發(fā)布時間：2018-05-13 02:42

  本文選題：表皮松解性掌跖角化癥 + KRT9�。� 參考：《浙江大學》2017年博士論文

【摘要】：背景:表皮松解性掌跖角化癥(epidermolyticpalmoplantarkeratoderma,EPPK。OMIM:144200)屬于一種較為常見的常染色體顯性皮膚遺傳病,是高度遺傳異質(zhì)性的掌跖角化癥最為常見的亞型。EPPK的主要致病基因為表達角蛋白9(keratin9,K9)分子的KRT9基因,少數(shù)病例由KRT1基因突變引起。EPPK患者出生后數(shù)周到數(shù)月內(nèi)即逐漸開始出現(xiàn)典型的臨床癥狀,并持續(xù)終生。主要臨床表現(xiàn)為整個手掌和腳掌表皮的彌漫性角質(zhì)增厚,色黃,在增厚的表皮周邊有明顯的紅斑緣,許多患者伴多汗、臭汗癥。某些患者還伴發(fā)指節(jié)墊、斷指或先天性指屈曲等癥狀。目前,EPPK尚無有效的治療手段。國內(nèi)外的絕大多數(shù)研究報道尚聚焦于EPPK的變異組學。目的:在完善小鼠KRT9基因突變敲入模型表型分析的基礎(chǔ)上,探索一種可行、高效的EPPK個性化基因治療方案,為EPPK的后續(xù)臨床基因治療、用藥治療等研究奠定基礎(chǔ)。方法:(1)本課題組前期已報道發(fā)現(xiàn)幾個南方漢族EPPK家系特有一種KRT9基因 indel 突變類型:c.500_500delAinsGGCT(p.Tyr167delinsTrpLeu),并據(jù)此通過基因打靶技術(shù)構(gòu)建了 Krt9/c.434delAinsGGCT(p.Tyr144delinsTrpLeu)突變敲入小鼠模型。本研究進一步對突變小鼠的足底皮膚進行深入的表型和病理學分析(HE染色、電鏡觀察等),以確定模型小鼠的EPPK樣病理改變;并從分子角度分析突變小鼠的足底皮膚組織K9蛋白的變化,以及K9蛋白的突變對其他蛋白質(zhì)表達的影響。(2)分別構(gòu)建人和小鼠的螢火蟲熒光素酶報告基因野生型和c.434delAinsGGCT突變型螢火蟲熒光素酶慢病毒表達載體,即 pfLUC-Homo-exlKRT9/c.500delAinsGGCT/wt(pfLUC-ex1KRT9-mutant/wt)和pfLUC-Mus-ex1Krt9-c.434delAinsGGCT/wt(pfLUC-ex1Krt9-mutant/wt),以熒光素酶報告實驗為依據(jù),按照EPPK患者和c.434delAinsGGCT突變小鼠的角蛋白9基因序列設計16條候選siRNA分子,篩選并驗證功能性和特異性相對最好的靶向治療siRNA,以達到敲降突變的Krt9等位基因表達量而對正常Krt9等位基因無影響或影響極小的目的。依據(jù)所篩選的siRNA序列,構(gòu)建特異性靶向治療shRNA慢病毒載體(即lv-sh-K9mut-8),以人永生化角質(zhì)形成細胞系HaCat為對象,用lv-sh-K9mut-8對其進行轉(zhuǎn)染,觀察有無脫靶效應。(3)將慢病毒載體候選靶向shRNA(lv-sh-K9mut-8)注射治療12周齡的Krt9+/mut突變雜合小鼠,觀察EPPK樣病理癥狀是否得到緩解等表型變化;從組織病理和分子水平上分析治療后的角化增生癥狀是否得到改善;分析靶向shRNA(lv-sh-K9mut-8)是否引發(fā)了不良的免疫反應。結(jié)果:(1)Krt9/c.434delAinsGGCT(p.Tyr144delinsTrpLeu)突變敲入雜合性成年小鼠的前、后爪在11～12周時出現(xiàn)穩(wěn)定、明顯的EPPK樣病理表型。HE染色顯示足墊凸起處表皮增厚、表皮擴張和角化過度癥狀,乳頭狀瘤狀增生,顯著的顆粒細胞增多癥和棘皮癥,以及具有不典型形狀的表皮上部中的空泡化細胞。透射電鏡超微結(jié)構(gòu)分析顯示,角化細胞的細胞裂解和表皮的基底層中的纖維絲的異常聚集。免疫熒光染色顯示,K9突變小鼠足墊中PCNA和p63陽性細胞的數(shù)量和染色強度增加;應激反應和創(chuàng)傷愈合角蛋白K6和K16在Krt9+/mut和Krt9mut/mut小鼠中的表達顯著高于Krt9+/+野生型小鼠;表達于基底上層的角蛋白K1和K10因K9的功能障礙而顯著增高,并具有局部擴張和異常聚集的現(xiàn)象。免疫印跡試驗(Westernblot)分析顯示,Krt9+/+和Krt9+/mut小鼠的K2表達量與野生型小鼠相比III減少了 60%。(2)以Krt9/c.434delAinsGGCT 和 KRT9/c.500delAinsGGC 小插缺突變?yōu)榘邢騭iRNA治療的研究對象,經(jīng)16條候選siRNA分子驗證之后,發(fā)現(xiàn)si-K9mut-8的功能性和特異性相對效應最好,僅敲降突變K9分子,而對正常K9蛋白影響極小。因此,以si-K9mut-8分子的堿基序列設計合成了慢病毒表達shRNA載體(lv-sh-K9mut-8),轉(zhuǎn)染HaCat細胞系,進一步明確lv-sh-K9mut-8對突變KRT9基因的靶向抑制效率。實驗結(jié)果表明,si-K9mut-8無明顯的脫靶效應。(3)Krt9+/mut突變雜合小鼠第10天和20天各1次的候選靶向shRNA注射治療之后,肉眼可見病鼠的角化過度癥狀減輕,顯示了表型上的治療改觀;檢測治療后的突變小鼠的前爪足墊突起處組織的增殖蛋白標記(PCNA、p63、involucrin和filaggrin)和細胞骨架角蛋白分子(K10、K1、K6、K16、K14、K5和K2e),發(fā)現(xiàn)shRNA治療部分修復了病鼠的病理學改變,而沒有引起明顯的不良免疫應激反應。結(jié)論:靶向特異性shRNA可能是一種具有較大潛力的EPPK個性化基因治療方案,并可為其他單基因遺傳性皮膚病的基因治療提供思路和借鑒。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R758.5

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