建立常染色体显性多囊肾病小型猪模型

发布时间：2018-03-11 11:13

本文选题：常染色体显性多囊肾病　切入点：小型猪　出处：《中国农业大学》2014年博士论文　论文类型：学位论文

【摘要】：常染色体显性多囊肾病(Autosomal Dominant Polycystic Kidney Disease, ADPKD)是一种常见的肾脏显性遗传疾病,由PKD1或者PKD2基因突变引起,发病率大约1/400-1/1000。病理症状主要表现为肾脏中产生充满液体的囊泡,并且随着病情的发展,这些囊泡的数量会逐渐增多,体积也会逐渐增大。大约一半的ADPKD患者在50～60岁期间会产生终末期肾病。到目前为止,除了肾脏透析或者肾脏移植,仍然没有药物或者治疗方法能够治愈ADPKD.通过对小鼠ADPKD疾病模型的研究,人们对于ADPKD的发病机理有了比较深刻的认识。但是,小鼠ADPKD模型还没有能够帮助人类寻找到一种有效的治疗药物或者治疗方法。相对于小鼠和人类在肾脏结构等方面的较大差异,小型猪和人类的肾脏具有更高的相似性,这使小型猪成为构建肾脏疾病模型的极佳实验动物。因此,本研究尝试建立ADPKD的小型猪模型,为研究ADPKD致病机理和开发治疗手段提供一种更能真实反映人类ADPKD的新模型。 SBM小鼠是一种在肾脏过表达c-Myc基因的ADPKD模型。以此为基础,本研究尝试通过在小型猪肾脏中过表达c-Myc基因来建立ADPKD模型。使用小鼠肾脏特异表达基因Ksp-cadherin的启动子和猪c-Myc基因的cDNA,构建了pKsp-c-Myc-b转基因载体,然后通过体细胞核移植得到了7头c-Myc转基因猪。其中,3头转基因猪出生后很快死亡,尸检没有发现肾脏有明显病变。Western Blot显示c-Myc基因在3号转基因猪的脑、心、肝、肾都有明显过表达。3号转基因猪的肾脏免疫组化染色结果显示：肾脏管腔上皮细胞的c-Myc基因明显过表达,但是除了管腔略微扩张,没有出现囊泡等明显病理症状。在存活的转基因猪生长到1、4、6月龄时,测定转基因猪和同窝野生型猪的血尿素氮(BUN)和血肌酐(Scr)：1月龄时转基因猪的BUN明显高于野生型猪,但是4、6月龄时没有显著差异；转基因猪和野生型猪的Scr在三个时间点都没有明显差异。在10号转基因猪生长到13月龄时,取其肾脏进行检测：WesternBlot显示其肾脏中c-Myc、Erkl/2总蛋白和磷酸化Erkl/2表达水平和野生型猪没有差异,HE染色也没有发现肾脏存在明显病变。因此,pKsp-c-Myc-b转基因小型猪未能表现出常染色体显性多囊肾病的病理症状,说明SBM小鼠有其独特性。为了在小型猪上建立含有PKD2错义突变(亚等位基因)的ADPKD疾病模型,本研究筛选了针对猪PKD2基因第9号外显子的7对TALEN,尝试将小型猪polycystin-2蛋白的第658号亮氨酸突变为色氨酸。通过测序和EcoRV酶切两种方法证明：T-93、T-931和T-934三对(?)ALEN有效,其中T-93的突变效率最高。37℃细胞培养条件下,T-93的突变效率是6%-7%；如果采取30℃低温培养,T-93的突变效率提高到15%～17%。T-93、T-931和T-934的靶点基本一致,区别在于T-931和T-934的识别序列更长,但是,T-931和T-934的突变效率明显低于T-93。将T-93和ssDNA同源模板一起核转染中国实验用小型猪胎儿成纤维细胞后,细胞活力很差,大量死亡,因此还需要继续优化条件。为了建立小型猪1KD2基因敲除模型,本研究利用CRISPR-Cas9技术筛选了针对猪PKD2基因不同外显子的11个靶点：6个靶点有效,其中pX330-1效率最高,达到了11%,其突变位点在第1号外显子,预期能有效造成polycystin-2蛋白失活。已有研究表明CDH16基因在人、小鼠和兔上是一种肾脏特异表达基因。为了在小型猪上实现肾脏特异性基因敲除,需要使用肾脏特异的启动子。因此,本研究对猪CDH16基因进行了鉴定：猪CDH16基因包含18个外显子；在中国实验用小型猪上,CDH16基因在肾脏的转录水平最高,同时在肺中也检测到了少量的转录本；双荧光素酶实验证明猪CDH16基因启动子在LLC-PK1细胞上有启动子活性。综上所述,Ksp-c-Myc-b转基因小型猪未能表现出常染色体显性多囊肾病的病理症状。成功筛选得到的TALEN和CR1SPR-Cas9靶点为构建PKD2基因错义突变或者PKD2基因敲除小型猪打下了基础。猪CDH16基因启动子能够用于构建CRISPR-Cas9的肾脏表达载体,实现小型猪PKD2基因肾脏特异敲除。
[Abstract]:Autosomal dominant polycystic kidney disease (Autosomal Dominant Polycystic Kidney Disease, ADPKD) is a common autosomal dominant kidney disease, caused by PKD1 or PKD2 gene mutation, the incidence rate is about 1/400-1/1000. the pathological symptoms of fluid filled vesicles produced in the kidney, and with the development of disease, the number of vesicles will be gradually increased also, the volume will gradually increase. About half of the ADPKD patients develop end-stage renal disease in 50~60 years. So far, in addition to kidney dialysis or a kidney transplant, still no treatment or drug can cure ADPKD. through the study of mouse model of ADPKD disease, the pathogenesis of ADPKD have a more profound understanding of. However, the mouse model of ADPKD has not been able to help people to find an effective drug treatment or treatment. Compared with mice and Large differences in the structure of the human kidney, pig and human kidney have higher similarity, which becomes an excellent experimental miniature pig animal model establishment of kidney disease. Therefore, this study attempts to establish pig models of ADPKD, provides a new model to better reflect the real human on ADPKD ADPKD the development of pathogenesis and treatment.
SBM mouse is an expression of c-Myc gene in the kidney of the ADPKD model. Based on this, this study attempts to establish the ADPKD model through the over expression of c-Myc gene in the kidney of miniature pigs. The promoter of mouse kidney specific expression of Ksp-cadherin gene and porcine c-Myc gene of cDNA, constructed the pKsp-c-Myc-b transgenic vector, then 7 the head of c-Myc transgenic pig obtained by somatic cell nuclear transplantation. Among them, 3 transgenic pigs soon after birth death, the autopsy found no obvious renal lesion of.Western Blot showed that the c-Myc gene in 3 transgenic pig brain, heart, liver, kidney and kidney have immune group was overexpression of.3 transgenic pig was showed c-Myc gene: kidney tubules epithelial cells significantly over expressed, but slightly lumen expansion, vesicles did not appear obvious pathological symptoms. In the survival of transgenic pigs grow up to 1,4,6 months of age, Blood urea nitrogen determination of transgenic pigs and littermate wild-type pigs (BUN) and serum creatinine (Scr):1 month old transgenic pig BUN was higher than that of wild type pigs, but 4,6 months of age had no significant difference; transgenic pigs and wild type porcine Scr have no obvious difference in the three time growth. From 13 month old to 10 transgenic pigs, kidneys were detected: WesternBlot c-Myc of the kidney, the total protein of Erkl/2 and phosphorylated Erkl/2 expression levels did not differ between wild type and pig, HE staining was also found no obvious renal lesions. Therefore, pKsp-c-Myc-b transgenic pigs failed to showed pathological symptoms of autosomal dominant polycystic kidney disease, SBM mice has its uniqueness.
In order to establish the PKD2 containing missense mutations in the pig (suballele) model of ADPKD disease, this study screened for 7 TALEN of porcine PKD2 gene exon ninth, try to polycystin-2 protein in miniature swine No. 658th leucine mutation of tryptophan. By sequencing and EcoRV restriction analysis proved that the two kinds of methods T-93, T-931 and T-934 three on ALEN (?), the T-93 mutation of the highest efficiency.37 C cell culture conditions, mutation efficiency of T-93 is 6%-7%; if you take the 30 low temperature culture, improve the mutation efficiency of T-93 to 15% ~ 17%.T-93, target T-931 and T-934 are basically the same, the difference lies in the recognition sequence longer, T-931 and T-934 but the mutation efficiency of T-931 and T-934 were significantly lower than that of T-93. T-93 and ssDNA homologous template together Chinese nuclear transfection experimental miniature pig fetal fibroblast cells, cell viability is very poor, a large number of deaths, therefore also need to Continue to optimize the conditions.
In order to establish the porcine 1KD2 gene knockout model, this study uses CRISPR-Cas9 technique to screen the 11 target for porcine PKD2 gene exon 6: different targets, the pX330-1 efficiency is the highest, reaching 11%, the mutation in exon first, is expected to be effective to polycystin-2 protein inactivation.
Studies have shown that CDH16 gene in human, mouse and rabbit is a kidney specific gene expression. In order to realize the kidney specific gene knockout in pig kidney, need to use specific promoter. Therefore, the research for the identification of porcine CDH16 gene: porcine CDH16 gene contains 18 exons; in China experimental miniature pigs, CDH16 gene transcription level was highest in the kidney, while the lung was also detected in a small amount of transcripts; double luciferase reporter assay showed that the porcine CDH16 gene promoter promoter activity in LLC-PK1 cells.
In summary, Ksp-c-Myc-b transgenic pigs failed to show symptoms of autosomal dominant polycystic kidney disease. Screened from TALEN and CR1SPR-Cas9 targets for the construction of PKD2 gene missense mutation or PKD2 gene knockout pigs. This is the foundation of the pig CDH16 gene promoter can be used to construct CRISPR-Cas9 expression vector for porcine kidney, PKD2 kidney specific gene knockout.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692;R-332

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