分子伴侣BiP结构域分析及对肝癌细胞增殖和凋亡的影响

Analysis on Molecular Chaperone BiP Domain and Influence on Proliferation and Apoptosis in Hepatoma Cells

结合免疫球蛋白(binding immunoglobulin protein,BiP),是Hsp70(70 kilodalton heatshock proteins)蛋白家族的成员之一,是内质网应激(endoplasmic reticulum stress,ERS)主要的调节器。为了研究BiP的分子结构与生物学功能的关系,首先,分析BiP的DNA序列和蛋白质空间构象,接着利用重叠PCR方法分别克隆ATPase结构域缺失体和peptide-binding结构域缺失体(BiPa和BiPp),成功构建带myc标签的真核表达载体,转染LO2细胞和SMMC-7721细胞,应用免疫印迹方法检测其在细胞内的表达;运用MTT法、BrdU免疫组化法及流式细胞仪分别检测细胞的增殖和凋亡;检测BiP及两个组成结构域对SMMC-7721细胞增殖和凋亡的影响。酶切、电泳和DNA测序结果显示,BiP全长和两个缺失体的真核表达载体构建成功;免疫印迹检测到BiP三种真核载体在LO2细胞和SMMC-7721细胞中均能正确表达;MTT和BrdU免疫组化结果表明,BiP全长和缺失体BiPa、BiPp三种真核载体均能有效促进SMMC-7721的增殖,各组间细胞增殖率结果分析提示ATPase功能域可能抑制细胞增殖,而peptide-binding结构域可能促进细胞的增殖;流式细胞仪结果显示,转染BiP、BiPa、BiPp三种真核载体均可以促进SMMC-7721的凋亡,缺失体BiPa与全长BiP组间凋亡率差异不显著;而缺失体BiPp组细胞凋亡率明显低于全长BiP组,两组差异具有显著性(P<0.05);提示ATPase功能域与细胞凋亡可能不相关,而peptide-binding结构域可能促进细胞的凋亡。上述结果表明,BiP的两个组成结构域:ATPase功能域与peptide-binding结构域,独立存在或者共同存在时,调控细胞增殖与凋亡具有差异性;当两个结构域共同存在时,它们会协同调控细胞的增殖与凋亡,具体的分子机制还需要进一步的实验研究和探讨。

Binding immunoglobulin protein （BiP） lator for Endoplasmic Reticulum Stress （ERS）. To study is a resident member of Hsp70 family and master regu- the relationship of Bip domain with the physiological function, BiP DNA sequence and protein structure were analysed, two deletion mutants of BiP and BiP full-length eukaryotic expression vectors with myc tag were successfully constructed with the method of overlapping PCR mutagenesis respectively. BiPa deletion mutant is BiP mutant with ATPase domain deleted, BiPp deletion mutant is BiP mutant with peptide-binding domain deleted. After these eukaryotic expression vectors were transfected into LO2 and SMMC-7721 cells, they were identified the expression by Western blot. Then cell proliferation and apoptosis were analysed by MTT assay, BrdU immunohistochemistry and flow cytometry （FCM） respectively, next the relationship between cell proliferation/apoptosis and BiP/BiPa/BiPp were also analysed. Both the full-length BiP and the deletion mutants BiPa, BiPp can effectively improve SMMC-7721 cells proliferation by MTT assay and BrdU immuno-histochemistry. Besides, every group of cell proliferation rate results showed that ATPase domain might be inhibit cell growth, while peptide-binding domain might be increase cell growth. FCM results showed that both the full-length BiP and the deletion mutants BiPa, BiPp can enhance SMMC-7721 cells apoptosis. The difference of apoptosis rate between group BiPa and group BiP were no significance, however, the apoptosis rate of group BiPp was clearly lower than group BiP （P〈0.05）. It was showed that ATPase domain might be unconcerned with cell apoptosis, while peptide-binding domain might be increase cell apoptosis. These two domains, ATPase and peptide-binding, can coregulate cell proliferation and cell poptosis when they combined with each other. The molecular mechanism of them need to be studied deeply.