【作者】 赖颖晖；

【导师】 赖永榕；

【作者基本信息】 广西医科大学 ， 血液内科， 2010， 博士

【摘要】 背景针对β-地中海贫血的α珠蛋白链相对过剩这一主要病理基础,有研究报道应用反义寡核苷酸技术调控人α-珠蛋白基因表达,结果显示α-反义寡核苷酸能有效地抑制体外培养的重型β-地中海贫血红系细胞α-珠蛋白基因表达,改善珠蛋白基因α/β+γ的比例失衡。然而,对于人α-反义寡核苷酸治疗β-地中海贫血的体内试验研究尚未见报道。目的建立人源化水平较高的人源非肥胖糖尿病/重症联合免疫缺陷(NOD/SCID)小鼠模型,在此基础上建立人源β-地中海贫血小鼠模型,并构建介导人α-反义寡核苷酸的慢病毒载体,探讨慢病毒载体介导α-反义寡核苷酸对人源β-地中海贫血小鼠模型的作用。方法1、将人正常骨髓或脐血单个核细胞分别移植给经不同方案和剂量预处理的NOD/SCID小鼠,移植后小鼠腹腔注射重组人细胞因子。观察小鼠存活情况,移植6周后检测小鼠骨髓及外周血人-CD45+细胞比例。2、将β-地中海贫血患者的骨髓单个核细胞,经尾静脉输注给经全身照射预处理的NOD/SCID小鼠,移植后小鼠腹腔注射重组人细胞因子。观察小鼠体重等一般状况,移植5周后取小鼠骨髓及外周血,检测人CD45+细胞比例,并行血红蛋白电泳、珠蛋白肽链聚丙烯酰胺凝胶电泳、人β-地贫基因分析及透射电镜观察小鼠外周血及骨髓红系细胞内α-珠蛋白链的沉积情况,取小鼠的肝脏和脾脏行大体、普通病理及铁染色检查。3、针对人α-反义寡核苷酸序列,通过双限制性内切酶消化和连接的方法构建pGCSIL-vshRNA-GFP载体质粒,接着该质粒转化感受态的大肠杆菌E.coliDH5α,通过PCR及基因测序鉴定阳性克隆,再经Lipofectamine 2000将pGCSIL-vshRNA-GFP,pHelper 1.0和pHelper 2.0三质粒系统共转染293 T细胞包装病毒,通过绿色荧光蛋白的表达测定收集的病毒滴度。此外,同样方法制备随机序列慢病毒载体并包装纯化。4、将介导α-反义寡核苷酸的慢病毒载体,感染β-地中海贫血患者骨髓CD34+细胞后,再输注给经全身照射预处理的NOD/SCID小鼠,并以输注未经感染的β-地贫骨髓细胞、感染随机序列慢病毒的β-地贫骨髓细胞、正常骨髓CD34+细胞及空白小鼠为对照。观察小鼠体重等一般状况,移植6周后,取小鼠骨髓及外周血,检测人CD45+细胞比例,并行血红蛋白电泳及人β-地贫基因分析,取小鼠的肝脏和脾脏行大体、普通病理及铁染色检查。结果1、全身照射(加或不加环磷酰胺)骨髓移植组和全身照射(加或不加环磷酰胺)脐血移植组小鼠输注人单个核细胞数分别为(0.656 0±0.008 9)×106和(4.077 5±0.845 0)×106。全身照射/环磷酰胺组死亡率均为100%,全身照射组死亡率均为33.3%,且全身照射剂量越大,死亡率越高。移植6周后,小鼠骨髓中人CD45+细胞的比例,全身照射脐血移植组13号和6号小鼠分别为59.61%和21.46%,而全身照射骨髓移植组2号和8号小鼠其比例均为0,空白对照组小鼠比例均为0。2、β-地中海贫血患者骨髓细胞移植NOD/SCID小鼠5周后,检测小鼠骨髓和外周血人CD45+细胞比例分别为:β-地贫/HbE骨髓移植组:7.21%、4.08%(+47天),7.37%、6.03%(+61天),β-地贫骨髓移植组:8.17%、3.43%(+36天),16.82%、8.89%(+45天)。移植组小鼠血红蛋白电泳可见HbA2条带,肽链分析显示α链的条带要比β链的条带浓和宽,β-地贫基因检测结果与供者相符,部分红系细胞内出现电子致密物(过剩α-珠蛋白肽链)沉积。移植组小鼠脾脏长度与其体重的比值比空白对照组的大[(0.5762±0.0451)vs(0.3628±0.0340)mm/g,P=0.000],肝脾出现较明显的铁沉积。3、重组质粒的外源基因PCR鉴定正确,基因测序结果与所需要的α-ASON序列完全一致,浓缩后慢病毒滴度为5×109TU/ml。4、慢病毒所感染的β-地贫骨髓细胞移植NOD/SCID小鼠,移植后6周各组小鼠与移植前体重比较:正常骨髓移植组和空白对照组体重增加或显著增加,而其余小鼠体重减轻或不增加。移植后6周各移植组小鼠骨髓和外周血能检测到人CD45+细胞(0.01-8.39%),且输注β-地中海贫血患者细胞(不论是否输注慢病毒感染细胞)的小鼠可检测到与供者相符的人β-地贫突变基因,输注β-地贫患者细胞的小鼠脾脏长度与其体重的比值比正常骨髓移植及空白对照组的大([0.6848±0.0886)v(s0.5573±0.0088)mm/g,P=0.016]。在α-ASON-LV感染β-地贫骨髓细胞移植组,肝脾(尤其脾)铁沉积比β-地贫骨髓移植组和NC-GFP-LV感染β-地贫骨髓细胞移植组的程度轻。结论在输注造血干细胞数量达阈剂量的基础上,尽可能提高预处理的强度,并使用人细胞因子可提高人源NOD/SCID小鼠模型的人源化水平。将β-地中海贫血患者骨髓单个核细胞移植给NOD/SCID小鼠,可建立人源化β-地中海贫血小鼠模型。所构建介导α-反义寡核苷酸的慢病毒载体感染人β-地贫骨髓细胞植入NOD/SCID小鼠,有可能减轻人源β-地中海贫血小鼠模型的铁沉积。慢病毒载体介导α-反义寡核苷酸对人源β-地中海贫血小鼠模型的作用有待进一步研究。更多还原

【Abstract】 Objective To generate humanizedβ-thalassemia mice models and construct recombinant lentiviral(LV) vectors carrying humanα-antisense oligonucleotide, to explore the role of lentiviral vectors carrying humanα-antisense oligonucleotide in humanizedβ-thalassemia mice models.Methods 1.The human bone marrow or cord blood mononuclear cells were transplanted into NOD/SCID mice pretreated with the different schemes and dosage. After transplantation, human recombinant growth factors consisting of rhEPO and rhGM-CSF were injected into the mice intraperitoneally. The survival status of the mice was recorded to calculate death rate. Six weeks after the transplantation, flow cytometric detection of human cells in murine tissues (bone marrow and blood) was performed with monoclonal antibodies against human CD45.2.The bone marrow mononuclear cells ofβ-thalassemia patients were transplanted into the sublethally irradiated NOD/SCID mice. After transplantation, human recombinant growth factors consisting of rhEPO and rhGM-CSF were injected into the mice intraperitoneally. Five weeks after the transplantation, flow cytometric detection of human cells in murine tissues (bone marrow and blood) was performed with monoclonal antibodies against human CD45. Electrophoresis of hemoglobin, analysis of hemoglobin chains and detection ofβ-thalassemia gene mutations were performed in murine tissues (bone marrow or blood). Theα-globin chain precipitates in erythroid cells were observed in electron microscopy.In addition,spleen and liver of the mice were removed, observed,embedded,and sectioned.Specimens were stained with hematoxylin and eosin and iron stains (Prussian blue).3.According to the humanα-antisense oligonucleotide sequence, pGCSIL- vshRNA-GFP plasmid was constructed by double restriction enzyme digestion and ligation, and then the plasmid was transformed into E.coliDH5α. Purified pGCSIL-vshRNA-GFP plasmids from the positive clones was confirmed by PCR and sequencing. 293T cells were cotransfected with lentiviral vector pGCSIL-vshRNA-GFP, pHelper 1.0 and pHelper 2.0 by Lipofectamine 2000 to produce lentivirus. The titer of virus was tested according to the expression level of green fluorescent protein. In addition, with the same methods, recombinant lentiviral vectors for NC sequence were constructed.4.The bone marrow CD34+ cells ofβ-thalassemia patients were transduced by lentiviral vectors carrying humanα-antisense oligonucleotide. Then the transduced cells were transplanted into the sublethally irradiated NOD/ SCID mice by tail vein injection,while non- transducedβ-thalassemia cells,β-thalassemia cells transduced by NC-GFP-LV and normal bone marrow cells were used as controls. After transplantation, the general conditions such as body weight of the mice were observed. Six weeks after the transplantation, flow cytometric detection of human cells in murine tissues (bone marrow and blood) was performed with monoclonal antibodies against human CD45. Electrophoresis of hemoglobin and detection ofβ-thalassemia gene mutations were performed in murine tissues (bone marrow or blood). In addition, spleen and liver of the mice were removed, observed, embedded, and sectioned. Specimens were stained with hematoxylin and eosin and iron stains. Results 1.The death rates of the mice in TBI/CY bone marrow transplantation group and TBI/CY cord blood transplantation group were all 100%.While the death rates of the mice in TBI bone marrow transplantation group and TBI cord blood transplantation group were all 33.3%. In TBI cord blood transplantation group, the percentage of human CD45+ cells in the bone marrow of the No. 13 and the No.6 mouse was 59.61% and 21.46%, respectively at week 6 after transplantation. While in TBI bone marrow transplantation group and control group, the percentage of that of each mouse was 0, respectively.2.Five weeks after transplantation, in theβ-thalassemia/HbE bone marrow transplantation group, the percentages of human CD45+ cells in murine tissues (bone marrow and blood) were 7.21% and 4.08% in +47 day, 7.37% and 6.03% in +61 day,respectively. And in theβ-thalassemia bone marrow transplantation group , those were 8.17% and 3.43% in +36 day, 16.82% and 8.89% in +45 day,respectively. There were HbA2 bands in hemoglobin electrophoresis of the transplantation group mice. Theirα-globin chain bands were thicker and wider than theβ-chain in hemoglobin chains analysis. In the transplantation group mice, the results ofβ-thalassemia gene mutations correspond with the donor. There wereα-globin chain precipitates in some erythroid cells.The ratios of the spleen length to body weight of the transplantation group mice were greater than those of control group[(0.5762±0.0451)vs(0.3628±0.0340)mm/g,P=0.000]. The iron deposition was observed in liver and spleen of the transplantation group mice.3.The exogenous gene sequence of the recombinant plasmids was completely in accordance with that of the humanα-antisense oligonucleotide. The titer of concentrated virus was 5×109TU/ml.4.Six weeks after the transplantation, the mice transplanted with normal bone marrow cells and blank mice became heavier, and other mice lost or maintained their weight. In bone marrow and blood of the mice being transplanted with human bone marrow cells, human CD45+ cells could be detected. In mice being transplanted withβ-thalassemia bone marrow cells (non-transduced or transduced by LV), the results ofβ-thalassemia gene mutations correspond with the donor. The ratio of the spleen length to body weight of the mice being transplanted withβ-thalassemia bone marrow cells (non-transduced or transduced by LV) were greater than that of other mice([0.6848±0.0886)v(s0.5573±0.0088)mm/g,P=0.016]. The iron deposition observed in liver and spleen of the mice being transplanted withα-ASON- LV-transducedβ-thalassemia bone marrow cells was less than that of the mice transplanted with non-transduced or NC-GFP-LV-transducedβ-thalassemia bone marrow cells.Conclusions The method whichβ-thalassemia bone marrow cells were transplanted into the sublethally irradiated NOD/SCID mice can generate humanizedβ-thalassemia mice models succesfully. The iron deposition of humanizedβ-thalassemia mice models was possibly reduced by lentiviral vectors carrying humanα-antisense oligonucleotide. The role of lentiviral vectors carrying humanα-antisense oligonucleotide in humanizedβ-thalassemia mice need to be observed continually.更多还原