TGF-β1对话Hedgehog-Gli1信号调控肾小管上皮细胞表型转化和胶原累积

该研究旨在探讨Hedgehog-Gli1(HH)信号在肾小管上皮细胞表型转化(epithelial-mesenchymal transition,EMT)和胶原累积中的作用及与TGF-β1信号的对话机制。该实验通过体外培养大鼠肾小管上皮细胞NRK-52E,以溶剂作为对照组,以1~50 ng/m L重组蛋白sonic hedgehog(Shh)或5 ng/m L TGF-β1作为诱导组,以加入或不加入HH信号特异性阻断剂环靶明(cyclopamine,Cyp)5μmol/L为干预组。细胞培养24 h,采用ELISA、q RT-PCR、免疫细胞荧光染色和Western blot等方法检测HH信号相关分子(Ptch1、Smo和Gli1)、TGF-β1、EMT相关分子(Rac1蛋白、肌成纤维细胞标志物α-SMA和上皮细胞标志物E-cadherin)、III型胶原m RNA或蛋白的表达。结果发现,外源性Shh上调Smo和Gli1表达,抑制Ptch1表达,继而激活HH信号;HH信号活化抑制肾小管上皮细胞E-cadherin的表达,上调α-SMA、III型胶原和TGF-β1的表达。环靶明干预后,Smo表达下调,进而抑制HH信号、EMT和胶原累积,并下调TGF-β1的表达。应用TGF-β1诱导小管上皮细胞EMT,同时也上调HH信号分子Smo和Gli1的表达,下调Ptch1的表达,提示TGF-β1可诱导HH信号活化。综上所述,HH信号和TGF-β1均参与了肾小管上皮细胞EMT和胶原累积过程。HH信号活化可促进TGF-β1的表达,同时TGF-β1能激活HH信号,推测TGF-β1与HH信号可能存在交叉对话以调控EMT和胶原累积。     

转化生长因子β1和snail1参与糖尿病大鼠肾小管上皮细胞向间充质细胞转变

本文旨在观察转化生长因子β1（transforming growth factor-β1,TGF-β1）和锌指转录因子Snail1在糖尿病（diabetes mellitus,DM）大鼠肾组织中的表达,并初步探讨它们与肾小管上皮细胞向间充质细胞转变的关系。链脲佐菌素（streptozotocin,STZ）诱发大鼠DM,按病程分为2、4、8、12、16、20、24周、16周胰岛素治疗（16wA）、20,周胰岛素治疗（20wA）和24周胰岛素治疗（24wA）组（n=6）。其中胰岛素治疗组动物从第13周起用胰岛素控制血糖至正常水平,每一时点均设鼠龄匹配的正常对照组。测定各组血糖、24h尿蛋白、血肌酐（serum creatinine,Scr）、肾脏指数。PAS染色光镜观察肾脏病理学改变。免疫组织化学检测肾脏Snail1、TGF-β1、α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）、E-钙黏素和纤连蛋白（fibronectin,FN）的表达;Western blot检测肾皮质Snail1、TGF-β1和E-钙黏素蛋白表达。RT-PCR检测肾皮质Snail1和E-钙黏素mRNA表达。结果显示：（1）DM各组大鼠的血糖、24h尿蛋白、Scr、肾脏指数均较正常对照组明显升高（P〈0．05,P〈0．01）,胰岛素治疗组大鼠上述指标均较DM组显著降低（P〈0．01）。（2）TGF-β1和Snail1免疫组织化学阳性染色见于DM各组大鼠肾小管,正常对照组未见阳性表达,胰岛素治疗组大鼠弱阳性表达,并随治疗时间延长而减少。从16周开始在DM大鼠肾小管上皮细胞可见α-SMA蛋白阳性表达,胰岛素治疗组大鼠未见α-SMA蛋白表达;DM组大鼠E-钙黏素蛋白阳性染色明显少于正常对照组。（3）DM组大鼠肾皮质TGF-β1和Snail1蛋白以及Snail1 mRNA表达较正常对照组显著增高（P〈0．01）,胰岛素治疗组大鼠则显著低于DM组（P〈0．01）;DM组E-钙黏素mRNA和蛋白表达与TGF-β1和Snail1呈相反变化。结果提示,TGF-β1和Snail1可能参与DM大鼠肾小管上皮细胞向间充质细胞转变,胰岛素治疗可抑制两者表达并阻断肾小管上皮细胞向间充质细胞转变。     

转化生长因子β1和Snaill参与糖尿病大鼠肾小管上皮细胞向间充质细胞转变

本文旨在观察转化生长因子β1(transforming growth factor-β1,TGF-β1)和锌指转录因子Snaill在糖尿病(diabetesmellitus,DM)大鼠.肾组织中的表达,并初步探讨它们与肾小管上皮细胞向间充质细胞转变的关系.链脲佐菌素(streptozotocin,STZ)诱发大鼠DM,按病程分为2、4、8、12、16、20、24周、16周胰岛素治疗(16wA)、20周胰岛素治疗(20wA)和24周胰岛索治疗(24wA)组(n=6).其中胰岛素治疗组动物从第13周起用胰岛素控制血糖至正常水平,每一时点均设鼠龄匹配的正常对照组.测定各组血糖、24 h尿蛋白、血肌酐(serum creatinine,Scr)、肾脏指数.PAS染色光镜观察肾脏病理学改变.免疫组织化学检测肾脏SnMll、TGF-β1、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、E-钙黏素和纤连蛋白(fibronectin,FN)的表达;Western blot检测肾皮质SnailI、TGF-β1和E-钙黏素蛋白表达.RT-PeR检测肾皮质Snaill和E-钙黏素mRNA表达.结果显示:(1)DM各组大鼠的血糖、24 h尿蛋白、Scr、肾脏指数均较正常对照组明显升高(P<0.05,P<0.01),胰岛素治疗组大鼠上述指标均较DM组显著降低(P<0.01).(2)TGF-β1和Snmll免疫组织化学阳性染色见于DM各组大鼠肾小管,正常对照组未见阳性表达,胰岛素治疗组大鼠弱阳性表达,并随治疗时间延长而减少.从16周开始在DM大鼠肾小管上皮细胞可见α-SMA蛋白阳性表达,胰岛素治疗组大鼠未见α-SMA蛋白表达;DM组大鼠E-钙黏素蛋白阳性染色明显少于正常对照组.(3)DM组大鼠肾皮质TGF-β和Snaill蛋白以及Snaill mRNA表达较正常对照组显著增高(P<0.01),胰岛素治疗组大鼠则显著低于DM组(P<0.01);DM组E.钙黏素mRNA和蛋白表达与TGF-β1和Snaill呈相反变化.结果提示,TGF-βl和Snaill可能参与DM大鼠肾小管上皮细胞向问充质细胞转变,胰岛素治疗可抑制两者表达并阻断肾小管上皮细胞向间充质细胞转变.     

高脂喂养大鼠肾小管上皮细胞SREBP-1、TGF-β1、α-SMA的表达和细胞外基质沉积

目的:探讨高脂喂养对大鼠肾脏小管上皮细胞SREBP-1、TGF-β1、α-SMA表达和细胞外基质(ECM)的影响。方法:高脂饲料喂养大鼠12周后,油红O检测肾脏脂质沉积,Masson染色检测肾小管间质细胞外基质沉积,免疫组化、Western blot和原位杂交检测SREBP-1、TGF-β1、α-SMA和FN的表达。结果:高脂喂养后大鼠体重明显增加,血糖、甘油三酯和胰岛素均升高,油红O检测显示大鼠肾小管上皮细胞内出现明显脂滴。SREBP-1蛋白和mRNA在肾小管上皮细胞内表达,高脂组高于正常对照组,分别是正常组的1.88倍和1.85倍;TGF-β1和α-SMA也定位于肾小管上皮细胞胞浆并出现上调。Masson染色显示高脂喂养大鼠肾间质ECM沉积增多,纤维粘连蛋白FN检测也显示模型组表达强于对照组。结论:高脂饮食喂养可能通过上调肾脏小管上皮细胞SREBP-1表达使细胞内脂滴沉积,并进一步诱导TGF-β1、α-SMA合成而导致细胞外基质堆积。     

IgA肾病组织中巨噬细胞浸润与肾间质纤维化、EMT的关系及临床意义

目的探讨IgA肾病组织中巨噬细胞与肾小管间质纤维化(Tubulointerstitial fibrosis,TIF)及上皮细胞间充质转化(Epithelial-Mesenchymal Transition,EMT)的关系及其临床意义。方法采用HE、PAS、PASM、Masson染色评估56例IgA肾病肾穿刺组织肾小管间质病理变化,根据肾小管萎缩及间质纤维化程度分为轻度、中度、重度三组,另设对照组5例,来自正常肾脏组织。采用免疫组织化学方法检测CD68、CD206及EMT相关蛋白TGF-β1、Vimentin、SMA、E-Cadherin的水平,并收集患者详细临床资料。结果 CD68、CD206蛋白在TIF重度组中的水平高于中度组,在TIF中度组中的水平高于TIF轻度组;CD68阳性巨噬细胞、CD206阳性巨噬细胞计数与TGF-β1、Vimentin及SMA蛋白的水平正相关;与E-Cadherin蛋白的水平呈负相关;与肾功能指标血肌酐、尿素氮及尿蛋白呈正相关。TGF-β1、Vimentin及SMA的水平与肾小管间质纤维化程度及肾功能指标血肌酐、尿素氮及尿蛋白正相关;E-Cadherin蛋白的表达与肾小管间质纤维化程度及肾功能指标血肌酐、尿素氮及尿蛋白呈负相关。结论巨噬细胞可能参与了IgA肾病的肾小管间质损伤,尤其是TGF-β1诱导的肾小管EMT过程。     

氰酸盐诱导氧化应激促进肾小管上皮细胞上皮–间充质转化

该研究探讨氰酸盐(cyanate)诱导肾小管上皮细胞氧化应激损伤和促进肾纤维化的作用。氰酸盐作用HK-2肾小管上皮细胞后, CCK8法检测其对细胞活力的影响;倒置显微镜观察细胞形态的改变; DCFH-DA法检测细胞ROS水平;细胞免疫荧光和Western blot分别检测E-cadherin、Fibronectin、α-SMA的表达; Western blot检测TGF-β的表达水平。结果显示, 2 mmol/L氰酸盐明显下调HK-2细胞的活力(P<0.05),细胞形态变为长梭形。氰酸盐作用24 h后, HK-2细胞内ROS水平呈浓度依赖性升高。免疫荧光和Western blot结果均显示,氰酸盐作用24 h后, HK-2的Fibronectin、α-SMA表达升高, E-cadherin表达下降; TGF-β的表达水平随氰酸盐浓度升高而上调(P<0.05)。以上结果表明,氰酸盐诱导肾小管上皮细胞产生过量ROS,上调TGF-β水平促进细胞上皮–间充质细胞转化(epithelia-mesenchymal transition, EMT)。     

TGF-β1对乳腺癌细胞系Snail表达的影响

目的通过TGF-β1诱导乳腺癌MCF-7发生上皮-间质转化(epithelial-mesenchymal transition,EMT)后检测锌指转录因子Snail表达的改变,探讨Snail在EMT及乳腺癌发生发展中的作用。方法常规培养乳腺癌细胞株MCF-7后,用TGF-β1诱导其发生EMT,用Transwell侵袭小室法进行细胞体外侵袭能力检测;用免疫组织化学方法及免疫荧光检测E-cadherin、Vi mentin、Snail的表达;用real ti me PCR检测E-cadherin、Vi mentin、Snail mRNA的表达。结果TGF-β1处理72h后的MCF-7细胞穿透能力明显增强。E-cadherin蛋白及mRNA表达减少,Vi mentin、Snail蛋白及mRNA表达增加。结论E-cadherin、Vi mentin是细胞发生EMT的重要生物学标志,Snail可能在转录水平上调控E-cadherin、Vi mentin蛋白的表达,Snail在EMT和乳腺癌的发生发展中起着重要的作用。     

TGF-β1在自发性高血压大鼠肾损害中作用的研究

目的研究转化生长因子-β1(transforming growth factor-β1,TGF-β1)在自发性高血压大鼠(spontaneously hypertertensive rat,SHR)肾脏的表达及其与肾损害的关系.方法以同龄雄性正常血压(Wistar Kyoto,WKY)和自发性高血压大鼠为研究对象,分别于12周龄和24周龄时检测两种大鼠尾动脉血压、肾功能及β2微球蛋白(β2-MG),并采用免疫组织化学的方法检测TGF-β1在肾脏中的表达.结果同WKY组比较, SHR组24周时β2-MG显著增高(P<0.01);而且尾动脉血压显著性增高;而尿素氮和血肌酐的差异无显著性(P>0.01).TGF-β1在WKY组肾小管的表达无或极微量;在SHR组的肾小球有少量表达,但在肾小管的表达显著,且随高血压病程的进展, TGF-β1的表达显著性增加(P<0.01).结论 TGF-β1在自发性高血压大鼠肾小管的表达显著增加,与肾损害的各项指标呈正相关.     

miR-21对TGF-β1诱导的肾小管上皮细胞间质转分化的影响

目的:观察miR-21在转化生长因子β1(TGF-β1)诱导的人肾小管上皮细胞(HK-2细胞)上皮间质转分化(EMT)中的作用,并探讨miR-21参与调控HK-2细胞EMT的可能靶点。方法:体外培养的HK-2细胞分为6组:正常对照组、转化生长因子β1(TGF-β1)模型组、miR-21 mimic阴性组、miR-21 mimic组、miR-21 inhibitor阴性组和miR-21 inhibitor组。细胞经4 ng/ml TGF-β1处理建立EMT模型,检测miR-21和EMT相关指标的表达变化,利用基因转染技术,将miR-21 mimic质粒或miR-21 inhibitor质粒转染经TGF-β1处理的HK-2细胞,使细胞过表达或抑制表达miR-21,在此基础上观察细胞EMT相关指标的变化以及磷酸酯酶(PTEN)基因的影响。结果:(1)与正常组相比,模型组的miR-21含量显著升高(P0.05),上皮表型标志物E-cadherin的mRNA和蛋白表达水平均显著降低(P0.01),间质表型标志物α平滑肌肌动蛋白(α-SMA)mRNA和蛋白水平也显著升高(P0.05,P0.01);(2)转染miR-21 mimic后,与miR-21 mimic阴性对照组相比,miR-21含量显著升高(P0.01),PTEN、E-cadherin的mRNA和蛋白水平显著降低(P0.05,P0.01),α-SMA mRNA和蛋白水平显著升高(P0.05,P0.01);转染miR-21 inhibitor后,与miR-21 inhibitor阴性对照组相比,miR-21含量显著降低(P0.01),PTEN、E-cadherin的mRNA和蛋白含量显著升高(P0.05,P0.01),α-SMA mRNA、蛋白水平显著降低(P0.05,P0.01)。结论:miR-21在TGF-β1诱导的HK-2细胞EMT发生中具有重要作用,并且可能通过靶基因PTEN参与EMT相关分子的表达调控。     

糖尿病大鼠血糖控制前后肾小管上皮细胞VEGF、TGF-β1及Smad蛋白表达的动态研究

目的动态观察链脲佐菌素（STZ）诱导的糖尿病大鼠血糖控制前后肾小管上皮细胞（TEC）中血管内皮生长因子（VEGF）、转化生长因子β1（TGF-β1）、Smad2/3、Smad4的表达情况,探讨四者在糖尿病大鼠TEC表型转变和肾间质纤维化中可能发挥的作用及相互关系。方法实验动物随机分为5组,依病程长短分为①A组（2周组）,②B组（4周组）,③C组（8周组）,④D组（16周组）,⑤E组（24周组）,每组分别设有正常对照组（N组）和糖尿病组（a组）;另外,16周、24周两组加设胰岛素治疗组（b组）。采用尾静脉注射STZ法复制糖尿病大鼠模型;免疫组织化学方法检测肾小管VEGF、TGF-β1、Smad2/3、Smad4及α-平滑肌肌动蛋白（-αSMA）和纤连蛋白（FN）的表达;Western blot检测肾皮质VEGF和TGF-β1蛋白;PAS染色光镜观察肾小管基底膜变化及细胞外基质沉积情况等形态学改变;生化方法测定血糖、血肌酐及24小时尿蛋白量。结果正常对照组VEGF、TGF-β1及Smad2/3、Smad4在肾小管均有少量表达,-αSMA在肾小管无表达;糖尿病组肾小管前述四者的表达均显著高于正常对照组,且从16周开始肾小管上皮细胞可见α-SMA蛋白阳性表达;糖尿病16周时肾小管VEGF、TGF-β1、Smad2/3、Smad4两两之间呈正相关;随糖尿病进展,α-SMA及FN在肾小管表达增多,24h尿蛋白增多,肾脏肥大指数增大,而VEGF、TGF-β1二者都分别和-αSMA、FN、24h尿蛋白及肾脏肥大指数呈正相关性;胰岛素治疗后,VEGF、TGF-β1、Smad2/3、Smad4及FN的表达都比糖尿病组明显下降,且各指标之间的正相关性依然存在,-αSMA蛋白则呈阴性表达。结论糖尿病肾病大鼠肾小管上皮细胞表达的VEGF、TGF-β1及Smad2/3、Smad4参与了TEC表型转变和肾间质纤维化的发生,并且VEGF和TGF-β1相互作用,共同促进了肾脏损害。胰岛素对DN大鼠TEMT和肾间质纤维化的影响可能部分是通过间接阻断VEGF、TGF-β1和Smad2/3、Smad4在TEC中的合成来实现的。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.