含蛋白转导域的SARA/SBD融合蛋白对腹膜透析大鼠腹膜结构和功能的作用

目的:探讨含蛋白转导域的SARA融合蛋白(PTD-SAR/SBD)对腹膜透析大鼠腹膜纤维化的抑制作用。方法:每日腹腔注射4.25%葡萄糖腹膜透析液(PDF)制备腹膜透析大鼠模型。28只Sprague-Dawley大鼠随机分为正常对照组(n=8);腹膜透析模型组(n=10);PTD-SARA/SBD蛋白干预组(n=10)。4周后行腹膜平衡试验,检测超滤量、葡萄糖吸收率;留取壁层腹膜组织行HE染色;免疫组织化学方法检测大鼠壁层腹膜间皮细胞转分化指标E-cadherin和Twist的表达;Western blotting测定E-cadherin、twist,以及CollagenⅠ、TGF-β1、P-Smad3、t-Smad3的表达。结果:①与正常对照组比较,模型组大鼠壁层腹膜增厚,糖转运率增加,超滤量降低(P0.01);免疫组织化学与western blotting检测结果显示E-cadherin表达下调,Twist表达上调;CollagenⅠ、P-Smad3、TGF-β1表达增加。②与模型组比较,PTD-SARA/SBD蛋白干预组大鼠壁层腹膜糖转运率降低,超滤量增加(P0.05);E-cadherin表达上调,Twist表达下调;CollagenⅠ、TGF-β1、P-Smad3表达减少,各组t-Smad3无明显变化。结论:PTD-SARA/SBD融合蛋白通过抑制TGF-β/Smads信号通路部分逆转腹膜间皮细胞转分化从而改善腹膜结构和功能,为防治腹膜透析所致腹膜纤维化奠定基础。     

当归黄芪提取物对慢性腹膜功能衰竭大鼠腹膜功能、结构及TGF-β1表达的影响

目的:观察当归黄芪提取物(EAA)对大鼠腹膜功能、结构及转化生长因子β1(TGF-β_1)表达的影响。方法:50只SD大鼠随机分为正常对照组、模型对照组、阳性对照组、EAA高、低剂量组(n=10)。除正常对照组外,其余4组大鼠每天腹腔注射4.25%高糖腹透液100 ml/kg,连续40 d,并在第8天、10天、12天腹腔注射大肠杆菌脂多糖(LPS)5 mg/kg,建立大鼠慢性腹膜功能衰竭模型,造模的同时灌胃给予相应药物。通过称大鼠体重和观察活动状态,测定超滤量、腹膜转运功能参数、壁层腹膜厚度、壁层腹膜TGF-β1的表达,研究EAA对大鼠腹膜功能、结构的保护作用及TGF-β1表达的影响。结果:EAA能升高高糖腹膜透析液联合LPS诱发的腹膜功能衰竭大鼠超滤量和腹膜转运功能参数;使大鼠腹膜增厚程度降低,TGF-β1表达减少。结论:EAA对大鼠腹膜功能、结构具有一定的保护作用且能抑制TGF-β_1表达。     

含蛋白转导域的SARA/SBD融合蛋白的原核表达、纯化及生物学活性鉴定

目的:SARA/SBD是纤维化形成过程中的负性调节因子。原核表达、纯化含反式激活蛋白(TAT)蛋白转导域(PTD)的TAT PTD-SARA/SBD融合蛋白,并鉴定其生物学活性。方法:将TAT PTD-SARA/SBD基因克隆入带His标签的原核表达载体pET-44a(+)中,转化大肠杆菌BL21,IPTG诱导表达,表达产物经Ni2+-NTA亲和层析柱纯化后,SDS-PAGE和Western印迹鉴定目的蛋白;用人腹膜间皮细胞系(HPMC),通过免疫细胞化学方法检测其穿膜能力,及与TGF-β1信号通路中Smad2因子的共定位情况。结果:用基因工程方法表达和纯化了TAT PTD-SARA/SBD融合蛋白,目的蛋白约占菌体总蛋白的20%左右,且以可溶形式表达,经Ni2+-NTA纯化后,所获蛋白纯度高于95%(HPLC归一法);功能学实验结果显示该蛋白能穿过胞膜,主要定位于胞核,且与Smad2因子具有核内共定位。结论:表达了TAT PTD-SARA/SBD融合蛋白,该蛋白具有生物学活性。     

高表达trip-1蛋白对大鼠肾小管上皮细胞转分化的影响

目的:研究上调大鼠肾小管上皮细胞中trip-1蛋白的表达量对TGF-β1诱导的上皮细胞转分化的影响.方法:包装人TRIP-1基因重组腺病毒,用其感染NRK52E细胞36h上调内源性trip-1蛋白表达量,之后用10 ng/ml的TGF-β1对细胞进行刺激诱导,72h后做Western Blot检测细胞中E-cadherin蛋白和α-SMA蛋白表达量.结果:①包装的人TRIP-1基因重组腺病毒感染细胞能够有效上调细胞中trip-1蛋白的表达量.②上调细胞中trip-1蛋白表达量,对TGF-β1引起的NRK52E细胞中E-cadherin蛋白表达水平降低有所抑制,但对TGF-β1引起的NRK52E细胞中α-SMA蛋白表达水平升高没有明显调节作用.结论:上调大鼠肾小管上皮细胞中trip-1蛋白的表达量在一定程度上抑制了TGF-β1诱导的NRK52E细胞转分化.     

大黄素对TGF-β1诱导的肾小管上皮细胞间质转分化的影响

目的:探讨大黄素对TGF-β1诱导的人肾小管上皮细胞(HK-2)间质转分化的影响。方法:不同浓度大黄素分别作用于TGF-β1诱导HK-2细胞24 h和48 h,通过细胞增殖实验确定最佳大黄素最佳给药浓度。TGF-β1诱导HK-2细胞24 h后收集细胞用于免疫印迹Western blot和实时荧光定量PCR(RT-PCR)分析。Western印迹法分别检测纤维化相关蛋白Collagen IV的表达,和肾小管上皮细胞向间充质细胞转分化关键蛋白α-SMA和E-Cadherin的表达;RT-PCR法检测肾小管上皮细胞向间充质细胞转分化关键蛋白α-SMA的表达。结果:由细胞增殖实验结果表明40μM大黄素是最佳给药浓度。Western结果表明,与模型组相比,大黄素组下调纤维化相关蛋白Collagen IV的表达,大黄素组与模型组蛋白差异有统计学意义(P0.05)。与模型组相比,大黄素组下调α-SMA蛋白表达水平,而上调E-Cadherin蛋白表达,差异有统计学意义(P0.05)。RT-PCR结果表明,与模型组相比,大黄素组降低α-SMA mRNA的含量,大黄素组与模型组α-SMA mRNA含量差异有统计学意义(P0.05)。结论:大黄素可通过抑制TGF-β1诱导的HK-2细胞间质转分化,从而发挥延缓肾间质纤维化的过程。     

青蒿琥酯基于TGF-β/Smad通路减轻2型糖尿病大鼠脾脏纤维化的机制研究CSCD

探讨青蒿琥酯(artesunate,ART)对2型糖尿病(type 2 diabetes mellitus,T2DM)大鼠脾脏纤维化的防治作用,并明确其相关分子机制,为T2DM脾脏损伤防治提供实验依据。50只SD实验大鼠分为正常组和模型组,模型组高糖高脂喂养8周后,注射链脲佐菌素构建T2DM模型,再随机分为糖尿病组、二甲双胍治疗组、青蒿琥酯治疗组、联合治疗组,连续用药干预4周后取大鼠脾脏,称脾脏重量;HE、Masson染色观察脾脏病理变化;免疫组织化学法和Western blot检测脾脏组织中转化生长因子β1(transforming growth factor-β,TGF-β1)、重组人蛋白2(smad family member 2,Smad2)、重组人蛋白7(smad family member 7,Smad7)、Ⅰ型胶原(CollagenⅠ)表达;qRT-PCR检测脾脏组织中TGF-β1、Smad2、Smad7、CollagenⅠ的mRNA表达量。与正常组比较,糖尿病组大鼠脾脏指数下降,脾脏细胞排列紊乱,大量纤维化增生。与糖尿病组比较,各用药干预组的脾脏损伤和纤维化减轻,脾脏组织中TGF-β1、Smad2、CollagenⅠ蛋白和mRNA含量显著降低、Smad7明显上升。以上结果说明ART能够减轻T2DM大鼠脾脏损伤,抑制脾脏纤维化增生,其机制可能与抑制TGF-β/Smad信号通路有关。     

血管平滑肌细胞中ERK通路与TGF-β_1/Smad通路的相互作用

目的:探讨血管平滑肌细胞(VSMC)中TGF-β/Smad与ERK信号转导通路是否存在相互调节关系。方法:原代培养的大鼠胸主动脉平滑肌细胞,分四组:①对照组,②TGF-β1组,③ERK阻断剂(PD98059)组和④TGF-β1+ERK阻断剂(PD98059)组。分别用Western blot法检测VSMC内Smad2/3、ERK1/2蛋白表达及磷酸化Smad2/3、磷酸化ERK1/2蛋白含量,RT-PCR方法测VSMC中Smad2、Smad3mRNA的表达。结果:①与对照组相比,TGF-β1组P-Smad2/3、P-ERK1/2蛋白含量增多(P0.05),ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量减少(P0.05),TGF-β1+ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量无差异;与TGF-β1组相比,TGF-β1+ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量减少(P0.05)。各组间Smad2/3、ERK1/2蛋白表达无差异。②各组的Smad2、Smad3mRNA表达无差异。结论:TGF-β1诱导的Smad2/3蛋白磷酸化依赖ERK通路激活,但ERK通路对Smad2/3蛋白和mRNA表达水平无影响。     

多配体蛋白多糖-1在慢性阻塞性肺疾病大鼠肺上皮间质转化中的作用及机制*

目的:探讨多配体蛋白多糖-1(syndecan-1)在慢性阻塞性肺疾病(COPD)大鼠肺上皮间质转化(EMT)中的作用及机制。方法:选择清洁级SD雄性大鼠30只,随机分为假手术组(暴露气管后注射生理盐水,n=10),COPD组(注射1 mg/ml脂多糖后进行烟熏,造模完成后转染100 μl空载病毒,n=10),syndecan-1过表达组(注射1 mg/ml脂多糖后进行烟熏。造模完成后转染100 μl携带大鼠syndecan-1基因的重组腺病毒载体Ad-CMV-GFP-SDC1,n=10),每天1次,连续处理2周。处理结束后检测各组大鼠肺功能并取肺组织;采用HE染色法观察肺组织损伤情况;采用免疫组化检测各组大鼠肺组织syndecan-1、波形蛋白(vimentin)、E-钙黏蛋白(E-cadherin)的蛋白表达;采用Western blot检测转化生长因子-β1(TGF-β1)、Smad2/3和p-Smad2/3蛋白表达水平;采用qRT-PCR法检测各组大鼠肺组织中vimentin、E-cadherin、TGF-β1和Smad2/3 mRNA表达水平。结果:与假手术组相比,COPD组大鼠气道黏膜脱落,管腔狭窄,气道壁较多炎性细胞浸润,肺气肿严重,每分钟呼气量(V_E)、最大呼气流量(PEF)和0.3s用力呼气容积(FEV_0.3)和肺组织E-cadherin mRNA表达水平均显著降低(P＜0.05),而vimentin、TGF-β1、Smad2/3 mRNA表达水平及TGF-β1、Smad2/3和p-Smad2/3的蛋白表达水平均显著升高(P＜0.05)。与COPD组相比,syndecan-1过表达组气道黏膜脱落及气道壁炎性细胞浸润数减少,管腔狭窄、肺气肿情况均有所改善,V_E、PEF、FEV_0.3和肺组织E-cadherin mRNA表达水平显著升高(P＜0.05),而vimentin、TGF-β1、Smad2/3 mRNA表达水平及TGF-β1、Smad2/3和p-Smad2/3的蛋白表达水平均显著降低(P＜0.05)。结论:COPD大鼠体内TGF-β/Smad信号通路活化且存在肺EMT;过表达syndecan-1可抑制TGF-β/Smad信号通路,减轻EMT,改善COPD大鼠肺组织损伤。     

大黄素靶向Notch通路抗腹膜透析大鼠腹膜纤维化

长期腹膜透析治疗经常并发腹膜纤维化,本文旨在探讨大黄素对腹膜透析相关性腹膜纤维化大鼠的治疗作用及其相关的细胞、分子机制。在体内实验中,以腹腔注射4.25%葡萄糖腹膜透析液(100 m L/kg)诱发大鼠腹膜透析相关性腹膜纤维化动物模型,于2周、4周、6周时进行模型大鼠腹膜平衡实验,并测量腹膜厚度、应用HE染色和Masson染色进行组织病理学评估,ELISA法测定血浆中III型前胶原氨基端肽(PIIINP)水平。选取6周模型大鼠,用大黄素腹腔注射进行治疗,观察治疗后上述指标的变化,同时以Real-time PCR方法检测大鼠腹膜Notch1、Jagged-1、Hes-1基因表达水平,Western blot方法检测大鼠腹膜组织Notch1、Jagged-1、Hes-1、Notch胞内结构域(Notch intracellular domain,NICD)蛋白表达。在体外实验中,培养大鼠腹膜间皮细胞,使其过表达或沉默Notch1,Western blot检测大黄素对腹膜间皮细胞Hes-1、Hey的诱导表达作用。HE染色显示:随着造模时间的延长,间皮细胞减少、部分脱落,间质有炎细胞浸润,6周时Masson染色显示模型组腹膜明显增厚(P0.01),可见大量胶原沉积。与对照组相比,6周模型组大鼠血浆PIIINP水平显著升高(P0.01)。与6周模型组相比,大黄素处理可增加模型大鼠腹膜组织中间皮细胞数量,抑制腹膜增厚(P0.01),改善胶原沉积,降低血浆PIIINP水平(P0.05),并在基因、蛋白水平下调Notch1、Jagged-1、Hes-1的表达(P0.05或P0.01),且下调NICD蛋白水平(P0.01);体外实验显示:与正常细胞大黄素处理组相比,大黄素对过表达Notch1间皮细胞的Hes-1、Hey表达具有抑制作用(P0.05),而对Notch1沉默间皮细胞的Hes-1、Hey表达无明显影响(P0.05)。以上结果表明,在高浓度葡萄糖腹膜透析液所致的大鼠腹膜纤维化模型中有Notch通路的活化,大黄素可能通过抑制Notch通路改善腹膜纤维化程度。     

自制腹透管腹膜透析相关性大肠埃希菌腹膜炎大鼠模型的建立及腹膜AQP1表达研究

目的通过自制腹透管建立一个便利的腹膜透析相关性腹膜炎大鼠模型,研究水孔通道蛋白1 (aquaporin 1,AQP1)在腹膜炎大鼠腹膜表达情况。方法将60只SD大鼠分为3组,第1组为空白组;第2组为穿刺组,以传统方式直接向大鼠腹腔注射腹膜透析液及大肠杆菌菌液;第3组为置管组,采用医用18G静脉留置针,打侧孔消毒灭菌后,从左下腹迷路穿刺置入腹腔后拔出穿刺针芯,贴无菌膜后固定,消毒留置针硅胶帽后注入大肠杆菌,建立腹膜透析相关性大肠埃希菌腹膜炎大鼠模型。建模后饲养大鼠72 h处死,取大鼠壁层腹膜行病理切片观察腹膜炎细胞浸润情况,同时采用免疫组化法检测AQP1在大鼠壁层腹膜的表达情况。结果穿刺组大鼠存活率为50%,置管组大鼠存活率为85%,两组大鼠的存活率比较差异有统计学意义(P0.05),且两组大鼠壁层腹膜病理切片光镜下均观察到大量炎细胞浸润。空白组、置管组的AQP1表达均明显低于穿刺组,差异均有统计学意义(均P0.05)。结论医用留置针置入大鼠腹腔可建立腹膜透析相关性腹膜炎大鼠模型;AQP1在腹膜透析相关性腹膜炎大鼠的壁层腹膜中表达上调,与大鼠的腹膜炎病情变化程度密切相关。     

Expression, purification, and functional characterization of recombinant PTD-SARA

The Smad anchor for receptor activation (SARA) protein is a binding partner for Smad2/3 that plays an important role in the fibrotic promoting signaling pathway initiated by transforming growth factor-β1 (TGF-β1). The C-terminal 665-750 aa of SARA comprises the Smad-binding domain (SBD). By direct interaction through the SBD, SARA inhibits Smad2/3 phosphorylation and blocks the interaction between Smad2/3 and Smad4, thereby restrains the process of fibrosis. In this study, we constructed a SARA peptide aptamer based on the SBD sequence. The recombinant SARA aptamer, fused with a protein transduction domain (PTD-SARA), was cloned, purified from E. coli, and characterized for the first time. The full-length PTD-SARA coding sequence, created with E. coli favored codons, was cloned into a pQE-30 vector, and the recombinant plasmid was transformed into an M15 strain. After Isopropyl β-D-1-Thiogalactopyranoside (IPTG) induction and Ni(2+) affinity purification, recombinant PTD-SARA was further identified by immunoblotting and protein N-terminal sequencing. Epifluorescence microscopy revealed that the recombinant PTD-SARA was transferred into the cytoplasm and nucleus more efficiently than SARA. Moreover, the recombinant PTD-SARA was found to up-regulate the level of E-cadherin and down-regulate the levels of α-SMA and phospho-Smad3 more efficiently than SARA (P < 0.05). Our work explored a method to obtain recombinant PTD-SARA protein. The recombinant PTD-SARA fusion protein could enter HK2 cells (an immortalized proximal tubule epithelial cell line) more efficiently than the SARA protein and reverse the renal epithelial-to-mesenchymal transdifferentiation process that was induced by TGF-β1 more effectively than the SARA protein. Recombinant PDT-SARA is likely to be a potential candidate for clinical prevention and treatment of renal fibrosis.     

尿毒症皮肤瘙痒患者采取腹膜透析与血液透析治疗的效果探究

目的：探究尿毒症皮肤瘙瘁患者采取腹膜透析与血液透析治疗的临床效果,并为该病的临床治疗提供经验积累。方法：选取我院肾内科于2005年1月-2012年12月收治的52例尿毒症皮肤瘙痒患者,利用随机数字表法进行分组,分别设为研究组和对照组。其中对照组实施血液透析治疗,而研究组开展腹膜透析治疗。记录两组在治疗前和治疗后第8周末血生化客观指标及皮肤瘙痒程度,并做好对比。结果：两组在治疗前的皮肤瘙痒评分、β2-MG、PTH、p3,BUN、SCr值差异无统计学意义（P〉0．05）;治疗后,研究组皮肤瘙瘁评分为（3．4±0．8）分,对照组为（5．8±0．9）分,差异有统计学意义（P〈0．05）。治疗后,研究组β2-MG、PTH及Ps．均低于对照组,差异有统计学意义（P〈0．05）。结论：尿毒症皮肤瘙痒患者的主要致敏因子为大中分子,采取腹膜透析能够有效清除大分子物质,进而达到瘙痒程度的有效改善。     

Calcitriol decreases TGF-β1 and angiotensin II production and protects against chlorhexide digluconate-induced liver peritoneal fibrosis in rats

Peritoneal fibrosis is a major complication of peritoneal dialysis that can lead to ultrafiltration failure. This study investigates the protective effects of calcitriol on chlorhexidine digluconate-induced peritoneal fibrosis in rats. Peritoneal fibrosis was induced in Sprague-Dawley rats by daily administration of 0.5 mL 0.1% chlorhexidine digluconate in normal saline via peritoneal dialysis for 1 week. Rats received daily intravenous injections of calcitriol (low-dose, 10 ng/kg; or high-dose, 100 ng/kg) for 1 week. After 7 days, conventional 4.25% Dianeal (30 mL) was administered via peritoneal dialysis over 4 h. Peritoneal solute transport was calculated from the dialysate concentration relative to its concentration in the initial infused dialysis solution (D₄/D_{0 glucose}) for glucose, and the dialysate-to-plasma concentration ratio (D₄/P_{4 urea}) at 4 h for urea. Rats were then sacrificed and the liver peritoneum was harvested for immunohistochemical analysis via microscopy. After dialysis, the D₄/P_{4 Urea} level was reduced; increases were observed in the D₄/D_{0 glucose} level and the levels of active transforming growth factor-β1 and angiotensin II in serum and dialysate; the liver peritoneum and muscle peritoneum was markedly thickened, and the expression of α-SMA, fibronectin, collagen, vascular endothelial growth factor, angiotensin II, transforming growth factor-β1, and phosphorylated Smad2/3 (P-Smad2/3)-positive cells in the liver peritoneum was elevated in the peritoneal fibrosis group compared with the vehicle group. Calcitriol decreased the serum and dialysate active transforming growth factor-β1 and angiotensin II level, decreased the thickness of the liver peritoneum and muscle peritoneum, and decreased the expression of α-SMA, fibronectin, collagen, vascular endothelial growth factor, angiotensin II, transforming growth factor-β1, and P-Smad2/3-positive cells in liver peritoneum cells. High-dose calcitriol exhibited better protective effects against peritoneal fibrosis than did the lower dose. Calcitriol protected against chlorhexidine digluconate-induced peritoneal fibrosis in rats by decreasing transforming growth factor-β1 and angiotensin II production.     

Beneficial effects of enalapril on chlorhexidine digluconate-induced liver peritoneal fibrosis in rats

Peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The present study was designed to investigate the protective effects of enalapril on chlorhexidine digluconate-induced liver PF by decreasing transforming growth factor-β1 (TGF-β1) production in rats. PF was induced in Sprague-Dawley rats by daily administration of 0.5 ml 0.1% chlorhexidine digluconate in normal saline via PD tube for one week. Rats received daily intravenous injections of low dose enalapril (1 mg/kg), or high dose enalapril (2.5 mg/kg), for one week. After 7 days, conventional 4.25% Dianeal (30 ml) was administered via a PD catheter with a dwell time of 4 h and assessment of peritoneal function. At the end of dialysis, the rats were sacrificed and liver peritoneum was harvested for microscopic examination and immunohistochemistry. There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D?/P?(urea) level was reduced, the D?/D? glucose level, serum and the dialysate TGF-β1 level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, collagen and vascular endothelial growth factor (VEGF) were elevated in the PF group compared with the vehicle group. High dose of enalapril decreased the serum and dialysate TGF-β1 levels, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen and VEGF-positive cells in the liver peritoneum. Low dose of enalapril did not protect against chlorhexidine digluconate-induced PF in the rat. Enalapril protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-β1 production.     

HSC的活化、增殖与TGF-β1及其Ⅰ型受体在中晚期纤维化肝组织中的改变及护肝片对其的影响

目的 探讨护肝片对中、晚期纤维化大鼠肝组织肝星状细胞（HSC）的活化与增殖及转化生长因子-β1（TGF-β1）及其工型受体（TβRⅠ）表达的影响。方法 采用12．5％CCh诱导的大鼠肝纤维化模型,自造模之日起,大鼠分组灌胃给药（护肝片921mg·kg^-1）或溶媒,每日一次,直至8或13周末,分别处死动物,取左叶肝组织石蜡包埋,制作组织芯片,免疫组化S-P法检测大鼠肝组织α-平滑肌肌动蛋白（α-SMA）、TGF-β1及TβRⅠ蛋白的表达,并用Meta Morph图像分析系统计数α-SMA阳性细胞数、对TGF-β1。及TβRⅠ蛋白表达量进行定量分析。结果 1．模型复制8周和13周,模型组的肝损伤及其纤维化分级均明显高于正常组（P〈0．01）,护肝片组的肝损伤及其纤维化分级均轻于模型组。2．模型复制8周和13周,模型组活化的HSC（即α-SMA阳性细胞）数量较正常组明显增多、TGF-β1及TβRⅠ蛋白的表达较正常组明显增强（P〈0．01）;3．护肝片显著抑制8、13周纤维化肝组织HSC的活化与增殖和TGF-β1及TβRⅠ蛋白的表达（P〈0．01）。结论 抑制HSC的活化与增殖和TGF-β1及TβRⅠ的表达可能是护肝片抗肝纤维化作用的靶点之一。     

肝组织中NF-κB、TGF-β1及其Ⅰ型受体mRNA和HSC在肝纤维化中的改变及护肝片对其的影响

目的探讨中、晚期纤维化大鼠肝组织中肝星状细胞(HSC)的活化与增殖、核转录因子-κB(NF-κB)及转化生长因子-β1(TGF-β1)及其Ⅰ型受体(TβRⅠ)表达的改变及护肝片对其的影响。方法采用12.5%CCl4诱导的大鼠肝纤维化模型,自造模之日起,大鼠分组灌胃给药(护肝片921mg/kg)或溶媒,每日一次,直至8或13周末,分别处死动物,取左叶肝组织石蜡包埋,制作组织芯片。免疫组化S-P法检测大鼠肝组织α-平滑肌肌动蛋白(-αSMA)和NF-κB p65蛋白的表达,原位杂交检测TGF-β1及TβRⅠmRNA的表达;并用MetaMorph图像分析系统计数-αSMA阳性细胞数,对NF-κB p65蛋白、TGF-β1及TβRⅠmRNA的表达量进行定量分析。结果 1.模型复制8周和13周,模型组的肝损伤及其纤维化分级均明显高于正常组(P<0.01),护肝片组的肝损伤及其纤维化分级均轻于模型组。2.模型复制8周和13周,模型组活化的HSC(即-αSMA阳性细胞)数量较正常组明显增多,NF-κB p65蛋白、TGF-β1及TβRⅠmRNA的表达均较正常组明显增强(P<0.01);3.护肝片显著抑制8、13周纤维化肝组织HSC的活化与增殖和NF-κB p65蛋白、TGF-β1及TβRⅠmRNA的表达(P<0.01)。结论抑制HSC的活化与增殖和NF-κB p65蛋白与TGF-β1及TβRⅠmRNA的表达可能是护肝片抗肝纤维化作用的靶点之一。     

糖尿病肾病腹膜透析血管内皮功能变化及内皮功能不全的相关因素探讨

目的：探讨分析糖尿病肾病腹膜透析患者血管内皮功能的变化。方法：将来我院行腹膜透析的糖尿病肾病患者56例作为观察组,非糖尿病肾病患者64例作为对照组,测量血压及血容量,采用血流介导的肱动脉扩张法测定血流介导的血管扩张。结果：观察组患者的收缩压、脉压、细胞外液均明显高于对照组,血管扩张则显著低于对照组（P〈0．05）;血管扩张与身高、体重、收缩压、细胞外液均呈负相关（P〈0．05）;细胞外液、有无糖尿病肾病是血管扩张的独立预测因素。结论：糖尿病肾病腹膜透析患者具有严重的内皮功能不全,其中细胞外液、有无糖尿病肾病是血管扩张的独立预测因素。     

松果菊苷对db/db糖尿病小鼠心肌的保护作用

糖尿病心肌病（diabetic cardiomyopathy, DCM）是指发生于糖尿病患者,不能用冠心病、高血压性心脏病及其他心脏病变来解释的心肌疾病。目前,DCM的病因和发病机制尚未完全阐明,且缺乏特异性治疗手段。中药管花肉苁蓉提取物松果菊苷（echinacoside, ECH）对心肌细胞具有保护作用。以db/m小鼠为正常对照组（db/m组）,db/db小鼠分为模型组（db/db组）和ECH干预组（db/db+ECH组）,探讨了ECH对糖尿病db/db小鼠心肌的影响及机制。db/db+ECH组小鼠给予松果菊苷灌胃,db/m组和db/db组小鼠给予0.9%氯化钠溶液灌胃。心脏超声观察心脏功能,Masson染色观察组织胶原纤维含量,逆转录聚合酶链式反应检测Ⅰ型胶原和Ⅲ型胶原mRNA的表达,蛋白质免疫印迹技术检测转化生长因子-β1（transforming growth factor-β1, TGF-β1）、phospho-Smad2（p-Smad2）和phospho-Smad3（p-Smad3）的表达。结果显示,ECH能够改善db/db小鼠左心室肥大和心脏功能,降低胶原沉积（P<0.05）。ECH能够降低Ⅰ型和Ⅲ型胶原mRNA的表达（P<0.01）,下调TGF-β1、p-Smad2和p-Smad3蛋白的表达（P<0.05）。ECH对糖尿病心肌的保护作用可能与负反馈调节TGF-β1/Smads信号通路相关,研究结果为DCM的早期干预提供了新思路。     

Valsartan decreases TGF-β1 production and protects against chlorhexidine digluconate-induced liver peritoneal fibrosis in rats

Peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The present study was designed to investigate the protective effects of valsartan on chlorhexidine digluconate-induced PF by decreasing TGF-β1 production in rats. PF was induced in Sprague–Dawley rats by daily administration of 0.5 ml 0.1% chlorhexidine digluconate in normal saline via peritoneal dialysis (PD) tube for 1 week. Rats received daily intravenous injections of low dose valsartan (1 mg/kg) or high dose valsartan (3 mg/kg) for 1 week. After 7 days, conventional 4.25% Dianeal (30 ml) was administered via a PD catheter with a dwell time of 4 h and assessed of peritoneal function. At the end of dialysis, rats were sacrificed and the liver peritoneum was harvested for microscopically and immunohistochemistry. There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D₄/P_4Urea level was reduced, the D₄/D₀ glucose level, serum and dialysate transforming growth factor-β1 (TGF-β1) level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, collagen, and vascular endothelial growth factor (VEGF) were elevated in the PF group compared with the vehicle group. High dose of valsartan decreased the serum and dialysate TGF-β1 level, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen, and VEGF-positive cells in liver peritoneum. The low dose of valsartan did not protect against chlorhexidine digluconate-induced PF in rat. Valsartan protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-β1 production.     

转化生长因子β1质粒对新鲜同种异体骨移植的免疫影响

目的：研究局部含注射转化生长因子β1（TGF-β1）基因的质粒时大鼠同种异体骨移植的免疫排斥反应的影响。方法：将64只Wister大鼠完全随机分成4组,每组16只,切取SD大鼠的胫骨移植到Wister大鼠人工建模后形成的胫骨缺损区。TGF-β1组于移植骨局部一次性注射含有TGF-β1基因的质粒（40μg／只）;空质粒组于移植局部注射空质粒;免疫抑制剂组术前3天开始腹腔内注射环孢素A（10mg／kg）直至处死;异体移植组仅行异体骨移植,不予特殊处理。术后3、6、12周行光镜和电镜检查。结果：异体移植组和空质粒组骨基质排列紊乱,骨细胞消失,基质区内见块状低电子密度区。TGF-β1组和免疫抑制剂组可见骨细胞和完整的成骨细胞,骨小管状态良好。混合淋巴细胞培养（MLC）结果显示TGF-β1组（0-331±0．017）与免疫抑制剂组（0．501±0．004）差异无统计学意义（P〉0．05）,TGF-β1组与空质粒组（1．104±0．023）和异体移植组（1．206±0．019）差异有统计学意义（P〈0．05）。结论：新鲜骨移植过程中局部注射TGF-β1质粒可发挥免疫抑制作用,降低宿主的免疫排斥反应：