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The effect of fasting or calorie restriction on mitophagy induction: a literature review

JOURNAL OF CACHEXIA SARCOPENIA AND MUSCLE 2020年第6期11卷 1447-1458页

作者： Mehrabani, Sanaz Bagherniya, Mohammad Askari, Gholamreza Read, Morgayn, I Sahebkar, Amirhossein Isfahan Univ Med Sci Food Secur Res Ctr Esfahan Iran Isfahan Univ Med Sci Sch Nutr & Food Sci Dept Clin Nutr Esfahan Iran Isfahan Univ Med Sci Sch Nutr & Food Sci Dept Community Nutr Esfahan Iran Univ Otago Sch Med Sci Dept Pharmacol Dunedin New Zealand FDA Halal Res Ctr IRI Tehran Iran Mashhad Univ Med Sci Biotechnol Res Ctr Pharmaceut Technol Inst Mashhad Razavi Khorasan Iran Mashhad Univ Med Sci Neurogen Inflammat Res Ctr Mashhad Razavi Khorasan Iran Polish Mothers Mem Hosp Res Inst PMMHRI Lodz Poland

Mitochondrial dysfunction can be a major cause of a wide range of age-related diseases. Maintaining the normal homeostasis of mitochondria population plays an important role in ensuring people's health, which is done through the mitophagy process. Among the various stimuli for the onset of mitophagy, caloric restriction (CR) is one of the strongest non-genetic triggers for initiating the mitophagy process. The primary objective of this paper is to review the literature assessing the effect of CR on mitophagy. Medline, Web of Science, Scopus, and Google Scholar databases was searched from inception to 1 August 2019. Reference lists from all selected articles were also examined for additional relevant studies. The evidence regarding the effect of fasting or CR on mitophagy is still limited. In addition, the methodological approaches of the studies are too heterogeneous in terms of types of food restriction, study duration, and targeted tissues. Most of the studies showed that fasting or CR induced mitophagy and mitophagy-related markers such as Binp3 and Parkin. However, some studies demonstrated that mitophagy occurred both in fasting and fed state with no significant differences or may be induced in fed state. Study on the muscle tissue of subjects after exercise showed that mitophagy was upregulated in the fed state. It has been demonstrated that mitophagy in the muscle was lowered in the absence of AMP-dependent kinase and fibroblast growth factor 21 genes, both in fasted and fed conditions. Current evidence overwhelmingly suggests that CR and fasting induce mitophagy and mitophagy-related markers. Based on the current evidence that we reviewed here, it could be concluded that fasting or CR has a promising role as a novel and practical approach in the prevention of age-related diseases without any side effects by inducing mitophagy in different organs of the body. More studies will be required in future to clarify the relationship between food deprivation and mito

关键词： Mitophagy PINK1 bnip3 Fasting Calorie restriction

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Human Milk Fortification Increases bnip3 Expression Associated With Intestinal Cell Death In Vitro

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JOURNAL OF PEDIATRIC GASTROENTEROLOGY AND NUTRITION 2015年第5期61卷 583-590页

作者： Diehl-Jones, William Archibald, Alyssa Gordon, Joseph W. Mughal, Wajihah Hossain, Zakir Friel, James K. Athabasca Univ Fac Hlth Disciplines Athabasca AB Canada Univ Manitoba Dept Biol Sci Winnipeg MB Canada Univ Manitoba Dept Human Anat & Cell Sci Winnipeg MB Canada Univ Manitoba Fac Nursing Winnipeg MB Canada Univ Manitoba Dept Human Nutr Sci Winnipeg MB Canada

Objectives: The aim of the present study was to determine the in vitro effect(s) of a bovine-based human breast milk fortifier (HMF) on human intestinal cells. HMF increases the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein (bnip3) and cell death;the prostaglandin analogue misoprostol will rescue this effect. Methods: Cultured intestinal cells were exposed to in vitro-digested human breast milk (BM) + HMF. Intracellular oxidation, cell damage/cell death, and bnip3 expression were measured after exposure. Results: In vitro-digested BM+ HMF significantly increased intracellular oxidation, cell damage, and cell death in enterocyte cell cultures compared with either saline or BM controls, an effect that was rescued by the prostaglandin analogue, misoprostol. bnip3 transcript and bnip3 protein levels were significantly increased in vitro after treatment with BM_HMF. We also provide evidence that transfection of enterocytes with bnip3 increases cell death, an effect that is rescued by a nonfunctional bnip3 splice variant. Conclusions: Our data support the hypothesis that HMF increases intestinal bnip3 in vitro, and that the gene product triggers cell death. We suggest that misoprostol is a promising therapy, which may reduce intestinal cell death.

关键词： bnip3 breast milk cell death human milk fortifier intestinal cells

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白桦脂酸减轻LPS诱导心脏损伤的线粒体自噬相关机制研究

白桦脂酸减轻LPS诱导心脏损伤的线粒体自噬相关机制研究

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作者：孟祥飞浙江省医学科学院

学位级别：硕士

脓毒症是机体对感染反应失调引起的危及生命的多器官功能障碍综合征。心脏损伤是重症监护病房脓毒症患者的常见并发症,已成为患者高死亡率的首要原因之一。线粒体自噬在清除受损线粒体,维持线粒体稳态,保护细胞活性中发挥重要作用。由... 详细信息

脓毒症是机体对感染反应失调引起的危及生命的多器官功能障碍综合征。心脏损伤是重症监护病房脓毒症患者的常见并发症,已成为患者高死亡率的首要原因之一。线粒体自噬在清除受损线粒体,维持线粒体稳态,保护细胞活性中发挥重要作用。由脓毒症引发的线粒体损伤和线粒体自噬不足加剧了脓毒症心脏损伤,脂多糖(lipopolysaccharide,LPS)是引起脓毒症病理生理结果的主要因素,常被用于构建脓毒症心脏损伤的模型。白桦脂酸(betulinic acid,BA)是一种五环三萜酸,大量动物实验和细胞实验证实其可以减轻多种疾病导致的心脏损伤。我们早期研究发现BA可通过抗炎和抗氧化应激等多种途径减轻脓毒症血管损伤,然而BA能否改LPS诱导的心脏损伤,其机制如何,尚不清楚。目的:1.给予腹腔注射LPS诱导心脏损伤的大鼠BA预处理,明确BA对LPS大鼠心脏的保护作用。2.采用线粒体自噬抑制剂Mdivi-1解析BA保护心脏的线粒体自噬及相关机制。方法:雄性SD大鼠随机分为四组:对照组(Control),LPS组(LPS),BA(25mg/kg,5 d,i.g.)预处理组(BA+LPS),线粒体自噬抑制剂(5 mg/kg,i.p.)+BA预处理组(Mdivi-1+BA+LPS)。BA预处理5天后,第6天腹腔注射LPS(10mg/kg),8 h后对大鼠进行心超检测,苏木素-伊红染色(hematoxylin-eosin staining,HE)染色检测心脏组织形态,酶联免疫吸附法测定心脏损伤标志物血清心肌肌钙蛋白I(cardiac troponin I,c Tn I)、肌酸激酶同工酶MB(creatine kinase-MB,CK-MB)和炎症因子白介素1β(interleukin-1β,IL-1β)、白介素6(interleukin-6,IL-6)、肿瘤坏死因子α(tumor necrosis factorα,TNF-α),比色法检测心脏线粒体呼吸链复合物体(I-III)酶活性,超氧化物阴离子荧光探针(Dihydroethidium,DHE)染色检测心脏组织活性氧自由基(reactive oxygen species,ROS),免疫荧光共定位检测线粒体自噬水平,Western blot测定心脏组织自噬相关蛋白微管相关蛋白轻链3抗体(microtubule-associated protein light chain 3,LC3)、溶酶体相关膜蛋白1(lysosomal associated membrane protein 1,Lamp1)、选择性自噬接头蛋白p62(sequestosome 1)、PTEN诱导假定激酶1(PTEN induced putative kinase1,PINK1)、Parkin和BCL2/腺病毒E1B 19k Da蛋白相互作用蛋白3(BCL2 and adenovirus E1B 19 k Da interacting protein 3,bnip3)等表达,线粒体膜通道孔检测试剂盒检测心脏组织线粒体通透性转换孔(mitochondrial permeability transition pore,m PTP)开放程度。结果:BA预处理显著提高了LPS组大鼠左心室射血分数和左室短轴缩短率,改善了心肌组织结构,明显降低了血清心脏损伤标志物c Tn I、CK-MB和炎症因子TNF-α、IL-1β、IL-6含量(P<0.05),明显提高了心脏线粒体呼吸链复合体(I-III)酶活性(P<0.01),显著抑制了心脏线粒体活性氧的积聚(P<0.01),明显增加了心脏组织LC3 II与Lamp1的表达(P<0.01),也明显增加了心脏线粒体PINK1、Parkin和LC3 II的表达(P<0.05),显著下调了心脏线粒体bnip3和p62的表达(P<0.01),同时显著减少了心脏线粒体通透性转换孔含量和开放(P<0.05)。线粒体自噬抑制剂Mdivi-1逆转了上述BA对LPS大鼠心脏的保护作用(P<0.05)。结论:***减轻了LPS处理大鼠心脏损伤。***通过增强心脏线粒体自噬减轻了LPS诱导的心脏损伤,激活PINK1/Parkin通路和抑制bnip3可能参与了该种保护作用。

关键词： LPS诱导的心脏损伤线粒体自噬 PINK1/Parkin通路 bnip3

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Expression of bnip3 and its correlations to hypoxia-induced autophagy and clinicopathological features in salivary adenoid cystic carcinoma

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CANCER BIOMARKERS 2015年第4期15卷 467-475页

作者： Chen, Zhanwei Wu, Haiwei Huang, Shengyun Li, Wengang Zhang, Shizhou Zheng, Peihui Zhou, Xiaoqing Liu, Wenlei Zhang, Dongsheng Shandong Univ Shandong Prov Hosp Dept Oral & Maxillofacial Surg Jinan 250021 Shandong Peoples R China

BACKGROUND: The expression of Bcl-2/adenovirus E1B 19 kDa-interacting protein3 (bnip3) has been explored in many human malignancies, but not in adenoid cystic carcinoma (ACC). OBJECTIVE: This study investigated the clinical significance of expression of bnip3 in ACC tissues and cells and elucidated its correlations to hypoxia-induced autophagy. METHODS: Immunohistochemical and immunofluorescence staining were used to explore bnip3, HIF-1 alpha and LC3 expression. RESULTS: bnip3 was positively expressed in 41 cases (63.1%), and was significantly correlated with histological grade (P = 0.001). HIF-1 alpha was positively expressed in 52 cases (80.0%) and was significantly correlated with TNM stage (P = 0.023) and histological grade (P = 0.024). LC3 was positively expressed in 37 cases (56.9%) and was significantly correlated with TNM stage (P = 0.019). The expression of bnip3 was correlated with HIF-1 alpha expression (P = 0.011). The overall survival in the negative bnip3 expression group tended to be better than in the positive bnip3 expression (P = 0.011). In vitro experiment, bnip3 immunofluorescence staining was detected in cells treated with CoCl2 (for hypoxic condition). CONCLUSIONS: The data indicated that bnip3 plays a vital role in the tumorigenesis of adenoid cystic carcinoma and could be a new target for gene therapy of adenoid cystic carcinoma.

关键词： Adenoid cystic carcinoma hypoxia bnip3 HIF-1 alpha autophagy

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Transcriptional regulation of bnip3 by Sp3 in prostate cancer

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PROSTATE 2015年第14期75卷 1556-1567页

作者： Huang, Ying Shen, Pengfei Chen, Xueqin Chen, Zhibin Zhao, Tao Chen, Ni Gong, Jing Nie, Ling Xu, Miao Li, Xinglan Zeng, Hao Zhou, Qiao Sichuan Univ West China Hosp Dept Pathol & Lab Pathol Chengdu 610041 Peoples R China Fujian Prov Hosp Dept Pathol Fuzhou Peoples R China Sichuan Univ West China Hosp Dept Urol Chengdu 610041 Peoples R China

BACKGROUNDThe transcription factors Sp3/Sp1 are expressed in a various types of cancers and bnip3 is overexpressed in prostate cancer. Although it has been demonstrated that bnip3 is transcriptionally regulated by HIF-1 and is post-transcriptionally regulated by miR145, our previous data indicated that there might be some other transcription factors regulating bnip3 in prostate cancer. This study is conducted to investigate whether bnip3 expression is directly regulated by Sp3/Sp1 or not. MATERIALS AND METHODSBioinformatics analysis shows that bnip3 promoter contains several potential Sp3/Sp1 binding sites. And then it is demonstrated that SP3 could regulate the bnip3 transcriptionally by binding to the predicted sites by dual reporter gene assays, ChIP, and EMSA. The biological effects of SP3 regulating bnip3 on prostate cancer cells proliferation are measured by MTT, TUNEL, and flow cytometry. RESULTSOur data show that Sp3 but not Sp1, is positively related to bnip3 overexpression in prostate cancer. Sp3 can directly regulate bnip3 transcription by mainly binding to the Sp3 binding sites (-624 approximate to-615 and -350 approximate to-343) of bnip3 promoter. Knockdown of Sp3 by RNA interference could reduce cells growth and lead to cells apoptosis in PC-3 and DU145. Sp3-dependent bnip3 overexpression might be an important mechanism to promote prostate cancer cells proliferation. CONCLUSIONThis is the first study to provide direct evidence of Sp3-dependent bnip3 expression. Sp3 might be the major transcriptional regulator of bnip3 in prostate cancer and it is worthy to further study. The regulation of bnip3 by Sp3 may be a new cancer-specific therapeutic target in prostate cancer. Prostate 75:1556-1567, 2015. (c) 2015 Wiley Periodicals, Inc.

关键词： prostate cancer Sp3 bnip3 transcriptional regulation proliferation

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Mitophagy defects arising from bnip3 loss promote mammary tumor progression to metastasis

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EMBO REPORTS 2015年第9期16卷 1145-1163页

作者： Chourasia, Aparajita H. Tracy, Kristin Frankenberger, Casey Boland, Michelle L. Sharifi, Marina N. Drake, Lauren E. Sachleben, Joseph R. Asara, John M. Locasale, Jason W. Karczmar, Gregory S. Macleod, Kay F. Univ Chicago Ben May Dept Canc Res Chicago IL 60637 USA Comm Canc Biol Chicago IL USA Comm Mol Metab & Nutr Chicago IL USA Comm Mol Pathogenesis & Mol Med Chicago IL USA Univ Chicago Biomol NMR Facil Chicago IL 60637 USA Beth Israel Deaconess Med Ctr Div Signal Transduct Boston MA 02215 USA Harvard Univ Sch Med Boston MA USA Cornell Univ Div Nutr Sci Ithaca NY USA Univ Chicago Dept Radiol Chicago IL 60637 USA

bnip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for bnip3 in a clinically relevant mouse model of mammary tumorigenesis. bnip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of bnip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1 alpha and Hif target genes, including those involved in glycolysis and angiogenesistwo processes that are also markedly increased in bnip3-null tumors. Glycolysis inhibition attenuates the growth of bnip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that bnip3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunctioncaused by defects in mitophagycan promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.

关键词： bnip3 breast cancer glycolysis HIF-1 alpha invasive carcinoma mitophagy ROS

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Mitochondrial autophagy: Origins, significance, and role of bnip3 and NIX

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BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2015年第10期1853卷 2775-2783页

作者： Ney, Paul A. New York Blood Ctr Dept Cell & Mol Biol Lindsley F Kimball Res Inst New York NY 10065 USA

Mitochondrial autophagy (mitophagy) is a core cellular activity. In this review, we consider mitophagy and related cellular processes and discuss their significance for human disease. Strong parallels exist between mitophagy and xenophagy employed in host defense. These mechanisms converge on receptors in the innate immune system in clinically relevant scenarios. Mitophagy is part of a cellular quality control mechanism, which is implicated in degenerative disease, especially neurodegenerative disease. Furthermore, mitophagy is an aspect of cellular remodeling, which is employed during development bnip3 and NIX are related multi-functional outer mitochondrial membrane proteins. bnip3 regulates mitophagy during hypoxia, whereas NIX is required for mitophagy during development of the etythroid lineage. Recent advances in the field of bnip3- and NIX-mediated mitophagy are discussed. This article is part of a Special Issue entitled: Mitophagy. (C) 2015 Elsevier B.V. All rights reserved.

关键词： bnip3 NIX Mitochondrion Autophagy Necrosis Erythrocyte

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黏附蛋白33（TvAP33）介导阴道毛滴虫黏附宿主细胞的机制研究

黏附蛋白33（TvAP33）介导阴道毛滴虫黏附宿主细胞的机制研究

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作者：邓洋阳新乡医学院

学位级别：硕士

背景阴道毛滴虫（Trichomonas vaginalis,***）是一种兼性厌氧的胞外寄生原虫,常寄生于人体泌尿生殖系统。女性感染滴虫后常表现为滴虫性阴道炎、宫颈炎等症状,男性感染后多表现为尿道炎、附睾炎等。此外,阴道毛滴虫感染还与不孕不育及... 详细信息

背景阴道毛滴虫（Trichomonas vaginalis,***）是一种兼性厌氧的胞外寄生原虫,常寄生于人体泌尿生殖系统。女性感染滴虫后常表现为滴虫性阴道炎、宫颈炎等症状,男性感染后多表现为尿道炎、附睾炎等。此外,阴道毛滴虫感染还与不孕不育及女性宫颈癌和男性前列腺癌相关。阴道毛滴虫作为胞外寄生虫首先黏附到宿主细胞表面,利用伪足穿行包绕和鞭毛的定向机械运动紧紧固定在宿主细胞上,进而在定植部位形成特有的微环境,产生吞噬及细胞脱落因子损伤宿主细胞。滴虫在黏附宿主细胞过程中,由其分泌的表面蛋白介导。阴道毛滴虫黏附蛋白33（TvAP33）作为滴虫表面黏附蛋白之一,在黏附宿主细胞和致病中发挥着重要作用,但其作用机制尚不明确。目的1.明确黏附蛋白TvAP33在阴道毛滴虫黏附和致病中的作用。2.发现宿主细胞中BCL2 interacting protein 3（bnip3）与TvAP33相互作用,并探寻TvAP33与bnip3相互作用在阴道毛滴虫黏附宿主细胞中的作用,为寻找抗阴道毛滴虫的药物靶点和阴道毛滴虫病的诊断分子提供科学依据。方法1.采用RNA干扰技术降低阴道毛滴虫滋养体中TvAP33的表达,采用qPCR和Western blot检测TvAP33的mRNA转录和蛋白表达水平。*** TrackerTMOrange标记阴道毛滴虫滋养体,使用荧光显微镜观察黏附于人阴道上皮细胞（VK2/E6E7）上的滋养体。3.采用MTT检测法、流式细胞技术、台盼蓝染色法,分析干扰TvAP33表达后的滋养体对VK2/E6E7细胞的增殖、凋亡和死亡的影响。4.采用TvAP33多克隆抗体被动免疫小鼠,腹腔感染阴道毛滴虫滋养体;TvAP33多克隆抗体封闭阴道毛滴虫滋养体后腹腔感染小鼠。观察小鼠生存状态。5.采用免疫荧光分析rTvAP33蛋白与VK2/E6E7细胞的结合。6.实验室前期已成功构建VK2/E6E7细胞的cDNA文库,利用酵母双杂交系统筛选出与TvAP33互作的分子,采用免疫共沉淀和免疫共定位分析TvAP33与bnip3的相互作用。7.采用RNA干扰技术降低VK2/E6E7细胞中bnip3的表达;采用qPCR和Western blot检测bnip3的mRNA转录和蛋白表达水平。*** TrackerTMOrange标记阴道毛滴虫滋养体,使用荧光显微镜观察TvAP33表达降低后滋养体黏附于低表达bnip3的VK2/E6E7细胞的情况。9.通过MTT检测TvAP33表达降低后,滋养体对抑制低表达bnip3的VK2/E6E7细胞增殖的影响。10.通过台盼蓝染色分析TvAP33表达降低后,滋养体对感染低表达bnip3的VK2/E6E7细胞死亡的情况。结果***和Western blot结果表明成功敲低阴道毛滴虫滋养体中TvAP33的mRNA转录和蛋白表达水平。2.荧光显微镜下观察到降低TvAP33表达的滋养体黏附于VK2/E6E7细胞的数量明显减少（P<0.001）。3.降低TvAP33的表达后,阴道毛滴虫滋养体抑制VK2/E6E7细胞的增殖作用减弱;诱导VK2/E6E7细胞凋亡和死亡率明显降低（P<0.001）。***33抗体阻断及封闭滋养体后腹腔感染小鼠,小鼠生存时间显著延长（P<0.01）。5.免疫荧光分析表明rTvAP33可以与VK2/E6E7细胞结合。6.免疫共沉淀和免疫共定位指出TvAP33与宿主细胞中bnip3相互作用。***和Western blot结果证实成功敲低VK2/E6E7细胞中bnip3的mRNA转录和蛋白表达水平。8.当阴道毛滴虫滋养体正常表达TvAP33时,VK2/E6E7细胞中bnip3的表达不影响阴道毛滴虫黏附VK2/E6E7细胞（P>0.05）;而滋养体中TvAP33表达降低时,VK2/E6E7细胞中bnip3表达下降,黏附VK2/E6E7细胞的阴道毛滴虫数量显著下降（P<0.05）。9.阴道毛滴虫滋养体正常表达TvAP33时,VK2/E6E7细胞中bnip3的表达不影响阴道毛滴虫抑制VK2/E6E7细胞增殖（P>0.05）;而滋养体中TvAP33表达降低时,VK2/E6E7细胞中bnip3表达下降,阴道毛滴虫抑制VK2/E6E7细胞增殖的作用减弱（P<0.001）。***2/E6E7细胞中bnip3的表达不影响阴道毛滴虫感染导致VK2/E6E7细胞死亡（P>0.05）。结论***33介导阴道毛滴虫滋养体黏附宿主细胞和致病。***33和bnip3的相互作用介导了阴道毛滴虫对宿主细胞的黏附和致病性。因此,TvAP33有可能作为药物靶点或疫苗候选抗原抗阴道毛滴虫感染。

关键词：阴道毛滴虫黏附蛋白33 bnip3 相互作用致病性

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bnip3在胰腺癌中的表达及调控凋亡的作用和机制

BNIP3在胰腺癌中的表达及调控凋亡的作用和机制

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作者：张旭苏州大学

学位级别：硕士

目的:Bcl-2/腺病毒E1B19kDa结合蛋白3(bnip3)是Bcl-2蛋白家族BH3-only亚家族的非典型成员,尽管研究表明bnip3作为缺氧诱导因子的靶基因在多种疾病过程中承担着重要的角色,比如心肌缺血,细胞自噬,细胞凋亡,但是关于bnip3功能不同的研究... 详细信息

目的:Bcl-2/腺病毒E1B19kDa结合蛋白3(bnip3)是Bcl-2蛋白家族BH3-only亚家族的非典型成员,尽管研究表明bnip3作为缺氧诱导因子的靶基因在多种疾病过程中承担着重要的角色,比如心肌缺血,细胞自噬,细胞凋亡,但是关于bnip3功能不同的研究往往得出相互矛盾的结果。本实验旨在探讨bnip3在胰腺癌组织标本中的表达及其临床意义,研究bnip3在胰腺癌细胞株中的表达及其调控胰腺癌细胞凋亡的作用和机制。方法:(1)利用免疫荧光(IF)及免疫组织化学法(IHC)检测bnip3在70对胰腺癌组织和相应癌旁组织中的定位及表达,并分析bnip3的表达与临床病理因素、其他凋亡相关蛋白及肿瘤凋亡及增殖的关系;(2)利用逆转录聚合酶联反应(RT-PCR)和Western blot检测bnip3在胰腺癌细胞株PANC-1,Bx Pc-3,CaPan-1,SW1990、CFPAC-1、Patu-8988中的表达情况,分别用siRNA-bnip3转染胰腺癌细胞株靶向沉默bnip3的表达,用bnip3全基因质粒载体转染胰腺癌细胞株上调其表达,利用RT-PCR和Western blot确认RNAi靶向沉默及质粒过表达bnip3的效果;(3)沉默bnip3及上调其表达后,利用细胞凋亡检测、线粒体膜电位检测,细胞活性氧检测,Western blot检测凋亡相关蛋白的表达,研究bnip3调控胰腺癌细胞凋亡的作用及机制;(4)利用甲基化PCR检测6株胰腺癌细胞的甲基化状态,去甲基化药物处理细胞,分析bnip3甲基化和其表达的相关性;(5)利用CHIP实验分析bnip3甲基化对缺氧诱导因子和其启动子区结合的影响。结果:(1)bnip3在胰腺癌组织中的阳性率明显低于癌旁组织,且两者的差异有统计学意义(χ2=19.457,P<0.001),两者阳性表达率分别为35.7%和72.9%;bnip3的表达与临床病理因素的分析结果显示,bnip3的低表达与肿瘤大小、肿瘤分期及淋巴结转移密切相关,差异有统计学意义(P<0.05),而与性别、年龄、肿瘤部位、肿瘤分级无明显相关,差异无统计学意义(P>0.05)。胰腺癌组织中bnip3的表达和促凋亡蛋白表达Bax正相关,和抑凋亡蛋白Bcl-2表达负相关,其低表达与肿瘤细胞增殖活跃、凋亡减少有关;(2)RT-PCR和Western blot检测结果显示,常氧条件下bnip3的mRNA和蛋白水平在六株胰腺癌细胞株中除一株低表达外,其余的5株均表达沉默,且缺氧条件培养并未诱导其表达。(3)通过上调及下调bnip3表达发现bnip3可以调控线粒体途径相关凋亡蛋白的表达,和诱导细胞活性氧的产生、线粒体膜电位的下降及细胞凋亡。(4)甲基化PCR检测证实bnip3启动子区的高甲基化和其在胰腺癌中的低表达密切相关,bnip3启动子区的甲基化影响了缺氧诱导因子HIF-1α和其启动子区缺氧反应元件的结合。结论:bnip3在胰腺癌组织中低表达,bnip3的低表达与肿瘤大小、肿瘤分期、淋巴结转移密切相关。在体外实验中bnip3启动子区的高甲基化抑制了HIF-1α和bnip3的结合并使其表达沉默,进而为胰腺癌细胞逃避bnip3诱导的线粒体途径促进细胞凋亡作用提供了条件。由实验结果可以推断,恢复胰腺癌中bnip3的表达则可以增强肿瘤细胞对缺氧诱导凋亡的敏感性,有望成为潜在的治疗靶点。

关键词： bnip3 甲基化胰腺癌凋亡

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线粒体自噬蛋白与胎儿多普勒在子痫前期的相关性研究

线粒体自噬蛋白与胎儿多普勒在子痫前期的相关性研究

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作者：孟迪吉林大学

学位级别：硕士

目的:通过测定正常孕妇与子痫前期(preeclampsia,PE)患者线粒体自噬蛋白BCL2腺病毒E1B19k Da相互作用蛋白3(Bcl-2 and adenovirus E1B19k Da interacting protein3,bnip3)在血清及胎盘中的表达水平,同时测量胎儿多普勒脐动脉(umbilical artery,UA)的相关血流动力力学指标,探究bnip3的表达与疾病发生的关系及其与胎儿脐动脉血流参数对评估胎儿状况的价值。方法:根据纳入标准,从2021年4月至2022年11月就诊于吉林大学白求恩第二医院并住院行剖宫产的孕妇中,选取无合并症正常妊娠及子痫前期的孕妇各24例。收集终止妊娠前孕妇外周静脉血进行离心并取其上清液后分装至Ep管中,-80℃保存。剖宫产胎盘娩出后,在非出血、钙化及坏死部位,取2块大小约1.0cm×1.0cm×1.0cm的胎盘组织,用生理盐水冲洗胎盘组织并用无菌纱布将其水分吸干,一块放置于10%的福尔马林溶液中,一块放置于液氮过夜后转移至-80℃冰箱冻存。采用酶联免疫吸附实验(ELISA)检测血清中bnip3的浓度;蛋白印迹法(Western-Blot)及免疫组化检测胎盘组织中bnip3蛋白的表达水平。应用Voluson E10的腹部超声检查方法,获取并比较两组胎儿脐动脉血流动力学参数,主要包括脐动脉的搏动指数(pulsation index,PI)、阻力指数(resistance index,RI)和收缩期峰值血流速度/舒张末期血流速度(peak systolic flow velocity/end diastolic flow velocity,S/D)。根据结果并结合入组患者的临床相关资料,应用Graph Pad Prism 9.2.0及SPSS27.0进行统计学分析。结果:1.通过ELISA实验结果统计分析发现,子痫前期组与正常对照组相比血清bnip3的浓度降低,差异有显著统计学意义(P<0.0001)。2.通过Western-Blot实验结果统计分析发现,子痫前期组胎盘组织中bnip3蛋白的表达较正常对照组降低,差异有统计学意义(P<0.05)。3.通过免疫组化染色结果分析发现,bnip3在合体滋养层细胞可见明显表达,细胞滋养细胞层仅有微弱的表达。在正常对照组中呈高表达,子痫前期组中则呈低表达。子痫前期组中胎盘组织bnip3的表达较正常对照组降低,差异有显著统计学意义(P<0.01)。4.通过腹部彩色超声多普勒测量胎儿脐动脉血流动力学指标,结果统计分析发现子痫前期组PI、RI、S/D等脐动脉血流参数较正常组升高,PI差异有显著统计学意义(P<0.01),RI、S/D差异有统计学意义(P<0.05)。5.通过相关性分析发现,血清bnip3浓度与胎盘组织bnip3蛋白相对表达水平呈正相关。胎儿脐动脉血流动力学参数PI、RI、S/D与新生儿Apgar评分、新生儿体质量呈负相关。血清bnip3浓度及胎盘组织bnip3蛋白相对表达水平与孕妇血压呈负相关,脐动脉血流动力学参数PI、RI、S/D与孕妇血压呈正相关。外周血清bnip3的浓度与脐动脉PI、RI、S/D呈负相关。以上均具有统计学意义(P<0.05)。胎盘组织bnip3蛋白相对表达水平与脐动脉血流动力学参数PI、RI、S/D相关性无统计学意义(P>0.05)。结论:1.血清及胎盘组织中bnip3表达具有相关性,其在子痫前期患者中表达水平降低可能与线粒体自噬功能紊乱、疾病的发生发展有关。针对bnip3的研究有望为临床诊断、治疗PE提供理论依据及潜在的治疗靶点,但其具体分子机制还需进一步探讨。2.胎儿脐动脉血流动力学指标与新生儿体质量、新生儿Apgar评分具有相关性,对PE患者胎儿状况的评估具有一定的价值,在一定程度上可预测妊娠结局,为临床干预及终止妊娠时机的选择提供依据。3.线粒体自噬蛋白bnip3的表达水平是否与胎儿脐动脉血流动力学之间存在相关性尚不明确,仍需进一步的研究。

关键词：子痫前期 bnip3 线粒体自噬胎儿多普勒超声脐动脉多普勒

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