的改变。采用统计软件包SPSS10.0进行统计分析。结果1．RT-PCR法显示：p53、AS共转染成功并在HL60细胞中稳定表达。2．细胞集落实验显示：目的基因转染后，细胞集落形成数均有减少(p＜0.05)；与空转染对照组比较p＜0.05，与单一转染组比较p＜0.05(n=3)，其中共转染p53、AS双基因组最为明显(p＜0.05)，且集落变小。3．MTT细胞生长曲线检测显示：与空转染对照组比较p＜0.05，与单一转染组比较p＜0.05，可见单独转染p53、AS基因与共转染p53、AS基因的细胞生长与空载体转染细胞比较均变慢、倍增时间延长(p＜0.05)；共转染p53、AS基因对HL60细胞的生长抑制作用明显高于单独转染组(p＜0.05)。4．流式细胞仪检测显示：在转染空载体、p53、AS、共转染p53、AS基因的细胞组中，后3种方式的转染均能引起细胞出现不同程度的凋亡峰；与单独转染所引起的凋亡峰相比，共转染基因所所引起的凋亡峰更为明显；共转染p53、AS基因诱导HL60细胞凋亡时互为协同作用。5．免疫组化检测显示：转染前细胞的胞浆内出现大量棕黄色颗粒，VEGF表达为强阳性，而转染后仅少数细胞的胞浆内出现浅黄色，VEGF表达水平下降；转染前细胞的胞浆内出现大量强染色特异性颗粒，bcl-2表达为强阳性，但转染后细胞的胞浆内几乎不出现特异性颗粒，bcl-2表达水平明显下降；转染前细胞的胞浆内少有棕黄色颗粒，bax阳性表达低，但转染后细胞的胞浆内出现大量棕黄色颗粒，bax表达水平明显升高。结论p53、AS共转染具有协同抑制HL60细胞的生长，共同促进细胞凋亡，使HL60细胞的VEGF、bcl-2表达水平下降，bax表达水平明显升高，通过影响bcl-2／bax的比率可能是其机制之一。更多还原

【Abstract】 ObjectiveTo observe the inhibitory effect and the expression levels of VEGF、bax、bcl-2 of combined p53 and AS transfecting in HL60 cell. To discuss the possibility on treatment in acute leukemia of combined genes.MethodsTransfect the p53 and AS into HL60 cell with lipofectamine. After transfection, RT-PCR was used to verify whether the target gene had been transfected and expressed or not.Cell clones trial, MTT growth curve and flow cytometry assay were used analyze to the inhibitory effect of combined p53 and AS transfecting in HL60 cell. RT-PCR and immunohistochemistry staining were used to analyze the expression levels of VEGF、bax、bcl-2 of combined p53 and AS transfecting in HL60 cell. The date were analysised by spss10.0.Results1. RT-PCR show: p53 and AS had been transfected and expressed in HL60 cell.2. Cell clones trial show: After transfection, the number of all cell clones were reduce(p<0.05), compared with the empty gene transfect group p<0.05, compared with the single gene transfect group p<0.05(n=3), compared with was evidence(p<0.05) and the cell clones became small.3. MTT growth curve show: After transfection, compared with the empty gene transfect group p<0.05, compared with the single gene transfect group p<0.05, compared with the empty gene transfect group, the cells in the single gene(p53 or AS)transfect group and the two genes(p53 and AS) transfect group grew slowly and longer(p<0.05).They were suppressedby the two genes and inhibition of the combined genes was more powerful than a single one(p<0.05).4. Flow cytometry assay show: In the single gene(p53 or AS)transfect group and the two genes(p53 and AS) transfect group could apper differ peak value of apoptosis, compared with the single gene transfect group, it was more evidence in the two genes(p53 and AS) transfect group, the inhibition effect of cooperate with combined p53 and AS transfecting in HL60 cell.5. Immunohistochemistry staining show: After transfection, the expression levels of VEGF and bcl-2 were lower, the expression levels of bax was higher than before transfection.ClonculsionsThe inhibition effect of cooperate with combined p53 and AS transfecting in HL60 cell. After transfection, the expression levels of VEGF and bcl-2 were lower, the expression levels of bax was higher. It may be one of the mechanism by the effect of bcl-2/bax rate.更多还原