【摘要】：第一部分貴州麻江藍莓營養(yǎng)成分的研究目的:1.探討2016年貴州麻江藍莓營養(yǎng)成分,并比較與10年前同品種藍莓營養(yǎng)成分的比較的情況。方法:以直接滴定法、索氏脂肪提取器測定法、分光光度法及酸堿滴定法分別對藍莓中的糖、脂肪、蛋白質(zhì)及酸進行檢測;以反向色譜分離法、酸性滴定法、正向色譜分離法、反向色譜分離法分別對藍莓中維生素A、C、D、E的含量進行檢測;改良的Marklund方法(改良的鄰苯三酚自氧化法)檢測SOD含量;以溶劑浸提法提取后高效液相色譜法(HPLC-MS)對藍莓花青素進行鑒定。結(jié)果:貴州省麻江縣2016年種植的圓藍品種的藍莓蛋白質(zhì)、總糖的含量以及糖酸比均降低,差異有統(tǒng)計學意義(P0.05),而脂肪、總酸的含量無明顯變化,差異無統(tǒng)計學意義(P0.05)。與2007年比較,2016年貴州省麻江縣種植的圓藍品種的SOD,藍莓花青素、維生素C、維生素E的含量有顯著性增加,差異有統(tǒng)計學意義(P0.05),而維生素A、維生素D的含量無明顯變化,差異無統(tǒng)計學意義(P0.05);與2007年比較,2016年藍莓中所含的Na+、Ca2+均顯著性增加,差異有統(tǒng)計學意義(P0.05),而K+及Mg2+的含量無差異,無統(tǒng)計學意義(P0.05)。結(jié)論:研究表明,貴州省麻江圓藍品種藍莓具有較高營養(yǎng)價值,種植10年后藍莓的三大營養(yǎng)物質(zhì)成分含量有所降低,與當年降雨量增多及果樹樹齡增大有關(guān);麻江圓藍藍莓中的花青素、維生素C、維生素E的含量較高,且明顯高于2007年栽種初產(chǎn)果實中相關(guān)指標的含量,這與藍莓成熟后花青素富集及成熟藍莓鮮果中鈉、鈣離子水平增加對藍莓花青素穩(wěn)定性增加而發(fā)生的增色效應有關(guān)。第二部分藍莓對肝纖維化大鼠肝臟組蛋白乙�；揎椉凹毎饣康�:探討藍莓對四氯化碳(CCl4)致大鼠肝纖維化后大鼠血清的肝功能、肝纖維化三項,肝組織中細胞外基質(zhì)及組蛋白乙�；揎梐c H3K9、ac H3K14、ac H3K18位點修飾水平的影響。方法:50只雄性SD大鼠,180±20g,隨機分為對照組(Control group)、模型組(Hepatic Fibrosis group)、藍莓治療組(Blueberry Treatment group)、藍莓預防組(Blueberry Prevention group)、自然恢復組(Natural Recovery group),每組10只。除正常組外,其余各組用40%CCl4花生油溶液0.3 m L/100 g皮下注射,間隔三天注射一次以制備大鼠肝纖維化模型,正常組大鼠皮下注射等量花生油溶液,實驗12周末處死正常組與肝纖維化組大鼠,實驗16周末處死剩余組大鼠;取各組大鼠血清檢測其肝功能、肝纖維化三項指標;取各組大鼠肝臟,測定肝臟指數(shù);取左肝葉組織常規(guī)固定,HE染色、Masson染色觀察組織病理改變情況;蛋白質(zhì)免疫印跡法(Western blot,WB)檢測并比較各組大鼠右肝葉組織α-SMA、TIMP-1、Ⅰ型膠原表達情況和ac H3K9、ac H3K14、ac H3K18修飾水平變化。結(jié)果:1.關(guān)于肝指數(shù),與正常組比較,肝纖維化組大鼠肝臟指數(shù)增加(P0.05),與肝纖維化組比較,經(jīng)藍莓汁灌胃后,藍莓治療組與藍莓預防組大鼠的肝指數(shù)降低,差異有統(tǒng)計學意義(P0.05);與自然恢復組比,藍莓預防組肝指數(shù)降低,有顯著性差異(P0.05),而藍莓治療組無明顯變化,無明顯差異(P0.05);與藍莓治療組比較,藍莓預防組肝指數(shù)降低,有顯著性差異(P0.05)。2.根據(jù)肝纖維化分級,病理學檢查提示注射CCl4花生油溶液的肝纖維化組大鼠肝組織內(nèi)大量炎性細胞浸潤,肝臟纖維結(jié)締組織明顯增多,部分區(qū)域有假小葉形成,明顯高于正常組大鼠,Masson染色觀察提示CCl4花生油溶液注射后,肝纖維化組大鼠肝臟中膠原等細胞外基質(zhì)表達明顯增加,說明肝纖維化造模成功;與模型組比較,藍莓治療組及藍莓預防組大鼠肝臟纖維化分級顯著性降低,有顯著性差異(P0.01);與自然恢復組比較,藍莓預防組大鼠肝臟纖維化分級顯著性降低,有顯著性差異(P0.01),而藍莓治療組大鼠肝臟纖維化分級無明顯變化,無明顯統(tǒng)計學差異(P0.05)。與藍莓治療組比較,藍莓預防組肝臟纖維化分級有明顯變化,有顯著性差異(P0.05)。3.關(guān)于肝功能:(1)與正常組比較,模型組AST顯著增高(P0.01);與模型組比較,藍莓治療組及藍莓預防組AST顯著降低(P0.01);與自然恢復組比較,藍莓預防組顯著降低(P0.01),藍莓治療組降低(P0.05);與藍莓治療組比較,藍莓預防組有降低(P0.05);(2)與正常組比較,模型組ALT顯著增高(P0.01);與模型組比較,藍莓治療組及藍莓預防組ALT顯著下降(P0.01);與自然恢復組比較,藍莓預防組ALT顯著降低(P0.01),藍莓治療組無顯著性差異(P0.05);與藍莓治療組比較,藍莓預防組ALT降低(P0.05)。3.關(guān)于肝纖維化指標,(1)與正常組比較,模型組HA顯著增高(P0.01);與模型組比較,藍莓治療組及藍莓預防組HA顯著降低(P0.01);與自然恢復組比較,藍莓預防組顯著降低(P0.01),藍莓治療組降低(P0.05);與藍莓治療組比較,藍莓預防組降低(P0.05);(2)與正常組比較,余各組大鼠LN顯著性增加(P0.05)。(3)與正常組比較,模型組Ⅳ-C顯著增高(P0.01);與模型組比較,藍莓治療組及藍莓預防組Ⅳ-C顯著降低(P0.01);與自然恢復組比較,藍莓治療組及藍莓預防組顯著降低(P0.05);與藍莓治療組比較,藍莓預防組降低(P0.05);4.與正常組比較,模型組肝組織中的α-SMA、?型膠原和TIMP-1蛋白表達均增加,有顯著性差異(P0.01);與模型組比較:藍莓治療組、藍莓預防組及自然恢復組肝組織中的α-SMA、?型膠原和TIMP-1表達降低,有顯著性差異(P0.01);與自然恢復組比較,藍莓治療組及藍莓預防組肝組織中的α-SMA、?型膠原和TIMP-1表達降低,有顯著性差異(P0.05),以藍莓預防組降低較為明顯。與藍莓治療組比較,藍莓預防組肝組織中的的α-SMA、?型膠原和TIMP-1表達降低。5.與正常組比較,模型組肝組織中的ac H3K9、ac H3K14、ac H3K18修飾水平均降低,有顯著性差異(P0.01);與模型組比較,藍莓治療組、藍莓預防組及自然恢復組肝組織中的ac H3K9、ac H3K14、ac H3K18修飾水平均升高,有顯著性差異(P0.01);與自然恢復組比較,藍莓治療組及藍莓預防組的ac H3K9、ac H3K14、ac H3K18修飾水平均升高,有顯著性差異(P0.05),以藍莓預防組升高較為明顯。與藍莓治療組比較,藍莓預防組肝組織中的ac H3K9、ac H3K14、ac H3K18修飾水平升高。結(jié)論:證實了口服藍莓可以顯著改善肝纖維化病理改變,降低血清肝功能指標ALT、AST及血清肝纖維化相關(guān)指標,改善肝臟內(nèi)細胞外基質(zhì)沉積和代謝,預防性使用藍莓對肝纖維化的改善作用更好。ac H3K9、ac H3K14、ac H3K18組蛋白乙�；揎椄淖兛赡軈⑴c了肝纖維化的發(fā)生及發(fā)展,并與藍莓改善肝纖維化發(fā)生機制有關(guān)。第三部分藍莓提取的花青素在體外對激活型大鼠肝星狀細胞株組蛋白乙�；揎椀挠绊懞驮鲋车蛲龅淖饔媚康�:探討藍莓花青素對大鼠肝星狀細胞株(HSCs-T6)增殖與凋亡的影響,并對藍莓花青素處理后的HSCs-T6細胞外基質(zhì)蛋白及組蛋白乙�；揎梐c H3K9、ac H3K14、ac H3K18位點修飾水平的影響。方法:采用藥理學的方法提純藍莓中的花青素。HSCs-T6常規(guī)復蘇培養(yǎng)后,分別加入藍莓花青素50ug/m L、100ug/m L、150ug/m L、200ug/m L。使用實時無標記細胞分析儀(RTCA x CELLigence)動態(tài)觀察不同濃度藍莓花青素(50ug/m L、100ug/m L、150ug/m L、200ug/m L)處理HSCs-T6 72 h后細胞增殖的影響,確定合適的花青素濃度及作用時間;根據(jù)(RTCA)實驗結(jié)果選擇合適的時間后加入Annexin V-FITC/PI雙染后,熒光顯微鏡下觀察不同濃度藍莓花青素處理后的HSCs-T6細胞形態(tài)變化;流式細胞儀檢測不同濃度花青素處理HSCs-T6細胞凋亡的情況;western blotting檢測藍莓花青素最適濃度處理HSCs-T6細胞36 h,細胞的α-SMA、Ⅰ型膠原及TIMP-1表達,western blotting檢測HSCs-T6細胞ac H3K9、ac H3K14、ac H3K18修飾水平變化。結(jié)果:與對照組相比,不同濃度藍莓花青素處理HSCs-T6細胞36h,均能明顯抑制HSCs-T6細胞的增殖,呈劑量依賴性;濃度為50μmol/L時,HSCs-T6細胞的存活率低于50%(P0.05);免疫熒光實驗顯示,隨著藍莓花青素濃度增加并處理HSCs-T6 36 h,HSCs-T6凋亡數(shù)量逐漸增加,更多的HSCs-T6細胞核被PI染成紅色,且呈濃度依賴性。流式細胞儀檢測的凋亡實驗中,與對照組相比,不同濃度的藍莓花青素處理HSCs-T6細胞36 h,隨著藍莓花青素濃度增加凋亡率增加,差異有統(tǒng)計學意義(P0.01);與對照組相比,50ug/m L濃度的藍莓花青素處理HSCs-T6細胞36h,細胞的α-SMA、Ⅰ型膠原、TIMP-1表達明顯降低(P0.05);與對照組相比,50 ug/m L濃度的藍莓花青素處理HSCs-T6細胞36 h,細胞的ac H3K9、ac H3K14、ac H3K18修飾水平增加(P0.01);結(jié)論:藍莓花青素可以抑制大鼠激活型HSC細胞株HSCs-T6增殖,促進細胞凋亡,調(diào)節(jié)ECM合成和分解代謝。藍莓花青素是藍莓改善肝纖維化的重要有效成分之一;藍莓花青素可上調(diào)HSCs-T6細胞中ac H3K9、ac H3K14、ac H3K18組蛋白乙�；揎椝�,通過改變其組蛋白乙�；揎棇CM代謝相關(guān)蛋白表達來達到減少ECM沉積是藍莓花青素改善肝纖維化的機制之一。
[Abstract]:Objective: 1. To study the nutritional components of blueberry in Majiang, Guizhou in 2016, and compare the nutritional components with that of the same variety of blueberry 10 years ago. Methods: Sugar, fat and protein in blueberry were determined by direct titration, Soxhlet fat extractor, spectrophotometry and acid-base titration, respectively. The contents of vitamin A, C, D and E in blueberry were determined by reverse chromatography, acidic titration, forward chromatography and reverse chromatography, respectively; the content of SOD was detected by improved Marklund method (improved pyrogallol autoxidation method); the content of SOD was determined by solvent extraction followed by high performance liquid chromatography (HPLC-MS). Results: The contents of protein, total sugar and sugar-acid ratio of blueberries planted in 2016 in Majiang County of Guizhou Province were significantly lower than those in 2016 (P 0.05), while the contents of fat and total acid had no significant difference (P 0.05). The contents of SOD, blueberry anthocyanin, vitamin C and vitamin E increased significantly (P 0.05), but the contents of vitamin A and vitamin D did not change significantly (P 0.05); compared with 2007, the contents of Na +, Ca2 + in 2016 blueberry increased significantly (P 0.05), while the contents of K + increased significantly (P 0.05). There was no significant difference in the contents of Mg2 + and Mg2 + (P 0.05). Conclusion: The results showed that the blueberry varieties of Majiang Round Blue had high nutritional value, and the contents of the three nutrients of blueberry decreased after planting for 10 years, which was related to the increase of rainfall and the age of fruit trees. The content of vegetable E was higher than that of the related indexes in the primary fruits in 2007, which was related to the enrichment of anthocyanin and the increase of sodium and calcium in the fresh fruits of mature blueberries. Objective: To investigate the effects of blueberry on serum liver function, liver fibrosis, extracellular matrix and histone acetylation modified AC H3K9, AC H3K14, AC H3K18 loci in rats with hepatic fibrosis induced by carbon tetrachloride (CCl4). Mol group, Hepatic Fibrosis group, Blueberry Treatment group, Blueberry Prevention group, Natural Recovery group, 10 rats in each group. Rats in the normal group were subcutaneously injected with the same amount of peanut oil solution, rats in the normal group and the liver fibrosis group were sacrificed at the end of the 12th week and rats in the remaining group at the end of the 16th week. E staining and Masson staining were used to observe the histopathological changes; Western blot (WB) was used to detect and compare the expression of alpha-SMA, TIMP-1, type I collagen and the changes of modification levels of AC H3K9, AC H3K14 and AC H3K18 in the right hepatic lobe of rats in each group. Results: 1. Compared with the normal group, the liver index of rats in the liver fibrosis group increased. In addition (P 0.05), compared with the liver fibrosis group, the liver index of the blueberry treatment group and the blueberry prevention group decreased significantly (P 0.05); compared with the natural recovery group, the liver index of the blueberry prevention group decreased significantly (P 0.05), while the blueberry treatment group did not change significantly (P 0.05). Compared with the control group, the liver index of the blueberry prevention group decreased significantly (P 0.05). 2. According to the liver fibrosis grading, pathological examination showed that a large number of inflammatory cells infiltrated in liver tissue, fibrous connective tissue increased significantly, and pseudolobules formed in some areas of the liver of the rats injected with CCl4 peanut oil solution, which was significantly higher than that of the normal group, Ma. Son staining showed that the expression of extracellular matrix (ECM) such as collagen in liver of rats with hepatic fibrosis was significantly increased after CCl4 peanut oil injection, indicating that the model of hepatic fibrosis was successfully established. Compared with the blueberry group, the grade of liver fibrosis in the blueberry prevention group decreased significantly (P 0.01), but the grade of liver fibrosis in the blueberry treatment group did not change significantly (P 0.05). Compared with the blueberry treatment group, the grade of liver fibrosis in the blueberry prevention group changed significantly (P 0.05). 3. Can: (1) compared with the normal group, the model group AST significantly increased (P 0.01); compared with the model group, the blueberry treatment group and the blueberry prevention group AST significantly decreased (P 0.01); compared with the natural recovery group, blueberry prevention group significantly decreased (P 0.01), blueberry treatment group decreased (P 0.05); compared with the blueberry treatment group, blueberry prevention group decreased (P 0.05); (2) compared with the normal group, blueberry prevention group decreased (P 0.05); (2) compared with the blueberry Compared with the model group, ALT was significantly increased (P 0.01); compared with the model group, ALT in the blueberry treatment group and the blueberry prevention group was significantly decreased (P 0.01); compared with the natural recovery group, ALT in the blueberry prevention group was significantly decreased (P 0.01), but there was no significant difference in the blueberry treatment group (P 0.05); compared with the blueberry treatment group, ALT in the blueberry prevention group was significantly decreased (P 0.05). 3. (1) Compared with the normal group, the HA of the model group increased significantly (P 0.01); compared with the model group, the HA of the blueberry treatment group and the blueberry prevention group decreased significantly (P 0.01); compared with the natural recovery group, the blueberry prevention group decreased significantly (P 0.01), the blueberry treatment group decreased (P 0.05); compared with the blueberry treatment group, the blueberry prevention group decreased (P 0.05); and (2) compared with the normal group, the rest of each group. LN increased significantly (P 0.05). (3) Compared with the normal group, model group IV-C increased significantly (P 0.01); compared with model group, blueberry treatment group and blueberry prevention group IV-C decreased significantly (P 0.01); compared with natural recovery group, blueberry treatment group and blueberry prevention group decreased significantly (P 0.05); compared with blueberry treatment group, blueberry prevention group decreased (P 0.05); 4. Compared with the normal group, the expression of alpha-SMA, collagen and TIMP-1 protein in the liver tissue of the model group increased significantly (P 0.01); Compared with the model group, the expression of alpha-SMA, collagen and TIMP-1 protein in the liver tissue of the blueberry treatment group, the blueberry prevention group and the natural recovery group decreased significantly (P 0.01); Compared with the natural recovery group, the expression of collagen and TIMP-1 protein in the blueberry was significantly lower (P 0.01). The expression of alpha-SMA, collagen and TIMP-1 in the liver tissue of the treatment group and the blueberry prevention group was significantly lower than that of the blueberry prevention group (P 0.05). Compared with the blueberry treatment group, the expression of alpha-SMA, collagen and TIMP-1 in the liver tissue of the blueberry prevention group was significantly lower. Compared with the model group, the levels of ACH3K9, ACH3K14 and ACH3K18 in the liver tissues of the blueberry treatment group, the blueberry prevention group and the natural recovery group were significantly higher (P 0.01); compared with the natural recovery group, the levels of ACH3K9, ACH3K14, ACH3 K18 in the blueberry treatment group and the blueberry prevention group were significantly higher (P 0.01). Compared with the blueberry treatment group, the levels of ACH3K9, ACH3K14 and ACH3K18 in the liver tissue of the blueberry prevention group increased. Conclusion: Oral blueberry can significantly improve the pathological changes of liver fibrosis and reduce the serum ALT, AST and blood levels of liver function indicators. The acetylation modification of ACH3K9, ACH3K14 and ACH3K18 histones may be involved in the occurrence and development of liver fibrosis and may be related to the mechanism of blueberry improving liver fibrosis. Effects of anthocyanin extracted from blueberry on histone acetylation and apoptosis of activated rat hepatic stellate cell line in vitro Objective: To investigate the effects of blueberry anthocyanin on proliferation and apoptosis of rat hepatic stellate cell line (HSCs-T6), and to modify the extracellular matrix protein and histone acetylation of HSCs-T6 treated with blueberry anthocyanin. Methods: Anthocyanins in blueberries were purified by pharmacological methods. HSCs-T6 was cultured by routine resuscitation. Anthocyanins 50ug/m L, 100ug/m L, 150ug/m L, 200ug/m L were added to blueberries respectively. Real-time labeless cell analyzer (RTCA x CELLigence) was used to observe the dynamic changes of blueberry flowers at different concentrations. The effect of anthocyanin (50ug/m L, 100ug/m L, 150ug/m L, 200ug/m L) on the proliferation of HSCs-T6 cells after 72 h treatment was determined to determine the appropriate anthocyanin concentration and action time. According to the experimental results of RTCA, the morphology of HSCs-T6 cells treated with different concentrations of blueberry anthocyanin was observed under fluorescence microscope after adding Annexin V-FITC/PI double staining. Flow cytometry was used to detect the apoptosis of HSCs-T6 cells treated with different concentrations of anthocyanin; Western blotting was used to detect the expression of alpha-SMA, collagen type I and TIMP-1 in HSCs-T6 cells treated with blueberry anthocyanin at the optimum concentration for 36 hours; Western blotting was used to detect the changes of the modification levels of AC H3K9, AC H3K14 and AC H3K18 in HSCs-T6 cells. Compared with HSCs-T6 cells treated with different concentrations of blueberry anthocyanin for 36 hours, the proliferation of HSCs-T6 cells was significantly inhibited in a dose-dependent manner; the survival rate of HSCs-T6 cells was less than 50% (P 0.05) when the concentration of blueberry anthocyanin was 50 micromol/L; immunofluorescence assay showed that with the concentration of blueberry anthocyanin increased and treated with HSCs-T6 for 36 hours, the number of apoptosis of HSCs-T6 cells increased gradually, and more. The nuclei of HSCs-T6 cells were stained red by PI in a concentration-dependent manner. In the apoptosis test of flow cytometry, compared with the control group, the apoptosis rate of HSCs-T6 cells treated with blueberry anthocyanin at different concentrations for 36 hours increased with the increase of blueberry anthocyanin concentration, and the difference was statistically significant (P 0.01); compared with the control group, the 50 ug/ml concentration of blueberry anthocyanin increased the apoptosis rate (P 0.01). The expression of alpha-SMA, collagen type I and TIMP-1 in HSCs-T6 cells treated with blueberry anthocyanin decreased significantly after 36 hours (P 0.05); compared with the control group, the expression of ACH3K9, ACH3K14 and ACH3K18 in HSCs-T6 cells treated with blueberry anthocyanin at 50 ug/ml for 36 hours increased (P 0.01); Conclusion: blueberry anthocyanin can inhibit the increase of HSCs-T6 in rat activated HSC cells. Blueberry anthocyanin is one of the important components of blueberry to improve liver fibrosis; blueberry anthocyanin can up-regulate the acetylation level of AC H3K9, AC H3K14 and AC H3K18 histones in HSCs-T6 cells, and can be achieved by altering the acetylation of histone to the expression of ECM metabolism-related proteins. Reducing ECM deposition is one of the mechanisms of blueberry anthocyanin improving liver fibrosis.
【學位授予單位】：貴州醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R575.2

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