PML(NLS~–)蛋白在NB4细胞及其裸鼠移植瘤中定位的验证

Localization of PML(NLS~–) Protein in NB4 Cells and Transplanted Tumors in Nude Mice

验证中性粒细胞弹性蛋白酶(neutrophil elastase,NE)切割PML-RARα后,PML(NLS–)蛋白的存在和定位。将质粒pCMV-HA-NE电转染NB4细胞,用Western blot法验证质粒转染成功;提取电转染质粒成功的NB4细胞的胞浆蛋白,用Western blot法检测NB4细胞中PML(NLS–)蛋白的表达;免疫荧光法和激光共聚焦检测电转染质粒成功的NB4细胞中PML(NLS–)蛋白的表达及定位;同时,建立NB4细胞、K562细胞和电转染质粒成功的NB4细胞裸鼠皮下瘤模型,用Western blot、免疫组化法检测PML(NLS–)蛋白在移植瘤组织细胞中的表达与定位。结果表明,Western blot检测电转染质粒pCMV-HA-NE的NB4细胞成功表达NE蛋白;NE酶成功切割PML-RARα,Western blot检测到电转染质粒pCMV-HA-NE的NB4细胞表达PML(NLS–)蛋白;免疫荧光和激光共聚焦均可检测到电转染质粒成功的NB4细胞中PML(NLS–)蛋白定位于细胞胞浆;Western blot和免疫组化法检测到电转染质粒成功的NB4细胞裸鼠移植瘤中的PML(NLS–)蛋白的表达且定位于细胞胞浆,而NB4和K562细胞裸鼠皮下瘤中PML蛋白主要定位于胞核。综上所述,该文成功将质粒pCMV-HA-NE电转染NB4细胞并用Western blot、免疫荧光、激光共聚焦、免疫组化验证PML(NLS–)蛋白存在于NB4细胞胞浆,这一现象可以为急性早幼粒细胞白血病的临床早期诊断与治疗提供新的依据。

To verify the existence and location of PML （NLS-） protein after PML-RARa cleaved by neu- trophil elastase （NE）, the plasmid pCMV-HA-NE was electroporated into NB4 cells and NE protein was verified by Western blot. We extracted the cytoplasm protein of NB4-HA-NE cells and detected the PML （NLS^-） protein by Western blot. Immunofluorescence assay and confocal laser microscopy were performed to localize the PML （NLS^-） protein in the cytoplasm of NB4-HA-NE cells. Then, NB4, K562 and NB4-HA-NE cells were expressed in xenograft of nude mice. Western blot and immunohistochemistry were done to detect PML （NLS^-） protein in the cytoplasm of the NB4-HA-NE cells. The results indicated that NE protein was detected in the NB4 -HA-NE ceils and PML （NLS-） protein was verified in the NB4-HA-NE cells by Western blot, which demonstrated that PML-RARα was cut successfully by NE. Immunofluorescence assay and confocal laser microscopy showed that the PML （NLS^-） protein was localized in the cytoplasm of NI34-HA-NE ceils. Meanwhile, PML （NLS^-） protein in nude mice was expressed and localized in the cytoplasm of NB4-HA-NE cells manifested by Western blot and immunohistochemistry, while PML protein was almost localized in the nucleus of NB4 and K562 cells in nude mice. In summary, the pCMV-HA-NE plasmid was successfully electransfered into NB4 cells and PML （NLS-） protein was verified to be in the cytoplasm of NB4-HA-NE cells with the use of Western blot, immunofluorescence, confocal laser microscopy and immunohistochemical techniques, which could provide new evidence for early diagnosis and treatment of acute promyelocytic leukemia.