本文選題：常染色體顯性多囊腎病　切入點(diǎn)：小型豬　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：常染色體顯性多囊腎病(Autosomal Dominant Polycystic Kidney Disease, ADPKD)是一種常見的腎臟顯性遺傳疾病,由PKD1或者PKD2基因突變引起,發(fā)病率大約1/400-1/1000。病理癥狀主要表現(xiàn)為腎臟中產(chǎn)生充滿液體的囊泡,并且隨著病情的發(fā)展,這些囊泡的數(shù)量會(huì)逐漸增多,體積也會(huì)逐漸增大。大約一半的ADPKD患者在50～60歲期間會(huì)產(chǎn)生終末期腎病。到目前為止,除了腎臟透析或者腎臟移植,仍然沒有藥物或者治療方法能夠治愈ADPKD.通過對(duì)小鼠ADPKD疾病模型的研究,人們對(duì)于ADPKD的發(fā)病機(jī)理有了比較深刻的認(rèn)識(shí)。但是,小鼠ADPKD模型還沒有能夠幫助人類尋找到一種有效的治療藥物或者治療方法。相對(duì)于小鼠和人類在腎臟結(jié)構(gòu)等方面的較大差異,小型豬和人類的腎臟具有更高的相似性,這使小型豬成為構(gòu)建腎臟疾病模型的極佳實(shí)驗(yàn)動(dòng)物。因此,本研究嘗試建立ADPKD的小型豬模型,為研究ADPKD致病機(jī)理和開發(fā)治療手段提供一種更能真實(shí)反映人類ADPKD的新模型。 SBM小鼠是一種在腎臟過表達(dá)c-Myc基因的ADPKD模型。以此為基礎(chǔ),本研究嘗試通過在小型豬腎臟中過表達(dá)c-Myc基因來建立ADPKD模型。使用小鼠腎臟特異表達(dá)基因Ksp-cadherin的啟動(dòng)子和豬c-Myc基因的cDNA,構(gòu)建了pKsp-c-Myc-b轉(zhuǎn)基因載體,然后通過體細(xì)胞核移植得到了7頭c-Myc轉(zhuǎn)基因豬。其中,3頭轉(zhuǎn)基因豬出生后很快死亡,尸檢沒有發(fā)現(xiàn)腎臟有明顯病變。Western Blot顯示c-Myc基因在3號(hào)轉(zhuǎn)基因豬的腦、心、肝、腎都有明顯過表達(dá)。3號(hào)轉(zhuǎn)基因豬的腎臟免疫組化染色結(jié)果顯示：腎臟管腔上皮細(xì)胞的c-Myc基因明顯過表達(dá),但是除了管腔略微擴(kuò)張,沒有出現(xiàn)囊泡等明顯病理癥狀。在存活的轉(zhuǎn)基因豬生長(zhǎng)到1、4、6月齡時(shí),測(cè)定轉(zhuǎn)基因豬和同窩野生型豬的血尿素氮(BUN)和血肌酐(Scr)：1月齡時(shí)轉(zhuǎn)基因豬的BUN明顯高于野生型豬,但是4、6月齡時(shí)沒有顯著差異；轉(zhuǎn)基因豬和野生型豬的Scr在三個(gè)時(shí)間點(diǎn)都沒有明顯差異。在10號(hào)轉(zhuǎn)基因豬生長(zhǎng)到13月齡時(shí),取其腎臟進(jìn)行檢測(cè)：WesternBlot顯示其腎臟中c-Myc、Erkl/2總蛋白和磷酸化Erkl/2表達(dá)水平和野生型豬沒有差異,HE染色也沒有發(fā)現(xiàn)腎臟存在明顯病變。因此,pKsp-c-Myc-b轉(zhuǎn)基因小型豬未能表現(xiàn)出常染色體顯性多囊腎病的病理癥狀,說明SBM小鼠有其獨(dú)特性。 為了在小型豬上建立含有PKD2錯(cuò)義突變(亞等位基因)的ADPKD疾病模型,本研究篩選了針對(duì)豬PKD2基因第9號(hào)外顯子的7對(duì)TALEN,嘗試將小型豬polycystin-2蛋白的第658號(hào)亮氨酸突變?yōu)樯彼帷Ｍㄟ^測(cè)序和EcoRV酶切兩種方法證明：T-93、T-931和T-934三對(duì)(?)ALEN有效,其中T-93的突變效率最高。37℃細(xì)胞培養(yǎng)條件下,T-93的突變效率是6%-7%；如果采取30℃低溫培養(yǎng),T-93的突變效率提高到15%～17%。T-93、T-931和T-934的靶點(diǎn)基本一致,區(qū)別在于T-931和T-934的識(shí)別序列更長(zhǎng),但是,T-931和T-934的突變效率明顯低于T-93。將T-93和ssDNA同源模板一起核轉(zhuǎn)染中國(guó)實(shí)驗(yàn)用小型豬胎兒成纖維細(xì)胞后,細(xì)胞活力很差,大量死亡,因此還需要繼續(xù)優(yōu)化條件。 為了建立小型豬1KD2基因敲除模型,本研究利用CRISPR-Cas9技術(shù)篩選了針對(duì)豬PKD2基因不同外顯子的11個(gè)靶點(diǎn)：6個(gè)靶點(diǎn)有效,其中pX330-1效率最高,達(dá)到了11%,其突變位點(diǎn)在第1號(hào)外顯子,預(yù)期能有效造成polycystin-2蛋白失活。 已有研究表明CDH16基因在人、小鼠和兔上是一種腎臟特異表達(dá)基因。為了在小型豬上實(shí)現(xiàn)腎臟特異性基因敲除,需要使用腎臟特異的啟動(dòng)子。因此,本研究對(duì)豬CDH16基因進(jìn)行了鑒定：豬CDH16基因包含18個(gè)外顯子；在中國(guó)實(shí)驗(yàn)用小型豬上,CDH16基因在腎臟的轉(zhuǎn)錄水平最高,同時(shí)在肺中也檢測(cè)到了少量的轉(zhuǎn)錄本；雙熒光素酶實(shí)驗(yàn)證明豬CDH16基因啟動(dòng)子在LLC-PK1細(xì)胞上有啟動(dòng)子活性。 綜上所述,Ksp-c-Myc-b轉(zhuǎn)基因小型豬未能表現(xiàn)出常染色體顯性多囊腎病的病理癥狀。成功篩選得到的TALEN和CR1SPR-Cas9靶點(diǎn)為構(gòu)建PKD2基因錯(cuò)義突變或者PKD2基因敲除小型豬打下了基礎(chǔ)。豬CDH16基因啟動(dòng)子能夠用于構(gòu)建CRISPR-Cas9的腎臟表達(dá)載體,實(shí)現(xiàn)小型豬PKD2基因腎臟特異敲除。
[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.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692;R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前5條

1 馮媛媛;白雪源;賀津;葉建華;陳香美;;中國(guó)實(shí)驗(yàn)用小型豬血液指標(biāo)正常參考值分析[J];中國(guó)畜牧獸醫(yī);2013年06期

2 沈珊珊;李旭艷;謝院生;金美玲;崔少遠(yuǎn);季佳瑤;萬嘉嘉;劉穎;李清剛;白雪源;陳香美;;中國(guó)實(shí)驗(yàn)用小型豬腎小管的發(fā)育及各節(jié)段標(biāo)志物的表達(dá)[J];中國(guó)中西醫(yī)結(jié)合腎病雜志;2013年04期

3 裴德智;簡(jiǎn)述中國(guó)實(shí)驗(yàn)用小型豬[J];實(shí)驗(yàn)動(dòng)物科學(xué)與管理;1997年01期

4 于書敏,王傳武,趙德明,張慶才,裴德智;中國(guó)實(shí)驗(yàn)小型豬培育和病原凈化[J];實(shí)驗(yàn)動(dòng)物科學(xué)與管理;2003年02期

5 ;中國(guó)實(shí)驗(yàn)用小型豬的培育、開發(fā)與應(yīng)用[J];中國(guó)實(shí)驗(yàn)動(dòng)物學(xué)雜志;1991年01期

，

本文編號(hào)：1597914