rAAV9-CAG-Hamp在铁过载小鼠模型中治疗的安全性和有效性评估

发布时间：2018-02-04 05:07

本文关键词： 铁过载铁调素膜转运铁蛋白 rAAV9-hamp 治疗　出处：《广西医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:铁调素表达下调是引起非输血依赖型地贫铁过载的主要原因,目前认为可通过增加铁调素的表达用于治疗铁过载疾病。我们通过构建重组铁调素-腺相关病毒载体,并转染到铁过载小鼠模型体内,观察其在铁过载小鼠中对铁代谢的影响,并探讨其治疗效果和相关作用机制,为非输血依赖型地贫患者继发性铁过载的基因治疗奠定基础。方法:1.构建铁过载小鼠模型,16周后通过病理HE染色观察肝脏铁过载情况。2.包含目的基因小鼠hamp cDNA的腺相关病毒载体的构建、包装、纯化后获得滴度滴度1×1012vg/ml。3.铁过载小鼠随机分为A、B、C、D、E组5组,A、B、C组分别注射浓度为的:2.5×108vg/ml、2.5×109vg/ml、2.5×1010vg/ml的rAAV9-hamp、D组为2.5×1010vg/ml的含AAV-CAG、E组为PBS,注射体积分别为100μl。分别在第2天、14天、28天、56天分别处理5只小鼠,获取相应的组织标本。采用荧光检测腺相关病毒转染效率、流式细胞计数法测定外周全血CD8+细胞比例、Real-Time PCR检测肝脏hamp mRNA表达情况、western blot检测fpn蛋白表达情况以及亚铁嗪法、化学发光微粒子免疫检测法测定血清铁及血清铁蛋白(serum ferritin,SF)浓度。结果:1.铁过载小鼠模型肝脏、心脏HE染色见大量有明显的铁沉积。2.荧光显微镜下见注射含CAG基因的AAV的小鼠肝脏中荧光蛋白表达强度要明显高于PBS组;在第56天时,其表达强度无明显减弱。3.各组注射AAV的小鼠外周血CD8+细胞比例与PBS组相比未见有明显升高。4.低浓度实验组的hamp mRNA表达量在第2天、14天和56天时与E组比明显升高(p0.05),但其升高水平较中浓度组(A组)低;中浓度实验组(B组)的表达量在各时间段中的表达均明显升高(p0.05);高浓度组(C组)仅在第2天、14天、28天表达升高(p0.05),而D组的hamp mRNA表达变化与E组均无差异(p0.05)。膜转运铁蛋白(ferroportin,fpn1)的蛋白浓度在第2天时无明显降低;14天之后,三组实验组的蛋白浓度开始降低;第56天时,各实验组蛋白仍有降低趋势。各小组各时间段血清铁变化中以B组在第56天与E组相比下降幅度最大,下降水平达E组的48.6%(p0.05),而血清铁蛋白浓度均无明显改变(p0.05)。结论:我们的研究结果表明rAAV9-hamp可以在小鼠肝脏中成功转染,并使hamp基因在肝脏细胞中长期稳定表达;以中浓度hamp mRNA表达水平较为稳定,且表达水平明显高于PBS对照组;hamp高表达可降低十二指肠的fpn1蛋白浓度,抑制肠道铁吸收。rAAV9-hamp的成功转染及hamp的成功以及稳定的高表达可为我们下一步在hbb th3地贫鼠模型中治疗继发性铁过载提供依据。
[Abstract]:Objective: the down-regulation of iron modulin expression is the main cause of iron overload caused by non-transfusion dependent thalassemia. It is suggested that the recombinant iron-adeno-associated virus vector can be used to treat iron overload disease by increasing the expression of iron modulin. We have constructed the recombinant iron modulin-adeno-associated virus vector and transfected it into the iron overload mouse model. To observe the effect of iron metabolism on iron metabolism in iron overload mice and to explore its therapeutic effect and related mechanism. To lay a foundation for gene therapy of secondary iron overload in patients with non-transfusion dependent thalassemia. After 16 weeks, the liver iron overload was observed by HE staining. 2. Construction and packaging of adeno-associated virus vector containing the target gene hamp cDNA in mice. The titer of iron overload mice was 1 脳 1012vg / ml. The mice were randomly divided into 5 groups. Group C was injected with 2.5 脳 10 ~ 8vg / ml rAAV9-hamp at a concentration of 2.5 脳 10 ~ (8) vg 路ml ~ (-1), and 2.5 脳 10 ~ (9) vg 路ml ~ (-1) 路ml ~ (-1) of rAAV9-hamp. Group D (2.5 脳 10 ~ (10) v / ml) with AAV-CAGN E was treated with PBS.The injection volume was 100 渭 l, respectively, on the second day, 14 days and 28 days, respectively. Five mice were treated for 56 days and the corresponding tissue samples were obtained. The transfection efficiency of adeno-associated virus was detected by fluorescence and the percentage of peripheral blood CD8 cells was measured by flow cytometry. Real-Time PCR was used to detect the expression of hamp mRNA in liver and western blot to detect the expression of fpn protein. The serum iron and serum ferritin SFs were determined by chemiluminescence microparticle immunoassay. A large number of iron deposits were observed in the heart by HE staining. Under fluorescence microscope, the expression intensity of fluorescent protein in the liver of mice injected with AAV containing CAG gene was significantly higher than that in PBS group. On day 56. The percentage of CD8 cells in peripheral blood of mice injected with AAV in each group was not significantly increased compared with that in PBS group. 4. Hamp in low concentration experimental group. The expression of mRNA was on the second day. On the 14th and 56th day, compared with group E, the level of increase was significantly higher than that of group E, but the level of elevation was lower than that of group A. The expression of P0. 05 in the middle concentration group (group B) was significantly higher than that in the control group (P < 0. 05). In group C, the expression of P0. 05 was increased only on day 14 and day 28 (P < 0. 05). However, there was no difference between group D and group E in the expression of hamp mRNA. The protein concentration of fpn1) did not decrease at the 2nd day. After 14 days, the protein concentration of the three experimental groups began to decrease. On the 56th day, the protein of each experimental group still had the tendency to decrease. The change of serum iron in group B was the biggest in comparison with group E on the 56th day. The level of decrease reached 48.6% of group E (p0.05). However, serum ferritin concentration did not change p0.05. Conclusion: our results show that rAAV9-hamp can be successfully transfected in mouse liver. Hamp gene was stably expressed in liver cells for a long time. The expression level of hamp mRNA in medium concentration was stable, and the expression level was significantly higher than that in PBS control group. High expression of hamp could decrease the concentration of fpn1 protein in duodenum. Inhibition of the successful transfection of intestinal iron absorption. RAAV9-hamp, the success of hamp and the stable overexpression of hbb may be the next step in hbb. The treatment of secondary iron overload in th3 thalassemia rat model provides evidence.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R556.61

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