【作者】 叶建华；

【导师】 李宁；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2014， 博士

【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,H&E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。更多还原

【Abstract】 Autosomal dominant polycystic kidney disease (ADPKD) is a common dominant renal disorder caused by mutations in either PKD1or PKD2, with an incidence of1/400～1/1000. Its key feature is fluid-filled cysts in bilateral kidneys, and as the illness deteriorates, both number and volume of cysts increase. Approximately half of ADPKD patients develop end stage renal disease (ESRD) at their fifth or sixth decade of life. Until now, there is no effective therapy for ADPKD except for renal dialysis or kidney transplantation. The research in mouse models of ADPKD has promoted people’s understanding about the pathogenesis of ADPKD to a large extent; however, mouse models have not yet brought about an effective therapy. Contrasted to the obvious disparity between mouse and people in kidney, the mini-pig owns more similarities with human in kidney, which makes the mini-pig an excellent model to study renal diseases. Herein, I try to establish the ADPKD mini-pig model in this study, so that it will serve as a novel model faithfully recapitulating human ADPKD, and promote the study of etiology as well as development of effective therapeutic interventions.The SBM mice represent a transgenic model of ADPKD by overexpressing c-Myc in kidneys. Therefore, c-Myc was overexpressed in pig’s kidneys in an effort to imitate the SBM mice. The pKsp-c-Myc-b transgene vector was constructed using mouse Ksp-cadherin promoter and the cDNA of pig’s c-Myc gene. After somatic cell nuclear transfer (SCNT), seven c-Myc transgenic piglets were delivered. Three transgenic piglets died soon after birth, displaying no obvious abnormality in kidneys at necropsy. Western Blot analysis showed that c-Myc was overexpressed in the brain, heart, liver and kidney of c-Myc transgenic piglet (No.3). Immunohistochemical staining of kidney sections revealed high level of c-Myc within the epithelial cells of renal tubules in piglet No.3. However, except for slight dilation of renal tubules, no cysts were observed. When c-Myc transgenic pigs were1,4,6months old, blood sample were collected to measure blood urea nitrogen (BUN) and serum creatinine (Scr). At1month, c-Myc transgenic pigs had significantly higher BUN level than the wild-type littermates, but this difference did not persist to4months or6months. No difference in Scr was detected between transgenic pigs and wild-type pigs at all three time points. When c-Myc transgenic pig No.10turned13months old, kidney sample was collected. Western Blot results revealed that there was no difference in the protein level of c-Myc, total ERK1/2and phosphorylated ERK1/2between transgenic pig No.10and a wild-type pig. No noticeable renal abnormalities were found through H&E staining of kidney sections from transgenic pig No.10. Therefore, c-Myc transgenic pigs did not develop ADPKD, implying the uniqueness of SBM mice.In order to introduce a missense mutation (hypomorphic allele) into pig PKD2gene, seven pairs of TALEN targeting the9th exon of pig PKD2gene were screened for changing the658th amino acid of polycystin-2from Leu to Trp. Both sequencing and EcoR V digestion analysis demonstrated that T-93, T-931and T-934were able to cause mutations in target sites, while T-93had the highest efficiency. Upon37℃cell culture condition, T-93yielded mutation frequency of6%～7%, and if cells were applied to transient hypothermia (30℃) after nucleofection, the mutation frequency rose to15%～17%. T-93, T-931and T-934had almost the same target site, except that the binding sites of T-931and T-934was several base pairs longer than those of T-93. The extended binding sites led to decreased mutation frequency. Once T-93and single-stranded DNA (ssDNA) donor template were co-transfected into Chinese experimental mini-pig fetal fibroblasts, the cell viability dropped dramatically, so further optimization was needed to improve cell condition.Moreover, CRISPR-Cas9technique was exploited to knock out the pig PKD2gene. Within all11target sites on different exons of PKD2,6targets sites were mutated, while pX330-1had the highest mutation rate of11%. Because the target site of pX330-1was on the first exon, it was expected to end in complete inactivation of polycystin-2.As reported in human, mouse and rabbit, CDH16is specifically expressed in the kidney. A kidney-specific promoter is needed to knock out PKD2gene in a kidney specific manner. Therefore, the pig CDH16gene was identified in this study. The pig CDH16gene had18exons, and its transcripts were detected in both the kidney and lung of Chinese experimental mini-pig, with relatively higher transcription level in the kidney. Dual-luciferase assay proved that the pig CDH16promoter was able to drive transcription in LLC-PK1cells.In summary, pKsp-c-Myc-b transgenic mini-pig did not develop ADPKD. Screened TALEN and CRISPR-Cas9could be used to introduce hypomorphic PKD2allele or knock out PKD2gene in mini-pig. At the same time, the identified pig CDH16promoter could be utilized to re-construct current CRIPSR-Cas9vector and constrain Cas9expression mainly in the kidney, thus knocking out pig PKD2gene in a kidney specific manner.更多还原