本文選題：腎陽虛 + 腎陰虛�。� 參考：《福建中醫(yī)藥大學》2017年博士論文

【摘要】：目的比較腎陰虛、腎陽虛與非腎虛組患者皮質(zhì)骨及松質(zhì)骨骨組織蛋白雙向電泳(2-DE)圖譜的差異,篩選與腎陰虛、腎陽虛證密切相關(guān)的蛋白質(zhì),在Western Blot和RT-PCR技術(shù)驗證部分差異蛋白的基礎(chǔ)上,應(yīng)用ELISA技術(shù)靶定血清中與骨組織差異表達一致的蛋白質(zhì),一方面從骨組織差異蛋白表達層面闡釋"腎主骨"理論的科學內(nèi)涵,另一方面探討腎陰虛、腎陽虛證潛在血清標志物并豐富它們的客觀診斷依據(jù)。方法1.獲取腎陰虛、腎陽虛及非腎虛組患者皮質(zhì)骨、松質(zhì)骨及血液作為研究樣本,提取并制備各自的骨組織總蛋白研究樣品;采用2-DE技術(shù)得到患者皮質(zhì)骨及松質(zhì)骨骨組織蛋白質(zhì)組圖譜。2.采用PD Quest軟件對腎陰虛、腎陽虛及非腎虛組患者骨組織蛋白質(zhì)組圖譜進行組內(nèi)一致性分析,并以蛋白點體積變化達1.5倍為標準,對組間差異表達的蛋白點進行靶定。3.采用基質(zhì)輔助激光解吸電離飛行時間質(zhì)譜(MALDI-TOF-MS)技術(shù)定性差異表達的蛋白并檢索生物信息學數(shù)據(jù)庫,確定并分析差異蛋白名稱及其功能。4.在初步確定差異蛋白功能的基礎(chǔ)上,結(jié)合導師團隊前期腎陰虛、腎陽虛大鼠模型骨組織差異蛋白質(zhì)組研究結(jié)果,組間共有的差異蛋白對"腎主骨"理論的研究意義,以及后期血清標志物篩選的依據(jù)等因素,故選定腎陰虛、腎陽虛組共有的6個差異表達蛋白:皮質(zhì)骨中α-烯醇化酶(Eno1)、線粒體熱休克蛋白70(Hsp70)、載脂蛋白A-I(ApoA-I),松質(zhì)骨中骨保護素(OPG)、骨形態(tài)發(fā)生蛋白4(BMP-4)及GDP解離抑制因子β(Arhgdib),采用Western Blot和RT-PCR技術(shù)對它們的表達情況進行驗證。5.應(yīng)用ELISA技術(shù)檢測經(jīng)過上述驗證的相關(guān)蛋白在各自血清標本中表達情況。6.所有數(shù)據(jù)用SPSS19.0軟件進行分析統(tǒng)計。統(tǒng)計數(shù)據(jù)用平均值±標準差(x±S)表示。組內(nèi)比較采用配對t檢驗和秩和檢驗,組間比較采用One-wαy ANOVA和LSD檢驗,P0.05有顯著性差異,P0.01有極顯著性差異。結(jié)果1.2-DE獲得的腎陰虛、腎陽虛及非腎虛組患者骨組織樣本蛋白質(zhì)組凝膠圖譜背景清晰,組內(nèi)蛋白一致性及辨識度均較好;其中皮質(zhì)骨、松質(zhì)骨可辨識總蛋白點數(shù)目分別為490個、582個。2.經(jīng)腎陰虛、腎陽虛及非腎虛組患者骨組織蛋白質(zhì)組凝膠圖譜兩兩比對,并經(jīng)質(zhì)譜鑒定、生物信息學檢索顯示已知蛋白,分別為:與非腎虛組比較,腎陰虛證患者骨組織有26個蛋白出現(xiàn)差異表達:①皮質(zhì)骨與松質(zhì)骨中共有的7個蛋白:α-烯醇化酶、載脂蛋白A-Ⅰ、骨橋蛋白和骨形態(tài)發(fā)生蛋白2等4個蛋白上調(diào),GDP解離抑制因子β、翻譯控制腫瘤蛋白和骨形態(tài)發(fā)生蛋白4等3個蛋白下調(diào);②皮質(zhì)骨中波形蛋白、Ⅰ型膠原α-1鏈、脫氧尿苷三磷酸酶、維生素D結(jié)合蛋白3等4個蛋白上調(diào)表達,線粒體熱休克蛋白70、增殖細胞核抗原、心肌營養(yǎng)素樣細胞因子1與線粒體熱休克蛋白90等4個蛋白下調(diào)表達;③松質(zhì)骨中膜聯(lián)蛋白A1、β微管蛋白2C、線粒體熱休克蛋白60、丙酮酸激酶同工酶、破骨細胞生成抑制因子與胰島素樣生長因子結(jié)合蛋白3等6個上調(diào)表達的蛋白,網(wǎng)鈣蛋白3、熱休克蛋白B2、N端中段骨鈣素、過氧化物酶體增殖物激活受體γ共激活因子-1、類固醇受體輔助激活因子3等5個下調(diào)表達的蛋白。與非腎虛組比較,腎陽虛證患者骨組織有24個蛋白出現(xiàn)差異表達:①皮質(zhì)骨與松質(zhì)骨共有7個蛋白:線粒體熱休克蛋白90表達上調(diào),骨形態(tài)發(fā)生蛋白4、骨橋蛋白、GDP解離抑制因子β、酪氨酸激酶受體、丙酮酸激酶同工酶、蛋白質(zhì)二硫鍵異構(gòu)酶A3等6個蛋白表達下調(diào);②皮質(zhì)骨中載脂蛋白A-Ⅰ、α烯醇化酶、線粒體熱休克蛋白70等3個蛋白表達上調(diào),膜聯(lián)蛋白A1、膜聯(lián)蛋白A3、過氧化物酶體增殖物激活受體γ共激活因子-1、Ⅰ型膠原交聯(lián)氨基末端肽以及骨形態(tài)發(fā)生蛋白2等5個蛋白表達下調(diào);③松質(zhì)骨中抗酒石酸酸性磷酸酶、線粒體熱休克蛋白60、破骨細胞生成抑制因子、波形蛋白等4個蛋白表達上調(diào),N端中段骨鈣素、Ⅰ型膠原α-1鏈、線粒體ATP合酶亞基α、網(wǎng)鈣蛋白3、原肌球蛋白α-4等5個蛋白表達下調(diào)。腎陰虛與腎陽虛證患者骨組織有23個差異表達蛋白:①皮質(zhì)骨方面,以腎陰虛組為參考,腎陽虛組線粒體熱休克蛋白70、線粒體熱休克蛋白90、線粒體ATP合酶亞基α、錨蛋白重復序列、小分子熱休克蛋白和骨形態(tài)發(fā)生蛋白4等6個蛋白上調(diào)表達,骨橋蛋白、α烯醇化酶、過氧化氧化還原蛋白2、成纖維細胞生長因子受體3、骨唾液酸蛋白、GDP解離抑制因子β等6個蛋白下調(diào)表達;②松質(zhì)骨方面,以腎陰虛組為參考,腎陽虛組抗酒石酸酸性磷酸酶、線粒體熱休克蛋白60、錨蛋白重復序列和轉(zhuǎn)化生長因子-α等4個蛋白表達上調(diào),骨唾液酸蛋白、丙酮酸透激酶同工酶、膜聯(lián)蛋白A3、過氧化氧化還原蛋白2、蛋白酶體β亞基9型、磷酸甘油酸激酶1和骨形態(tài)發(fā)生蛋白4等7個蛋白表達下調(diào)。3.扣除腎陰虛、腎陽虛及非腎虛組兩兩比較中重復出現(xiàn)20個蛋白,剩下41個差異蛋白。41個差異蛋白質(zhì)根據(jù)生物信息學檢索可知,其中大部分蛋白與骨組織構(gòu)成、骨代謝以及骨組織有機物質(zhì)轉(zhuǎn)運等功能密切相關(guān),如Ⅰ型膠原α-1鏈、骨形態(tài)發(fā)生蛋白4等蛋白與骨組織結(jié)構(gòu)與細胞支架構(gòu)成相關(guān);α-烯醇化酶、成纖維細胞生長因子受體3等蛋白可以在骨細胞代謝過程發(fā)揮催化、調(diào)節(jié)等作用;維生素D結(jié)合蛋白3、網(wǎng)鈣蛋白3等蛋白與物質(zhì)轉(zhuǎn)運密切相關(guān)。"腎"可能通過調(diào)節(jié)這些與骨組織關(guān)系密切的蛋白表達而發(fā)揮"主骨"作用。4.Western Blot 及 RT-PCR 檢測皮質(zhì)骨 Eno1、Hsp70、ApoA-Ⅰ,松質(zhì)骨 OPG、BMP-4及Arhgdib等蛋白,結(jié)果顯示,與非腎虛組相比,腎陰虛組皮質(zhì)骨Eno1、ApoA-Ⅰ呈高表達,皮質(zhì)骨Hsp70、松質(zhì)骨BMP-4、Arhgdib、OPG呈低表達;腎陽虛組皮質(zhì)骨Eno1、Hsp70及ApoA-Ⅰ呈高表達,松質(zhì)骨BMP-4、Arhgdib、OPG呈低表達。與腎陰虛組相比,腎陽虛組皮質(zhì)骨中Hsp70和松質(zhì)骨OPG呈高表達,皮質(zhì)骨Eno1、松質(zhì)骨BMP-4呈低表達,而皮質(zhì)骨ApoA-Ⅰ及松質(zhì)骨Arhgdib未有顯著性差異。5.Eno1、Hsp70、ApoA-Ⅰ、OPG、BMP-4 和 Arhgdib 等蛋白血清 ELISA 檢測結(jié)果顯示,相對于非腎虛組,腎陰虛組Hsp70、BMP-4和Arhgdib含量降低,Eno1、ApoA-Ⅰ、OPG含量升高,腎陽虛組ApoA-Ⅰ、BMP-4和Arhgdib含量降低,Eno1、OPG和Hsp70含量升高。相對于腎陰虛組,腎陽虛組Hsp70升高,BMP-4和Arhgdib降低。其中Hsp70、BMP-4在血清表達趨勢與骨組織一致;腎陰虛組血清APoA-Ⅰ升高,與骨組織中表達一致,而腎陽虛組血清與骨組織不同;腎陰虛、腎陽虛組血清Eno1、Arhgdib、OPG檢測結(jié)果與它們在骨組織表達不一致。結(jié)論1.較之非腎虛組,腎陰虛證者皮質(zhì)骨存在Ⅰ型膠原α-1鏈、脫氧尿苷三磷酸酶等15個差異蛋白,松質(zhì)骨中有膜聯(lián)蛋白A1、β微管蛋白2C等18個差異蛋白,腎陽虛證者皮質(zhì)骨存在骨形態(tài)發(fā)生蛋白4、骨橋蛋白等15個差異蛋白,松質(zhì)骨有線粒體熱休克蛋白60、破骨細胞生成抑制因子等16個差異蛋白,這些差異表達蛋白大多數(shù)都與骨組織結(jié)構(gòu)構(gòu)成、骨代謝調(diào)節(jié)以及物質(zhì)轉(zhuǎn)運等功能相關(guān),提示它們可能都參與"腎主骨"過程。"腎"對這些蛋白差異表達的調(diào)控,可能是"腎主骨"理論的科學內(nèi)涵之一。2.由腎陰虛與腎陽虛證患者骨組織差異蛋白分析可知,皮質(zhì)骨中線粒體熱休克蛋白90、線粒體ATP合酶亞基α等12個存在表達差異,松質(zhì)骨中波形蛋白、破骨細胞生成抑制因子等11個也存在表達差異,這些是腎陰虛、腎陽虛證的微觀實質(zhì)之一,是腎陰虛、腎陽虛狀態(tài)骨組織結(jié)構(gòu)及功能差異的物質(zhì)基礎(chǔ)。3.腎陰虛狀態(tài)下Hsp70低表達、ApoA-Ⅰ高表達,腎陽虛狀態(tài)下Hsp70高表達,經(jīng)2-DE、Western Blot、RT-PCR及血清ELISA等多種方法檢測,結(jié)果相互印證且表達趨勢一致,它們可作為腎陰虛、腎陽虛證血清潛在標志物,同時可以補充腎虛證臨床診斷的依據(jù)。
[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.
【學位授予單位】：福建中醫(yī)藥大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R223

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