表皮松解性掌拓角化症基因突变敲入小鼠的表型分析及靶向shRNA治疗的探索
本文选题:表皮松解性掌跖角化症 + KRT9 ; 参考:《浙江大学》2017年博士论文
【摘要】:背景:表皮松解性掌跖角化症(epidermolyticpalmoplantarkeratoderma,EPPK。OMIM:144200)属于一种较为常见的常染色体显性皮肤遗传病,是高度遗传异质性的掌跖角化症最为常见的亚型。EPPK的主要致病基因为表达角蛋白9(keratin9,K9)分子的KRT9基因,少数病例由KRT1基因突变引起。EPPK患者出生后数周到数月内即逐渐开始出现典型的临床症状,并持续终生。主要临床表现为整个手掌和脚掌表皮的弥漫性角质增厚,色黄,在增厚的表皮周边有明显的红斑缘,许多患者伴多汗、臭汗症。某些患者还伴发指节垫、断指或先天性指屈曲等症状。目前,EPPK尚无有效的治疗手段。国内外的绝大多数研究报道尚聚焦于EPPK的变异组学。目的:在完善小鼠KRT9基因突变敲入模型表型分析的基础上,探索一种可行、高效的EPPK个性化基因治疗方案,为EPPK的后续临床基因治疗、用药治疗等研究奠定基础。方法:(1)本课题组前期已报道发现几个南方汉族EPPK家系特有一种KRT9基因 indel 突变类型:c.500_500delAinsGGCT(p.Tyr167delinsTrpLeu),并据此通过基因打靶技术构建了 Krt9/c.434delAinsGGCT(p.Tyr144delinsTrpLeu)突变敲入小鼠模型。本研究进一步对突变小鼠的足底皮肤进行深入的表型和病理学分析(HE染色、电镜观察等),以确定模型小鼠的EPPK样病理改变;并从分子角度分析突变小鼠的足底皮肤组织K9蛋白的变化,以及K9蛋白的突变对其他蛋白质表达的影响。(2)分别构建人和小鼠的萤火虫荧光素酶报告基因野生型和c.434delAinsGGCT突变型萤火虫荧光素酶慢病毒表达载体,即 pfLUC-Homo-exlKRT9/c.500delAinsGGCT/wt(pfLUC-ex1KRT9-mutant/wt)和pfLUC-Mus-ex1Krt9-c.434delAinsGGCT/wt(pfLUC-ex1Krt9-mutant/wt),以荧光素酶报告实验为依据,按照EPPK患者和c.434delAinsGGCT突变小鼠的角蛋白9基因序列设计16条候选siRNA分子,筛选并验证功能性和特异性相对最好的靶向治疗siRNA,以达到敲降突变的Krt9等位基因表达量而对正常Krt9等位基因无影响或影响极小的目的。依据所筛选的siRNA序列,构建特异性靶向治疗shRNA慢病毒载体(即lv-sh-K9mut-8),以人永生化角质形成细胞系HaCat为对象,用lv-sh-K9mut-8对其进行转染,观察有无脱靶效应。(3)将慢病毒载体候选靶向shRNA(lv-sh-K9mut-8)注射治疗12周龄的Krt9+/mut突变杂合小鼠,观察EPPK样病理症状是否得到缓解等表型变化;从组织病理和分子水平上分析治疗后的角化增生症状是否得到改善;分析靶向shRNA(lv-sh-K9mut-8)是否引发了不良的免疫反应。结果:(1)Krt9/c.434delAinsGGCT(p.Tyr144delinsTrpLeu)突变敲入杂合性成年小鼠的前、后爪在11~12周时出现稳定、明显的EPPK样病理表型。HE染色显示足垫凸起处表皮增厚、表皮扩张和角化过度症状,乳头状瘤状增生,显著的颗粒细胞增多症和棘皮症,以及具有不典型形状的表皮上部中的空泡化细胞。透射电镜超微结构分析显示,角化细胞的细胞裂解和表皮的基底层中的纤维丝的异常聚集。免疫荧光染色显示,K9突变小鼠足垫中PCNA和p63阳性细胞的数量和染色强度增加;应激反应和创伤愈合角蛋白K6和K16在Krt9+/mut和Krt9mut/mut小鼠中的表达显著高于Krt9+/+野生型小鼠;表达于基底上层的角蛋白K1和K10因K9的功能障碍而显著增高,并具有局部扩张和异常聚集的现象。免疫印迹试验(Westernblot)分析显示,Krt9+/+和Krt9+/mut小鼠的K2表达量与野生型小鼠相比III减少了 60%。(2)以Krt9/c.434delAinsGGCT 和 KRT9/c.500delAinsGGC 小插缺突变为靶向siRNA治疗的研究对象,经16条候选siRNA分子验证之后,发现si-K9mut-8的功能性和特异性相对效应最好,仅敲降突变K9分子,而对正常K9蛋白影响极小。因此,以si-K9mut-8分子的碱基序列设计合成了慢病毒表达shRNA载体(lv-sh-K9mut-8),转染HaCat细胞系,进一步明确lv-sh-K9mut-8对突变KRT9基因的靶向抑制效率。实验结果表明,si-K9mut-8无明显的脱靶效应。(3)Krt9+/mut突变杂合小鼠第10天和20天各1次的候选靶向shRNA注射治疗之后,肉眼可见病鼠的角化过度症状减轻,显示了表型上的治疗改观;检测治疗后的突变小鼠的前爪足垫突起处组织的增殖蛋白标记(PCNA、p63、involucrin和filaggrin)和细胞骨架角蛋白分子(K10、K1、K6、K16、K14、K5和K2e),发现shRNA治疗部分修复了病鼠的病理学改变,而没有引起明显的不良免疫应激反应。结论:靶向特异性shRNA可能是一种具有较大潜力的EPPK个性化基因治疗方案,并可为其他单基因遗传性皮肤病的基因治疗提供思路和借鉴。
[Abstract]:Background: epidermolysis palmar keratosis (epidermolyticpalmoplantarkeratoderma, EPPK.OMIM:144200) is one of the most common autosomal dominant skin hereditary diseases. It is the most common subtype.EPPK of highly genetic heterozygous keratataris.EPPK because of the KRT9 gene of the expression of keratin 9 (keratin9, K9). In several cases, typical clinical symptoms began to occur in the weeks to months after the birth of the KRT1 gene. The main clinical manifestations were diffuse keratinocyte thickening of the entire palmar and palmar epidermis, yellow, obvious reddish margins around the thickened epidermis, and many patients with perspiration and peridrosis. At present, there is no effective treatment for EPPK. Most of the research reports at home and abroad are still focused on the variant group of EPPK. Objective: on the basis of improving the phenotypic analysis of KRT9 gene mutation in mice, a feasible and efficient EPPK individualized gene therapy prescription is explored. The case has laid the foundation for the follow-up clinical gene therapy of EPPK and the treatment of drug use. Methods: (1) a few indel mutations of the KRT9 gene in the EPPK family of the Han nationality were reported in the earlier period of our group: c.500_500delAinsGGCT (p.Tyr167delinsTrpLeu), and Krt9/c.434delAinsGGCT (p.Tyr) was constructed by gene targeting. 144delinsTrpLeu) mutated into the mouse model. This study further analyzed the plantar skin of the mutant mice by deep phenotype and pathological analysis (HE staining, electron microscopy) to determine the EPPK like pathological changes in the model mice, and to analyze the changes in the K9 protein in the foot bottom skin tissue of the mutant mice and the mutation of the K9 protein from the molecular angle. The effects on the expression of other proteins. (2) the firefly luciferase reporter gene wild type and c.434delAinsGGCT mutant luciferase Lentivirus Expression Vector, pfLUC-Homo-exlKRT9/c.500delAinsGGCT/wt (pfLUC-ex1KRT9-mutant/wt) and pfLUC-Mus-ex1Krt9-c.434delAinsGGCT/wt (pfLUC-ex1Krt9-), were constructed respectively. Mutant/wt), based on the luciferase report test, 16 candidate siRNA molecules were designed according to the nucleotide sequence of the keratin 9 gene of the EPPK patients and c.434delAinsGGCT mutant mice, and the target therapy siRNA was screened and verified to achieve the Krt9 allele expression of the knockdown mutation to the normal Krt9 allele. The specific target therapy shRNA lentivirus vector (lv-sh-K9mut-8) was constructed based on the selected siRNA sequence, and the human immortalized keratinocyte line HaCat was used as the target, and lv-sh-K9mut-8 was used to transfect it. (3) the candidate target of the lentivirus carrier to shRNA (lv-sh-K9mut-8) was injected. Treatment of 12 weeks old Krt9+/mut mutant heterozygous mice was used to observe whether the EPPK like pathological symptoms were remission and other phenotypic changes; whether the symptoms of keratosis after treatment were improved from the histopathological and molecular levels; and whether the target shRNA (lv-sh-K9mut-8) caused a bad immune response. Results: (1) Krt9/c.434delAinsGGCT (p.Tyr1 44delinsTrpLeu) before the mutation was knocked into the heterozygous adult mice, the posterior claw was stable at 11~12 weeks. The obvious EPPK like pathological phenotypic.HE staining showed the thickening of the epidermis, the epidermis expansion and hyperkeratosis, the papilloma hyperplasia, the prominent granulosa cell increasing and the acanthosis, and the epidermis with the atypical shape. The ultrastructural analysis showed that the cell lysis of keratinocytes and the abnormal aggregation of fibrous filament in the basal layer of the epidermis. Immunofluorescence staining showed that the number and intensity of PCNA and p63 positive cells in the foot mats of K9 mutant mice increased, and the stress response and wound healing keratin K6 and K16 were in Krt9+/mut The expression in the Krt9mut/mut mice was significantly higher than that in the Krt9+/+ wild type mice; the keratin K1 and K10 expressed in the upper basement were significantly higher because of the dysfunction of K9, and had local dilatation and abnormal aggregation. The Westernblot analysis showed that the K2 expression of Krt9+/+ and Krt9+/mut mice was compared to those of the wild type mice. I reduced 60%. (2) with Krt9/c.434delAinsGGCT and KRT9/c.500delAinsGGC small insertion mutation as the target of siRNA therapy. After 16 candidate siRNA molecules, it was found that the functional and specific relative effects of si-K9mut-8 were best, only knockdown K9 molecules, but minimal to normal K9 protein. Therefore, si-K9mut-8 molecules were used. The base sequence was designed to synthesize the shRNA vector (lv-sh-K9mut-8) and transfect the HaCat cell line to further clarify the target inhibition efficiency of lv-sh-K9mut-8 to the mutant KRT9 gene. The experimental results showed that there was no clear Miss effect of si-K9mut-8. (3) the candidate target of Krt9+/mut mutation heterozygous mice was treated with shRNA for 1 times and 20 days each. After the treatment, the naked eye could be seen that the hyperkeratokeratosis of the rats was relieved and the phenotypic treatment was improved; the proliferating protein markers (PCNA, p63, involucrin and filaggrin) and the cytoskeleton keratin molecules (K10, K1, K6, K16, K14, K5 and K2e) were detected in the mutant mice after the treatment. The pathological changes of mice did not cause obvious adverse immune responses. Conclusion: targeted specific shRNA may be a potential EPPK individualized gene therapy scheme, and can provide ideas and reference for gene therapy of other single gene hereditary dermatosis.
【学位授予单位】:浙江大学
【学位级别】:博士
【学位授予年份】:2017
【分类号】:R758.5
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