摘要
为研究牦牛(Bos grunniens) B细胞淋巴瘤2相关蛋白A1 (B cell lymphoma 2 related protein A1, BCL2A1)的原核表达及体外活性。采用原核表达载体构建、细胞划痕实验、CCK-8法、透射电镜和实时荧光定量PCR等方法。结果显示,成功构建pET-32a-BCL2A1重组质粒,表达获得约33 kDa的BCL2A1。终浓度为0.02μg/mL、0.2μg/mL、2.0μg/mL的BCL2A1均能使HepG2细胞活性显著降低(P <0.05),并在一定程度上抑制细胞的迁移。2.0μg/mL BCL2A1导致HepG2细胞核固缩,胞质中高密度物质聚集,溶酶体吞噬形成致密的凋亡小体等。此外,细胞凋亡相关基因CASP9的mRNA水平在2.0μg/mL组中显著上调(P <0.05),CASP8的mRNA水平在0.2μg/mL和2.0μg/mL组中显著上调(P <0.05),而CASP3和Cyt c的mRNA水平在3个浓度处理组均显著上调(P <0.05)。这提示BCL2A1可能通过凋亡途径影响HepG2细胞活性,为进一步探索牦牛BCL2A1基因功能积累资料。
The aim of this study was to explore the expression and vitro viability of Bos grunniens B cell lymphoma 2 related protein A1(BCL2A1). To this end, prokaryotic expression vector construction, cell scratch test, CCK-8 kit,transmission electron microscope(TEM) and RT-qPCR were used in this experiment. The results showed that recombinant plasmid pET-32 a-BCL2A1 was successfully constructed and a 33 kDa protein was expressed. The viability of HepG2 cells was significantly decreased after treating with BCL2A1(0. 02 μg/mL, 0. 2 μg/mL, and 2. 0 μg/mL)(P <0. 05), and cell migration was inhibited to some extent. HepG2 cells that were treated with BCL2 A1(2. 0 μg/mL)showed karyopyknosis, the electron-dense nuclear material characteristically aggregated in cytoplasm, and lysosome phagocytic organelles formed apoptotic bodies. In addition, the mRNA level of apoptosis-related gene CASP9 was significantly upregulated by 2. 0 μg/mL BCL2A1(P < 0. 05), mRNA level of CASP8 was significantly upregulated by 0. 2 μg/mL and 2. 0 μg/mL BCL2 A1(P < 0. 05), mRNA level of CASP3 and Cyt c were significantly upregulated at different concentration treatment groups(P < 0. 05). It is indicated that BCL2A1 may affect the viability of HepG2 cells through the apoptosis pathway. These results benefit for further study of the function of yak’s BCL2A1.
作者
梅群弟
王海鹏
王利
傅芳
李娟
郑姚
张玲
MEI Qundi;WANG Haipeng;WANG Li;FU Fang;LI Juan;ZHENG Yao;ZHANG Ling(Key Laboratory of Animal Science of State Ethnic Affairs Commission,Southwest Minzu University,Chengdu 610041,China)
出处
《兽类学报》
CAS
CSCD
北大核心
2022年第2期196-203,共8页
Acta Theriologica Sinica
基金
西南民族大学中央高校基本科研业务费专项资金项目(2021NYYXS90)
四川省留学人员科技活动择优资助项目(2019)。
