Ubiquitin-specific protease 21 promotes tumorigenicity and stemness of colorectal cancer by deubiquitinating and stabilizing ZEB1

BACKGROUND Colorectal cancer(CRC)is one very usual tumor together with higher death rate.Ubiquitin-specific protease 21(USP21)has been confirmed to take part into the regulation of CRC progression through serving as a facilitator.Interestingly,the promotive function of USP21 has also discovered in the progression of CRC.ZEB1 has illustrated to be modulated by USP7,USP22 and USP51 in cancers.However,the regulatory functions of USP21 on ZEB1 in CRC progression need more invest-igations.AIM To investigate the relationship between USP21 and ZEB1 in CRC progression.METHODS The mRNA and protein expressions were assessed through RT-qPCR,western blot and IHC assay.The interaction between USP21 and ZEB1 was evaluated through Co-IP and GST pull down assays.The cell proliferation was detected through colony formation assay.The cell migration and invasion abilities were determined through Transwell assay.The stemness was tested through sphere formation assay.The tumor growth was evaluated through in vivo mice assay.RESULTS In this work,USP21 and ZEB1 exhibited higher expression in CRC,and resulted into poor prognosis.Moreover,the interaction between USP21 and ZEB1 was further investigated.It was demonstrated that USP21 contributed to the stability of ZEB1 through modulating ubiquitination level.In addition,USP21 streng-thened cell proliferation,migration and stemness through regulating ZEB1.At last,through in vivo assays,it was illustrated that USP21/ZEB1 axis aggravated tumor growth.CONCLUSION For the first time,these above findings manifested that USP21 promoted tumorigenicity and stemness of CRC by deubiquitinating and stabilizing ZEB1.This discovery suggested that USP21/ZEB1 axis may provide novel sights for the treatment of CRC.

Association of 370-371insACA, 494T〉C, and 1423C〉T haplotype in ubiquitin-specific protease 26 gene and male infertility： a meta-analysis

Whether the 370-371insACA, 494T〉C, and 1423C〉T haplotype in ubiquitin-specific protease 26 （USP26） gene is associated with male infertility is controversial. To clarify this issue, we conducted a meta-analysis based on the most recent studies. Eligible studies were screened by using PubMed and Embase. Pooled odd ratio （OR） with 95% confidence interval （CI） was calculated with fixed effect models. Ten studies with 1603 patients and 2505 controls were included, Overall, the results indicated that there was an association between the haplotype and male infertile risk （OR = 1.74, 95% CI： 1.09-2.77）. The OR calculated based on the five studies in Asia and three in Europe was 1.96 （95% CI： 1,05-3.67） and 1.54 （95% Ch 0.75-3.16） respectively, however, the OR was 0.86 （95% Ch 0.05-15,29） based on the two investigations in America. In addition, the data from the patients with azoospermia （AZO） showed an increased pooled OR of 2.35 （95% Cl： 1.22-4.50）. In contrast, the studies with oligoasthenoteratozoospermia （OAT） exhibited that the pooled OR was 0,97 （95% Ch 0.43-2.16）. Our analyses indicate that there is an association of alteration in USP26 with male infertility, especially in AZO and Asian population.

Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility

Aim： To study the incidence of single nucleotide polymorphisms in ubiquitin-specific protease 26 （USP26） gene and its involvement in idiopathic male infertility in China. Methods： Routine semen analysis was performed. Infertility factors such as immunological, infectious and biochemical disorders were examined to select patients with idiopathic infertility. DNA was isolated from peripheral blood of the selected patients and control population, which were examined for mutations using polymerase chain reaction-single strand conformation polymorphism analysis. Furthermore, nucleotide sequences were sequenced in some patients and controls. Results： Of 41 infertile men, 9 （22.0%, P = 0.01） had changes in USP26 gene on the X chromosome. A compound mutation （364insACA; 460G→A） was detected in 8 patients （19.5%, P = 0.01） and a 1044T→A substitution was found in 1 patient （2.4%, P 〉 0.05）. All three variations led to changes in the coding amino acids. Two substitutions predict some changes： 460G→ A changes a valine into an isoleucine, and 1044T → A substitutes a leucine for a phenylalanine. Another insertion of three nucleotides ACA causes an insertion of threonine. No other changes were found in the remaining patients and fertile controls. Conclusion： The USP26 gene might be of importance in male reproduction. Mutations in this gene might be associated with male infertility, and might negatively affect testicular function. Further research on this issue is in progress.

Ubiquitin-specific protease 22 enhances intestinal cell proliferation and tissue regeneration after intestinal ischemia reperfusion injury

BACKGROUND Intestinal ischemia reperfusion(I/R) injury is a serious but common pathophysiological process of many diseases, resulting in a high mortality rate in clinical practice. Ubiquitin-specific protease 22(USP22) acts as regulator of cell cycle progression, proliferation, and tumor invasion. Depleted USP22 expression has been reported to contribute to arrested cell cycle and disrupted generation of differentiated cell types in crypts and villi. However, the role of USP22 in intestinal damage recovery has not been investigated. Therefore, elucidation of the underlying mechanism of USP22 in intestinal I/R injury may help to improve the tissue repair and patient prognosis in clinical practice.AIM To investigate the role of USP22 in intestinal cell proliferation and regeneration after intestinal I/R injury.METHODS An animal model of intestinal I/R injury was generated in male Sprague-Dawley rats by occlusion of the superior mesenteric artery followed by reperfusion.Chiu's scoring system was used to grade the damage to the intestinal mucosa. An in vitro model was developed by incubating rat intestinal epithelial IEC-6 cells in hypoxia/reoxygenation conditions in order to simulate I/R in vivo. siRNA and overexpression plasmid were used to regulate the expression of USP22. USP22,Cyclin D1, and proliferating cell nuclear antigen(PCNA) expression levels were measured by Western blot analysis and immunohistochemistry staining. Cell survival(viability) and cell cycle were evaluated using the Cell Counting Kit-8and flow cytometry, respectively.RESULTS USP22 expression was positively correlated with the expression levels of PCNA and Cyclin D1 both in vivo and in vitro, which confirmed that USP22 was involved in cell proliferation and intestinal regeneration after intestinal I/R injury. Decreased levels of Cyclin D1 and cell cycle arrest were observed in the USP22 knockdown group(P < 0.05), while opposite results were observed in the USP22 overexpression group(P < 0.05). In addition, increased expression of USP22 was related to improved intestinal pathology or IEC-6 cell viability after I/R or hypoxia/reoxygenation. These results suggested that USP22 may exert a protective effect on intestinal I/R injury by regulating cell proliferation and facilitating tissue regeneration.CONCLUSION USP22 is correlated with promoting intestinal cell proliferation and accelerating intestinal tissue regeneration after intestinal I/R injury and may serve as a potential target for therapeutic development for tissue repair during intestinal I/R injury.

Ubiquitin-specific protease 15 contributes to gastric cancer progression by regulating the Wnt/β-catenin signaling pathway

BACKGROUND Ubiquitin-specific protease 15(USP15)is an important member of the ubiquitinspecific protease family,the largest deubiquitinase subfamily,whose expression is dysregulated in many types of cancer.However,the biological function and the underlying mechanisms of USP15 in gastric cancer(GC)progression have not been elucidated.AIM To explore the biological role and underlying mechanisms of USP15 in GC progression.METHODS Bioinformatics databases and western blot analysis were utilized to determine the expression of USP15 in GC.Immunohistochemistry was performed to evaluate the correlation between USP15 expression and clinicopathological characteristics of patients with GC.A loss-and gain-of-function experiment was used to investigate the biological effects of USP15 on GC carcinogenesis.RNA sequencing,immunofluorescence,and western blotting were performed to explore the potential mechanism by which USP15 exerts its oncogenic functions.RESULTS USP15 was up-regulated in GC tissue and cell lines.The expression level of USP15 was positively correlated with clinical characteristics(tumor size,depth of invasion,lymph node involvement,tumor-node-metastasis stage,perineural invasion,and vascular invasion),and was related to poor prognosis.USP15 knockdown significantly inhibited cell proliferation,invasion and epithelialmesenchymal transition(EMT)of GC in vitro,while overexpression of USP15 promoted these processes.Knockdown of USP15 inhibited tumor growth in vivo.Mechanistically,RNA sequencing analysis showed that USP15 regulated the Wnt signaling pathway in GC.Western blotting confirmed that USP15 silencing led to significant down-regulation ofβ-catenin and Wnt/β-catenin downstream genes(c-myc and cyclin D1),while overexpression of USP15 yielded an opposite result and USP15 mutation had no change.Immunofluorescence indicated that USP15 promoted nuclear translocation ofβ-catenin,suggesting activation of the Wnt/β-catenin signaling pathway,which may be the critical mechanism promoting GC progression.Finally,rescue experiments showed that the effect of USP15 on gastric cancer progression was dependent on Wnt/β-catenin pathway.CONCLUSION USP15 promotes cell proliferation,invasion and EMT progression of GC via regulating the Wnt/β-catenin pathway,which suggests that USP15 is a novel potential therapeutic target for GC.

Emerging potential of ubiquitin-specific proteases and ubiquitinspecific proteases inhibitors in breast cancer treatment

Breast cancer is the most frequently diagnosed cancer in women,accounting for 30%of new diagnosing female cancers.Emerging evidence suggests that ubiquitin and ubiquitination played a role in a number of breast cancer etiology and progression processes.As the primary deubiquitinases in the family,ubiquitin-specific peptidases(USPs)are thought to represent potential therapeutic targets.The role of ubiquitin and ubiquitination in breast cancer,as well as the classification and involvement of USPs are discussed in this review,such as USP1,USP4,USP7,USP9X,USP14,USP18,USP20,USP22,USP25,USP37,and USP39.The reported USPs inhibitors investigated in breast cancer were also summarized,along with the signaling pathways involved in the investigation and its study phase.Despite no USP inhibitor has yet been approved for clinical use,the biological efficacy indicated their potential in breast cancer treatment.With the improvements in phenotypic discovery,we will know more about USPs and USPs inhibitors,developing more potent and selective clinical candidates for breast cancer.

Inhibition of Ubiquitin-specific Protease 4 Attenuates Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells via Transforming Growth Factor Beta Receptor Type Ⅰ

Objective Ubiquitin-specific protease 4(USP4)facilitates the development of transforming growth factor-beta 1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)in various cancer cells.Moreover,EMT of renal tubular epithelial cells(RTECs)is required for the progression of renal interstitial fibrosis.However,the role of USP4 in EMT of RTECs remains unknown.The present study aimed to explore the effect of USP4 on the EMT of RTECs as well as the involved mechanism.Methods In established unilateral ureteral obstruction(UUO)rats and NRK-52E cells,immunohistochemistry and Western blot assays were performed.Results USP4 expression was increased significantly with obstruction time.In NRK-52E cells stimulated by TGF-β1,USP4 expression was increased in a time-dependent manner.In addition,USP4 silencing with specific siRNA indicated that USP4 protein was suppressed effectively.Meanwhile,USP4 siRNA treatment restored E-cadherin and weakened alpha smooth muscle actin(α-SMA)expression,indicating that USP4 may promote EMT.After treatment with USP4 siRNA and TGF-β1 for 24 h,the expression of TGF-β1 receptor type I(TβRI)was decreased.Conclusion USP4 promotes the EMT of RTECs through upregulating TβRI,thereby facilitating renal interstitial fibrosis.These findings may provide a potential target of USP4 in the treatment of renal fibrosis.

USP7-MDM2-p53信号轴对子宫内膜癌细胞增殖、凋亡和细胞周期的影响

目的:探讨泛素特异性蛋白酶7(USP7)调节Mdm2 p53结合蛋白同源物(MDM2)-p53轴对子宫内膜癌细胞增殖、凋亡和细胞周期的影响。方法:Western blot检测人子宫内膜癌组织、癌旁组织、人子宫内膜上皮细胞hEEC及人子宫内膜癌细胞系Ishikawa、HEC-1-A、KLE中USP7蛋白表达。将Ishikawa细胞分为NC组、P22077(USP7抑制剂)组、pcDNA组、pcDNA-MDM2组、P22077+pcDNA组、P22077+pcDNA-MDM2组,CCK-8法和克隆形成实验检测Ishikawa细胞增殖;流式细胞术检测Ishikawa细胞凋亡与细胞周期变化;Western blot检测Ishikawa细胞中USP7、细胞周期蛋白D1(CyclinD1)、周期素依赖性激酶2(CDK2)、Bcl-2相关X蛋白(Bax)、MDM2、p53蛋白表达。以RG7388(MDM2抑制剂)或PFT-α(p53抑制剂)与20μmol/L P22077共处理Ishikawa细胞48 h以验证USP7-MDM2-p53信号轴上下游关系。结果:USP7蛋白在子宫内膜癌组织和细胞中高表达,且Ishikawa细胞中USP7蛋白表达量最高,因此,选择Ishikawa细胞为研究对象。与NC组比较,P22077组Ishikawa细胞OD 450值、克隆形成率、S期和G 2/M期细胞数、USP7、CyclinD1、CDK2、MDM2蛋白表达降低,细胞凋亡率、G_(0)/G_(1)期细胞数、p53、Bax蛋白表达升高(P<0.05);与NC组、pcDNA组比较,pcDNA-MDM2组Ishikawa细胞OD 450值、克隆形成率、S期和G 2/M期细胞数、USP7、CyclinD1、CDK2、MDM2蛋白表达升高,细胞凋亡率、G_(0)/G_(1)期细胞数、p53、Bax蛋白表达降低(P<0.05);与P22077组、P22077+pcDNA组比较,P22077+pcDNA-MDM2组Ishikawa细胞OD 450值、克隆形成率、S期和G 2/M期细胞数、USP7、CyclinD1、CDK2、MDM2蛋白表达升高,细胞凋亡率、G_(0)/G_(1)期细胞数、p53、Bax蛋白表达降低(P<0.05)。p53为USP7-MDM2通路下游分子。结论:抑制USP7表达可能通过下调MDM2来激活p53进而抑制Ishikawa细胞增殖、促进细胞凋亡及周期停滞。

抑制泛素特异性蛋白酶7改善血管紧张素Ⅱ诱导的心肌细胞肥大

目的探究泛素特异性蛋白酶7(USP7)抑制剂FT671在血管紧张素Ⅱ(AngⅡ)诱导的H9c2细胞肥大中的作用和潜在机制。方法将H9c2细胞随机分为四组:对照组、FT671组、AngⅡ组、FT671+AngⅡ组。利用α-actinin细胞免疫荧光染色检测心肌细胞表面积;Western blot检测心房钠尿肽(ANP)、脑钠肽(BNP)、B细胞淋巴瘤-2(Bcl-2)、B细胞淋巴瘤-2相关X蛋白(Bax)、白细胞介素-6(IL-6)、单核细胞趋化蛋白-1(MCP-1)、肿瘤坏死因子-α(TNF-α)、NOD样受体热蛋白结构域相关蛋白3(NLRP3)以及NADPH氧化酶4(Nox4)的蛋白表达水平;RT-PCR检测ANP、BNP、β-肌球蛋白重链(β-MHC)、IL-6、MCP-1、TNF-α的mRNA表达;TUNEL染色检测细胞凋亡情况;细胞计数试剂8(CCK8)实验检测各组细胞活力;活性氧(ROS)染色检测细胞内氧化损伤水平。结果与对照组相比,AngⅡ组USP7蛋白表达明显增加,而使用FT671后,USP7表达明显被抑制。与AngⅡ组相比,FT671+AngⅡ组心肌细胞面积减小;ANP、BNP、Bax的蛋白表达减少,ANP、BNP、β-MHC、Bax、IL-6、MCP-1以及TNF-α的mRNA表达减少;Bcl-2的蛋白和mRNA表达均增加。与AngⅡ组相比,FT671+AngⅡ组TUNEL阳性细胞明显减少,心肌细胞活力增强,ROS生成减少,Nox4、NLRP3蛋白表达减少,差异具有统计学意义(P<0.05)。结论FT671通过抑制Nox4/ROS/NLRP3炎症通路减轻AngⅡ诱导的心肌细胞中的氧化应激和炎症反应,从而减轻心肌细胞肥大。

泛素特异性蛋白酶7功能和在结直肠癌中的研究进展

泛素特异性蛋白酶7(ubiquitin-specific protease 7,USP7)是一种广泛参与各种细胞过程的去泛素化酶。USP7在调控细胞增殖与凋亡,细胞分裂、分化,DNA损伤修复,表观遗传学调控等生物学过程中有关键作用。本文对USP7的结构及其广泛的生物学功能进行综述。

1例USP7基因自发突变致Hao-Fountain综合征的病例特点分析并文献复习

回顾分析德州市妇女儿童医院1例因泛素特异性蛋白酶7(USP7)基因自发突变所致Hao-Fountain综合征患儿的临床资料,该女性患儿因语言及智力发育落后1年就诊,查体表情淡漠,眼神交流少,眼距宽,小下颌,眼球略凹陷,眼窝略深。基因检测USP7基因有1个杂合突变,确诊为Hao-Fountain综合征。USP7基因突变属于杂合突变,导致USP7蛋白缺失,不能完成正常的细胞周期,考虑该基因的突变与神经发育障碍有关。患儿后期随访尤为重要,应定期随访患儿生长发育水平,早期发现异常,及早进行科学干预,尽可能提高患儿生活质量。

Ubiquitin-specific protease 24 promotes EV71 infection by restricting K63-linked polyubiquitination of TBK1

TANK-binding kinase 1(TBK1)is an essential protein kinase for activation of interferon regulatory factor 3(IRF3)and induction of the type I interferons(IFN-I).Although the biochemical regulation of TBK1 activation has been studied,little is known about how enterovirus 71(EV71)employs the deubiquitinases(DUBs)to regulate TBK1 activation for viral immune evasion.Here,we found that EV71 infection upregulated the expression of ubiquitinspecific protease 24(USP24).Further studies revealed that USP24 physically interacted with TBK1,and can reduce K63-linked polyubiquitination of TBK1.Knockdown of USP24 upregulated TBK1 K63-linked polyubiquitination,promoted the phosphorylation and nuclear translocation of IRF3,and in turn improved IFN-I production during EV71 infection.As a consequence,USP24 knockdown dramatically inhibited EV71 infection.This study revealed USP24 as a novel regulator of TBK1 activation,which promotes the understanding of immune evasion mechanisms of EV71 and could provide a potential strategy for treatment of EV71 infection.

MMP7和SLPI在甲状腺乳头状癌中的表达及意义

目的:探讨基质金属蛋白酶7(MMP7)和分泌型白细胞蛋白酶抑制剂(SLPI)在甲状腺乳头状癌(PTC)中的表达及意义。方法:基于肿瘤基因组图谱(TCGA)数据库中甲状腺癌基因表达数据下载样本567例,包括癌组织509例,正常组织58例。提取基因矩阵数据并整理,利用R语言edge R包筛选出两组差异表达基因,通过Cytoscape中MCODE插件筛选出潜在关键基因20个。选定一个关键基因MMP7,通过UALCAN数据库,挖掘到密切相关的基因SLPI。选取2020年1月至2021年6月山东第一医科大学附属滨州市人民医院收治的69例PTC患者,采用免疫组织化学染色SABC法检测PTC组织和相应癌旁组织中MMP7、SLPI蛋白的表达,分析二者与PTC患者临床病理因素间的关系及二者间的相互关系。结果:MMP7与SLPI存在相关性(r=0.5,P<0.05)。免疫组织化学结果显示,MMP7和SLPI在PTC组织中的阳性表达率均高于癌旁组织[82.6%(57/69)vs 29.0%(20/69),71.0%(49/69)vs15.9%(11/69)],差异均有统计学意义(χ2=40.222、42.579,P均<0.01)。PTC组织中MMP7和SLPI的表达与TNM分期、被膜外侵犯和淋巴结转移均有关(P<0.05)。MMP7和SLPI蛋白在PTC组织中表达呈正相关(r=0.381,P=0.001)。结论:MMP7和SLPI蛋白两者可能相互作用参与PTC的发生与发展。

Effect of protease inhibitor from Agaricus bisporus on glucose uptake and oxidative stress in 3T3-L1 adipocytes

Objective:To explore the effect of the protease inhibitor from Agaricus bisporus(J.E.Lange)Imbach(AbPI)on glucose uptake and oxidative stress in 3 T3-L1 adipocytes.Methods:Adipocytes were differentiated and stained with OilRed-O staining to confirm adipogenesis.The toxic/protective effect of AbPI on the adipocytes was determined by MTT assay,intracellular reactive oxygen species generation through flow cytometry,and morphologically through confocal microscopy using propidium iodide,4,6-diamino-2-phenylindol dihydrochloride,and 2’,7’-dichlorofluorescein diacetate dyes.The uptake of fluorescent glucose analog,2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose by adipocytes was also studied through confocal microscopy.Results:MTT assay showed that the cell survival rate was(28.00±3.00)%,(92.33±2.60)%,and(71.34±2.10)%in the presence of 2 mM H2O2,AbPI alone,and AbPI and H2O2 both,respectively,in comparison to the control.Oil-Red-O staining indicated that Ab PI enhanced adipogenesis.AbPI stimulated the glucose uptake by adipocytes similar to the drug rosiglitazone,and showed insulinsensitizing effect in the presence of insulin,but failed to stimulate the uptake in the absence of insulin.Intracellular reactive oxygen species generation was reduced in differentiating adipocytes upon Ab PI treatment.Confocal microscopy showed that the damaged cell population rose to 3.50%,117.84%,and 261.50%in the presence of Ab PI alone,AbPI with H2O2,and H2O2 alone,respectively.Conclusions:The protease inhibitor enhances glucose uptake by adipocytes and exhibits a cytoprotective effect on them.

TTF-1、CK7、Napsin A联合检验对诊断肺腺癌的价值研究

目的:探究肺腺癌采用甲状腺转录因子-1(TTF-1)、细胞角蛋白7(CK7)、天冬氨酸蛋白酶(Napsin A)进行联合检验的价值。方法:选取2021年1月—2022年11月许昌北海医院收治的非小细胞肺癌患者100例作为研究对象,根据病理诊断结果分为肺鳞癌组、肺腺癌组,各50例。使用免疫组化法,检测患者病理组织中TTF-1、CK7、Napsin A阳性表达率,分析TTF-1、CK7、Napsin A单独检验与联合检验的准确率、敏感性和特异性及有无淋巴结转移患者联合检验阳性、1项阴性情况。结果:肺腺癌组TTF-1、CK7、Napsin A阳性表达率高于鳞癌组,差异有统计学意义(P<0.001);联合检验准确率、敏感度及特异度均高于三项指标单独检验,差异有统计学意义(P<0.05);肺腺癌淋巴结转移患者联合检验阳性率高于未转移患者,差异有统计学意义(P<0.05);淋巴结有转移患者有1项阴性率低于未转移患者,差异有统计学意义(P=0.003)。结论:TTF-1、CK7、NapsinA单项检测,能够帮助患者鉴别肺腺癌,通过联合检测,可获得较高的准确率,同时,肺腺癌患者淋巴结转移者中,阳性表达率较高,可为临床诊断提供参考。

Ubiquitin-specific protease 47 regulates intestinal inflammation through deubiquitination of TRAF6 in epithelial cells

Deubiquitinates(DUBs) alter the stabilities, localizations or activities of substrates by removing their ubiquitin conjugates,which are closely related to the development of inflammatory response. Here, we show that ubiquitin-specific protease 47(USP47) prevents inflammation development in inflammatory bowel disease(IBD). Compared with wild-type mice, Usp47 knockout mice are more susceptible to dextran sodium sulfate(DSS)-induced acute and chronic colitis with higher inflammatory cytokines expression and severe intestinal tissue damage. Chimeric mouse experiments suggest that non-hematopoietic cells mainly contribute to the phenotype. And, DSS-induced colitis of the Usp47 knockout mice depends on commensal bacteria.Mechanistically, down-regulation of USP47 aggravates the activation of NF-κB signaling pathway by increasing the K63-linked poly-ubiquitination of tumor necrosis factor receptor-associated factor 6(TRAF6) in intestinal epithelial cells. Furthermore, the expression of USP47, negatively correlated with the degree of inflammation, is lower at colonic inflammatory lesions than that non-inflammatory sites from the intestine from ulcerative colitis(UC) and Crohn's disease(CD) patients. These data, taken together, indicate that USP47 regulates intestinal inflammation through de-ubiquitination of K63-linked poly-ubiquitination TRAF6 in intestinal epithelial cells.

SPINK7对IL-22介导的角质细胞异常增殖及炎症应答的影响

目的:探讨SPINK7对IL-22刺激后角质形成细胞HaCaT的过度增殖及炎症应答的影响及其可能的机制。方法:采用IL-22刺激体外培养的HaCaT细胞。采用RT-PCR检测SPINK7和细胞炎症因子的mRNA表达;Western blot检测SPINK7,cyclin D1、survivin和Wnt/β-catenin通路相关蛋白的表达;MTT法检测细胞增殖能力的变化;ELISA法检测上清液中IL-23、TNF-α和IL-17A的表达水平。结果:IL-22处理的HaCaT细胞中,SPINK7的mRNA和蛋白表达量均显著升高。此外,沉默SPINK7明显抑制IL-22诱导的细胞增殖,同时抑制凋亡相关蛋白cyclin D1及survivin的表达水平。SPINK7沉默显著降低IL-22刺激诱导的促炎性细胞因子IL-23、TNF-α和IL-17A的mRNA表达水平和分泌量。机制研究发现SPINK7沉默能够抑制IL-22激活的Wnt/β-catenin信号通路。结论:沉默SPINK7能够抑制IL-22诱导的细胞增殖和炎症应答反应,其机制可能与抑制Wnt/β-catenin信号通路有关,这为银屑病的诊断提供了新的标志物和治疗靶点。

siRNA干扰USP7表达对喉癌细胞生物学特性的影响

目的通过小干扰RNAs(siRNA)干扰泛素特异性蛋白酶7(USP7)在喉癌细胞中的表达,来探讨USP7对喉癌细胞生物学特性的影响。方法自行设计并使用高效siRNA在喉癌HEP2细胞株特异性干扰USP7表达,然后利用CCK-8法、Transwell小室迁移实验和流式细胞术观察USP7干扰后对喉癌细胞增殖、迁移能力和凋亡的影响。结果自行设计的siRNA可高效抑制喉癌HEP2细胞USP7mRNA及蛋白表达,显著抑制喉癌细胞的增殖、迁移能力,促进喉癌细胞的凋亡。结论 siRNAUSP7可以显著抑制喉癌细胞增殖和迁移能力,并促进凋亡,提示USP7可能是晚期喉癌治疗的一个重要靶标。

米曲霉HDF-7固定化菌体发酵产蛋白酶条件的优化

为了在工业化生产豆酱的过程中,缩短发酵周期,提高原料利用率,并降低生产成本,保证产品质量,利用从传统豆酱中分离得到的米曲霉(Aspergilleas oryzae)HDF-7作为出发菌株,经过海藻酸钠包埋法固定后,对其最佳固定化菌球的发酵产蛋白酶条件进行了优化。结果表明,固定化菌球以3%的接种量接入到中性蛋白酶发酵培养液中增殖至168 h后,其蛋白酶酶活力最高,为(18.49±0.65)U/mL,利用该增殖菌球单批次发酵到24 h时,其蛋白酶活力表现最高。并可以重复分批发酵7次而蛋白酶活力保持不变。本研究通过固定化米曲霉(Aspergilleas oryzae)HDF-7菌体并优化其发酵生产蛋白酶的条件,可以缩短发酵时间,提高菌体的利用效率,并保持酶活稳定。该研究为蛋白酶的规模化生产提供了试验基础。

培养基成分对海洋假单胞菌7-11产蛋白酶的影响

对海洋假单胞菌 7 1 1 ( pseudomonassp .7 1 1 )产蛋白酶进行了培养基的优化 ,初步确定了促使蛋白酶产率提高的培养基的配方 .经过 2 6℃、1 40r/min、1 2h培养 ,蛋白酶的相对产量可达 2 0 1 .7IU/mL ,比培养基优化前的 2 6.8IU/mL提高了 64 6%