PCSK9基因D374Y突變體轉(zhuǎn)基因豬的制備與分析
發(fā)布時(shí)間：2018-08-03 21:53
【摘要】：【目的】前蛋白轉(zhuǎn)化酶枯草溶菌素9(proprotein convertase subtilisin/Kexin type 9,PCSK9)基因是人類高膽固醇血癥(autosomal dominant hypercholesterolemia,ADH)的主效基因之一,其獲得型突變與人類家族性高膽固醇血癥有直接的關(guān)系。PCSK9-D374Y突變體對(duì)低密度脂蛋白受體(low density lipoprotein receptor,LDLR)的降解能力比野生型蛋白強(qiáng)十倍,增加了患高膽固醇血癥的風(fēng)險(xiǎn),從而加速動(dòng)脈粥樣硬化的進(jìn)程。豬心血管系統(tǒng)和血脂代謝方面與人類非常相近,成為研究動(dòng)脈粥樣硬化疾病的理想模型之一。然而自然發(fā)病的豬缺乏,且誘導(dǎo)病征發(fā)生緩慢。因此擬利用體細(xì)胞克隆技術(shù)制備PCSK9獲得型突變體轉(zhuǎn)基因豬,以模擬動(dòng)脈血管的病理學(xué)變化,加速發(fā)病進(jìn)程,為動(dòng)脈粥樣硬化的研究提供理想的動(dòng)物模型。【方法】研究使用人PCSK9基因D374Y突變體載體,用電轉(zhuǎn)染的方法將其整合到五指山小型豬近交系胎兒成纖維細(xì)胞中,并通過體細(xì)胞核移植技術(shù)獲得了人PCSK9基因D374Y突變體轉(zhuǎn)基因豬個(gè)體。通過Southern-blot、實(shí)時(shí)熒光定量PCR、Western-blot等方法,分別從DNA、RNA、蛋白的水平檢測了人PCSK9基因在轉(zhuǎn)基因豬肝臟中的整合表達(dá)情況。同時(shí),通過組織化學(xué)染色與H.E.染色的方法對(duì)轉(zhuǎn)基因豬進(jìn)行了組織學(xué)檢測�！窘Y(jié)果】轉(zhuǎn)基因陽性細(xì)胞集落在藥篩的第3天開始出現(xiàn),至第7天形成較大的單克隆點(diǎn),且PCR檢測結(jié)果顯示擴(kuò)增產(chǎn)物可以拼接為完整片段,說明外源片段在基因組中具有完整性;將篩選得到的陽性細(xì)胞作為體細(xì)胞克隆的供體細(xì)胞,通過體細(xì)胞核移植技術(shù)獲得了轉(zhuǎn)基因豬個(gè)體。PCR及Southern-blot檢測結(jié)果顯示,D374Y-PCSK9基因可以完整的插入豬的基因組中,且有串聯(lián)重復(fù)現(xiàn)象;RT-PCR和QPCR檢測結(jié)果表明,人PCSK9基因能在豬肝臟內(nèi)正常轉(zhuǎn)錄且不影響豬內(nèi)源性PCSK9基因的轉(zhuǎn)錄,且在其它內(nèi)臟器官,如心、脾、肺、腎也能檢測人PCSK9基因的表達(dá),而豬內(nèi)源性PCSK9基因在這些組織中表達(dá)量很低;Western-blot檢測結(jié)果與RNA水平的檢測類似。這些結(jié)果說明人D374Y-PCSK9基因成功整合到豬基因豬中,且能夠正常轉(zhuǎn)錄與翻譯。通過組織化學(xué)染色發(fā)現(xiàn),與野生型豬肝臟相比,克隆豬肝臟中LDLR蛋白水平極顯著低于野生型。另外,對(duì)克隆豬進(jìn)行H.E.染色后發(fā)現(xiàn)其肝臟組織有明顯的病理學(xué)變化,該結(jié)果說明,LDLR水平的急劇下降有可能是導(dǎo)致肝臟病變的原因�！窘Y(jié)論】成功獲得了人PCSK9基因D374Y突變體的克隆豬;與野生型豬肝臟相比,克隆豬肝臟中LDLR水平顯著降低,并且克隆豬肝臟發(fā)生了明顯病變。
[Abstract]:[objective] the proprotein invertase lysogenin 9 (proprotein convertase subtilisin/Kexin type 9 / PCSK9 gene is one of the major genes of (autosomal dominant hypercholesterolemia. Its acquired mutation was directly related to human familial hypercholesterolemia. PCSK9-D374Y mutant was 10 times more capable of degrading low density lipoprotein receptor (low density lipoprotein receptor LDLR than wild-type protein, which increased the risk of hypercholesterolemia. Thus speeding up the process of atherosclerosis. The cardiovascular system and lipid metabolism of pigs are very similar to those of human beings, and become one of the ideal models for the study of atherosclerotic diseases. However, naturally occurring pigs are deficient and slow to induce symptoms. Therefore, we intend to use somatic cell cloning technique to prepare PCSK9 acquired mutant transgenic pigs in order to mimic the pathological changes of arterial vessels and accelerate the pathogenesis of the disease. To provide an ideal animal model for the study of atherosclerosis. [methods] the D374Y mutant vector of human PCSK9 gene was used to integrate the D374Y mutant vector into fetal fibroblasts of Wuzhishan mini-pig inbred line by electrotransfection. Human PCSK9 gene D374Y mutant transgenic pig was obtained by somatic cell nuclear transfer technique. The integrative expression of human PCSK9 gene in transgenic porcine liver was detected by Southern-blot and real-time fluorescence quantitative PCR Western-blot. At the same time, by histochemical staining and H. E. The staining method was used to detect the histology of transgenic pigs. [results] the colony of transgenic positive cells began to appear on the third day of screening, and formed a large monoclonal spot on the 7th day. The results of PCR showed that the amplified products could be spliced into complete fragments, indicating that the foreign fragments had integrity in the genome, and the selected positive cells were taken as donor cells cloned from somatic cells. By means of somatic cell nuclear transfer technique, the results of .PCR and Southern-blot analysis showed that the gene of D374Y-PCSK9 could be inserted into the genome of pig completely, and there were tandem repeats in the genome. The results of RT-PCR and QPCR analysis showed that D374Y-PCSK9 gene could be inserted into pig genome completely. Human PCSK9 gene can be transcribed normally in pig liver without affecting the transcription of porcine endogenous PCSK9 gene, and the expression of human PCSK9 gene can also be detected in other visceral organs, such as heart, spleen, lung and kidney. However, the expression of porcine endogenous PCSK9 gene in these tissues was very low. The result of Western-blot analysis was similar to that of RNA level. These results suggest that human D374Y-PCSK9 gene is successfully integrated into porcine pigs and can be transcribed and translated normally. Histochemical staining showed that the level of LDLR protein in cloned pig liver was significantly lower than that in wild type pig liver. In addition, H. E. The pathological changes of liver tissue were found after staining, which indicated that the sharp decrease of LDLR level might be the cause of liver lesion. [conclusion] the cloned pig of human PCSK9 gene D374Y mutant was successfully obtained. Compared with wild-type pig liver, the LDLR level in cloned pig liver was significantly lower and the cloned pig liver had obvious pathological changes.
【作者單位】： 中國農(nóng)業(yè)科學(xué)院北京畜牧獸醫(yī)研究所;
【基金】：國家自然科學(xué)基金(31572378) 國家高技術(shù)研究發(fā)展計(jì)劃(863計(jì)劃,2012AA020603)
【分類號(hào)】：R-332;R543.5
本文編號(hào)：2163105
[Abstract]:[objective] the proprotein invertase lysogenin 9 (proprotein convertase subtilisin/Kexin type 9 / PCSK9 gene is one of the major genes of (autosomal dominant hypercholesterolemia. Its acquired mutation was directly related to human familial hypercholesterolemia. PCSK9-D374Y mutant was 10 times more capable of degrading low density lipoprotein receptor (low density lipoprotein receptor LDLR than wild-type protein, which increased the risk of hypercholesterolemia. Thus speeding up the process of atherosclerosis. The cardiovascular system and lipid metabolism of pigs are very similar to those of human beings, and become one of the ideal models for the study of atherosclerotic diseases. However, naturally occurring pigs are deficient and slow to induce symptoms. Therefore, we intend to use somatic cell cloning technique to prepare PCSK9 acquired mutant transgenic pigs in order to mimic the pathological changes of arterial vessels and accelerate the pathogenesis of the disease. To provide an ideal animal model for the study of atherosclerosis. [methods] the D374Y mutant vector of human PCSK9 gene was used to integrate the D374Y mutant vector into fetal fibroblasts of Wuzhishan mini-pig inbred line by electrotransfection. Human PCSK9 gene D374Y mutant transgenic pig was obtained by somatic cell nuclear transfer technique. The integrative expression of human PCSK9 gene in transgenic porcine liver was detected by Southern-blot and real-time fluorescence quantitative PCR Western-blot. At the same time, by histochemical staining and H. E. The staining method was used to detect the histology of transgenic pigs. [results] the colony of transgenic positive cells began to appear on the third day of screening, and formed a large monoclonal spot on the 7th day. The results of PCR showed that the amplified products could be spliced into complete fragments, indicating that the foreign fragments had integrity in the genome, and the selected positive cells were taken as donor cells cloned from somatic cells. By means of somatic cell nuclear transfer technique, the results of .PCR and Southern-blot analysis showed that the gene of D374Y-PCSK9 could be inserted into the genome of pig completely, and there were tandem repeats in the genome. The results of RT-PCR and QPCR analysis showed that D374Y-PCSK9 gene could be inserted into pig genome completely. Human PCSK9 gene can be transcribed normally in pig liver without affecting the transcription of porcine endogenous PCSK9 gene, and the expression of human PCSK9 gene can also be detected in other visceral organs, such as heart, spleen, lung and kidney. However, the expression of porcine endogenous PCSK9 gene in these tissues was very low. The result of Western-blot analysis was similar to that of RNA level. These results suggest that human D374Y-PCSK9 gene is successfully integrated into porcine pigs and can be transcribed and translated normally. Histochemical staining showed that the level of LDLR protein in cloned pig liver was significantly lower than that in wild type pig liver. In addition, H. E. The pathological changes of liver tissue were found after staining, which indicated that the sharp decrease of LDLR level might be the cause of liver lesion. [conclusion] the cloned pig of human PCSK9 gene D374Y mutant was successfully obtained. Compared with wild-type pig liver, the LDLR level in cloned pig liver was significantly lower and the cloned pig liver had obvious pathological changes.
【作者單位】： 中國農(nóng)業(yè)科學(xué)院北京畜牧獸醫(yī)研究所;
【基金】：國家自然科學(xué)基金(31572378) 國家高技術(shù)研究發(fā)展計(jì)劃(863計(jì)劃,2012AA020603)
【分類號(hào)】：R-332;R543.5
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