共刺激分子B7-2基因转染抑制乳腺癌生长和转移的实验研究
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江苏省卫生厅科技基金资助(J200624)


Experimental study on inhibiting growth and metastasis in human breast cancer by B7-2 gene transfection
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    摘要:

    目的:研究B7-2基因转染乳腺癌细胞对人乳腺癌肺转移的影响。方法:设计B7-2内引物,在内引物的5′端引入XbaⅠ、KpnⅠ酶切位点;在基因组B7-2编码基因两端设计外引物。分离人外周血单个核细胞,孵育成簇后提取总RNA,通过RT得到DNA,分别以外引物和内引物PCR扩增出B7-2基因,PCR产物经双酶切装入真核载体pcDNA3.1(+)构建成重组质粒B7-2pcDNA3.1,对重组质粒进行扩增。将重组质粒和空载体转染MD-MBA-231细胞,用FITC anti-human B7-2 antibody标记两组转染细胞和原细胞,流式细胞检测B7-2表达。将B7-2基因转染乳腺癌MD-MBA-231细胞,将MD-MBA-231/B7-2、MD-MBA-231/pcDNA3.1和MD-MBA-231分别接种于BALB/c小鼠皮下,观察B7-2基因转染对肺转移的影响。结果:获得B7-2重组基因,重组基因转染组的B7-2表达阳性率比空载体组和原细胞组分别高出36.77%和51.88%,种植MD-MBA-231/B7细胞株的BALB/c小鼠成瘤率及肺转移率显著低于对照组,HE染色发现淋巴细胞广泛浸润于MD-MBA-231/B7种植的肿瘤及其肺转移灶。结论:B7-2基因通过提高免疫原性抑制乳腺癌的生长及肺转移。

    Abstract:

    Objective:To investigated the inhibitory effect of B7-2 on pulmonary metastasis by B7-2 gene transfection into breast cancer cells. Methods:A pair of PCR inter primers for B7-2 KpnⅠ was designed based on the sequence registered in GenBank. XbaⅠand KpnⅠ sites were introduced into the 5′ ends of the primers,respectively. Adequate parts out of each end of B7-2 gene in genome were taken as a pair of outer primers. Mononuclear cells were isolated from human peripheral blood,incubated until clustering. Reverse transcript total RNA that isolated from those clustering cells. B7-2 gene was amplified with outer and inner primers with PCR. The PCR fragments were digested with the above two enzymes and then cloned into pcDNA3.1(+) vector to construct recombinant plasmid called B7-2pcDNA. The correct sequencing B7-2pcDNA clone was amplified and transtected into MD-MBA-231 cells. Another group of MD-MBA-231 cells were transfected with idling pcDNA3.1(+). Both transfected groups and the control group were marked with FITC anti-human B7-2 antibody and then detected by flow cytometric analysis. MD-MBA-231/B7, MD-MBA-231/PCDNA3.1, and MD-MBA-231 cells were inoculated into BALB/c mice respectively. The influence of B7-2 on the tumorous metastasis was also observed. Results:The B7-2 recombinant gene was constructed and its length was 952 bp. The B7-2 coding sequence within it was 100% homology with B7-2 homo gene previously registered in GenBank. The flow cytometric analysis shew that B7-2pcDNA group was 36.77% and 51.88% higher than pcDNA3.1(+) and control ones in B7-2 express rate,respectively. The tumor growth rate of MD-MBA-231/B7 xenograft was significantly slower than MD-MBA-231 xenograft in BALB/c mice. MD-MBA-231/B7 cells in orthotopic implantation developed significantly less pulmonary metastasis than MD-MBA-231/pcDNA3.1 and MD-MBA-231 cells. Histologic findings showed that leukocytes were intensively infiltrated in both the MD-MBA-231/B7-2 tumors and its metastatic lesions,however,were scarcely observed in the lesions associated with MD-MBA-231 and MD-MBA-231/pcDNA3.1 cells. Conclusion:B7-2 may play an important role in inhibiting lymph node metastasis by the mechanism of enhanced immunogenicity,and that B7-2 gene transduction might be effective against lymph node metastases of breast cancer.

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刘月仙,夏添松,李俊芳,王 水.共刺激分子B7-2基因转染抑制乳腺癌生长和转移的实验研究[J].南京医科大学学报(自然科学版),2007,(11):1228-1232

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  • 收稿日期:2007-04-19
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