Zheng Yu-Dan, Sun Zheng-Jiang, Ma Kun-Peng, Wang Yi-Qiang, Lin Dan-Dan
Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215000, Jiangsu Province, China.
Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215000, Jiangsu Province, China,E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022 Jun;30(3):688-694. doi: 10.19746/j.cnki.issn.1009-2137.2022.03.005.
To express matrix remodeling associated 7 (MXRA7) in the human acute myeloid leukemia SHI-1 cell line and to assess the role of MXRA7 in the biological function of SHI-1 cells.
The full-length cDNA sequence of human MXRA7 was synthesized and subcloned into the lentivirus shuttle vector pRRL-Venus. SHI-1 cells were transfected with the lentivirus which was packaged with 293T cells. The YFP-positive cells were sorted by flow cytometry and the stable cell lines were obtained by expanded culture. The expression and distribution of MXRA7 in SHI-1 cells were verified by real-time qPCR, Western blot and laser confocal techniques. Cell proliferation and cell cycle were measured by flow cytometry, and apoptosis was determined by Annexin V and 7-AAD staining. The expression of apoptosis related proteins were detected by Western blot.
The stable SHI-1 cell line overexpressing MXRA7 was established successfully. Laser confocal analysis confirmed that MXRA7 was expressed in the cytoplasm of SHI-1 cells. Compared with the control cell line, the overexpression of MXRA7 showed no effect on the cell proliferation and cell cycle, but reduced the percentage of apoptosis cells induced by methotrexate. Moreover, the expression of BCL-2 protein was increased by overexpression of MXRA7, which can inhibit cell apoptosis.
The SHI-1 stable cell line overexpressing MXRA7 was established successfully, and MXRA7 could inhibit drug-induced apoptosis through increasing the expression of BCL-2 protein.
在人急性髓系白血病SHI-1细胞系中表达基质重塑相关蛋白7（MXRA7），并评估MXRA7在SHI-1细胞生物学功能中的作用。
合成人MXRA7的全长cDNA序列，并亚克隆到慢病毒穿梭载体pRRL-Venus中。用293T细胞包装慢病毒后转染SHI-1细胞。通过流式细胞术分选YFP阳性细胞，并经扩大培养获得稳定细胞系。采用实时定量PCR、蛋白质免疫印迹和激光共聚焦技术验证MXRA7在SHI-1细胞中的表达及分布。通过流式细胞术检测细胞增殖和细胞周期，采用膜联蛋白V和7-氨基放线菌素D染色法检测细胞凋亡。通过蛋白质免疫印迹检测凋亡相关蛋白的表达。
成功建立了过表达MXRA7的SHI-1稳定细胞系。激光共聚焦分析证实MXRA7在SHI-1细胞的细胞质中表达。与对照细胞系相比，MXRA7的过表达对细胞增殖和细胞周期无影响，但降低了甲氨蝶呤诱导的凋亡细胞百分比。此外，MXRA7的过表达增加了BCL-2蛋白的表达，从而抑制细胞凋亡。
成功建立了过表达MXRA7的SHI-1稳定细胞系，MXRA7可通过增加BCL-2蛋白的表达抑制药物诱导的细胞凋亡。
