求助两篇论文全文
各位战友，我现在急需寻求“NRE对高脂血症模型大鼠降脂作用的蛋白质组学研究”和“小檗碱降低血清胆固醇的作用与分子机理研究”论文全文，不知哪位战友可帮帮忙，我的邮箱是[email]wangmin8782@163.com[/email]先谢谢了 作者姓名：孔维佳
论文题目：小檗碱降低血清胆固醇的作用与分子机理研究
作者简介：孔维佳，男， 1972年12月出生，2001年8月师从于中国协和医科大学蒋建东教授，于2004年12月获博士学位。
中 文 摘 要
血液中胆固醇特别是低密度脂蛋白胆固醇（LDL-c）浓度的升高是引起动脉粥样硬化和冠心病的最主要危险因素，降低血液中胆固醇的浓度可明显减少冠心病的发病率和死亡率。体内大部分胆固醇结合于低密度脂蛋白（LDL），经肝细胞表面的低密度脂蛋白受体（LDLR）代谢和清除。LDLR是位于细胞表面的跨膜受体，其表达受到胆固醇和其它许多非固醇类物质在转录水平或转录后水平的调控，上调肝脏LDLR的表达是降低血液胆固醇浓度的最主要手段。目前临床应用最广泛的能上调肝脏LDLR表达的药物是他汀类。他汀类药物竞争性抑制羟甲基戊二酸单酰辅酶A（HMG-CoA）还原酶的活性，使细胞内胆固醇合成减少，结果通过固醇调控元件结合蛋白（SREBPs）途径使LDLR基因的转录增加。总体来讲他汀类具有出色的降胆固醇疗效，但是也有部分患者因不良反应（如肝功能损害、肌病等）不能耐受其治疗，另有部分患者单用他汀类疗效欠佳而需联合用药。因此，筛选新型降胆固醇药物一直是调血脂药物的研究重点。
通过对传统中草药物的筛选，我们首次发现小檗碱（Berberine，BBR）能上调肝细胞LDLR的表达，并且在体内具有明确的降血脂疗效。BBR是从黄连和黄柏等中药中提取出来的生物碱，是具有明确化学结构的天然化合物，具有多种药理作用。BBR在我国已经有很长时间的临床应用历史，过去主要作为抗菌药物治疗细菌性腹泻，其疗效和安全性均得到普遍认可。
为了研究BBR降胆固醇作用的分子机制，我们设计了一系列体内外实验。BBR作用于体外培养的人肝癌细胞系Bel-7402和HepG2细胞后能显著上调LDLR mRNA的表达。BBR的作用是时间和剂量依赖性的，其上调LDLR表达的作用在处理细胞2小时后即可出现，并可维持至少24小时。BBR增加LDLR表达的作用不受培养基中胆固醇浓度高低的影响。BBR作用后能使细胞表面LDLR蛋白的表达水平明显增加，并显著增强细胞对荧光素标记的DiI-LDL的吞噬作用。BBR上调LDLR表达的作用是特异性的，因为它对HMG-CoA还原酶、LDLR相关蛋白（LRP）和载脂蛋白ApoB等其它脂质代谢相关基因的表达均无影响。
BBR上调LDLR表达的机制与他汀类药物不同，因为BBR对 HMG-CoA还原酶的催化活性无影响。BBR作用后不能使细胞内SREBP-2活性形式的含量增多，说明SREBP-2在BBR上调LDLR表达的过程中不起作用。BBR与洛伐他汀联合作用于HepG2细胞后LDLR表达水平的上调程度较两者单用均更为明显，说明两者具有相加效应。
以带有LDLR基因启动子和萤火虫荧光素酶Luc报告基因的质粒转染细胞后再用BBR处理，结果BBR对LDLR启动子的转录活性没有影响。BBR作用后能使细胞中LDLR mRNA的降解速度明显减慢，使其半寿期（T1/2）延长2倍以上。说明BBR主要在转录后水平通过增加LDLR mRNA的稳定性来上调其表达。BBR对HMG-CoA还原酶mRNA的降解速度没有影响。
BBR增加LDLR mRNA稳定性的机制与其3’非翻译区（3’UTR）有关。将LDLR 3’UTR全长或不同片段插入到pLuc中Luc报告基因的下游3’端形成重组质粒，将这些质粒转染细胞后用BBR处理，再分析含有LDLR 3’UTR的Luc mRNA水平。结果显示，插入3’UTR后Luc mRNA的基础表达水平明显下降。BBR反应元件位于LDLR 3’UTR的5’端，是包含ARE和UCAU序列的片段（UTR-2）。为了进一步分析UTR-2中BBR的作用序列，我们分别对其中的ARE和UCAU序列进行了删除。结果显示ARE或UCAU序列删除后都能使BBR的作用不同程度消失，说明它们都是BBR增加LDLR mRNA稳定性所必需的。
BBR在体外能迅速激活细胞的胞外信号调节激酶（ERK）信号转导途径，ERK的激活作用在时间上提前于LDLR的表达增加。BBR对ERK的激活也呈剂量依赖性。ERK的激活是BBR上调LDLR表达所必需的，用特异性阻断剂U0126阻断ERK信号转导能使BBR上调LDLR表达的作用完全消失。
BBR 0.5 g每天2次口服给药治疗3个月后能使32例初治的高胆固醇血症患者血清总胆固醇（TC）、甘油三酯（TG）和LDL-c浓度分别下降29%、35%和25%。BBR对其中Ⅱa和Ⅱb型患者的降胆固醇疗效类似。所有患者以BBR治疗后未出现明显不良反应。BBR还能使患者的肝功能得到改善。
BBR 100 mg/kg/d口服治疗10天后能使高血脂金黄地鼠的血清TC和LDL-c浓度分别下降37%和42%，并使地鼠肝脏中LDLR mRNA和蛋白的平均表达水平分别上调2.5倍和1.6倍，而且能显著减少肝组织中中性脂肪的含量。BBR在地鼠体内也能激活ERK信号转导途径，腹腔注射给药同样能显著降低胆固醇水平。BBR口服给药后动物粪便中脂类物质的含量较对照组无明显变化，提示口服BBR不影响肠道中胆固醇等脂类物质的吸收。
综上所述，我们的研究包括了从分子水平到细胞水平，从动物实验到临床试验等一系列工作。所有证据都说明BBR是一种安全可靠的新型降胆固醇药物，其作用机制与他汀类完全不同。我们认为BBR可单独应用或与他汀类联合应用于临床治疗高胆固醇血症。
关键词： 小檗碱，高胆固醇血症，低密度脂蛋白胆固醇，低密度脂蛋白受体，细胞信号转导，mRNA稳定性
New application of berberine: lipids lowering effect
and molecular mechanism
Kong Weijia
ABSTRACT
Elevation of serum cholesterol especially low-density lipoprotein cholestrol (LDL-c) levels is by far one of the most relative risk factors that cause atherosclerosis and coronary heart disease (CHD). Lowering serum LDL-c levels by medication can reduce the mortality of CHD significantly. The majority of cholesterol in the blood are taken up and cleared by hepatic low-density lipoprotein receptor (LDLR). LDLR is a transmembrane receptor located on cell surface, the expression level of which is tightly regulated by sterols and various non-sterol mediators both on the transcription level and on the post-transcription level. Upregulation of hepatic LDLR is the most effective method to lower serum LDL-c levels. The most widely-used LDLR upregulator at present time are statins. Statins can inhibit the enzymatic activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase competitively, then imhibit cell cholesterol synthesis, which in turn upregulate the transcription of LDLR gene through the sterol responsive element binding protein (SREBPs) pathway. In general, statins have excellent clinical result in treating hypercholesterolaemia. However, a small part of patients can not tolerate statins because of adverse effects such as liver function damage and myopathy, yet another part of patients need other lipid-altering agents because desirable LDL-c levels can not be achieved by statins alones. So, ongoing efforts for screening new types of LDLR upregulators have being done for a long time.
By screening the pool of traditional Chinese herbals, we found for the first time that berberine (BBR), an alkaloid isolated form herbals such as Coptis chinensis, can upregulate hepatic LDLR expression, and lower cholesterol levels in vivo. BBR has definite chemical structure and has various pharmacological effects. BBR has been used in China in clinical practice for a long time to treat diarrhea, the effectiveness and safety of the drug has been widely accepted.
We have designed a series of in vitro and in vivo experiments to study the mechanism by which BBR upregulates LDLR expression. BBR can upregulate the expression of LDLR mRNA to a significant extent in the human hepatoma cell line Bel-7402 and HepG2 cells. The activity of BBR is time and dose-dependent, we can find the upregulation of LDLR mRNA just after 2 hours’ BBR treatment, and the effect can last for at least 24 hours. The upregulation of LDLR by BBR is independent of medium cholesterol levels. Also, BBR treatment can increase the LDLR protein levels on the cell surface and enhance the uptake of DiI-LDL by cells. The upregulation of LDLR by BBR is specific, because BBR can not influence the expression levels of other important genes participating in lipid metabolism, such as HMG-CoA reductae, LDLR related protein (LRP) or ApoB genes.
The mechanism BBR upregulates LDLR expression is different from that of statins, since BBR can not inhibit the enzymatic activity of HMG-CoA reductase. BBR treatment can not increase the cellular active form of SREBP-2, indicating that SREBP-2 does not play a role in the upregulation of LDLR by BBR. BBR and lovastatin have additive effect in upregulating LDLR expression.
To study if BBR can enhance the transcriptional activity of LDLR gene promoter, we used a plasmid containing the LDLR promoter sequence and the luciferase (Luc) reporter to transfect HepG2 cells, then treated the cells with BBR and assay the reporter. We found that BBR has no influence on LDLR promoter activity. We also found that BBR can reduce the turnover rate of LDLR mRNA significantly without influencing the turnover of HMG-CoA reductase mRNA. BBR can prolong the half life (T1/2) of LDLR mRNA by more than 2-fold. These results indicate that BBR upregulates the expression of LDLR on post-transcriptional level by stabilizing LDLR mRNA.
BBR increases the stability of LDLR mRNA through its 3’ untranslated region (3’UTR). We inserted the whole LDLR 3’UTR or its different segments into the 3’ end of the Luc reporter driven by PCMV in the pLuc plasmid. The recombinant plasmids were transfected in to HepG2 cells and treated by BBR, then, the levels of chimeric Luc mRNAs containing LDLR 3’UTR were analyzed. We find that the basal level of wild type Luc mRNA is greatly compromised by insertion of LDLR 3’UTR. BBR can upregulate the chimeric Luc mRNAs containing the whole 3’UTR or a segment termed UTR-2 locating at the 5’ end of the LDLR 3’UTR, which containing 3 ARE elements and 4 UCAU motifs. To elucidate the role of these sequences in the BBR activity, we make deletions of the ARE and UCAU. We found that deletion of either sequence can impair the activity of BBR to a great extent, indicating that they are all needed for BBR to increase LDLR mRNA stability, they are BBR responsive elements.
BBR can activate extracellular-signal regulated kinase (ERK) signaling pathway in liver cells very rapidly and dose dependently in vitro. The activation of ERK is earlier than the upregulation of LDLR. The activation of ERK is absolutely necessary for BBR to upregulate LDLR, since blocking the ERK pathway by a specific inhibitor U0126 can totally abolish the effect of BBR to upregulate LDLR.
We treated 32 previously untreated hypercholesterolemic patients with BBR. 0.5 gram of BBR orally treated twice a day for 3 months can effectively lower their serum total cholesterol (TC), triglyceride (TG), and LDL-c levels by 29%, 35%, and 25%, respectively. BBR has equal effects in lowering cholesterol levels in type Ⅱa and Ⅱb of hypercholesterolemic patients. We did not find any significant adverse effects in BBR treated patients. We found that BBR can partially improve the liver function in treated patients.
We also studied the effect of BBR in hyperlipidemic hamsters. 100 mg/kg/d of BBR orally treated for 10 days can reduce the hamsters’ serum TC and LDL-c levels by 37% and 42%, respectively. We also found direct links between the cholesterol lowering effect of BBR and the upregulation of LDLR in the liver. BBR treatment upregulates the expression levels of LDLR mRNA and protein in hamster livers by 2.5 and 1.6-fold, respectively. The ERK signalling pathway in the treated livers is also activated. BBR treatment can reduce the fat storage caused by high fat and high cholesterol (HFHC) diet in the hamster livers to significant extent, which explains improvement of liver function observed in patients. BBR treated intraperitoneally can also lower serum cholesterol levels effectively. In addition, fecal lipid amounts in BBR treated animals are close to control animals, indicating that BBR orally treatment does not influence the absorption of lipids in the intestine.
Altogether, evidences from molecular mechanism study to cell level research, from animal in vivo test to clinical trial all prove that BBR is a promising and safe new cholesterol-lowering drug, the mechanism of which is distinct from statins. We propose that BBR can be used as monotherapy or in combination with statins in treating hypercholesterolemic patients.
Key words: Berberine, Hypercholesterolemia, Low-dwnsity lipoprotein cholesterol, Low-density lipoprotein receptor, Cell signaling pathways, mRNA stability 小檗碱降低血清胆固醇的作用与分子机理研究
这是一篇二○○七年全国优秀博士学位论文，我觉得对我们了解LDL的新进展有帮助，特转贴。
血液中胆固醇特别是低密度脂蛋白胆固醇（LDL-c）浓度的升高是引起动脉粥样硬化和冠心病的最主要危险因素，降低血液中胆固醇的浓度可明显减少冠心病的发病率和死亡率。体内大部分胆固醇结合于低密度脂蛋白（LDL），经肝细胞表面的低密度脂蛋白受体（LDLR）代谢和清除。LDLR是位于细胞表面的跨膜受体，其表达受到胆固醇和其它许多非固醇类物质在转录水平或转录后水平的调控，上调肝脏LDLR的表达是降低血液胆固醇浓度的最主要手段。目前临床应用最广泛的能上调肝脏LDLR表达的药物是他汀类。他汀类药物竞争性抑制羟甲基戊二酸单酰辅酶A（HMG-CoA）还原酶的活性，使细胞内胆固醇合成减少，结果通过固醇调控元件结合蛋白（SREBPs）途径使LDLR基因的转录增加。总体来讲他汀类具有出色的降胆固醇疗效，但是也有部分患者因不良反应（如肝功能损害、肌病等）不能耐受其治疗，另有部分患者单用他汀类疗效欠佳而需联合用药。因此，筛选新型降胆固醇药物一直是调血脂药物的研究重点。
通过对传统中草药物的筛选，我们首次发现小檗碱（Berberine，BBR）能上调肝细胞LDLR的表达，并且在体内具有明确的降血脂疗效。BBR是从黄连和黄柏等中药中提取出来的生物碱，是具有明确化学结构的天然化合物，具有多种药理作用。BBR在我国已经有很长时间的临床应用历史，过去主要作为抗菌药物治疗细菌性腹泻，其疗效和安全性均得到普遍认可。
为了研究BBR降胆固醇作用的分子机制，我们设计了一系列体内外实验。BBR作用于体外培养的人肝癌细胞系Bel-7402和HepG2细胞后能显著上调LDLR mRNA的表达。BBR的作用是时间和剂量依赖性的，其上调LDLR表达的作用在处理细胞2小时后即可出现，并可维持至少24小时。BBR增加LDLR表达的作用不受培养基中胆固醇浓度高低的影响。BBR作用后能使细胞表面LDLR蛋白的表达水平明显增加，并显著增强细胞对荧光素标记的DiI-LDL的吞噬作用。BBR上调LDLR表达的作用是特异性的，因为它对HMG-CoA还原酶、LDLR相关蛋白（LRP）和载脂蛋白ApoB等其它脂质代谢相关基因的表达均无影响。
BBR上调LDLR表达的机制与他汀类药物不同，因为BBR对 HMG-CoA还原酶的催化活性无影响。BBR作用后不能使细胞内SREBP-2活性形式的含量增多，说明SREBP-2在BBR上调LDLR表达的过程中不起作用。BBR与洛伐他汀联合作用于HepG2细胞后LDLR表达水平的上调程度较两者单用均更为明显，说明两者具有相加效应。
以带有LDLR基因启动子和萤火虫荧光素酶Luc报告基因的质粒转染细胞后再用BBR处理，结果BBR对LDLR启动子的转录活性没有影响。BBR作用后能使细胞中LDLR mRNA的降解速度明显减慢，使其半寿期（T1/2）延长2倍以上。说明BBR主要在转录后水平通过增加LDLR mRNA的稳定性来上调其表达。BBR对HMG-CoA还原酶mRNA的降解速度没有影响。
BBR增加LDLR mRNA稳定性的机制与其3’非翻译区（3’UTR）有关。将LDLR 3’UTR全长或不同片段插入到pLuc中Luc报告基因的下游3’端形成重组质粒，将这些质粒转染细胞后用BBR处理，再分析含有LDLR 3’UTR的Luc mRNA水平。结果显示，插入3’UTR后Luc mRNA的基础表达水平明显下降。BBR反应元件位于LDLR 3’UTR的5’端，是包含ARE和UCAU序列的片段（UTR-2）。为了进一步分析UTR-2中BBR的作用序列，我们分别对其中的ARE和UCAU序列进行了删除。结果显示ARE或UCAU序列删除后都能使BBR的作用不同程度消失，说明它们都是BBR增加LDLR mRNA稳定性所必需的。
BBR在体外能迅速激活细胞的胞外信号调节激酶（ERK）信号转导途径，ERK的激活作用在时间上提前于LDLR的表达增加。BBR对ERK的激活也呈剂量依赖性。ERK的激活是BBR上调LDLR表达所必需的，用特异性阻断剂U0126阻断ERK信号转导能使BBR上调LDLR表达的作用完全消失。
BBR 0.5 g每天2次口服给药治疗3个月后能使32例初治的高胆固醇血症患者血清总胆固醇（TC）、甘油三酯（TG）和LDL-c浓度分别下降29%、35%和25%。BBR对其中Ⅱa和Ⅱb型患者的降胆固醇疗效类似。所有患者以BBR治疗后未出现明显不良反应。BBR还能使患者的肝功能得到改善。
BBR 100 mg/kg/d口服治疗10天后能使高血脂金黄地鼠的血清TC和LDL-c浓度分别下降37%和42%，并使地鼠肝脏中LDLR mRNA和蛋白的平均表达水平分别上调2.5倍和1.6倍，而且能显著减少肝组织中中性脂肪的含量。BBR在地鼠体内也能激活ERK信号转导途径，腹腔注射给药同样能显著降低胆固醇水平。BBR口服给药后动物粪便中脂类物质的含量较对照组无明显变化，提示口服BBR不影响肠道中胆固醇等脂类物质的吸收。
综上所述，我们的研究包括了从分子水平到细胞水平，从动物实验到临床试验等一系列工作。所有证据都说明BBR是一种安全可靠的新型降胆固醇药物，其作用机制与他汀类完全不同。我们认为BBR可单独应用或与他汀类联合应用于临床治疗高胆固醇血症。
博士生：孔维佳。 导师：蒋建东。作者单位：中国协和医科大学。 论文全文应该在CNKI上有 [s:64] 作者:魏志锋
院校：吉林大学
关键字:NRE;高脂血症;蛋白质组学;质谱
学位：硕士
专业：内科学
摘要：目的:探讨新生鼠提取液(NRE)对高脂血症模型大鼠降血脂作用的具体靶标,明确高脂血症、脂肪肝在蛋白质水平的发生机理,进一步阐明NRE在蛋白质水平调整血脂代谢的作用机制。方法:⑴药效学实验:分别检测5组大鼠(空白对照组、模型对照组、小剂量NRE组、大剂量NRE、阳性药物-洛伐他汀对照组)血清中 TC、TG、HDL、LDL水平,并留取肝脏组织,观察肝组织中脂肪沉积情况。⑵蛋白质组学实验:应用等电聚焦/SDS聚丙烯酰胺双向凝胶电泳技术分离4 组大鼠(空白对照组、模型对照组、小剂量NRE组、大剂量NRE组)肝脏总蛋白,考马斯亮兰染色,PDQuest 2DE软件分析,对部分蛋白质点用基质辅助激光解析电离飞行时间质谱(matrix-assisted laserdesorption/ionization time of flying mass spectrometry,MALDI-TOF-MS)测定其胶内酶解后的肽指纹图谱,检索NCBInr或SwissProt数据库。结果:NRE可以明显降低血清TC、TG,LDL-C水平(P
Abstract:
Objective This study works on the target of NRE in order to illuminatethe proteinic mechanism , approach the regulation of lipidmetabomlism disorder,seek a proteinic regulation target for lipidmetabomlism,direct the development of descendens blood fatdrugs. Method: 1. 80 Wistar rats are provided by the Experimental AnimalCenter of Jilin University. Divide the rats randomly into 5 groups:blank group,model group,low-dose-NRE group,large-dose-NREgroup,positive drug control group. Detect the concentration ofplasma TC、TG、HDL、LDL in 5 groups respectively. Take the liverconstitution of rats in 5 groups to observe the state of fattydeposition in liver. 2. Extract the total protein of liver in 10 subjects in 4 groups( blank group , model group , low-dose-NRE group ,large-dose-NRE group ) . Using IEF/2-DE PAGE andMALDI-TOF-MS, we compare the abumdamces of protein in4groups. A total number of 54 protein spots with altered abundancesare detected and 11 of them were successfully identified byMALDI-TOF-MS. Result: 1. Changes of blood fat:The plasma TG、TC、LDL-c in the low-dose-NRE group andthe large-dose-NRE group are lower than it in experimentalhyperlipemia model group,plasma HDL-c is higher than it in modelgroup and positive group p 谢谢各位战友，我们学校数据库是有，但是下载下来只有一半，得不到全文
所以我才向各位战友求助，希望能帮帮忙，发到我邮箱，再次谢谢各位！
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