论文部分内容阅读
目的:通过建立稳定过表达溶酶体相关穿膜蛋白5(lysosomal associated protein transmembrane 5,Laptm5)3’不翻译区(3’UTR)的B细胞淋巴瘤38B9细胞株,探讨Laptm5 3’UTR对小鼠B细胞淋巴瘤38B9细胞株增殖、凋亡的影响。方法:荧光定量PCR及Western blotting检测小鼠B细胞及38B9细胞中Laptm5 miRNA及其蛋白质的表达水平;将小鼠Laptm5 3’UTR及其含有的microRNA结合位点突变的突变型基因片段构建入逆转录病毒表达载体p MSCV-PIG,包装成逆转录病毒,感染小鼠B细胞淋巴瘤细胞38B9,流式细胞术检测逆转录病毒感染率,细胞计数法及流式细胞术观察Laptm5 3’UTR或其突变型过表达后389B9细胞的增殖、凋亡情况。结果:相比小鼠B细胞,B细胞淋巴瘤细胞中Laptm5的miRNA及蛋白均显著降低(P<0.01)。成功获得稳定过表达Laptm5 3’UTR(突变型)的38B9细胞株。Laptm5 3’UTR细胞株比Laptm5 3’UTR突变型过表达细胞株的增殖能力显著减慢,凋亡率显著增加[(7.87±1.08)%vs(0.45±0.07)%,P<0.01]。结论:小鼠Laptm53’UTR具有抑制B细胞淋巴瘤增殖、促进其凋亡的作用,该作用可能与其影响相关microRNA的调控有关。
OBJECTIVE: To establish a B cell lymphoma 38B9 cell line stably overexpressing lysosomal associated protein transmembrane 5 (Laptm5) 3 ’untranslated region (3’UTR) Effect of Proliferation and Apoptosis in Murine B Cell Lymphoma 38B9 Cell Line. Methods: The expression level of Laptm5 miRNA and its protein in mouse B cells and 38B9 cells was detected by real-time PCR and Western blotting. The mutant gene fragments of Laptm5 3’UTR and its microRNA binding site were constructed into reverse The virus expression vector p MSCV-PIG was packaged into a retrovirus and infected with mouse B cell lymphoma cells 38B9. Flow cytometry was used to detect retroviral infection rate, cell counting method and flow cytometry to observe the expression of Laptm5 3’UTR or Proliferation and Apoptosis of 389B9 Cells after Mutant Overexpression. RESULTS: Laptm5 mRNA and protein were significantly decreased in B cell lymphoma cells compared with that of mouse B cells (P <0.01). The 38B9 cell line stably overexpressed Laptm5 3’UTR (mutant) was successfully obtained. The proliferation of Laptm5 3’UTR cell line was significantly slower than that of Laptm5 3’UTR mutant overexpression cell line [(7.87 ± 1.08)% vs (0.45 ± 0.07)%, P <0.01]. Conclusion: The mouse Laptm53’UTR can inhibit the proliferation and promote the apoptosis of B-cell lymphoma, which may be related to the regulation of its related microRNAs.