中文摘要：

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OligoDNA，与酶切后的GV248质粒连接形成表达载体，经筛选、鉴定后得3,13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AnnexinV／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。

英文摘要：

We studied the expression of Calreticulin （CALR） in patients with MDS （myelodysplastic syndrome） and also constructed RNAi lentiviral vector targeting CALR gene in SKM-1 to investigate the effect of CLAR-RNAi on proliferation and apoptosis of MDS cells. RT-PCR （reverse transcription PCR） was used to detect CALR expression in bone marrow specimens obtained from 34 MDS patients and 8 non-MDS patients. Three kinds of single-stranded primers were designed and synthesized, then annealed to double-stranded oligo sequences and subcloned into linear GV248 lentiviral plasmid to produce GV-CALR-RNAi lentiviral vector. After being identified by PCR and sequencing, recombinant lentiviral vector expressing CALR RNAi were obtained. This lentiviral vector was transferred into human SKM-1 cells, and the transfection efficiency was detected by flow cytometry. The interference efficiency of recombinant lentiviral vector was determined by reverse transcription PCR and Western blot, and the most effective vector was selected. CCK8 assay and Annexin V/7-AAD staining were conducted to observe the effects of CALR gene silencing on cell growth and apoptosis. CALR gene expression was significantly decreased （P〈0.01） in MDS patients when compared with healthy controls. Three kinds of CALR-RNAi-expressing lentiviral vectors were successfully constructed, and the transfection efficiency was more than 70%. Reverse transcription PCR and Western blot results showed that CALR-RNAi（3） lentiviral vector was the most effective vector for interfering the expression of CALR gene. Moreover, CALR-RNAi（3） could promote cell growth and inhibit apoptosis in SKM-1 cells （P〈0.05）. The percentage of SKM-1 cells in G0/G1 phase was decreased and it was increased in S phase. Results of Western blot showed that down regulation of CALR resulted in the decreased protein level of cleaved-caspase-3. CALR is an important tumor suppressor gene in MDS, and the CALR-RNAi（3） lentivirus vector will be helpful in studying the functional