miR-144在造血中的氧化应激初步研究

【作者】 王婷；

【导师】 郁多男；

【作者基本信息】 扬州大学 ， 生理学， 2019， 硕士

【摘要】 本研究首先构建了 miR-144和miR-451单独敲除的小鼠模型。分析了这两种动物模型的表型改变,并与miR-144/451双敲除小鼠作比较。旨在明确miR-144/451双敲除时候的红细胞抗氧化能力下降是由miR-144单敲除引起或miR-451单敲除引起还是两者协同作用。本课题利用野生型小鼠(wt)、miR-144/451基因敲除鼠(miR-144/451-/-,mko)、miR-144 和 miR-451(miR-144-/-和 miR-451-/-,miR-144 ko 和 miR-451 ko)单敲除小鼠作为体内研究对象,通过利用wt、mko、miR-144 ko和miR-451 ko共4组不同基因型的小鼠,进行以下实验:1)检测miR-144和miR-451在外周血、骨髓红细胞中表达趋势,进一步验证miR-144和miR-451基因表达缺失后小鼠贫血现象;2)通过体内外模型阐明miR-144和miR-451功能缺失后小鼠红细胞对各种应激的反应;3)通过体内实验进一步明确mko贫血小鼠与单基因敲除小鼠外周血中超氧化剂(ROS)水平,从而进一步阐明miR-144和miR-451的抗氧化作用。通过以上实验,本研究旨在明确miR-144和miR-451功能缺失后的表型改变,以及贫血的可能分子机制。研究内容分为以下三个部分:第一部分miR-144和miR-451基因单敲除小鼠模型的建立目的:构建miR-144和miR-451缺失的小鼠模型。方法:由上海邦耀生物科技有限公司帮助构建。首先利用麻省理工学院张峰教授实验室的公用网站设计先导RNA,然后利用crispr/cas9技术敲除包括miR-144前体在内的75bp以及miR-451前体在内的72bp,构建miR-144和miR-451单敲除小鼠。由公司提供的miR-144和miR-451单敲除杂合子小鼠,通过杂合子-杂合子杂交的方法获得纯合子利用PCR方法鉴定miR-144和miR-451的杂合子和纯合子。统计子代各种基因型小鼠的存活数量是否符合孟德尔遗传定律。结果:通过PCR鉴定证实这两种单敲除小鼠的miR-144和miR-451的表达量比野生型小鼠低约4000倍,说明miR-144和miR-451缺失。经杂合子-杂合子杂交的方法所得到的总量小鼠一共572只,其中,wt小鼠147只,miR-144-/+小鼠275只,miR-144ko小鼠150只,符合孟德尔遗传定律,说明敲除miR-144和miR-451的小鼠生存正常。miR-451单敲除小鼠子代和miR-144单敲除小鼠子代结果相类似,也符合孟德尔遗传定律。结论:通过crispr/cas9技术成功敲除miR-144和miR-451单个基因,敲除miR-144和miR-451的小鼠生存正常,小鼠子代各基因型小鼠生存符合孟德尔定律。第二部分miR-144在无应激状态下的造血研究目的:明确miR-144和miR-451单敲除小鼠有无表型改变,并且将4组实验小鼠做比较,确认原miR-144/451双敲除小鼠的表型改变是由miR-144所引起还是miR-451所引起,又或是由miR-144和miR-451两者协同作用所引起。方法:通过检测6-8周龄的4组基因型小鼠(wt,miR-144ko,miR-451 ko,mko)的外周血中各种血细胞指标,明确各种小鼠贫血情况;制作外周血细胞涂片,并进行瑞氏-吉姆萨染色,明确4组小鼠外周血中红细胞形态、数量;运用流式细胞技术检测4组小鼠外周血和骨髓中的红细胞,明确红细胞的分化成熟度;检测4种小鼠脾脏的形态学及组织学变化,明确各组小鼠的代偿性造血水平。结果:1)通过全血细胞仪分析提示,miR-144ko和wt小鼠外周血参数相近,而miR-451 ko和mko小鼠外周血参数相近;2)外周血细胞涂片瑞氏-吉姆萨染色结果提示:miR-144 ko小鼠红细胞大小较均匀,与wt相同,而miR-451 ko小鼠红细胞宽度有一定程度增加,形态也与mko相似;3)流式细胞仪检测结果提示:与wt组小鼠比较,miR-451 ko组小鼠的外周血中网织红细胞比例有一定程度升高,与mko相似,而miR-144 ko与wt相似;4)脾脏形态学及组织学结果提示:与wt组小鼠比较,miR-451 ko组小鼠的脾脏一定程度增大,重量也增加,组织学结果提示脾脏中红细胞数量一定程度增加,而miR-144 ko组小鼠与wt组小鼠无异。结论:miR-144功能缺失后小鼠表型改变不明显,而miR-451功能缺失后小鼠和mko小鼠表型改变相似,提示有轻度贫血现象。因此,我们猜测原miR-144/451双敲除小鼠的表型改变可能主要是由于miR-451功能缺失所引起的。第三部分miR-144在应激状态下的造血研究目的:检测在不同应激状态下miR-144和miR-451单敲除小鼠的表型改变。方法:通过物理、化学等方法建立小鼠贫血模型,观察各类贫血小鼠精神状况,皮肤黏膜情况以及存活情况,主要模拟急性失血性贫血、急性溶血性贫血等模型从而进一步明确应激后的表型改变。1)取6-8周龄的4组基因型小鼠(wt,miR-144ko,miR-451 ko,mko)各三只,通过眼眶内眦静脉窦连续三天放血模拟急性失血性贫血小鼠模型,然后检测外周血细胞参数,明确各种小鼠贫血情况;2)取6-8周龄的4组基因型小鼠(wt,miR-144 ko,miR-451 ko,mko)各三只,通过腹腔注射一定浓度的苯肼溶液模拟急性溶血性小鼠模型,通过眼眶内眦静脉窦采取小鼠外周血检测,记录相关指标变化,检测小鼠溶血严重程度;3)取4组基因型小鼠(wt,miR-144 ko,miR-451 ko,mko)通过眼眶内眦静脉窦采血得到外周血,制成细胞悬液,DCFH-DA孵育标记后,流式细胞仪检测外周血红细胞中的ROS表达;并予以过氧化氢(H2O2)作用红细胞后检测ROS。结果:1)急性贫血性小鼠模型的小鼠都出现一过性失血过多,行动变迟缓,急性溶血小鼠模型四组小鼠都出现溶血症状,各组小鼠的皮肤和黏膜呈现苍白状况,外周血红细胞数量、血红蛋白含量、红细胞压积降低,网织红细胞比例增高,小鼠表现为溶血状态,2)急性溶血性贫血模型小鼠的外周血流式细胞仪检测结果提示:腹腔注射苯肼溶液后4组小鼠的成熟红细胞全部急剧减少,网织红细胞明显增加,检测ROS水平,miR-451 ko组与mko组的ROS水平明显高于wt组和miR-144 ko组;3)流式细胞仪检测H2O2处理后4组小鼠外周血的结果提示,与wt组小鼠比较,mko组小鼠ROS水平明显升高,miR-451 ko组ROS水平与mko趋同,miR-144 ko组与wt相似。结论:miR-451功能缺失时小鼠氧化应激能力下降,表明抗氧化能力升高,而miR-144功能缺失小鼠与wt组相似,表明其抗氧化功能不明显。本实验模拟的急性失血性贫血模型小鼠以及溶血性贫血小鼠模型为临床人类急性失血性贫血和急性溶血性贫血时应激造血提供了相关支持性。更多还原

【Abstract】 This study first constructed mouse models of miR-144 and miR-451 single knockout.Phenotypic changes in these animals were analyzed and compared to miR-144/451 double knockout mice.The aim was to clarify whether the decrease in erythrocyte antioxidant capacity in miR-144/451 double knockout mice was caused by miR-144 single knockout or miR-451 single knockout or a synergistic effect.This study utilizes four genetypes of mice:wild-type mouse(wt),miR-144/451 knockout mice(miR-144/451-/-,mko),miR-144 and miR-451(miR-144-/-and miR-451-/-,miR-144 ko and miR-451 ko)single knockout mice and following experiments were designed:1)construction of miR-144 and miR-451 single knockout animals,validation of miR-144 and miR-451 expression,and after gene deletion,verifying the anemic phenotype of such mice;2)observation of the response of mouse erythrocytes to various stresses after miR-144 and miR-451 gene loss;3)determination of whether high reactive oxidant species(ROS)level in double miR-144/451 knockout animals are caused by miR-144 or miR-451.The project is divided into the following three parts:Part Ⅰ.Establishment of single knockout mouse model of miR-144 and miR-451 genesObjective:To construct a mouse model of miR-144 and miR-451 deletion.Method:Constructed by Shanghai Bangyao Company.First,the guide RNAs were designed using the public website of Professor Zhang Feng of the Massachusetts Institute of Technology,and then the 75 bp including the miR-144 precursor and the 72 bp of the miR-451 precursor were knocked out using the crispr/cas9 technique to construct the miR-144 and miR-451 single knockout mice.The miR-144 or miR-451 knockout heterozygous mice provided by the company were crossed to obtain homozygous animals.Genetyping was done by PCR.The number of survivors of various genotype mice in the progeny is counted to determine the accordance with Mendel’s law of inheritance.Results:The expression levels of miR-144 and miR-451 in the two single knockout mice were confirmed by PCR to be about 4000-fold lower than in wild-type mice.This indicates that miR-144 and miR-451 are deleted.A total of 572 mice were obtained by heterozygous crossing,and among them 147 are wt mice,275 are miR-144 heterozygous mice,and 150 are miR-144 homozygous ko mice,in line with Mendelian inheritance.Similar result was obtained for miR-451 single knockout mice.These results indicate that mice knocked out of miR-144 or miR-451 survived normally.Conclusion:The miR-144 and miR-451 single genes were successfully knocked out by crispr/cas9 technology.The mice knocked out of miR-144 and miR-451 survived normally.Part Ⅱ.Hematological study of miR-144 and miR-451 knockout mice at baselineObjective:To determine whether phenotypic changes were observed in miR-144 and miR-451 single knockout mice,and to confirm whether the phenotypic changes in the original miR-144/451 double knockout mice were caused by loss of miR-144 or miR-451,or caused by the synergy of both miR-144 and miR-451 loss.Methods:Blood cells from peripheral blood of 4 groups of mice(wt,miR-144 ko,miR-451 ko,mko)at the age of 6-8 weeks were used to determine the anemic phenotypes of various mice.Peripheral blood was prepared for complete blood count(CBC)analyses;the blood smear and the Wright-Gemsa staining were used to determine the morphology and quantity of red blood cells in the peripheral blood;flow cytometry was used to detect the reticulocytes in peripheral blood and bone marrows.The morphological changes of the spleens of the four types of mice were also examined by histology,and the compensatory hematopoietic levels of the mice in each group were determined.Results:1)Whole blood cytometry analysis indicated that the peripheral blood parameters of miR-144 ko and wt mice were similar,while the peripheral blood parameters of miR-451 ko and mko mice were similar;2)peripheral blood smear with Wright-Giemsa staining showed that the size of red blood cells from miR-144 ko mice is uniform and the same as wt,while the width of miR-451 red blood cells is increased to some extent,and the morphology is similar to that of mko.3)Flow cytometry analysis indicated that the proportion of reticulocytes in the peripheral blood of mice in the miR-451 ko group increased to some extent,similar to mko,while miR-144 ko was similar to wt;4)morphological and histological findings of spleen:compared with wt group mice,the spleen of the miR-451 ko group increased to a certain extent and the weight increased.The histological results indicated that the number of red blood cells in the spleen increased to some extent,while the miR-144 ko group was no different from the wt group.Conclusion:The phenotypic changes of mice after miR-144 depletion are not obvious,and the phenotypic changes of mice after miR-451 loss are similar,both with mild anemia.Therefore,we conclude that phenotypic changes in the original miR-144/451 double knockout mice are mainly due to the loss of miR-451 function.Part III.Hematological study of miR-144 and miR-451 knockout mice under different stress conditionsObjective:To detect phenotypic changes in miR-144 and miR-451 single knockout mice under different stress conditionsMethods:The mouse anemia models were established by physical and chemical methods to observe the mental state,skin mucosa and survival of various anemia mice.The model of acute hemorrhagic anemia and acute hemolytic anemia was simulated to further clarify the phenotype change.1)four groups of 6-8 weeks old mice(wt,miR-144 ko,miR-451 ko,mko)were used to simulate acute hemorrhagic anemia mice by exsanguination of the orbital iliac vein for three consecutive days,and peripheral blood cell parameters were determined in various mice;2)the acute hemolytic mouse model was simulated by intraperitoneal injection of a certain concentration of benzoquinone solution.The peripheral blood of the mice was taken through the orbital sputum,and the changes of related indexes were recorded to detect the hemolytic severity of the mice.3)peripheral blood was obtained from four groups of genotype mice(wt,miR-144 ko,miR-451 ko,mko)by sampling from the eyelids,and the cell suspension was prepared.After DCFH-DA labeling,flow cytometry was used to detect ROS levels in peripheral blood cells;Hydrogen peroxide(H2O2)was used to increase the oxidant stress on red blood cells.Results:1)The mice in the acute blood loss mouse model showed excessive transient blood loss.The mice in the acute hemolysis mouse model showed lysis of red cells.The skin and mucous membrane of each group showed paleness.Peripheral blood red blood cell count,hemoglobin content and hematocrit decreased while reticulocyte rate increased dramatically,indicating acute hemolysis,2)The mature red blood cells from all four groups of mice decreased sharply while reticulocytes increased significantly.The ROS levels in the miR-451 ko group and the mko group were significantly higher than those in the wt group and the miR-144 ko group.3)compared with the wt group,the ROS level of the mko group was significantly increased,the ROS level of the miR-451 ko group was similar to that of mko,and the miR-144 ko group was similar to wt.Conclusion:The anti-oxidative ability of red blood cells is decreased when miR-451 is absent,indicating that miR-451 elevates antioxidant capacity.ROS level in miR-144 knockout red cells is similar to that of the wt group,suggesting no antioxidant function for miR-144.更多还原