β微管蛋白基因在骨髓增生异常综合征向白血病转化中的意义

The expression of beta-tubulin gene in myelodysplastic syndrome evoluting to leukemia

目的 探讨β微管蛋白（IUBB）基因在骨髓增生异常综合征（MDS）向白血病转化（简称转白）中的作用.方法 基于复旦大学附属华山医院已建立的MDS患者巢式病例对照研究队列,从中选取符合转白危险因素配对条件的各11例患者建立病例组（发生转白的MDS）和对照组（未发生转白的MDS）.采用实时定量PCR检测两组患者骨髓单个核细胞中TUBB mRNA表达水平,并采用生长曲线测定（CCK-8法）、软琼脂克隆形成实验和透射电镜,在MDS转白细胞株（SKM-1细胞株）中观察TUBB基因沉默前后SKM-1细胞株的生物学特性的变化.结果 （1）实时定量PCR结果显示,病例组的TUBB mRNA相对表达水平显著高于对照组（2.91±0.41比0.90±0.23,P＜0.01）.（2）TUBB基因特异性小干扰RNA（TUBB-siRNA）转染组的SKM-1细胞在转染后24、48和72 h的吸光度值（A450/630nm）分别为0.299±0.045、0.526±0.034和0.652±0.035,均低于相同时点的阴性siRNA转染组（0.438±0.074、0.858±0.064和0.974±0.044,P值均＜0.05）,TUBB-siRNA转染组SKM-1细胞的增殖活性受到明显抑制作用.（3）软琼脂克隆形成实验显示,TUBB-siRNA转染组的细胞克隆形成率为（7.0±0.2）％,显著小于阴性siRNA转染组的（25.0±0.2）％（P＜0.01）.（4）透射电镜显示,转染TUBB-siRNA后,SKM-1细胞出现细胞质空泡、核碎裂等细胞凋亡征象.结论 TUBB基因可能通过影响MDS恶性克隆的增殖途径在MDS转白过程中起一定作用.

Objective Based on our previous established cohort of myelodysplastic syndrome （MDS）, we investigated the potential effect of beta-tubulin （TUBB） gene in the transformation of MDS into acute leukemia Methods From our nested case-control study cohort of MDS patients, we chose 11 paired transformed and nontransformed MDS patients.TUBB gene expression was tested by quantitative real-time PCR.TUBB-siRNA transfection was used to down-regulate TUBB gene expression in SKM-1 cell line.The function of TUBB gene in SKM-1 cell line was evaluated by cell proliferation, soft agar clone formation and electron microscope.Results TUBB gene expression in MDS patients in transformed group were significantly higher than that in control group （2.91 ± 0.41 vs 0.90 ± 0.23, P ＜0.01）.After TUBB-siRNA transfection, A450/630nm of SKM-1 cells at 24 h, 48 h and 72 h were 0.299 ± 0.045, 0.526 ± 0.034 and 0.652 ± 0.035, respectively, which were significantly decreased than those in negative-siRNA group （0.438 ±0.074, 0.858 ±0.064 and 0.974 ±0.044） （P ＜0.05）.Soft agar clone formation in TUBB-siRNA group was （7.0 ±0.2）％, which was significantly reduced than that of negative-siRNA group （25.0 ± 0.2）％ （P ＜ 0.01）.Electron microscope showed significant apoptotic signs in TUBB-siRNA group, including vacuoles in cytoplasm and karyorrhexis.Conclusion Our results indicate that TUBB gene may play a role in the transformation of MDS into acute leukemia by affecting the proliferation of malignant clones.