Efficient in vivo delivery of siRNA into brain capillary endothelial cells along with endogenous lipoprotein

The brain capillary endothelial cell (BCEC) is a major functional component of the blood-brain barrier and is an underlying factor in the pathophysiology of various diseases, including brain ischemia, multiple sclerosis, and neurodegenerative disorders. We examined gene silencing in BCECs by using endogenous lipoprotein to introduce short-interfering RNA (siRNA) in vivo. A cholesterol-conjugated 21/23-mer siRNA targeting organic anion transporter 3 (OAT3) mRNA (Chol-siOAT3) was intravenously injected into mice after its incorporation into extracted endogenous lipoproteins. Chol-siOAT3 was not delivered to neurons or glia, but was successfully delivered into BCECs and resulted in a significant reduction of OAT3 mRNA levels when injected after its incorporation into high-density lipoprotein (HDL). Efficient delivery was not achieved, however, when Chol-siOAT3 was injected without any lipoproteins, or after its incorporation into low-density lipoprotein (LDL). Investigations in apolipoprotein E (ApoE)-deficient and LDL receptor (LDLR)-deficient mice revealed that the uptake of HDL-containing Chol-siOAT3 was mainly mediated by ApoE and LDLR in mice. These findings indicate that siRNA can be delivered into BCECs in vivo by using endogenous lipoprotein, which could make this strategy useful as a new gene silencing therapy for diseases involving BCECs.     

'Localized amyloidosis': 123I-labelled SAP component scintigraphy and labial salivary gland biopsy

In apparently localized amyloidosis, there is no appropriate test to determine whether systemic deposits exist. We studied the value of serum amyloid P component (SAP) scintigraphy and labial salivary gland (LSG) biopsy on patients with apparently localized amyloidosis in 12 patients who had neither clinical nor biological evidence of systemic amyloidosis. All patients had an LSG biopsy and echocardiography. Iodine-123-labelled serum amyloid P component (123I-SAP) scintigraphy was performed in all patients. Whole-body scintigraphy was done, and tissue retention was evaluated at 24 h and 48 h. Of these 12 patients, three had amyloidosis in their LSG and had abnormal 123I-SAP scintigraphy; these three had a secondary clinical history of systemic amyloidosis. Three other patients had abnormal 123I-SAP scintigraphy without detectable systemic amyloid deposits, but one had a previous history of bilateral carpal tunnel syndrome treated with infiltration. 123I-SAP scintigraphy in association with LSG biopsy may be helpful in determining the localized or systemic character of amyloid disease.      

Laparoscopic features of primary biliary cirrhosis in AMA-positive and AMA-negative patients

BACKGROUND AND STUDY AIMS: Antimitochondrial antibody (AMA)-negative primary biliary cirrhosis (PBC) has been difficult to diagnose. Laparoscopic features of AMA-negative PBC were evaluated in comparison with those of AMA-positive PBC and autoimmune hepatitis. PATIENTS AND METHODS: 71 patients who fulfilled the diagnostic criteria for PBC were enrolled in the study; 48 were AMA-positive and 23 were AMA-negative. As a disease control, 46 autoimmune hepatitis patients were included. Both the frequency and specificity of each laparoscopic finding were evaluated. A laparoscopic scoring system was introduced, which used, common and uncommon laparoscopic findings, and was evaluated for the diagnosis of AMA-negative PBC. RESULTS: The characteristic laparoscopic findings for AMA-positive PBC were yellowish-white marking (92 %), dark-brown discoloration (73 %), gentle undulation (67 %), reddish patch (38 %), and yellowish-white nodules (32 %). On the other hand, laparoscopic findings such as trench-like depression, reddish markings, and wide and small depressions were uncommon in PBC compared with autoimmune hepatitis. The frequencies of characteristic and uncommon laparoscopic findings did not differ statistically between AMA-positive and AMA-negative PBC, but were different between AMA-positive or AMA-negative PBC and autoimmune hepatitis. Scores based on common and uncommon laparoscopic findings were 5.5 +/- 1.5 (mean +/- SD) in AMA-positive PBC, 5.6 +/- 2.0 in AMA-negative PBC, and - 0.30 +/- 0.5 in autoimmune hepatitis. CONCLUSION: The laparoscopic findings in AMA-negative PBC did not differ from those of AMA-positive PBC. A laparoscopic scoring system may be helpful in the diagnosis of AMA-negative PBC.     

转化生长因子β1在白细胞介素1β诱导大鼠肾小管上皮细胞转分化及大黄素干预中的作用

目的:大黄素对白细胞介素1β诱导NRK52E细胞转分化有显著抑制作用。实验拟进一步观察转化生长因子β1在白细胞介素1β诱导大鼠肾小管上皮细胞-肌成纤维细胞转分化及大黄素抑制作用中的意义。方法:实验于2006-10/2007-05在泸州医学院附属医院免疫实验室完成。⑴实验材料及分组:以培养的大鼠肾小管上皮细胞株(NRK52E)为观察对象,按如下分组分别添加不同处理因素:①对照组:仅加入体积分数为0.05小牛血清的高糖DMEM培养基。②白细胞介素1β诱导组:加含白细胞介素1β终浓度为10μg/L的高糖DMEM培养基。③SB431542阻断组:加含白细胞介素1β终浓度为10μg/L及SB431542终浓度为10μmol/L的高糖DMEM培养基。④白细胞介素1β 大黄素组:同时加分别含白细胞介素1β终浓度为10μg/L及大黄素终浓度为25mg/L的高糖DMEM培养基。⑵实验评估:培养48h后用倒置相差显微镜观察细胞形态,细胞免疫化学染色法检测肌酸激酶、α-平滑肌肌动蛋白及转化生长因子β1的表达。结果:①白细胞介素1β可诱导部分细胞由卵圆形转变为梭形,且肌酸激酶表达减弱(P<0.01),α-平滑肌肌动蛋白及转化生长因子β1表达显著增强(P<0.01)。②SB431542特异性抑制转化生长因子β1作用后,白细胞介素1β诱导的细胞形态改变受抑,同时肌酸激酶表达增强(P<0.01),α-平滑肌肌动蛋白表达减弱(P<0.01),但转化生长因子β1的表达却无明显变化。③大黄素对白细胞介素1β诱导的细胞形态改变及肌酸激酶、α-平滑肌肌动蛋白的表达有明显抑制作用,其抑制作用与SB431542的作用相比无显著差异;同时,大黄素对白细胞介素1β诱导的转化生长因子β1的表达也有明显抑制作用(P<0.01)。结论:转化生长因子β1可能介导了白细胞介素1β诱导大鼠肾小管上皮细胞-肌成纤维细胞转分化,并参与了大黄素抑制白细胞介素1β诱导大鼠肾小管上皮细胞转分化的作用。     

广东地区散发克罗恩病患者β防御素2启动子区基因突变检测

目的:对广东地区散发克罗恩病患者β防御素2启动子区基因突变进行筛查,探讨该基因在克罗恩病发病中的作用。方法:克罗恩病组为2002-01/2006-10于南方医院消化科确诊的克罗恩病患者45例,对照组为门诊健康志愿者50例。签定知情同意书后分别取静脉血5mL,提取基因组DNA,并根据设计好的引物序列及PCR反应条件进行目的片段的扩增,阴性对照使用蒸馏水代替DNA。扩增成功的目的基因片段用DNA纯化试剂盒进行纯化,并由上海英俊公司完成测序工作。利用DNAMAN软件将患者测序结果与正常对照结果进行比对,并与基因库数据对照(http://www.ncbi.nlm.nih.gov/BLAST/),SPSS13.0进行统计学分析。并根据结果分析其与克罗恩病患者病变特点的相关性。结果:①纳入的45例克罗恩病患者与50例健康志愿者在性别和年龄上无统计学意义,并全部进入实验分析。②45例克罗恩病患者中有4例在β防御素2启动子区第-233(G→C)位发现基因突变,密码子由AGG变成AGC,编码氨基酸由精氨酸变成丝氨酸,而50例对照组未发现此改变,二者比较具有统计学意义(χ2=4.34,P<0.05)。③4例突变的患者病变均位于小肠(χ2=10.81,P<0.01)且病情较重。结论:在克罗恩患者中存在着β防御素2启动子区基因突变携带者,与患者发病年龄、病变部位、病变程度明显相关,有必要对其功能进行进一步探讨。     

白细胞介素1及白细胞介素8在心肌缺血再灌注损伤中的动态演变及药物干预效果

目的:在心肌缺血再灌注损伤中,炎症细胞因子参与其过程的多个环节。实验拟验证白细胞介素1、白细胞介素8因子在此过程中的动态变化,并分析其与药物干预的关系。方法:实验于2005-10/2006-11在新乡医学院形态学实验室完成。①实验分组:选择健康Wistar成年大鼠70只,按随机数字表法分为3组:对照组(n=10)、模型组(n=30)和药物组(n=30)。后两组又分为缺血0.5h,再灌注2,4,8,12,24h6个时相点,每个时相点5只。对照组只设12h一个时相点作为总体对照。②实验方法:大鼠麻醉后,药物组在右股静脉注入甲泼尼龙(30mg/kg),对照组及模型组注入生理盐水(0.75mg/kg)。采用夹闭左冠状动脉前降支建立大鼠心肌缺血再灌注损伤模型。对照组只开胸不夹闭。③实验评估:在各时相点观察各组大鼠缺血再灌注后的心肌细胞改变;血清学检测白细胞介素1、白细胞介素8因子的动态表达。结果:①模型组缺血再灌注12h炎细胞浸润最显著,药物组炎细胞呈散在浸润。②模型组和药物组白细胞介素1、白细胞介素8因子质量浓度明显高于对照组[缺血再灌注8h为例,白细胞介素1分别为(99.21±14.37),(85.77±11.31),(21.87±10.32)ng/L;白细胞介素8分别为(794.85±24.07),(536.95±19.72),(103.94±11.59)ng/L,P<0.05],峰值分别在缺血再灌注4,8h;同时相点药物组白细胞介素1、白细胞介素8因子质量浓度明显低于模型组(P<0.05)。结论:白细胞介素1和白细胞介素8因子在心肌缺血再灌注损伤的炎症反应中发挥着重要作用;甲泼尼龙对缺血再灌注损伤心肌有保护作用。     

脑源性神经生长因子促进成年大鼠脑海马神经干细胞定向分化的浓度选择

目的：探讨培养基中表皮生长因子与血清两者浓度一定的条件下，不同浓度的脑源性神经生长因子对成年大鼠脑海马神经干细胞向神经元分化的影响。 方法：实验于2007—08在中国医科大学神经解剖研究室完成。①材料：清洁级雄性成年SD大鼠1只，由中国医科大学实验动物部提供，实验过程中对动物的处置符合动物伦理学标准。实验过程中应用的表皮生长因子、脑源性神经生长因子均由R＆D公司提供：（多实验方法：无菌条件下分离大鼠脑海马组织，剪碎后胰蛋白酶消化，过滤、离心、弃上清，加入含2％B27、20μg／L表皮生长因子、20μg／L碱性成纤维生长因子的DMEM／F12无血清条件培养基体外培养神经干细胞，传至第4代时利用有限稀释法进行单克隆培养，100倍镜下克隆球直径约为200μm时制备单细胞悬液，稀释后滴加于96孔板内，设立两组，全量新鲜培养基组加入刚配置未使用过的DMEM／F12无血清培养基100μL，半量条件培养基组加入上述曾用于神经干细胞培养且含有其代谢产物的1／2DMEM／F12无血清培养基100斗L。（劲实验评估：观察神经干细胞的单克隆培养增殖情况。对克隆球行巢蛋白、神经元特异性烯醇化酶、胶质原纤维酸性蛋白免疫细胞化学染色。按培养基中脑源性神经生长因子终浓度的不同将所培养细胞设立0，50，100，150，200μg／L组，各组均加入20μg／L表皮生长因子和体积分数为0．1的胎牛血清，1周后行神经元特异性烯醇化酶免疫细胞化学染色，检测神经干细胞向神经元分化情况。 结果：①神经干细胞单克隆培养结果：单克隆培养开始时神经干细胞增殖缓慢，半量条件培养基组细胞增殖速度快于全量新鲜培养基组，随着细胞数的增多，两组细胞增殖速度也相应加快，分别在培养后第12天、第15天形成直径为200μm的克隆球。②神经干细胞免疫细胞化学染色结果：单克隆培养后克隆球表达巢蛋白，诱导分化后神经元特异性烯醇化酶、胶质原纤维酸性蛋白均呈阳性表达：③各组神经干细胞分化为神经元的比例：与0μg／L脑源性神经生长因子组比较，50，100μg／L脑源性神经生长因子组的神经干细胞分化为神经元比例均明显增高（t=2．502～5．025，P〈0．05）；而浓度为150，200μg／L时均无明显变化（t=0．420～1．857，P〉0．05）。 结论：向含有B27、表皮生长因子、碱·性成纤维生长因子的DMEM／F12条件培养基中加入20μg／L表皮生长因子和体积分数为0．1胎牛血清的情况下，脑源性神经生长因子促使神经干细胞向神经元分化的较佳浓度为50μg／L。     

瘢痕疙瘩家系Fas基因的突变：2个家系10份标本分析

目的:观察瘢痕疙瘩家系样本中Fas基因有无突变,探讨Fas基因突变在瘢痕疙瘩形成中的意义。方法:实验于2005-01/05在上海基康公司完成。①标本来自南方医科大学南方医院整形外科2005年收集的A和B两个瘢痕疙瘩家系,所有参与观察的家系成员均签署知情同意书。②采用聚合酶链反应及基因测序技术,分别以A家系两例患者的瘢痕疙瘩组织为观察对象,以其周围正常皮肤及外周静脉血作为自身对照;其配偶的外周静脉血作为正常对照。并以B家系中两例患者的外周静脉血作为不同家系间的对照。共取10份样本,4份组织样本,6份静脉血标本。检测10份样本中Fas基因外显子1～9的基因序列。结果:①基因测序发现所检测的10个瘢痕疙瘩家系标本Fas基因的1～8外显子均未发现突变。②2份瘢痕疙瘩组织标本在第9外显子编码区的11bp,53bp两个位点上存在单个碱基的基因突变或多态性改变。结论:瘢痕疙瘩Fas基因外显子9区段的基因结构异常极有可能造成Fas蛋白的功能改变,从而导致身体局部瘢痕疙瘩的形成。     

瘢痕疙瘩发病机制的研究动态

目的:总结瘢痕疙瘩在分子遗传学和生物化学方面的发病机制,为临床诊断和治疗提供可靠的依据。资料来源:应用计算机检索Pubmed1980-01/2006-10相关瘢痕疙瘩发病机制方面的文献,检索词“keloids”,限定文献语言种类为English。同时计算机检索中国期刊网1980-01/2006-10相关瘢痕疙瘩发病机制方面的文献,检索词“瘢痕疙瘩”,限定文献语言种类为中文。资料选择:对资料进行初审,选取包括瘢痕疙瘩发病机制的文献,开始查找全文。纳入标准:与瘢痕疙瘩和瘢痕疙瘩成纤维细胞相关的分子生物学研究。排除标准:相关的综述文献。资料提炼:共检索到681篇关于瘢痕疙瘩发病机制的文献,最终纳入33篇符合标准的文献。资料综合:瘢痕疙瘩有着过度生长、侵犯邻近组织、手术切除后极易复发、治疗效果不佳等特点,成为当今医学界面临的一个重大难题。瘢痕疙瘩发病机制迄今未明,本文从遗传机制、成纤维细胞功能、生物活性因子和胶原代谢等方面综述了近几年来对瘢痕疙瘩病因研究的最新进展,发现利用基因检测技术成为探求病因的一个重要手段和发展趋势,也为未来基因治疗提供了可靠的实验室依据。结论:越来越多的研究在瘢痕疙瘩相关致病基因位点的定位和克隆方面展开,这不但能够查找出发病原因,还可以为易感人群的早期预防提供方向,为瘢痕疙瘩的基因治疗奠定一定的基础。