基于肾阴虚、肾阳虚状态人骨组织差异蛋白质组探讨“肾主骨”理论

发布时间：2018-06-22 03:22

本文选题：肾阳虚 + 肾阴虚　；参考：《福建中医药大学》2017年博士论文

【摘要】：目的比较肾阴虚、肾阳虚与非肾虚组患者皮质骨及松质骨骨组织蛋白双向电泳(2-DE)图谱的差异,筛选与肾阴虚、肾阳虚证密切相关的蛋白质,在Western Blot和RT-PCR技术验证部分差异蛋白的基础上,应用ELISA技术靶定血清中与骨组织差异表达一致的蛋白质,一方面从骨组织差异蛋白表达层面阐释"肾主骨"理论的科学内涵,另一方面探讨肾阴虚、肾阳虚证潜在血清标志物并丰富它们的客观诊断依据。方法1.获取肾阴虚、肾阳虚及非肾虚组患者皮质骨、松质骨及血液作为研究样本,提取并制备各自的骨组织总蛋白研究样品;采用2-DE技术得到患者皮质骨及松质骨骨组织蛋白质组图谱。2.采用PD Quest软件对肾阴虚、肾阳虚及非肾虚组患者骨组织蛋白质组图谱进行组内一致性分析,并以蛋白点体积变化达1.5倍为标准,对组间差异表达的蛋白点进行靶定。3.采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术定性差异表达的蛋白并检索生物信息学数据库,确定并分析差异蛋白名称及其功能。4.在初步确定差异蛋白功能的基础上,结合导师团队前期肾阴虚、肾阳虚大鼠模型骨组织差异蛋白质组研究结果,组间共有的差异蛋白对"肾主骨"理论的研究意义,以及后期血清标志物筛选的依据等因素,故选定肾阴虚、肾阳虚组共有的6个差异表达蛋白:皮质骨中α-烯醇化酶(Eno1)、线粒体热休克蛋白70(Hsp70)、载脂蛋白A-I(ApoA-I),松质骨中骨保护素(OPG)、骨形态发生蛋白4(BMP-4)及GDP解离抑制因子β(Arhgdib),采用Western Blot和RT-PCR技术对它们的表达情况进行验证。5.应用ELISA技术检测经过上述验证的相关蛋白在各自血清标本中表达情况。6.所有数据用SPSS19.0软件进行分析统计。统计数据用平均值±标准差(x±S)表示。组内比较采用配对t检验和秩和检验,组间比较采用One-wαy ANOVA和LSD检验,P0.05有显著性差异,P0.01有极显著性差异。结果1.2-DE获得的肾阴虚、肾阳虚及非肾虚组患者骨组织样本蛋白质组凝胶图谱背景清晰,组内蛋白一致性及辨识度均较好;其中皮质骨、松质骨可辨识总蛋白点数目分别为490个、582个。2.经肾阴虚、肾阳虚及非肾虚组患者骨组织蛋白质组凝胶图谱两两比对,并经质谱鉴定、生物信息学检索显示已知蛋白,分别为:与非肾虚组比较,肾阴虚证患者骨组织有26个蛋白出现差异表达:①皮质骨与松质骨中共有的7个蛋白:α-烯醇化酶、载脂蛋白A-Ⅰ、骨桥蛋白和骨形态发生蛋白2等4个蛋白上调,GDP解离抑制因子β、翻译控制肿瘤蛋白和骨形态发生蛋白4等3个蛋白下调;②皮质骨中波形蛋白、Ⅰ型胶原α-1链、脱氧尿苷三磷酸酶、维生素D结合蛋白3等4个蛋白上调表达,线粒体热休克蛋白70、增殖细胞核抗原、心肌营养素样细胞因子1与线粒体热休克蛋白90等4个蛋白下调表达;③松质骨中膜联蛋白A1、β微管蛋白2C、线粒体热休克蛋白60、丙酮酸激酶同工酶、破骨细胞生成抑制因子与胰岛素样生长因子结合蛋白3等6个上调表达的蛋白,网钙蛋白3、热休克蛋白B2、N端中段骨钙素、过氧化物酶体增殖物激活受体γ共激活因子-1、类固醇受体辅助激活因子3等5个下调表达的蛋白。与非肾虚组比较,肾阳虚证患者骨组织有24个蛋白出现差异表达:①皮质骨与松质骨共有7个蛋白:线粒体热休克蛋白90表达上调,骨形态发生蛋白4、骨桥蛋白、GDP解离抑制因子β、酪氨酸激酶受体、丙酮酸激酶同工酶、蛋白质二硫键异构酶A3等6个蛋白表达下调;②皮质骨中载脂蛋白A-Ⅰ、α烯醇化酶、线粒体热休克蛋白70等3个蛋白表达上调,膜联蛋白A1、膜联蛋白A3、过氧化物酶体增殖物激活受体γ共激活因子-1、Ⅰ型胶原交联氨基末端肽以及骨形态发生蛋白2等5个蛋白表达下调;③松质骨中抗酒石酸酸性磷酸酶、线粒体热休克蛋白60、破骨细胞生成抑制因子、波形蛋白等4个蛋白表达上调,N端中段骨钙素、Ⅰ型胶原α-1链、线粒体ATP合酶亚基α、网钙蛋白3、原肌球蛋白α-4等5个蛋白表达下调。肾阴虚与肾阳虚证患者骨组织有23个差异表达蛋白:①皮质骨方面,以肾阴虚组为参考,肾阳虚组线粒体热休克蛋白70、线粒体热休克蛋白90、线粒体ATP合酶亚基α、锚蛋白重复序列、小分子热休克蛋白和骨形态发生蛋白4等6个蛋白上调表达,骨桥蛋白、α烯醇化酶、过氧化氧化还原蛋白2、成纤维细胞生长因子受体3、骨唾液酸蛋白、GDP解离抑制因子β等6个蛋白下调表达;②松质骨方面,以肾阴虚组为参考,肾阳虚组抗酒石酸酸性磷酸酶、线粒体热休克蛋白60、锚蛋白重复序列和转化生长因子-α等4个蛋白表达上调,骨唾液酸蛋白、丙酮酸透激酶同工酶、膜联蛋白A3、过氧化氧化还原蛋白2、蛋白酶体β亚基9型、磷酸甘油酸激酶1和骨形态发生蛋白4等7个蛋白表达下调。3.扣除肾阴虚、肾阳虚及非肾虚组两两比较中重复出现20个蛋白,剩下41个差异蛋白。41个差异蛋白质根据生物信息学检索可知,其中大部分蛋白与骨组织构成、骨代谢以及骨组织有机物质转运等功能密切相关,如Ⅰ型胶原α-1链、骨形态发生蛋白4等蛋白与骨组织结构与细胞支架构成相关;α-烯醇化酶、成纤维细胞生长因子受体3等蛋白可以在骨细胞代谢过程发挥催化、调节等作用;维生素D结合蛋白3、网钙蛋白3等蛋白与物质转运密切相关。"肾"可能通过调节这些与骨组织关系密切的蛋白表达而发挥"主骨"作用。4.Western Blot 及 RT-PCR 检测皮质骨 Eno1、Hsp70、ApoA-Ⅰ,松质骨 OPG、BMP-4及Arhgdib等蛋白,结果显示,与非肾虚组相比,肾阴虚组皮质骨Eno1、ApoA-Ⅰ呈高表达,皮质骨Hsp70、松质骨BMP-4、Arhgdib、OPG呈低表达;肾阳虚组皮质骨Eno1、Hsp70及ApoA-Ⅰ呈高表达,松质骨BMP-4、Arhgdib、OPG呈低表达。与肾阴虚组相比,肾阳虚组皮质骨中Hsp70和松质骨OPG呈高表达,皮质骨Eno1、松质骨BMP-4呈低表达,而皮质骨ApoA-Ⅰ及松质骨Arhgdib未有显著性差异。5.Eno1、Hsp70、ApoA-Ⅰ、OPG、BMP-4 和 Arhgdib 等蛋白血清 ELISA 检测结果显示,相对于非肾虚组,肾阴虚组Hsp70、BMP-4和Arhgdib含量降低,Eno1、ApoA-Ⅰ、OPG含量升高,肾阳虚组ApoA-Ⅰ、BMP-4和Arhgdib含量降低,Eno1、OPG和Hsp70含量升高。相对于肾阴虚组,肾阳虚组Hsp70升高,BMP-4和Arhgdib降低。其中Hsp70、BMP-4在血清表达趋势与骨组织一致;肾阴虚组血清APoA-Ⅰ升高,与骨组织中表达一致,而肾阳虚组血清与骨组织不同;肾阴虚、肾阳虚组血清Eno1、Arhgdib、OPG检测结果与它们在骨组织表达不一致。结论1.较之非肾虚组,肾阴虚证者皮质骨存在Ⅰ型胶原α-1链、脱氧尿苷三磷酸酶等15个差异蛋白,松质骨中有膜联蛋白A1、β微管蛋白2C等18个差异蛋白,肾阳虚证者皮质骨存在骨形态发生蛋白4、骨桥蛋白等15个差异蛋白,松质骨有线粒体热休克蛋白60、破骨细胞生成抑制因子等16个差异蛋白,这些差异表达蛋白大多数都与骨组织结构构成、骨代谢调节以及物质转运等功能相关,提示它们可能都参与"肾主骨"过程。"肾"对这些蛋白差异表达的调控,可能是"肾主骨"理论的科学内涵之一。2.由肾阴虚与肾阳虚证患者骨组织差异蛋白分析可知,皮质骨中线粒体热休克蛋白90、线粒体ATP合酶亚基α等12个存在表达差异,松质骨中波形蛋白、破骨细胞生成抑制因子等11个也存在表达差异,这些是肾阴虚、肾阳虚证的微观实质之一,是肾阴虚、肾阳虚状态骨组织结构及功能差异的物质基础。3.肾阴虚状态下Hsp70低表达、ApoA-Ⅰ高表达,肾阳虚状态下Hsp70高表达,经2-DE、Western Blot、RT-PCR及血清ELISA等多种方法检测,结果相互印证且表达趋势一致,它们可作为肾阴虚、肾阳虚证血清潜在标志物,同时可以补充肾虚证临床诊断的依据。
[Abstract]:Objective to compare the difference in the bi-directional electrophoresis (2-DE) map of the cortical bone and the cancellous bone tissue in the kidney yang deficiency and the non kidney deficiency group, and to screen the proteins closely related to kidney yin deficiency and kidney yang deficiency syndrome. On the basis of Western Blot and RT-PCR techniques to verify the partial differential proteins, the differential table of serum and bone tissue was targeted by ELISA technology. On the one hand, it explains the scientific connotation of the theory of "kidney main bone" from the expression level of bone tissue difference protein. On the other hand, we discuss the deficiency of kidney yin, the potential serum markers of kidney yang deficiency syndrome and enrich their objective diagnosis basis. Method 1. the cortical bone, cancellous bone and blood of the patients with kidney yin deficiency, kidney yang deficiency and non kidney deficiency were studied. Sample, extraction and preparation of the samples of the total bone tissue protein of each bone, the protein group Atlas of the cortical bone and the cancellous bone tissue of the patients was obtained by 2-DE technology.2., and the PD Quest software was used to analyze the protein composition of the bone tissue in the kidney yin deficiency, the kidney yang deficiency and the non kidney deficiency group, and the change of the protein point volume was 1.5 times that of the group. Standard, the protein points differentially expressed between groups are targeted.3. using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) technology to identify the proteins expressed qualitatively and to retrieve the bioinformatics database, and to determine and analyze the name of the differential protein and its function.4. on the basis of the preliminary identification of the differential protein function. The study results of the difference protein group in the bone tissue of the kidney yang deficiency rat model, the research significance of the difference protein on the "kidney main bone" theory and the basis for the screening of the later serum markers, the 6 differentially expressed proteins of the kidney yin deficiency and the kidney yang deficiency group were selected: the alpha enolase (Eno1) in the cortical bone and the line granules in the cortical bone. Body heat shock protein 70 (Hsp70), apolipoprotein A-I (ApoA-I), bone protector in cancellous bone (OPG), bone morphogenetic protein 4 (BMP-4) and GDP dissociative inhibitory factor beta (Arhgdib). Western Blot and RT-PCR techniques were used to verify their expression. ELISA technology was used to detect related proteins in their serum specimens. .6. all data were analyzed with SPSS19.0 software. Statistical data were expressed with mean standard deviation (x + S). The group was compared with paired t test and rank sum test, and One-w alpha y ANOVA and LSD test were used among groups. P0.05 had significant difference, P0.01 had significant difference. The gelatin Atlas of the bone tissue samples in the non kidney asthenia group was clear, the protein consistency and identification in the group were all good, and the total protein points of the cortical bone and the cancellous bone were identified 490 respectively. The gel Atlas of the white matter group in the bone tissue of the kidney yang deficiency group and the kidney yang deficiency group was 22 comparison, and the mass spectrometry was identified by mass spectrometry, Compared with the non kidney deficiency group, there were 26 proteins in the bone tissue of the kidney yin deficiency syndrome: (1) there were 7 proteins in the cortical bone and the cancellous bone: alpha enolase, apolipoprotein A- I, osteopontin and bone morphogenetic protein 2 and so on 4 up-regulated proteins, and GDP depressor beta. 3 proteins, such as tumor protein and bone morphogenetic protein 4, were regulated, and 4 proteins, such as vimentin in cortical bone, type I collagen alpha -1 chain, deoxy uridine three phosphatase, vitamin D binding protein 3, were up-regulated, mitochondria heat shock protein 70, proliferating cell nuclear antigen, cardiac muscular dystrophy like cytokine 1 and mitochondrial heat shock protein 90 4 proteins and other proteins were downregulated; (3) the membrane associated protein A1, beta microtubule protein 2C, mitochondrial heat shock protein 60, pyruvate kinase isoenzyme, osteoclast inhibition factor and insulin-like growth factor binding protein 3, such as 6 up - regulated proteins, reticulin 3, heat shock protein B2, middle N end osteocalcin, peroxisome Colonies activated receptor gamma CO activation factor -1, steroid receptor auxiliary activator 3 and other 5 down-regulated proteins. Compared with the non kidney deficiency group, there were 24 proteins in the bone tissue of the patients with kidney yang deficiency: (1) there were 7 proteins in cortical bone and cancellous bone: up regulation of egg white 90 expression in mitochondrial heat shock, bone morphogenetic protein 4, osteopontin, The expression of GDP dissociation inhibitor beta, tyrosine kinase receptor, pyruvate kinase isoenzyme, protein two sulfur bond isomerase A3, and other 6 proteins were downregulated, and the expression of apolipoprotein A- I, alpha enolase, mitochondrial heat shock protein 70, and other 3 proteins were up-regulated in cortical bone, and annexin A1, annexin A3, peroxisome proliferator activated receptor gamma CO activation factor -1, type I collagen cross-linked amino terminal peptide and bone morphogenetic protein 2, and other 5 proteins were downregulated; (3) the anti tartaric acid phosphatase in the cancellous bone, the mitochondrial heat shock protein 60, the osteoclast inhibition factor, the vimentin and other 4 protein expressions, the middle N end osteocalcin, the type I collagen alpha -1 chain, and the mitochondrial ATP combination. The expression of 5 proteins, such as enzyme subunit alpha, net calcin 3 and promyosin alpha -4, were downregulated. There were 23 differentially expressed proteins in the bone tissue of kidney yin deficiency and kidney yang deficiency syndrome: (1) cortical bone, renal Yin deficiency group, kidney yang deficiency group, mitochondria heat shock protein 70, mitochondrial heat shock egg white 90, mitochondrial ATP synthase subunit alpha, and anchor protein repeat sequence, small 6 proteins, such as molecular heat shock protein and bone morphogenetic protein 4, were up-regulated, osteopontin, alpha enolase, peroxidation and redox protein 2, fibroblast growth factor receptor 3, bone sialic acid protein, GDP depressor beta, and other 6 proteins were downregulated; 2. The cancellous bone, kidney yin deficiency group as reference, kidney yang deficiency group anti tartar Acid phosphatase, mitochondrial heat shock protein 60, anchor protein repeat sequence and transforming growth factor - alpha, and other 4 proteins are up-regulated, bone sialic acid protein, pyruvate permeable kinase isoenzyme, annexin A3, peroxidation redox protein 2, proteasome beta subunit 9, glyceric acid kinase 1 and bone morphogenetic protein 4 expression, 7 protein expressions In the 22 comparison, 20 proteins were repeated in the 22 comparison of kidney yin deficiency, kidney yang deficiency and non kidney deficiency group. The remaining 41 protein.41 differential proteins were found according to bioinformatics retrieval. Most of the proteins were closely related to bone tissue, bone metabolism and organic matter transfer of bone tissue, such as type I collagen alpha -1 chain, bone shape Morphogenetic protein 4 and other proteins are associated with the structure of bone tissue and cell scaffolds; alpha enolase, fibroblast growth factor receptor 3 and other proteins can play a catalytic and regulatory role in the metabolic process of bone cells; vitamin D binding protein 3, reticulin 3 and other proteins are closely related to the transport of substances. "Kidney" may be regulated by these and bone. .4.Western Blot and RT-PCR detected cortical bone Eno1, Hsp70, ApoA- I, cancellous bone OPG, BMP-4 and Arhgdib and other proteins. The results showed that the cortical bone Eno1, ApoA- I in the kidney yin deficiency group were highly expressed, the cortical bone Hsp70, the cancellous bone, and the low expression; The Eno1, Hsp70 and ApoA- I of the cortical bone in Yang deficiency group were highly expressed, and the BMP-4, Arhgdib and OPG were low expression in the cancellous bone. Compared with the kidney yin deficiency group, the Hsp70 and cancellous bone OPG in the cortical bone of the kidney yang deficiency group were highly expressed, the cortical bone Eno1 and the cancellous bone BMP-4 showed low expression, while the cortical ApoA- I and the cancellous bone Arhgdib were not significant differences. The results of serum ELISA detection of MP-4 and Arhgdib showed that the content of Hsp70, BMP-4 and Arhgdib in kidney yin deficiency group was lower than that of non kidney deficiency group, Eno1, ApoA- I, OPG content increased, ApoA- I in kidney yang deficiency group, BMP-4 and Arhgdib content decreased. The serum expression trend of Hsp70 and BMP-4 was consistent with bone tissue, and the serum APoA- I increased in the kidney yin deficiency group and the expression in bone tissue, but the serum of kidney yang deficiency group was different from bone tissue, kidney yin deficiency and kidney yang deficiency group serum Eno1, Arhgdib, OPG detection results were not consistent with those in bone tissue. Conclusion 1. is more than the non kidney deficiency group and kidney yin deficiency syndrome. There were 15 differential proteins, such as type I collagen alpha -1 chain, deoxy uridine three phosphatase, and 18 differential proteins such as annexin A1 and beta microtubule protein 2C in the cancellous bone. There were 15 differential proteins such as bone morphogenetic protein 4, osteopontin and other 15 proteins, cancellous bone with mitochondrial heat shock protein 60, and osteoclast inhibition cause in the cancellous bone. 16 differential proteins, most of which are related to the structure of bone tissue, the regulation of bone metabolism and the transport of material, suggest that they may be involved in the process of "kidney main bone". The regulation of the differential expression of these proteins by "kidney" may be one of the scientific connotation of "kidney main bone" theory.2. from deficiency of kidney yin and kidney yang deficiency syndrome. The analysis of bone tissue differential protein of the patients showed that there were 12 differences in the expression of heat shock protein 90 and ATP synthase subunit alpha in the cortical bone. There were 11 differences in the expression of vimentin and osteoclast inhibition factor in the cancellous bone. These were one of the microcosmic essence of kidney yin deficiency and kidney yang deficiency syndrome, and the deficiency of kidney yin and kidney yang deficiency. .3. low expression of Hsp70, high expression of ApoA- I, high expression of Hsp70 in kidney yang deficiency, 2-DE, Western Blot, RT-PCR and serum ELISA were detected by various methods such as 2-DE, Western Blot, RT-PCR and serum ELISA, and the results were consistent with each other, and they could be used as the potential markers of kidney yin deficiency and kidney yang deficiency syndrome. The basis of clinical diagnosis of kidney deficiency syndrome can be supplemented.
【学位授予单位】：福建中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R223

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