本文關(guān)鍵詞： 鐵過載 鐵調(diào)素 膜轉(zhuǎn)運鐵蛋白 rAAV9-hamp 治療　出處：《廣西醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:鐵調(diào)素表達下調(diào)是引起非輸血依賴型地貧鐵過載的主要原因,目前認(rèn)為可通過增加鐵調(diào)素的表達用于治療鐵過載疾病。我們通過構(gòu)建重組鐵調(diào)素-腺相關(guān)病毒載體,并轉(zhuǎn)染到鐵過載小鼠模型體內(nèi),觀察其在鐵過載小鼠中對鐵代謝的影響,并探討其治療效果和相關(guān)作用機制,為非輸血依賴型地貧患者繼發(fā)性鐵過載的基因治療奠定基礎(chǔ)。方法:1.構(gòu)建鐵過載小鼠模型,16周后通過病理HE染色觀察肝臟鐵過載情況。2.包含目的基因小鼠hamp cDNA的腺相關(guān)病毒載體的構(gòu)建、包裝、純化后獲得滴度滴度1×1012vg/ml。3.鐵過載小鼠隨機分為A、B、C、D、E組5組,A、B、C組分別注射濃度為的:2.5×108vg/ml、2.5×109vg/ml、2.5×1010vg/ml的rAAV9-hamp、D組為2.5×1010vg/ml的含AAV-CAG、E組為PBS,注射體積分別為100μl。分別在第2天、14天、28天、56天分別處理5只小鼠,獲取相應(yīng)的組織標(biāo)本。采用熒光檢測腺相關(guān)病毒轉(zhuǎn)染效率、流式細胞計數(shù)法測定外周全血CD8+細胞比例、Real-Time PCR檢測肝臟hamp mRNA表達情況、western blot檢測fpn蛋白表達情況以及亞鐵嗪法、化學(xué)發(fā)光微粒子免疫檢測法測定血清鐵及血清鐵蛋白(serum ferritin,SF)濃度。結(jié)果:1.鐵過載小鼠模型肝臟、心臟HE染色見大量有明顯的鐵沉積。2.熒光顯微鏡下見注射含CAG基因的AAV的小鼠肝臟中熒光蛋白表達強度要明顯高于PBS組;在第56天時,其表達強度無明顯減弱。3.各組注射AAV的小鼠外周血CD8+細胞比例與PBS組相比未見有明顯升高。4.低濃度實驗組的hamp mRNA表達量在第2天、14天和56天時與E組比明顯升高(p0.05),但其升高水平較中濃度組(A組)低;中濃度實驗組(B組)的表達量在各時間段中的表達均明顯升高(p0.05);高濃度組(C組)僅在第2天、14天、28天表達升高(p0.05),而D組的hamp mRNA表達變化與E組均無差異(p0.05)。膜轉(zhuǎn)運鐵蛋白(ferroportin,fpn1)的蛋白濃度在第2天時無明顯降低;14天之后,三組實驗組的蛋白濃度開始降低;第56天時,各實驗組蛋白仍有降低趨勢。各小組各時間段血清鐵變化中以B組在第56天與E組相比下降幅度最大,下降水平達E組的48.6%(p0.05),而血清鐵蛋白濃度均無明顯改變(p0.05)。結(jié)論:我們的研究結(jié)果表明rAAV9-hamp可以在小鼠肝臟中成功轉(zhuǎn)染,并使hamp基因在肝臟細胞中長期穩(wěn)定表達;以中濃度hamp mRNA表達水平較為穩(wěn)定,且表達水平明顯高于PBS對照組;hamp高表達可降低十二指腸的fpn1蛋白濃度,抑制腸道鐵吸收。rAAV9-hamp的成功轉(zhuǎn)染及hamp的成功以及穩(wěn)定的高表達可為我們下一步在hbb th3地貧鼠模型中治療繼發(fā)性鐵過載提供依據(jù)。
[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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R556.61

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