【作者】 张涛；

【导师】 王利；

【作者基本信息】 重庆医科大学 ， 内科学（血液）， 2016， 硕士

【摘要】 目的:研究骨髓增生异常综合征(MDS)患者中钙网织蛋白基因(Calreticulin,CALR)的表达,并构建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。方法:收集重庆医科大学附属第一医院血液科2010年~2014年的34例MDS患者骨髓标本和8例非MDS患者骨髓标本。按照WHO分型诊断标准和MDS国际预后积分系统,将实验组34例患者分为高危组和低危组,对照组取另外8例同期非MDS患者的骨髓标本。所有骨髓标本通过分离单个核细胞提取总RNA,逆转录制备为cDNA,使用RT-PCR技术检测两组骨髓标本及SKM-1细胞中CALR的相对表达水平。首先查询GenBank中CALR(human)的碱基序列,然后使用RNAi软件设计3条RNAi靶序列、1条阴性对照序列。合成RNAi靶序列的单链DNA oligo,退火配对产生双链,T4 DNA连接酶连接酶切后的慢病毒载体,转化感受态大肠杆菌,PCR筛选阳性克隆并进行测序鉴定,完成RNAi慢病毒载体的制备。共转染293T细胞进行包装得到3种GV-CALR-RNAi重组慢病毒载体。病毒滴度采用逐孔稀释法进行测定。三种GV-CALR-RNAi重组慢病毒分别稳定转染至SKM-1细胞中,通过流式细胞术检测转染效率。后收集各组细胞,分别提取总rna及蛋白,通过rt-pcr检测各组细胞calrmrna表达及westernblot技术检测各组细胞calr蛋白质的表达以验证干扰效果,筛选出最佳靶向序列。使用含最佳靶向序列的慢病毒载体转染skm-1细胞,cck-8法检测干扰calr基因的表达对skm-1细胞生长的影响,流式细胞术观察其对细胞周期的影响,annexinv/7-aad双染法用流式细胞术观察对细胞凋亡的影响,westernblot检测凋亡蛋白caspase-3的水平。结果:应用rt-pcr技术检测34例低、高危mds标本的calr表达,绝大多数标本calr基因表达明显降低,显著低于正常对照组(p<0.01)。同时,检测到人mds细胞株skm-1细胞中calr表达较正常对照明显降低(p<0.01)构建的3组calr-rnai重组慢病毒转染人mds细胞株skm-1后,倒置显微镜下观察gfp荧光逐渐增强,至培养第5天表达最强,calr-rnai(2)、calr-rnai(3)慢病毒干扰组流式细胞术检测转染效率均在70%以上,而calr-rnai(1)慢病毒干扰组流式细胞术检测转染效率在50%以下。rt-pcr检测转染后第5天实验组calr-mrna表达水平,结果显示calr-rnai(3)慢病毒载体与阴性对照组及空白对照组相比能有效降低calrmrna表达水平[(0.40±0.11),p<0.01]。westernblot检测结果也证实,calr-rnai(3)能有效敲除calr蛋白的表达,确定为最佳靶点在SKM-1细胞中转染CALR-RNAi(3)慢病毒及阴性对照组慢病毒,通过CCK-8法检测SKM-1细胞的生长,发现CALR-RNAi(3)通过干扰CALR表达可促进SKM-1细胞生长,细胞生长第5天统计学差异显著(P<0.05)。流式细胞术检测实验组和对照组细胞周期情况,结果显示CALR-RNAi(3)组G0/G1期细胞比例(45.25±4.45)%低于空白对照组(54.12±1.36)%和阴性对照组(54.67±1.26)%,且差异具有统计学意义(P=0.019,P=0.014);而CALR-RNAi(3)组S期细胞比例细胞比例为(46.15±2.99),明显高于空白对照组和阴性对照组[(38.42±2.11)%和(36.23±3.25)%,P<0.05。AnnexinV/7-AAD双染法用流式细胞仪检测凋亡细胞,发现CALR-RNAi(3)组较阴性对照组和空白对照组细胞凋亡减少(图7),且有显著性差异(P<0.05)。用Western blot检测各组细胞中cleaved-caspase-3的表达。结果显示,与阴性对照组和空白对照组相比,cleaved-caspase-3的表达在CALR-RNAi(3)组中明显降低。结论: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。更多还原

【Abstract】 Objective: To study the expression of calreticulin(CALR) in patients with myelodysplastic syndrome(MDS), and construct RNAi lentiviral vector targeting CALR gene in SKM-1. Then investigate the effect of CLAR-RNAi on cells apoptosis and proliferation.Methods: Reverse transcription PCR( RT-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 digested by enzyme to produce GV-CALR-RNAi lentiviral vector. The lentiviral vector was cotransferred into 293 T cells. 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 CALR gene was determined by quantitative reverse transcription PCR and Western blotting, and the most effectivetargeted sequence was screened. CCK8 assay and Annexin V/7-AAD staining were conducted to observe the effects of CALR gene silencing on cell growth and apoptosis.Results: CALR gene expression was significantly decreased(P < 0.01)in 31 of 34 patients with MDS compared with controls. Three kinds of CALR-RNAi-expressing lentiviral vector was successfully constructed, and the transfection efficiency was more than 70%. Quantitative 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 that in S phase was increased. Western blot showed that downregulation of CALR resulted in the decreased expression level of cleaved-caspase-3.Conclusion: CALR is an important tumor suppressor gene in MDS,and the CALR-RNAi(3) lentivirus vector will be helpful in further studying the functional role of CALR gene in MDS.更多还原