【作者】 李传芬；

【导师】 王敏；

【作者基本信息】 山东师范大学 ， 动物学， 2019， 博士

【摘要】 目的:构建人源HtrA1^L364P转基因小鼠(Mut-hHtrA1^L364P小鼠)并对其进行表型分析和致病机理研究伴有皮质下梗死和白质病变的常染色体隐性遗传性脑动脉病（cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy,CARASIL）是一种罕见的常染色体隐性遗传的脑小血管病（cerebral small vessel diseases,CSVD）,该病患者临床表现为卒中、进行性认知功能下降、假性球麻痹、早发性秃顶、反复腰痛。头颅磁共振显示多发性脑梗死和广泛皮层下白质脱髓鞘。尸检患者脑组织可见小动脉动脉硬化,中膜发生玻璃样病变,内膜和内弹力层纤维化增厚以及管腔向心性狭窄。该疾病的全球发病率较低,目前有文献报道的共50余例,其致病基因由日本学者Hara确定为高温需求因子A1（High temperature requirement A1,Htr A1）。2007年本研究组在国内首次报道一CARASIL家系,其临床特征均符合CARASIL的症状,测序结果显示其Htr A1的c DNA上发生了T1091C突变,导致364位点上的亮氨酸（Leu,L）突变为了脯氨酸（Pro,P）。目前国内外尚没有成功建立CARASIL模式动物,这也限制了对CARASIL的研究。本研究拟依据前期报道的CARASIL家系中发现的Htr A1基因突变位点构建模式小鼠,进而对该病进行病理分析及致病机理研究。转基因鼠是目前常用的疾病模型之一,本研究拟利用CRISPR/Cas9技术在小鼠Htr A1基因信号肽后面定向插入人源HtrA1^L364P无信号肽序列,使其融合表达。一方面,人源HtrA1^L364P的插入可以敲除小鼠本底Htr A1,通过表型分析,可以研究人源HtrA1^L364P转基因鼠是否引起CARASIL疾病;另一方面,人源HtrA1^L364P融合于小鼠Htr A1信号肽之后,不仅使其表达定位不会发生改变,其5’端受调控机制也不会受到影响。因此,可以更好的模拟人类发病情况。通过建立人源HtrA1^L364P敲入转基因鼠(此模型鼠命名为Mut-hHtrA1^L364P小鼠),可以分析HtrA1^L364P的致病机理,作为CARASIL及CSVD等相关疾病的研究模型。方法:研究共分为两个部分。第一部分:Mut-hHtrA1^L364P小鼠的构建及鉴定首先,构建Donor载体。通过检索人源和鼠源Htr A1的CDS序列、蛋白序列和基因组序列,分析确认各自的信号肽区域;根据小鼠Htr A1蛋白信号肽的位置,选择小向导RNA（small guide RNA,sg RNA）靶点所在区域;在该区域中,设计三条sg RNA和Cas9共同转染细胞,通过T7E1酶切法验证sg RNA的细胞内活性,从而选择最优的sg RNA并据此克隆左右同源臂。同时,获取人源Htr A1模板,对其进行定点突变,突变序列选择在小鼠中高效表达脯氨酸的CCG密码子。利用PCR法扩增人源Htr A1的不含信号肽区域,并对人源Htr A1进行定点突变,获得人源HtrA1^L364P序列,并将其插入克隆的左右同源臂之间,得到CRISPR/Cas9敲入的Donor载体。其次,对健康雌性小鼠进行超数排卵后合笼,以外科手术方式获取见阴道栓小鼠的输卵管,从而分离得到受精卵;同时利用结扎的雄鼠同步得到假孕的母鼠。利用体外转录的方法获得sg RNA、Cas9和Donor的c RNA,将其混合后显微注射到获得的受精卵中,随后,挑选健康的受精卵并移植回假孕母鼠体内。待新生鼠出生后,剪取新生鼠脚趾并进行PCR鉴定,获得转基因阳性鼠。同时,将阳性鼠与同背景野生型（wide type,WT）小鼠进行杂交,获得转基因纯合鼠,建立Mut-hHtrA1^L364P小鼠种群,并通过精子冷冻的方式保存精子。分离Mut-hHtrA1^L364P小鼠的原代脑血管平滑肌细胞（Vascular smooth muscle cells,VSMCs）,利用细胞免疫荧光技术检测血管平滑肌22α（smooth muscle22α,SM22α）的表达来确认人为得到的VSMCs。在此基础上,利用免疫印迹（Western Blot）和免疫荧光技术等方式,对Mut-hHtrA1^L364P小鼠大脑VSMCs中人源HtrA1^L364P的表达进行检测。第二部分:Mut-hHtrA1^L364P小鼠的表型分析及致病机理研究首先,对Mut-hHtrA1^L364P小鼠进行表型分析:观察其外表形态和活动状态;进行行为学分析:包括食物迷宫和水迷宫;进行病理学检测:包括苏木素伊红染色（hematoxylin-eosin s,HE）,弹力纤维染色,特异性抗体:平滑肌肌动蛋白（smooth muscle actin,SMA）、肌动蛋白（Actin）、CD31、CD68标记的免疫组化染色以及观察细胞结构和亚细胞结构的电镜检测。其次,对Mut-hHtrA1^L364P小鼠出现的异常表型进行致病机理研究。分离其大脑VSMCs进行细胞增殖、凋亡及活性检测;对转化生长因子-β（transgenic factor-β,TGF-β）信号通路关键因子:TGF-β、SMAD Family Member 2（Smad2）、SMAD Family Member 3（Smad3）和SMAD Family Member 4（Smad4）进行免疫组化实验,对其进行定位定性半定量研究;对该组关键因子进行RT-PCR和Western Blot实验,定量检测Mut-hHtrA1^L364P小鼠大脑的m RNA和蛋白表达。结果:第一部分结果Mut-hHtrA1^L364P小鼠的构建:检索得到相关的序列信息,并分析各自信号肽所在的位置,通过T7E1酶切法,确认了sg RNA的最佳序列:TCGGGGACCGGCCGCTCGGC,该序列在细胞内活性最高。同时,通过测序,验证了构建Donor载体的序列。通过三批次显微注射,分别获得了15、6和13只新生鼠。对这些鼠进行PCR鉴定,得到了6只F0代阳性雌鼠,同背景WT小鼠杂交后继续繁殖喂养,基因测序鉴定得到纯合F2代,建立Mut-hHtrA1^L364P小鼠种群。最后,成功冷冻其精子。Mut-hHtrA1^L364P小鼠的鉴定:在成功分离的Mut-hHtrA1^L364P小鼠的VSMCs中,RT-PCR、Western Blot及免疫荧光等实验结果显示Mut-hHtrA1^L364P小鼠只表达人源突变型HtrA1^L364P,不表达自身本底的野生型Htr A1,其主要在VSMCs的细胞质中表达。第二部分结果Mut-hHtrA1^L364P小鼠的表型分析:与同龄WT小鼠相比较,Mut-hHtrA1^L364P小鼠明显的毛发稀少、白毛较多,活动状态笨拙迟缓。食物迷宫实验数据显示Mut-hHtrA1^L364P小鼠和WT小鼠初次找到食物所用时间分别为144.93±54.63 sec、60.93±53.60 sec,两组间有极显著差异（p<0.001）,两组找到食物发生错误次数分别为1.93±1.16 sec、1.53±0.83 sec,无显著差异（p>0.05）。水迷宫实验数据显示120 sec内Mut-hHtrA1^L364P小鼠和WT小鼠第一次接触逃生台的时间分别为44.02±25.05 sec、11.57±8.50 sec,在逃生台象限的穿梭次数分别为1.75±0.97 sec、5.83±2.25 sec,均有极显著差异（p<0.001）。病理学检测结果显示:HE、弹力纤维染色、SMA、Actin、CD31、CD68染色综合显示Mut-hHtrA1^L364P小鼠脑部部分小动脉血管失去正常结构,结构紊乱,管腔狭窄,弹力纤维变性;电镜结果显示Mut-hHtrA1^L364P小鼠比WT小鼠皮层和海马部位的衰老神经元较多、线粒体、高尔基体、内质网等细胞器出现不正常形态结构,神经突触减少,毛细血管、小静脉、小动脉出现管壁增厚、管腔狭窄等非正常现象,且有较多的脂褐素堆积、同时出现较多自噬体、变性的髓鞘等。Mut-hHtrA1^L364P小鼠的致病机理研究:MTT和CFSE检测结果显示,与WT小鼠相比较,Mut-hHtrA1^L364P小鼠来源的VSMCs细胞活力显著降低（p<0.01）其细胞增殖速度极显著减慢（p<0.001）。Annexin V-FITC/PI双染法和Tunel法结果显示Mut-HtrA1^L364P原代VSMCs的细胞凋亡率（FITC标记率）显著高于WT小鼠VSMCs（p<0.001）。RT-PCR实验结果显示,与WT小鼠组相比,Mut-hHtrA1^L364P小鼠中TGF-β的m RNA表达水平均极显著升高（p<0.001）,Smad2和Smad3的m RNA表达水平均显著升高（p<0.01）,Smad4的m RNA表达水平在两组间无明显差异（p>0.05）。Western Blot实验显示Mut-hHtrA1^L364P小鼠中TGF-β和Smad2蛋白表达水平与WT小鼠比显著升高（p<0.01）,Smad3蛋白表达水平升高（p<0.05）,Smad4的蛋白表达水平在两组间无显著差异（p>0.05）。结论:1.成功构建了只表达人源突变型HtrA1^L364P的Mut-hHtrA1^L364P小鼠。HtrA1^L364P在Mut-hHtrA1^L364P小鼠大脑VSMCs中成功表达。2.Mut-hHtrA1^L364P小鼠在行为学和病理学方面可以很好地模拟CARASIL发病,该鼠可作为研究CARASIL疾病的模型。3.在Mut-hHtrA1^L364P小鼠机体内发现TGF-β/Smad信号通路异常上调,参与了CARASIL疾病致病过程。更多还原

【Abstract】 Objective:To construct human HtrA1^L364P transgenic mice(Mut-hHtrA1^L364Pmice)and study their phenotype and pathogenesis.Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy（CARASIL）is a rare autosomal recessive cerebrovascular disease（CSVD）.The clinical manifestations were stroke,cognitive decline,pseudobulbar palsy,early baldness and recurrent low back pain.Brain magnetic resonance imaging showed multiple cerebral infarction and extensive subcortical demyelination of white matter.Arteriosclerosis of small arteries,vitreous lesions of the medial membrane,fibrosis of the intima and elastic layer,and concentric stenosis of the lumen were seen in the brain tissue of autopsy patients.The global incidence of the disease is low.There are more than 50 cases reported in the literature.The pathogenic gene of the disease was identified as high temperature requirement A1（HtrA1）by Hara,a Japanese scholar.In 2007,our group reported for the first time a CARASIL family in China.Its clinical features were consistent with the symptoms of CARASIL.Sequencing results showed that T1091C mutation occurred in the HtrA1gene,resulting in the mutation of leucine（Leu,L）to proline（Pro,P）at 364 loci.At present,there is no successful CARASIL model animal at home and abroad,which also limits the study of CARASIL.The aim of this study is to construct model animals based on HtrA1 gene mutation sites found in the reported families,and then to study the pathological typing and pathogenesis of the disease.Transgenic mice are one of the most commonly used disease models.In this study,we intend to use CRISPR/Cas9 technology to insert human HtrA1^L364P signal-free peptide sequence after HtrA1 gene signal peptide in mice and make it fused and expressed.On the one hand,the insertion of human HtrA1^L364P can knock out the background HtrA1 of mice.Through phenotypic analysis,we can study whether human HtrA1^L364P transgenic mice cause CARASIL disease.On the other hand,when human HtrA1^L364P was fused with mice HtrA1 signal peptide,not only the expression and localization of HtrA1 signal peptide would not change,but also the regulatory mechanism of its 5’end would not be affected.Therefore,it can better simulate the real situation of human disease.By establishing human HtrA1^L364P knock-in transgenic mice(named Mut-hHtrA1^L364Pmice),the pathogenic mechanism of HtrA1^L364P can be analyzed,which can be used as research models of related diseases such as CARASIL and CSVD.Methods:The study was divided into two parts.Part Ⅰ:Construction and identification of Mut-hHtrA1^L364P miceFirst,the Donor vector is constructed.The CDS,protein and genome sequences of human and mice HtrA1 were retrieved to identify their signal peptide regions.According to the location of HtrA1 protein signal peptide in mice,the target region of small guide RNA（sg RNA）was selected.In this region,three sg RNAs were designed and co-transfected with Cas9 to verify the intracellular activity of sg RNA by T7E1digestion,so as to select the optimal sg RNA and clone the left and right homologous arms accordingly.At the same time,human HtrA1 template was obtained and site-directed mutagenesis was carried out.The mutant sequence selected CCG codon that expressed proline efficiently in mice.The region of human HtrA1 containing no signal peptide was amplified by PCR method,and the human HtrA1 was mutated to obtain the human HtrA1^L364P sequence,which was inserted into the left and right homologous arms of the clone to obtain the Donor vector knocked in by CRISPR/Cas9.Secondly,after superovulation,the healthy female mice were closed in cages,and the oviducts of the mice with vaginal suppositories were obtained surgically,thus the fertilized eggs were separated.Meanwhile,the spurious pregnant females were obtained synchronously by ligated male rats.Obtain sg RNA,Cas9 and Donor c RNA by in vitro transcription,mix them and microinject them into the obtained fertilized eggs.Healthy fertilized eggs were then selected and transplanted back into the pseudo-pregnant female.After the newborn mice were born,the toe of the newborn mice was clipped and identified by PCR,and transgenic positive mice were obtained.At the same time,the positive mice were cross-bred with wide type（WT）mice in the same background to establish the Mut-hHtrA1^L364P mice mice population.Transgenic homozygous mice were obtained and preserved by sperm freezing.Vascular smooth muscle cells（VSMCs）from homozygous transgenic mice were isolated and the expression of SM22 alpha was detected by cellular immunofluorescence technique to confirm the artificial VSMCs.On this basis,real-time fluorescence quantification polymerase chain reaction（RT-PCR）,Western Blot,immunofluorescence technology and other methods were used to detect the expression and localization of HtrA1^L364P in transgenic homozygous mice VSMCs.Part Ⅱ:Phenotypic analysis and pathogenesis of Mut-hHtrA1^L364P miceFirstly,phenotypic analysis of Mut-hHtrA1^L364P mice was carried out:Observe its appearance and action state.Conduct behavioral analysis:including food maze and water maze;Pathological examination:Including routine hematoxylin-eosin staining（HE）,Nissl staining,special staining of elastic fibers,specific antibodies:smooth muscle actin（SMA）,actin（Actin）,CD31,CD68 labeled immunohistochemical staining and the cell structure and subcellular structure were observed by electron microscopy.Secondly,the VSMCs of Mut-HtrA1^L364P mice were tested for cell proliferation,apoptosis and activity.The key factors of TGF-βsignaling pathway（TGF-β,Smad2,Smad3,Smad4）were localized and qualitatively identified by immunohistochemistry.RT-PCR and Western Blot experiments were conducted to quantitatively detect the expression of genes and proteins in Mut-hHtrA1^L364P mice.Result:Part Ⅰ Results:Construction of Mut-hHtrA1^L364P mice:Relevant sequence information was retrieved and the location of each signal peptide was analyzed.The best sequence of sg RNA was confirmed by T7E1 digestion:TCGGGGACCGGCCGCTCGGC.This sequence has the highest intracellular activity.At the same time,the sequence of Donor vector was verified by sequencing.Fifteen,six and thirteen newborn mice were obtained by three batches of microinjection.Six F0-positive female mice were identified by PCR.After hybridization with background WT mice,they continued to breed and feed.Homozygous F2 mice were identified by gene sequencing,and Mut-hHtrA1^L364P mice population was established.Finally,the sperm was successfully frozen.Identification of Mut-hHtrA1^L364P mice:in the VSMCs ofHtrA1^L364P mice successfully isolated fromHtrA1^L364P mice,RT-PCR Western Blot,immunofluorescence and other experimental results showed that Mut-HtrA1^L364P mice only expressed human mutant HtrA1^L364P,but did not express their own background wild-type HtrA1,which was mainly expressed in the cytoplasm of VSMCs.Part Ⅱ resultsPhenotypic analysis of Mut-hHtrA1^L364P mice:compared with WT mice of the same age,Mut-hHtrA1^L364P mice showed obvious sparse hair,more white hair and clumsy and slow movement.The results of the food maze experiment showed that the time of finding food for Mut-hHtrA1^L364P mice and WT mice was 144.93±54.63 seconds and60.93±53.60 seconds,respectively,with a significant difference between the two groups（p<0.001）.The number of errors in finding food in the two groups was1.93±1.16 seconds and 1.53±0.83 seconds,respectively,with no significant difference（p>0.05）.The water maze experiment data showed that the first time ofHtrA1^L364P mice and WT mice to contact the escape table within 120 seconds was44.02±25.05 seconds,11.57±8.50 seconds,respectively,and the number of times of shuttle in the escape table quadrant was 1.75±0.97 seconds and 5.83±2.25 seconds,respectively,with extremely significant differences（p<0.001）.Pathological test results showed that there was no significant difference in the structure,number and distribution of neurons betweenHtrA1^L364P mice and WT mice.Staining with HE,elastic fiber,SMA,Actin,CD31 and CD68 comprehensively showed that some small arteries in the brain ofHtrA1^L364P mice lost normal structure,with structural disorder,stenosis of lumen and degeneration of elastic fiber.Electron microscopy showed that the cortex and hippocampus of Mut-hHtrA1^L364P mice aging neurons more than WT mice,mitochondria,golgi apparatus and endoplasmic reticulum and other organelles appear abnormal morphology,synapses,capillaries,venules,and small artery wall thickening and luminal stenosis abnormal phenomenon,and there are more fat brown pigment deposition,appears more autophagy body at the same time.Pathogenic mechanism ofHtrA1^L364P mice:MTT and CFSE results showed that compared with WT mice,the viability of VSMCs derived fromHtrA1^L364Pmice was significantly decreased（p<0.01）and the proliferation rate was significantly slowed down（p<0.001）.Annexin V-FITC/PI double staining and Tunel method showed that the apoptotic rate（FITC labeling rate）of primary VSMCs ofHtrA1^L364P was significantly higher than that of VSMCs of WT mice（p<0.001）.RT-PCR results showed that compared with WT mice,the expression of TGF-βwas significantly increased inHtrA1^L364P mice（p<0.001）,the expression of Smad2and Smad3 was significantly increased（p<0.01）,and the expression of Smad4 was not significantly different between the two groups（p>0.05）.Western Blot test showed that the expression levels of TGF-βand Smad2 inHtrA1^L364P mice were significantly higher than those in WT mice（p<0.01）,while the expression levels of Smad3 were significantly higher（p<0.05）,and there was no significant difference in the expression levels of Smad4 between the two groups（p>0.05）.Conclusion:1.Mut-hHtrA1^L364P mice was successfully constructed.The mice only expressed human mutant HtrA1^L364P,but not its own background wild-type HtrA1.2.Mut-hHtrA1^L364P mice can well simulate the pathogenesis of CARASIL in behavioral and pathological aspects,which can be used as a model to study the disease of CARASIL.3.In Mut-hHtrA1^L364P mice,it was found that TGF-β/Smad signal pathway was abnormally up regulated,which was involved in the pathogenesis of CARASIL disease.更多还原