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该文研究了钙网蛋白(Calreticulin,CALR)基因在骨髓增生异常综合征(myelodysplastic syndrome,MDS)患者中的表达,并构建了CALR-RNAi慢病毒载体,观察其对人MDS细胞株SKM-1细胞生长和凋亡的影响。反转录PCR(reverse transcription PCR,RT-PCR)检测34例低危、高危MDS患者、8例非MDS患者骨髓标本及细胞株中CALR的表达;设计3条含靶向沉默CALR基因的Oligo DNA,与酶切后的GV248质粒连接形成表达载体,经筛选、鉴定后得到3种GV-CALR-RNAi重组慢病毒载体。分别转染SKM-1细胞,流式细胞术检测转染效率,RT-PCR及Western blot验证干扰效果,筛选出最佳靶向序列。然后,使用含最佳靶向序列的慢病毒载体转染SKM-1,CCK-8法和Annexin V/7-AAD双染法分别观察其对细胞生长和凋亡的影响,Western blot检测caspase-3的表达。34例低危、高危MDS患者中有31例检测到CALR基因表达较正常组明显降低(P<0.01)。成功构建了3种CALR-RNAi慢病毒载体,流式细胞术检测平均转染效率在70%以上。RT-PCR及Western blot检测结果显示,CALR-RNAi(3)慢病毒载体敲除效率最高,为最佳靶点。结果发现,CALR-RNAi(3)转染SKM-1后可促进细胞生长、抑制细胞凋亡(P<0.05),同时CALR-RNAi(3)组的G0/G1期细胞减少,S期细胞增多。Western blot证实凋亡因子cleaved-caspase-3的水平降低。CALR基因在MDS中起到抑癌基因的作用,构建的GV-CALR-RNAi(3)重组慢病毒载体有利于进一步研究CALR基因在MDS中的作用机制。
In this paper, the expression of calreticulin (CALR) gene in patients with myelodysplastic syndrome (MDS) was studied. CALR-RNAi lentiviral vector was constructed and its effect on human MDS cell line SKM-1 Effects of Cell Growth and Apoptosis. Reverse transcription PCR (RT-PCR) was used to detect the expression of CALR in 34 low-risk and high-risk MDS patients and 8 non-MDS patients with bone marrow samples and cell lines. Three Oligo DNAs containing targeting silencing CALR gene were designed And ligated with GV248 plasmid to form expression vector. After screening and identification, three GV-CALR-RNAi recombinant lentiviral vectors were obtained. The transfected SKM-1 cells were transfected respectively, the transfection efficiency was detected by flow cytometry, the interference effect was verified by RT-PCR and Western blot, and the best targeting sequence was screened out. Then, SKM-1 was transfected with the lentiviral vector containing the best targeting sequence. The effects of CCK-8 and Annexin V / 7-AAD double staining on the cell growth and apoptosis were observed. Western blot was used to detect the expression of caspase- 3 expression. Among 34 low-risk and high-risk MDS patients, 31 cases detected CALR gene expression was significantly lower than the normal group (P <0.01). Three kinds of CALR-RNAi lentiviral vectors were successfully constructed. The average transfection efficiency of flow cytometry was above 70%. The results of RT-PCR and Western blot showed that CALR-RNAi (3) lentiviral vector had the highest knockdown efficiency and was the best target. The results showed that, CALR-RNAi (3) transfected SKM-1 can promote cell growth and inhibit apoptosis (P <0.05), while CALR-RNAi (3) group G0 / G1 phase cells, S phase cells increased . Western blot confirmed that the apoptosis factor cleaved-caspase-3 levels decreased. CALR gene plays a role of tumor suppressor gene in MDS. The construction of GV-CALR-RNAi (3) recombinant lentiviral vector is conducive to further study the mechanism of CALR gene in MDS.