Spink5基因条件性敲除小鼠模型的构建及表型分析

【摘要】 目的 构建Spink5基因条件性敲除小鼠模型并鉴定其表型。方法 采用CRISPR/Cas9技术构建基因型为Mb1cre/+Spink5floxp/floxp的B淋巴细胞Spink5基因条件性敲除小鼠（敲除组）,以基因型为Mb1+/+Spink5floxp/floxp的小鼠为对照组。提取小鼠脾脏单个核细胞并经流式细胞荧光技术分选B淋巴细胞及非B淋巴细胞,然后分别进行基因型鉴定及淋巴上皮Kazal型抑制物（LEKTI）蛋白表达的测定。切取小鼠皮肤行HE染色,测量表皮厚度,免疫荧光染色测定小鼠皮肤LEKTI蛋白荧光强度。组间比较采用配对t检验或两独立样本t检验。结果 基因型鉴定结果表明,成功构建稳定的B淋巴细胞Spink5基因条件性敲除小鼠模型。Western印迹显示,条件性敲除小鼠B淋巴细胞中LEKTI蛋白相对表达量为0.01 ± 0.02,显著低于非B淋巴细胞（0.66 ± 0.11,t = 9.99,P < 0.001）及对照组小鼠B淋巴细胞（1.08 ± 0.13,t = 13.78,P < 0.001）。39只敲除组小鼠中,4只出现皮肤干燥、散在鳞屑性肥厚型斑丘疹。敲除组皮损部位表皮厚度为（90.42 ± 21.31） μm,显著高于其非皮损部位［（29.71 ± 3.63） μm,t = 5.05,P = 0.002］及对照组表皮［（12.42 ± 2.21） μm,t = 6.74,P < 0.001］。免疫荧光检测显示,敲除组皮损处LEKTI蛋白荧光强度为46.21 ± 1.21,非皮损处为46.62 ± 2.13,与对照组（47.69 ± 1.71）差异均无统计学意义（P > 0.05）。结论 成功构建了B淋巴细胞Spink5基因条件性敲除小鼠模型,为进一步探究Netherton综合征皮肤屏障缺陷伴免疫功能异常的机制提供研究基础。     

金属硫蛋白基因对小鼠UVB照射的保护作用

目的:研究金属硫蛋白基因对UVB照射反应的影响.方法:敲除小鼠金属硫蛋白基因,背部皮肤剃毛后24 h照射不同剂量的UVB(0.05 J/cm2,0.70 J/cm2,1.40 J/cm2),照光前、后24 h测量小鼠背部皮肤厚度,皮肤组织病理观察照光后24 h日晒伤细胞形成情况.正常野鼠作对照.结果:小剂量UVB照射时两种小鼠水肿程度无显著性差异,大剂量UVB照射时金属硫蛋白基因敲除小鼠背部水肿显著高于野鼠.病理显示金属硫蛋白基因敲除小鼠较野鼠有更多的日晒伤细胞产生.结论:金属硫蛋白基因对急性UVB照射具有保护作用.     

湿疹及皮炎

20031971 金属硫蛋白基因对小鼠变应性接触性皮炎的影响/路雪艳(北大三院皮肤科)…//北京大学学报(医学版).-2003,35(2).-184～186 通过对比金属硫蛋白(MTs)基因敲除小鼠(MT-/-,不表达MT-Ⅰ及MT-Ⅱ)及野生型鼠(MT+/+)对致敏物的反应,以探讨MTs在小鼠变应性接触性皮炎中所起的作用。所采用的动物模型为鼠耳肿胀试验模型,以二硝基氟苯致敏小鼠,并在其右耳诱导接触性皮炎,分别对比两组小鼠激发后耳肿胀度。结果:野生型鼠(MT+/+)耳肿胀度明显高于基因敲除鼠(MT-/-),     

角质形成细胞异常诱发银屑病：基因工程动物模型的启示

银屑病的发病机制目前尚未完全阐明［1］。30年来,学术界一直对角质形成细胞和免疫细胞的功能异常在银屑病发病中孰为因果存在争论［2］。起初认为银屑病是由于角质形成细胞异常增殖引起的,但随着研究的深入,在皮损区发现Th1和Th17细胞浸润,遂将注意力转向T细胞介导的免疫反应,并逐渐将其作为银屑病发病机制的核心［1］。近年来,基因工程银屑病动物模型研究发现,角质形成细胞在银屑病皮肤炎症的启动和维持中均发挥关键作用。根据基因工程技术的特点,银屑病小鼠模型主要分为条件性基因敲除和先天性基因表达异常小鼠模型。前者是在小鼠成年以后,通过注射枸橼酸他莫昔芬特异性敲除角质形成细胞内的某些蛋白质而诱发银屑病,更接近于人类银屑病的发生过程。后者是通过特异性细胞内基因敲除或插入技术,使小鼠出生时即有角质形成细胞内蛋白质表达增多或缺失,更接近于先天性疾病的发病模式。本文简述几种银屑病基因工程动物模型以及相关研究,并结合近年我们团队取得的一些研究成果,分析并深入理解角质形成细胞在银屑病发病机制中的作用……     

腺病毒载体介导的PML生长抑制因子转染人角质形成细胞的实验研究

目的探讨腺病毒介导的PML基因对角质形成细胞(KC)的生长抑制作用。方法用原位杂交和免疫组织化学法分别检测PML基因重组腺病毒的转导效率,观察培养人KC中PMLmRNA和蛋白的表达量及表达时间,MTT法检测PML基因转染后人KC的生长状况,观察转染PML基因后KC的增殖能力的变化。结果腺病毒滴度为100MO I时被转染的人KC可达100%,Ad-PML基因转染的人KC能高效表达PMLmRNA和PML蛋白,表达时间可持续2周以上,PML基因对人KC体外生长具有明显抑制作用。结论腺病毒介导的PML基因能在人KC中高效稳定地表达,能显著抑制人KC的体外生长,PML生长抑制因子有可能用于银屑病等皮肤病的基因治疗。     

抑制树突细胞MMP-13基因对咪喹莫特诱导的小鼠银屑病样炎症模型的影响

目的：明确抑制树突细胞MMP-13基因对咪喹莫特诱导的小鼠银屑病样炎症模型的影响。方法：构建树突细胞MMP-13基因特异性敲除的小鼠模型（MMP-13^LOX/LOX）,应用咪喹莫特诱导小鼠银屑病样皮损的发生。将小鼠分为野生型对照组、MMP-13^LOX/LOX对照组、咪喹莫特诱导的野生型实验组和咪喹莫特诱导的MMP-13^LOX/LOX实验组,分析不同组别小鼠皮损临床表现并记录PASI评分,检测各组小鼠皮损中IL-1β、IL-6、IL-23和IL-17A表达。结果：与咪喹莫特诱导的野生型实验组小鼠比较,咪喹莫特诱导的MMP-13^LOX/LOX实验组银屑病皮损表现相比更加温和,仅有轻度的棘层肥厚和真皮细胞浸润,皮损中IL-1β、IL-6表达下调,而IL-23和IL-17A表达无明显改变。结论：抑制树突细胞MMP-13基因可降低炎症因子IL-6、IL-1β的表达及减弱小鼠皮肤银屑病炎症反应。     

CARD9基因敲除小鼠骨髓来源树突状细胞对阿萨希毛孢子菌临床株的免疫反应缺陷

目的初步探讨CARD9基因敲除小鼠骨髓来源树突状细胞(bone marrowderived dendritic cell,BMDC)对阿萨希毛孢子菌临床株(Trichosporon asahii,T.asahii)的免疫反应缺陷。方法体外培养野生型(wild type,WT)与CARD9基因敲除型(CARD9knockout,CARD9-/-)小鼠BMDC并分别与热灭活的T.asahii进行共培养,比较两者对菌体的黏附吞噬能力、表面共刺激分子的激活、细胞因子的表达以及两种小鼠感染菌株后的生存率。结果共培养24 h后,WT与CARD9-/-小鼠BMDC对T.asahii的黏附吞噬情况比较未见明显差异;经T.asahii刺激的CARD9-/-小鼠BMDC的CD80、CD86激活情况以及白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α表达水平均明显低于WT小鼠BMDC(P0.05);感染T.asahii的CARD9-/-小鼠的生存率明显低于WT小鼠(P0.05)。结论 CARD9-/-小鼠BMDC对T.asahii的免疫反应缺陷主要体现在共刺激分子的激活以及促炎细胞因子的表达,但并未影响其对T.asahii的吞噬识别。     

转录辅激活因子Mediator 1调控小鼠皮肤毛发再生的作用机制研究

目的:探讨转录辅激活因子Mediator 1（Med1）对小鼠皮肤毛发再生的影响及可能机制。方法:将C57BL/6J品系来源的Med1 flox/flox小鼠与K14-Cre小鼠交配,通过Cre-Loxp系统获得Med1表皮特异性敲除小鼠,即表达K14-Cre的Med1 flox/flox小鼠（...     

DnaJA4敲除加重咪喹莫特诱导的小鼠银屑病样皮肤炎症

目的 探讨DnaJA4对咪喹莫特诱导小鼠银屑病样皮肤炎症的影响.方法 4组小鼠(每组8只),即应用咪喹莫特(imiquimod,IMQ)诱导银屑病样皮炎的DnaJA4敲除(knockout,KO-IMQ)组小鼠及野生型(wild-type,WT-IMQ)小鼠,涂以等量凡士林的DnaJA4敲除(knockout,KO-b...     

角质形成细胞增生行为与p16INK4a蛋白表达和基因失活的相关性研究

目的：探讨角质形成细胞(KC)增生行为与p16^INK4a蛋白表达及其基因失活的相关性。方法：对20例皮肤鳞状细胞癌、24例基底细胞癌、18例Bowen病、12例光线性角化病以及8例脂溢性角化病共计82例患者中具不同增生行为的KC,分别应用免疫组化检测其p16^INK4a蛋白表达：应用聚合酶链反应(PCR)、PCR-单链构象多态性以及PCR-限制性内切酶分析方法分别检测p16^INK4a基因外显子1和外显子2的缺失、突变和甲基化。结果：p16^INK4a蛋白表达的阳性率和表达强度随着KC恶性程度的增加而降低,基因缺失和甲基化是皮肤癌p16^INK4a基因失活的主要方式,p16^INK4a蛋白失表达与其基因失活是一致的。结论：p16^INK4a基因失活在KC恶性转化中起重要作用;p16^INK4a基因有可能成为皮肤癌诊断与治疗的一种新靶位。     

GPHR-dependent functions of the Golgi apparatus are essential for the formation of lamellar granules and the skin barrier

The lumen of the Golgi apparatus is regulated to be weakly acidic, which is critical for its functions. The Golgi pH regulator (GPHR) is an anion channel essential for normal acidification of the Golgi apparatus, and is therefore required for its functions. The Golgi apparatus has been thought to be the origin of lamellar granules in the skin. To study the functional role(s) of GPHR in the skin, we established keratinocyte-specific GPHR-knockout mice using the Cre-loxP system. These mutant mice exhibited hypopigmented skin, hair loss, and scaliness. Histological examination of GPHR-knockout mice showed ballooning of the basal cells and follicular dysplasia. In addition, inflammatory cells were seen in the dermis. The expression of trans-Golgi network 46, a marker for lamellar bodies, and kallikrein 7, a protein within lamellar bodies, is diminished in GPHR-knockout mouse skin. Examination by electron microscopy revealed that keratinocytes produced aberrant lamellar bodies. The transepidermal water loss of these knockout mice was increased compared with wild-type mice. Moreover, expression of cathelicidin-related antimicrobial peptide (CRAMP) in the skin was diminished. These results suggest that GPHR is essential for the homeostasis of the epidermis including the formation of lamellar bodies and for the barrier function.     

T cells reactive to keratinocyte antigens are generated during induction of contact hypersensitivity in mice. A model for autoeczematization in humans?

BACKGROUND: The role of keratinocytes (KC) in contact hypersensitivity (CH) has been examined more with respect to cytokine secretion and tolerance induction and less as a source of antigenic proteins to which chemical haptens can conjugate. OBJECTIVE: To determine whether KC-derived proteins can serve as antigenic carriers for haptens such as dinitrofluorobenzene (DNFB). METHODS: We examined the capacity of draining lymph node cells from BALB/c mice sensitized to DNFB to proliferate in response to hapten or to hapten-conjugated protein extracts derived from a KC line (DNP-Pam KC extract). Using limiting dilution microculture of these lymph node cells, we established DNP-specific T cell clones as well as DNP-Pam KC extract-reactive T cell clones. We also examined the proliferative responses of the DNP-Pam KC extract-reactive clones and of lymph node cells from mice sensitized to different haptens. RESULTS: Lymph node cells from DNFB-sensitized mice proliferated well to hapten or to DNP-Pam KC extract. Six la(d)-restricted, alphabeta TCR-bearing, CD4+ clones were established: 4 proliferated specifically to soluble hapten (DNBS), whereas 2 proliferated in response to DNP-Pam KC extract. Surprisingly, the DNP-Pam KC extract-reactive clones proliferated as well to Pam KC extract without hapten. Lymph node cells from hapten-sensitized mice not only proliferated specifically in response to the hapten to which they were sensitized, but also proliferated to Pam KC extract without hapten. CONCLUSIONS: T cell clones generated during the induction of (CH) in mice include those reactive to hapten as well as those reactive to KC antigens independent of hapten. Analogous mechanisms in humans might account for autoreactive events such as id reactions associated with CH and angry back syndrome during patch testing.     

Conditional gene targeting and its application in the skin

The knockout mouse is one of the most useful experimental animal systems in which to clarify functions of identified genes in vivo. Numerous knockout mice have been produced, and studies in these models have revealed new aspects in various biological fields. In fair cases, however, mice died during embryogenesis due to complete gene disruption, making it difficult to elucidate functions of given genes in adulthood. To avoid this, conditional gene targeting has been performed and shown to be effective on numerous cases. Here, we will overview established strategies for conditional gene targeting and present the results of our recent studies using the Cre/loxP system in the skin.     

Selectable bicistronic vectors in skin gene therapy

Bicistronic vectors (BCV) are important tools for gene therapy applications allowing selection for increased expression of a desired gene by linking it to a selectable gene, such as the multi-drug resistance (MDR) gene. However, both the design of the BCV and the cell type to be transduced can have a strong impact on the vector performance in the target cells. To analyze which factors might influence the efficiency of BCV in achieving high gene expression levels in skin and to determine the best suited BCV for cutaneous transduction, both keratinocytes (KC) and fibroblasts (FB) were transduced with different BCV constructs, BGIM, BMIG and QGIM. In BGIM, expression of the BCV cassette encompassing the green fluorescent protein (GFP) gene connected to the MDR gene was driven by a retroviral LTR-promoter. In BMIG, the order of the two genes was reversed, while in QGIM the GFP- and MDR-gene were arranged similar as in BGIM, but expressed by a CMV- instead of an LTR-promoter. FACS-analysis revealed that the percentage of genetically modified cells varied substantially with 47.9% QGIM-, 35.5% BMIG- and 17.9% BGIM-transduced KC expressing both genes. For FB the numbers were 56.7% (QGIM), 38.4% (BMIG) and 8.3% (BGIM). Furthermore, the choice of BCV determined the intensity of GFP-expression with the highest levels measured in BGIM-, followed by QGIM- and then BMIG-transduced cells. Interestingly, highly efficient enrichment through colchicine selection was possible for QGIM- (up to 97.1% KC, 97.8% FB) and BMIG- (85.0% KC and 98.0% FB) but not BGIM- (29.9% KC and 18.6% FB) transduced cells. Finally, immunohistochemistry and FACS-analysis demonstrated, that colchicine selection of QGIM-transduced skin equivalents led to increased numbers of GFP-expressing KC (from 51.2% up to 72.3%) and enhanced GFP-intensity in the skin. These results show that BCV present a promising vector system to enhance the expression of a desired gene in skin but important parameters taken into account when employing a selectable BCV for skin gene therapy applications are the retroviral vector backbone, the order in which the genes are arranged, and the target cells to be transduced and selected.     

Deletion of mu- and kappa-opioid receptors in mice changes epidermal hypertrophy, density of peripheral nerve endings, and itch behavior

The mu- (MOR) and kappa- (KOR) opioid receptors have been implicated in the regulation of homeostasis of non-neuronal cells, such as keratinocytes, and sensations like pain and chronic pruritus. Therefore, we have studied the phenotype of skin after deletion of MOR and KOR. In addition, we applied a dry skin model in these knockout mice and compared the different mice before and after induction of the dermatitis in terms of epidermal thickness, epidermal peripheral nerve ending distribution, dermal inflammatory infiltrate (mast cells, CD4 positive lymphocytes), and scratching behavior. MOR knockout mice reveal as phenotype a significantly thinner epidermis and a higher density of epidermal fiber staining by protein gene product 9.5 than the wild-type counterparts. Epidermal hypertrophy, induced by the dry skin dermatitis, was significantly less developed in MOR knockout than in wild-type mice. Neither mast cells nor CD4 T(h)-lymphocytes are involved in the changes of epidermal nerve endings and epidermal homeostasis. Finally, behavior experiments revealed that MOR and KOR knockout mice scratch less after induction of dry skin dermatitis than wild-type mice. These results indicate that MOR and KOR are important in skin homeostasis, epidermal nerve fiber regulation, and pathophysiology of itching.     

Inactivation of autophagy leads to changes in sebaceous gland morphology and function

We have reported recently that inactivation of the essential autophagy‐related gene 7 (Atg7) in keratinocytes has little or no impact on morphology and function of the epidermal barrier in experimental animals. When these mice aged, mutant males, (Atg7 ΔKC), developed an oily coat. As the keratin 14 promoter driven cre/LoxP system inactivates floxed Atg7 in all keratin 14 (K14) expressing cells, including sebocytes, we investigated whether the oily hair phenotype was the consequence of changes in function of the skin sebaceous glands. Using an antibody to the GFP‐LC3 fusion protein, autophagosomes were detected at the border of sebocyte disintegration in control but not in mutant animals, suggesting that autophagy was (a) active in normal sebaceous glands and (b) was inactivated in the mutant mice. Detailed analysis established that dorsal sebaceous glands were about twice as large in all Atg7 ΔKC mice compared to those of controls (Atg7 F/F), and their rate of sebocyte proliferation was increased. In addition, male mutant mice yielded twice as much lipid per unit hair as age‐matched controls. Analysis of sebum lipids by thin layer chromatography revealed a 40% reduction in the proportion of free fatty acids (FFA) and cholesterol, and a 5‐fold increase in the proportion of fatty acid methyl esters (FAME). In addition, the most common diester wax species (58‐60 carbon atoms) were increased, while shorter species (54‐55 carbon atoms) were under‐represented in mutant sebum. Our data show that autophagy contributes to sebaceous gland function and to the control of sebum composition.     

The skin as a biofactory for systemic secretion of erythropoietin: potential of genetically modified keratinocytes and fibroblasts

Background: The skin is an interesting target tissue for gene therapy applications because of its ready accessibility. One possibility would be to utilize the genetically modified skin as a biofactory secreting a systemically needed product, such as erythropoietin (EPO). Methods: Keratinocytes (KC) and fibroblasts (FB) were transduced with a retroviral vector encoding human EPO. Gene transfer efficiency was assessed by real‐time PCR analysis and flow cytometry of transduced cells. In addition, EPO synthesis and secretion were analysed by quantifying the amount of RNA and secreted protein in both monolayer cultures and skin equivalents (SE). Results: When cultured as a monolayer, EPO‐KC synthesized significantly more EPO than EPO‐FB, as shown by quantitatively measuring the amount of secreted protein and RNA. This correlated with an increased EPO‐vector incorporation in KC compared with FB, demonstrated by determining both the percentage of transduced cells and the average transgene copy number per cell. In addition, in transduced cell cultures enriched to equally high percentages of EPO+ cells, KC showed a higher activity of EPO secretion than FB. Finally, when assembled in a SE, EPO‐KC secreted significantly higher amounts of EPO than EPO‐FB, although reduced secretory activity of EPO‐KC monolayers grown in high calcium concentrations suggested that in stratified epidermis differentiated KC secrete less EPO than non‐differentiated KC. Conclusion: In summary, while both transduced KC and FB are able to synthesize and secrete human EPO, KC show higher potential in serving as possible target cells for therapeutic substitution with EPO, probably because of improved transduction rates and increased secretory activity.     

Mouse Samd9l is not a functional paralogue of the human SAMD9, the gene mutated in normophosphataemic familial tumoral calcinosis

Normophosphataemic familial tumoral calcinosis, charac-terized by ectopic mineralization of skin, is caused by mutations in the SAMD9 gene located in human chromosome 7q21, next to a paralogous gene, SAMD9-like (SAMD9L). The mouse does not have a SAMD9 orthologue, Samd9, because it has been deleted during evolution owing to genomic rearrangements. It has been suggested that the mouse Samd9l gene serves as a functional paralogue of human SAMD9. In this study, we examined Samd9l knockout mice with respect to ectopic mineralization. We also crossed these mice with Abcc6(tm1JfK) mice, a model system to study pseudoxanthoma elasticum, to see whether the absence of the Samd9l gene modifies the mineralization process. Necropsy analysis of Samd9l(tm1Homy) mice revealed no evidence of ectopic mineralization, and deletion of the Samd9l gene in mice failed to modify the mineralization process on the Abcc6(tm1JfK) background. Collectively, the results suggest that mouse Samd9l is not a functional paralogue of human SAMD9.     

The role of mitogen- and stress-activated protein kinase 1 and 2 in chronic skin inflammation in mice

Mitogen- and stress-activated protein kinase 1 and 2 (MSK1/2) are two kinases phosphorylated by both ERK1/2 and p38 MAPK. Recently, MSK1 and 2 have been reported to act as negative regulators of acute inflammation. In this study, we investigated the role of MSK1/2 in chronic skin inflammation using an oxazolone-induced allergic contact dermatitis model in MSK1/2 knockout mice and wild-type mice. MSK1/2 knockout mice were demonstrated to have significantly increased inflammation compared with wild-type mice. This was measured by an increased ear thickness, elevated infiltration of neutrophils in the skin and increased inflammatory histological changes. Furthermore, we found significantly elevated levels of the proinflammatory cytokines Tumor necrosis factor-α (TNF-α), IL-1β and IL-6 at both mRNA and protein levels in MSK1/2 knockout mice compared with wild-type mice after oxazolone treatment. In addition, the mRNA expression of the chemokine Thymus and activation regulated chemokine (TARC) was demonstrated to be significantly elevated in oxazolone-treated MSK1/2 knockout mice compared with wild-type mice. The increased expression of TARC was paralleled by increased infiltration of cells positive for the TARC receptor, CCR4, in the dermis of MSK1/2 knockout mice. Our results indicate that MSK1/2 are involved in the activation of feedback mechanisms that dampen oxazolone-induced skin inflammation.