用mRNA差异显示方法初步分离小鼠乳癌MA891细胞转移相关基因
摘要
肿瘤转移是导致肿瘤病人死亡的最重要的原因，因而一直是肿瘤研究的热点。在过去的研究中人们已经发现许多因素与肿瘤转移相关，如粘附分子转移相关基因，侵袭酶及其拮抗剂及肿瘤血管生成等。这么多因子与肿瘤转移相关，说明人们还未发现影响肿瘤的关键因子或者对不同的肿瘤细胞有不同转移相关分子起作用。目前仍有许多实验室从事肿瘤转移相关因子的分离工作。
本实验室在过去的研究中发现IFNγ可增强MA891肿瘤细胞系(一种来源于TA2小鼠自发性乳腺癌，具有高度肺转移性的细胞系)肺转移，而IFNα则抑制其转移。这说明IFNγ与IFNα处理的MA891细胞存在着与肿瘤转移相关基因的改变。因此我们用mRNA差异显示技术(mRNAdifferential display)对两种不同处理的MA891细胞的mRNA进行比较，以期分离与肿瘤转移相关系基因。
我们分别用200u／ml重组人IFNα及IFNγ处理MA891细胞48小时后，提取总RNA，用所有T12MA／C／G／T与20种AP引物(arbitrary primer)配对，每个RNA样本进行4×20次逆转录PCR反应，理论上可覆盖95％以上的mRNA。为排除一些假阳性，每个逆转录PCR反应都重复一次。我们共分离12个差异带，依据引物对及RNA来源命名为T2α、T2γ、C4α、T5α1、T5α2、T6α、T11γ、G15γ、T15α、T15γ、T16γ、T17α。其中T15α、T15γ未能再扩增，我们对其余的10个片段进行了克隆及
Tumor metastasis is the major cause of death in cancer patients. It has been a "hot spot" in the field of cancer research. Many factors have been found to be associated with tumor metastasis, such as adhesive molecules, metastasis-related genes, invasive enzymes and their antagonists, and tumor angiogenesis. That so many factors are related to tumor metastasis implicates that the tumor metastasis is a very sophisticated process. Many laboratories are engaged in identification of rumor metastasis associated factors.
We have previously reported that in vitro treatment with interferons of MA891, a spontaneous mouse mammary tumor capable of metastasizing to the lungs upon subcutaneous inoculation, could modulate its metastatic potential. EFN-γ increases while IFN-α decreases lung metastasis, implying that metastasis related gene(s) are differentially expressed when the cells are treated with these two EFNs. To identify the metastasis related genes, mRNA differential display technique was adopted.
Total RNA was extracted from MA891 cells that had been treated with 200u/ml human rIFN-α or rIFN-γ for 48 h. All the primer pairs (T12MA/C/G/T × 20 arbitrary primers) were used in the differential display, which would cover theoretically over 95% of mRNA. All the RT-PCR were done twice to minimize false positive differentially expressed bands. Twelve bands were separated from the gel and their cDNA fragments were extracted which were referred to as T2α, T2γ, C4α, T5α1, T5α2, T6α, T11γ, G15γ, T15α, T15γ, T16γ, and T17α according to their primers and RNA sources. Except for T15α and T15γ that failed to reamplicate, the other 10 cDNA fragments were reamplicated, cloned, and analyzed by Northern blot. T2γ, C4α, T11γ, and G15γ were further shown to be differentially expressed
本实验室在过去的研究中发现IFNγ可增强MA891肿瘤细胞系(一种来源于TA2小鼠自发性乳腺癌，具有高度肺转移性的细胞系)肺转移，而IFNα则抑制其转移。这说明IFNγ与IFNα处理的MA891细胞存在着与肿瘤转移相关基因的改变。因此我们用mRNA差异显示技术(mRNAdifferential display)对两种不同处理的MA891细胞的mRNA进行比较，以期分离与肿瘤转移相关系基因。
我们分别用200u／ml重组人IFNα及IFNγ处理MA891细胞48小时后，提取总RNA，用所有T12MA／C／G／T与20种AP引物(arbitrary primer)配对，每个RNA样本进行4×20次逆转录PCR反应，理论上可覆盖95％以上的mRNA。为排除一些假阳性，每个逆转录PCR反应都重复一次。我们共分离12个差异带，依据引物对及RNA来源命名为T2α、T2γ、C4α、T5α1、T5α2、T6α、T11γ、G15γ、T15α、T15γ、T16γ、T17α。其中T15α、T15γ未能再扩增，我们对其余的10个片段进行了克隆及
Tumor metastasis is the major cause of death in cancer patients. It has been a "hot spot" in the field of cancer research. Many factors have been found to be associated with tumor metastasis, such as adhesive molecules, metastasis-related genes, invasive enzymes and their antagonists, and tumor angiogenesis. That so many factors are related to tumor metastasis implicates that the tumor metastasis is a very sophisticated process. Many laboratories are engaged in identification of rumor metastasis associated factors.
We have previously reported that in vitro treatment with interferons of MA891, a spontaneous mouse mammary tumor capable of metastasizing to the lungs upon subcutaneous inoculation, could modulate its metastatic potential. EFN-γ increases while IFN-α decreases lung metastasis, implying that metastasis related gene(s) are differentially expressed when the cells are treated with these two EFNs. To identify the metastasis related genes, mRNA differential display technique was adopted.
Total RNA was extracted from MA891 cells that had been treated with 200u/ml human rIFN-α or rIFN-γ for 48 h. All the primer pairs (T12MA/C/G/T × 20 arbitrary primers) were used in the differential display, which would cover theoretically over 95% of mRNA. All the RT-PCR were done twice to minimize false positive differentially expressed bands. Twelve bands were separated from the gel and their cDNA fragments were extracted which were referred to as T2α, T2γ, C4α, T5α1, T5α2, T6α, T11γ, G15γ, T15α, T15γ, T16γ, and T17α according to their primers and RNA sources. Except for T15α and T15γ that failed to reamplicate, the other 10 cDNA fragments were reamplicated, cloned, and analyzed by Northern blot. T2γ, C4α, T11γ, and G15γ were further shown to be differentially expressed
引文
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