本文選題：地中海貧血 + 鐵過(guò)載　； 參考：《廣西醫(yī)科大學(xué)》2017年博士論文

【摘要】：地中海貧血(thalassemia)是一種遺傳性慢性溶血性貧血,重型β-地中海貧血需要依靠長(zhǎng)期輸血和去鐵治療以維持生命。長(zhǎng)期輸血以及小腸上皮細(xì)胞對(duì)鐵的吸收增加,可導(dǎo)致地中海貧血病人體內(nèi)鐵過(guò)載。鐵過(guò)載引起肝臟、心臟、內(nèi)分泌等多個(gè)器官功能衰竭,是地中海貧血患者死亡的主要原因,去鐵治療是重度β-地中海貧血治療的重要組成部分。鐵在人體內(nèi)的可溶性極低(10-18 M),還原鐵Fe++與過(guò)氧化氫反應(yīng)生成的羥基自由基,在游離鐵濃度過(guò)高時(shí),高度反應(yīng)活性的自由基能夠破壞的DNA,脂質(zhì)和蛋白質(zhì),可損害全身各組織器官,具有潛在的毒性,因此保持人體的鐵穩(wěn)態(tài)顯得尤其重要。MicroRNAs(miRNAs)是一類非編碼RNA,最早在真核生物中發(fā)現(xiàn),miRNAs具有內(nèi)源性的特點(diǎn),可調(diào)控多種生物功能。Micro RNAs也是調(diào)節(jié)鐵穩(wěn)態(tài)的調(diào)控因子之一,miRNA對(duì)鐵代謝具有靶向調(diào)控作用。本課題從小腸是鐵元素吸收的主要器官這一角度出發(fā),通過(guò)構(gòu)建鐵過(guò)載動(dòng)物模型,篩選出小腸上皮細(xì)胞鐵代謝相關(guān)蛋白的差異表達(dá)miRNA,對(duì)mi RNA和靶基因的調(diào)控關(guān)系進(jìn)行驗(yàn)證,在ICE-6細(xì)胞中的過(guò)表達(dá)mi RNA,研究該mi RNA調(diào)控IEC-6細(xì)胞的鐵代謝功能。預(yù)計(jì)本研究可為鐵代謝的miRNA調(diào)控、以及地中海貧血患者的去鐵治療提供研究思路和方向。第一章 鐵過(guò)載小鼠模型的構(gòu)建目的 構(gòu)建鐵過(guò)載小鼠模型,為下一步的研究提供合格實(shí)驗(yàn)標(biāo)本方法 采用6-8周齡的KM雄性小鼠隨機(jī)分為4組,腹腔注射不同劑量右旋糖酐鐵,觀察小鼠生長(zhǎng)發(fā)育情況,每周稱重并記錄。造模結(jié)束后采集小腸、肝、脾、心臟、腎等組織及血清鐵,測(cè)量鐵元素,同時(shí)采集肝、脾、心臟、腎等組織制作HE和鐵染色切片。結(jié)果 鐵過(guò)載小鼠出現(xiàn)外觀改變,生長(zhǎng)發(fā)育落后。觀察各組織器官可見形態(tài)顏色及質(zhì)地改變,呈現(xiàn)明顯的鐵沉積,檢測(cè)結(jié)果顯示血清鐵蛋白及小腸、肝、脾、心臟、腎等組織鐵含量增加,程度和注射鐵的劑量成正比。HE切片發(fā)現(xiàn)組織器官有病理改變,鐵染色切片看見有不同程度的鐵沉積,與注射鐵的劑量成正比。結(jié)論 腹腔注射右旋糖酐鐵可以構(gòu)建符合后期實(shí)驗(yàn)要求的鐵過(guò)載小鼠。第二章 鐵過(guò)載小鼠小腸上皮細(xì)胞差異表達(dá)miRNA的篩選與驗(yàn)證研究一 鐵過(guò)載小鼠小腸上皮細(xì)胞差異表達(dá)miRNA的篩選目的 比較正常對(duì)照組和高鐵組小鼠小腸細(xì)胞的高通量小RNA測(cè)序結(jié)果,篩選出鐵代謝相關(guān)基因的差異表達(dá)miRNA。方法 提取對(duì)照組和高鐵組兩組小鼠小腸上皮細(xì)胞的總RNA,構(gòu)建RNA文庫(kù),選擇合格文庫(kù)進(jìn)行高通量小RNA測(cè)序。評(píng)估測(cè)序質(zhì)量、分析其特有序列,對(duì)測(cè)序數(shù)據(jù)進(jìn)行基因組比對(duì)、miRNA的差異分析、差異表達(dá)miRNA的靶基因預(yù)測(cè)、GO富集分析、KEGG代謝通路分析,篩選出差異表達(dá)miRNA。對(duì)差異表達(dá)的miRNA采用Tagretscan等多個(gè)數(shù)據(jù)庫(kù)進(jìn)行交叉分析比較,篩選出可能靶向調(diào)控小腸鐵代謝相關(guān)基因的miRNA。結(jié)果 共有正常組、對(duì)照組各3個(gè)文庫(kù)進(jìn)行測(cè)序,共篩選出9個(gè)差異表達(dá)miRNA,通過(guò)對(duì)這9個(gè)miRNA進(jìn)行比較分析,最終選擇miR-143,miR let-7d進(jìn)行下一步的驗(yàn)證。結(jié)論 對(duì)兩組標(biāo)本進(jìn)行小RNA高通量測(cè)序,對(duì)測(cè)序結(jié)果進(jìn)行生物信息學(xué)分析及靶基因預(yù)測(cè),篩選出小鼠小腸細(xì)胞鐵代謝相關(guān)miRNA。研究二 miR let-7d靶向調(diào)控Slc11a2基因及miR 143靶向調(diào)控Slc40a1基因的研究目的 探討miR let-7d靶向調(diào)控Slc11a2基因及miR 143靶向調(diào)控Slc40a1基因的關(guān)系方法 分別構(gòu)建Slc11a2基因和Slc40a1基因3’-UTR的野生型和突變型載體,同時(shí)合成mi R let-7d mimics和mi R 143 mimics,將mi RNA與構(gòu)建好的報(bào)告基因載體共轉(zhuǎn)染至293T細(xì)胞,通過(guò)檢測(cè)報(bào)告基因相對(duì)熒光值的改變,以此驗(yàn)證該mi RNA和該靶基因的調(diào)控關(guān)系。結(jié)果 雙熒光素酶的表達(dá)分析顯示,mi R143對(duì)Slc40al基因野生型載體的熒光值沒(méi)有下調(diào)作用。說(shuō)明在該實(shí)驗(yàn)?zāi)Ｐ拖?mi R143可能不能通過(guò)Slc40al基因上相應(yīng)的3’UTR位點(diǎn)調(diào)控報(bào)告基因的表達(dá)。而mi R let-7d對(duì)Slc11a2野生型載體的熒光值有明顯下調(diào)作用,下調(diào)差別有統(tǒng)計(jì)學(xué)意義(P0.01)。說(shuō)明在該實(shí)驗(yàn)?zāi)Ｐ拖?mi R let-7d可以通過(guò)Slc11a2基因上相應(yīng)的3’UTR位點(diǎn)調(diào)控報(bào)告基因的表達(dá)。結(jié)論 首次通過(guò)構(gòu)建動(dòng)物模型篩選出mi R let-7d,并證實(shí)其可以通過(guò)與Slc11a2基因上相應(yīng)的3’UTR位點(diǎn)調(diào)控報(bào)告基因的表達(dá),Slc11a2基因是miR let-7d的靶基因。miR143不能通過(guò)Slc40al基因上相應(yīng)的3’UTR位點(diǎn)調(diào)控報(bào)告基因的表達(dá),不能確定Slc40al是miR143的靶基因。第三章 miR let-7d靶向調(diào)控IEC-6細(xì)胞鐵代謝功能的研究目的 研究在ICE-6細(xì)胞中過(guò)表達(dá)mi R let-7d是否能靶向調(diào)控Slc11a2基因的m RNA和蛋白表達(dá),及調(diào)控ICE-6細(xì)胞對(duì)鐵的吸收功能。方法 在IEC-6細(xì)胞中過(guò)表達(dá)miR let-7d,熒光顯微鏡觀察IEC-6細(xì)胞的轉(zhuǎn)染效率,采用Western Blot實(shí)驗(yàn)及q RT-PCR實(shí)驗(yàn),檢測(cè)轉(zhuǎn)染后的Slc11a2蛋白表達(dá)量和擴(kuò)增溶解曲線。使用含100mM的Fe SO4高鐵DMEM-H培養(yǎng)基和普通DMEM-H培養(yǎng)基培養(yǎng)IEC-6細(xì)胞,并轉(zhuǎn)染mi R let-7d mimics到細(xì)胞內(nèi),完成轉(zhuǎn)染后收集細(xì)胞,對(duì)細(xì)胞進(jìn)行計(jì)數(shù)后,原子吸收法測(cè)定細(xì)胞鐵含量。另取培養(yǎng)板培養(yǎng)細(xì)胞進(jìn)行鐵染色,在倒置顯微鏡下觀察細(xì)胞染色改變。結(jié)果 轉(zhuǎn)染miR let-7d mimics后熒光顯微鏡下觀察ICE-6細(xì)胞的轉(zhuǎn)染效率在75%以上,說(shuō)明mi R let-7d轉(zhuǎn)染IEC-6細(xì)胞成功。過(guò)表達(dá)后的Western Blot結(jié)果顯示空白對(duì)照組和處理組的蛋白表達(dá)量有明顯區(qū)別,轉(zhuǎn)染組的Slc11a2蛋白條帶量減少。q RT-PCR檢測(cè)Slc11a2基因mRNA的表達(dá)量,提示轉(zhuǎn)染組mRNA的表達(dá)量下降,和空白對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義。結(jié)論 首次發(fā)現(xiàn)了miR let-7d可轉(zhuǎn)染IEC-6細(xì)胞,miR let-7d通過(guò)靶向調(diào)控Slc11a2基因,抑制IEC-6細(xì)胞對(duì)鐵的吸收。
[Abstract]:Thalassemia (thalassemia) is a hereditary chronic hemolytic anemia. Severe beta thalassemia needs to rely on long-term blood transfusions and iron removal to maintain life. Long-term blood transfusion and increased absorption of iron by small intestinal epithelial cells can lead to iron overload in patients with thalassemia. Iron overload causes liver, heart, endocrinology and so on. Organ failure is the main cause of death in patients with thalassemia. Iron removal is an important part of the treatment of severe beta thalassemia. The soluble iron is extremely low in the human body (10-18 M), the reduction of iron Fe++ and hydrogen peroxide in the hydroxyl radical, when the free iron concentration is too high, the highly reactive free radical can be found. The damaged DNA, lipid and protein can damage the organs and organs of the whole body and have potential toxicity. Therefore, it is particularly important to maintain the iron homeostasis of the human body..MicroRNAs (miRNAs) is a class of non coded RNA. The earliest found in eukaryotes that miRNAs has endogenous characteristics and can regulate a variety of biological functions.Micro RNAs as well as regulating iron homeostasis. One of the regulatory factors, miRNA has a targeted regulation of iron metabolism. From the point of view of the small intestine as the main organ of iron absorption, the differential expression of iron metabolism related proteins in small intestinal epithelial cells is screened by constructing an iron overload animal model, and the regulatory relationship between MI RNA and target genes is verified, and ICE-6 cells are used in ICE-6 cells. The overexpression of MI RNA, in which the MI RNA regulates the iron metabolism of IEC-6 cells, is expected to provide research ideas and directions for the regulation of miRNA in iron metabolism, as well as for the treatment of iron removal in thalassemia patients. KM male mice of 6-8 weeks of age were randomly divided into 4 groups. Different doses of iron dextran was injected into the abdominal cavity. The growth and development of the mice were observed and weighed and recorded every week. The small intestine, liver, spleen, heart, kidney and other tissues and serum iron were collected after the model was finished, and the iron elements were measured, and the liver, spleen, heart, kidney and other tissues were collected to make HE and iron staining. The results showed that the iron content of serum ferritin and small intestine, liver, spleen, heart, kidney and other tissues increased, the degree of range and the dose of injection iron were in direct proportion to.HE slices found in tissues and organs. There is a pathological change. Iron staining section shows a different degree of iron deposition and is proportional to the dose of the iron injection. Conclusion intraperitoneal injection of dextran iron can construct the iron overload mice that meet the requirements of the later experiment. Second the screening and verification of differential expression of miRNA in small intestinal epithelial cells of iron overload mice Screening of differential expression of miRNA in skin cells compared the high throughput small RNA sequencing results of small intestinal cells in normal control group and high iron group, screening the differential expression of iron metabolism related genes by differential expression miRNA. method to extract the total RNA of small intestinal epithelial cells of two groups of mice in the control group and the high iron group, construct the RNA library and select the qualified library for high flux. Small RNA sequencing, evaluation of the quality of sequencing, analysis of its unique sequence, genomic alignment of sequencing data, miRNA difference analysis, differential expression of miRNA target gene prediction, GO enrichment analysis, KEGG metabolic pathway analysis, screening and screening of differential expression miRNA. for miRNA acquisition of different expressions of Tagretscan and other databases for cross analysis and comparison. The miRNA. results that could be targeted to regulate the genes related to iron metabolism in the small intestine were selected. The control group was sequenced in 3 libraries, and 9 different expressions of miRNA were screened. Through the comparison and analysis of the 9 miRNA, the final selection of miR-143, miR let-7d was verified by the next step. Conclusion high throughput sequencing of the two groups of specimens was carried out. Bioinformatics analysis and target gene prediction of sequencing results were used to screen the iron metabolism related miRNA. study of mouse small intestinal cells two miR let-7d target regulation Slc11a2 gene and miR 143 targeted Slc40a1 gene, and the relationship between miR let-7d targeting Slc11a2 gene and miR 143 target to control Slc40a1 gene The wild type and mutant vector of Slc11a2 gene and Slc40a1 gene 3 '-UTR were constructed respectively, and MI R let-7d mimics and MI R 143 mimics were synthesized, and MI RNA and constructed reporter gene carrier were co transfected to the cell. The expression analysis of double luciferase showed that MI R143 had no down-regulation on the fluorescence value of the Slc40al gene, which indicated that MI R143 could not regulate the expression of the reporter gene by the corresponding 3 'UTR locus on the Slc40al gene. And the MI R let-7d decreased the fluorescence of the Slc11a2 wild type carrier. The difference was statistically significant (P0.01). Under this experimental model, MI R let-7d could regulate the expression of the reporter gene through the corresponding 3 'UTR locus on the Slc11a2 gene. Conclusion the first animal model was first screened for MI R let-7d, and it was confirmed that it could be regulated by the corresponding 3' UTR locus on the Slc11a2 gene. Because of the expression, the Slc11a2 gene is the target gene of miR let-7d,.MiR143 can not regulate the expression of the reporter gene by the corresponding 3 'UTR locus on the Slc40al gene, and the target gene of Slc40al is not determined. The aim of the third chapter miR let-7d to regulate the metabolic function of IEC-6 cells is to study the overexpression in the ICE-6 cells. To regulate the expression of M RNA and protein of Slc11a2 gene, and to regulate the absorption function of ICE-6 cells to iron. Methods the expression of miR let-7d in IEC-6 cells was overexpressed in IEC-6 cells. The transfection efficiency of IEC-6 cells was observed by fluorescence microscopy. Western Blot experiment and Q RT-PCR experiment were used to detect the expression of the protein and the expansion and dissolution curve of the transfected protein. IEC-6 cells were cultured with 100mM Fe SO4 high iron DMEM-H medium and ordinary DMEM-H medium, and IEC-6 cells were transfected with MI R let-7d mimics. After transfection, the cells were collected. After counting the cells, the iron content was measured by atomic absorption spectrometry. The culture plate culture cells were also stained with iron, and the cell staining changes were observed under the inverted microscope. Results after transfection of miR let-7d mimics, the transfection efficiency of ICE-6 cells was more than 75% under the fluorescence microscope, indicating that MI R let-7d transfected to IEC-6 cells successfully. The results of Western Blot after overexpression showed that the protein expression of the blank control group and the treated group was distincently different, and the Slc11a2 protein band in the transfected group was reduced by.Q. The expression of gene mRNA indicated that the expression of mRNA in the transfected group decreased, and there was a significant difference between the control group and the blank control group. The conclusion was that miR let-7d could be transfected to IEC-6 cells for the first time, and miR let-7d regulated the Slc11a2 gene by targeting and inhibited the absorption of iron by IEC-6 cells.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R556.61

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本文編號(hào)：1870948