雌二醇对肝纤维化大鼠Ⅰ,Ⅲ型胶原及TGFβ1表达的影响

目的:观察雌二醇对肝纤维化大鼠肝脏胶原沉积和转化生长因子β_1(TGF β_1)表达的影响,研究雌激素对肝纤维化形成的抑制作用,并探讨其可能的机制。方法:设立模型组、治疗对照组、雌二醇组和正常对照组,以四氯化碳复合因素诱导大鼠肝纤维化动物模型,雌二醇组在四氯化碳应用的同时皮下注射苯甲酸雌二醇1mg/kg,2次/wk,共8wk。大鼠肝脏HE染色与Masson染色,分级观察肝组织的炎性坏死与胶原纤维沉积变化,并观察对大鼠肝纤维化形成过程中肝脏表达Ⅰ,Ⅲ型胶原蛋白及对TGF β_1的影响。结果:与正常对照组比较,四氯化碳模型大鼠出现典型的肝纤维化表现,肝脏胶原纤维间隔广泛形成,肝小叶与肝窦内胶原增生沉积明显,Ⅰ,Ⅲ型胶原(0.58±0.26vs 6.34±2.24,1.07±0.49 vs 5.28±1.28,P值均<0.001)及TGF β_1基因表达明显增多;雌二醇应用可以明显减轻肝脏内胶原纤维增生沉积P<0.05),抑制肝脏Ⅰ、Ⅲ型胶原蛋白(2.47±0.76 vs 6.34±2.24,3.02±1.20 vs5.28±1.28,P值均<0.05)及TGF β_1的合成表达。结论:雌二醇可抑制肝纤维化大鼠肝脏Ⅰ,Ⅲ型胶原蛋白及TGF β_1的合成表达,发挥对肝纤维化的抑制作用。     

反义转化生长因子βⅡ型受体表达质粒对实验性肝纤维化的影响

目的 观察反义转化生长因子βⅡ型受作(TGF βRⅡ)表达质粒对实验性肝纤维化的影响。方法 运用重组DNA技术中构建反义TGF βRⅡ真核细胞表达质粒,采用猪血清腹腔注射制备免疫性大鼠肝纤维化模型,实验动物分为肝纤维化模型组、反义TGF βRⅡ治疗组、pCDNA3对照组及正常对照组。反义TGF βRⅡ质粒和pCDNA3空质粒与糖化多聚赖氨酸偶联后经尾静脉分别导入大鼠体内,通过northern blot、RT-PCR、Western blot检测外源导入质粒在肝组织中的表达,检测血清转化生长因子β1(TGFβ1)、肝组织羟脯氨酸测定,Ⅰ、Ⅲ型胶原免疫组织化学与Van Gieson染色观察反义TGF βRⅡ质粒对大鼠肝纤维化的影响。 结果 反义TGF βRⅡ表达质粒可在肝组织中获得确切表达,其表达可使反义治疗组血清TGF β1含量下降,反义TGF βRⅡ治疗组为(23.16 ± 3.13)ng/ml,模型组为(32.96±3.79)ng/ml,F=36.73,P<0.01。肝组织羟脯氦酸含量下降,治疗组为(0.17±0.01)mg/g,模型组为(0.30±0.03)mg/g,F=15.48,P<0.01。减少了肝组织Ⅰ、Ⅲ型胶原的沉积,治疗组Ⅰ型胶原为650.26±51.51,Ⅲ型胶原为661.5 8±55.28,模型组Ⅰ型胶原为1209.44±16.60,Ⅲ型胶原为1175.14±121.44,F值分圳为69.87、70.46,P<0.01。并促进反义治疗组肝脏病理形态一定程度的改善。     

排钱草总生物碱对免疫性肝纤维化大鼠Ⅰ、Ⅲ、Ⅳ型胶原及TGF-β1表达的影响

目的:观察排钱草总生物碱对免疫性肝纤维化大鼠肝脏胶原沉积和转化生长因子β1(TGF-β1)表达的影响,研究其对肝纤维化形成的抑制作用,并探讨可能的作用机制.方法:设立模型对照组、药物对照组、排钱草总生物碱高、中、低剂量组和正常对照组,以腹腔注射猪血清方法诱导大鼠肝纤维化动物模型.排钱草总生物碱高、中、低剂量组大鼠在注射猪血清的同时分别灌服排钱草总生物碱30mg/kg,20mg/kg,10mg/kg,1次/d,共8周.大鼠肝脏HE染色,观察各组大鼠肝组织的炎性坏死与胶原纤维沉积变化;免疫组化观察大鼠肝纤维化形成过程中腓钱草总生物碱对肝脏表达Ⅰ、Ⅲ、Ⅳ型胶原蛋白及TGF-β1的影响.结果:与正常对照组比较,模型组大鼠肝脏出现典型的肝纤维化表现,肝脏胶原纤维间隔广泛形成,肝小叶与肝窦内胶原增生沉积明显,Ⅰ,Ⅲ,Ⅳ型胶原(1.28±2.59 vs 13.82±6.55,1.00±1.22 vs 14.69±7.16,1.03±1.46 vs 12.44±6.89, P值均＜0.001)及TGF-β1表达明显增多.高、中、低剂量排钱草总生物碱均可以明显减轻大鼠肝脏内胶原纤维增生沉积(P＜0.05),抑制肝脏Ⅰ,Ⅲ,Ⅳ型胶原蛋白(P值均＜0.05)及TGF-β1的合成表达.结论:排钱草总生物碱通过抑制肝纤维化大鼠肝脏Ⅰ,Ⅲ,Ⅳ型胶原蛋白及TGF-β1的合成表达,起到良好地抗肝纤维化作用.     

SB203580对肝纤维化大鼠肝脏Ⅰ、Ⅲ型胶原蛋白表达的影响

目的:探讨SB203580对肝纤维化大鼠肝脏Ⅰ、Ⅲ型胶原蛋白表达的影响.方法:将32只♀SD大鼠分为4组:正常组(N组)8只、肝纤维化组(HF组)8只、DMSO溶剂干预组(D组)8只、P38MAPK抑制剂SB203580干预组(SB组)8只.采用四氯化碳复合因素法复制肝纤维化模型,肝纤维化模型制作结束后,D组给予2‰DMSO溶液3 mL/(kg?d),SB组给予SB203580溶液10 mg/(kg?d),N组、HF组给予同等剂量0.9%生理盐水3 mL/(kg?d)腹腔注射,连续注射4 d.干预结束后,宰杀大鼠留取肝脏,行肝组织病理切片HE染色评价肝纤维化分期,行Masson染色观察胶原纤维沉积情况,采用SP免疫组织化学法检测肝脏Ⅰ、Ⅲ型胶原蛋白表达,应用逆转录PCR法检测肝脏中Ⅰ、Ⅲ型胶原mRNA表达.结果:正常对照组(N组)、肝纤维化组(H F组)、S B203580溶剂组(D M S O组)和P38MAPK通路特异性抑制剂组(SB203580组)的肝纤维化分期平均秩分别为4.50、22.50、24.00和15.00;SSS评分分别为2.750±0.707、15.875±0.835、16.000±0.926和11.625±0.9 1 6;Ⅰ型胶原显色指数分别为1.5 7 5±0.249、7.650±0.621、7.725±0.501和4.625±0.495;Ⅲ型胶原显色指数分别为2.375±0.518、4.025±0.446、4.075±0.544和3.375±0.167;Ⅰ型胶原mRNA表达分别为0.020±0.003、0.012±0.002、0.009±0.002和0.016±0.005;Ⅲ型胶原mRNA表达分别为0.412±0.772、0.773±0.137、0.799±0.116和0.572±0.862.HF组与N组相比,Ⅰ、Ⅲ型胶原及其mRNA表达升高(均P<0.001),与肝纤维化分期结果一致(P<0.001);D组与HF组无明显差异(均P>0.05);SB组与HF组相比,Ⅰ、Ⅲ型胶原表达降低(Ⅰ型胶原显色指数P<0.001,Ⅲ型胶原显色指数P=0.041);Ⅰ型胶原mRNA表达降低(P=0.005),同时Ⅲ型胶原mRNA表达亦降低(P=0.005),肝纤维化分期下降(P=0.015).结论:P38MAPK抑制剂SB203580阻断P38MAPK通路具有降低肝脏Ⅰ、Ⅲ型胶原表达的作用,能够延缓肝纤维化的发生发展.     

肝纤维化几种病理染色方法及意义归纳

目的:归纳肝组织各种病理切片染色方法,探讨不同方法在大鼠肝纤维化病理诊断中的意义。方法:将实验大鼠分为正常组和模型组,每组各8只,模型组腹腔注射30%CCl4橄榄油溶液2. 0ml/kg,1次/4天,共7次,建立肝纤维化模型。取材肝组织制作病理切片,分别进行HE染色、弹力胶原纤维染色、网状纤维染色、Masson(马松)三色染色、天狼星红染色。结果:正常组大鼠肝组织各种染色均呈现正常形态。模型组大鼠肝脏切片HE染色示肝细胞变性、坏死,呈现大面积假小叶组织结构,属坏死后肝硬变;弹力胶原纤维染色示大量胶原纤维沉积,汇管区周围胶原纤维沉积,纤维条索较粗且染色着色较深,包裹,已形成假小叶;马松染色示大量蓝色胶原纤维沉积,自汇管区周围向外延伸,纤维条索较粗且染色着色较深,表明胶原纤维较多,已形成假小叶;网状纤维染色示网状纤维失去正常分布状态,自汇管区周围网状纤维塌陷、融合、增粗、包裹并已形成假小叶;天狼星红染色示各型胶原纤维自汇管区向外扩展,偏光镜下观察可见胶原沉积、扩展已形成假小叶,其中大量红色I型胶原纤维、绿色III型胶原,少量疏网状II型胶原纤维和淡黄色IV型胶原纤维。结论:HE染色可以观察细胞形态,弹力胶原纤维染色、网状纤维染色、Masson(马松)三色染色均能诊断肝纤维化,而天狼星红染色可以对肝纤维化组织中各型胶原纤维进行分型。     

护肝解纤汤治疗肝纤维化及其作用机制的初步研究

目的 观察护肝解纤汤治疗大鼠肝纤维化效果及有关细胞因子的变化,初步探讨护肝解纤汤的作用机制.方法 100只雄性SD大鼠随机分组后,以四氯化碳腹腔内注射的方法建立大鼠肝纤维化模型,造模后施以由低、中、高剂量姜黄等组成的护肝解纤汤,以小柴胡汤和复方鳖甲软肝片为阳性对照.12周后处死大鼠,留取肝左叶同一部位新鲜肝组织,行苏木素-伊红、Masson染色,免疫组织化学染色检测肝组织Ⅰ、Ⅲ型胶原,转化生长因子β1(TGF β1)、血小板衍生长因子(PDGF-BB)的变化.多组数据间的比较采用SPSS12.0统计软件进行单因素方差分析.结果 护肝解纤汤能降低病理评分,改善肝纤维化病理损伤,抑制Ⅰ、Ⅲ型胶原合成并促使其降解,降低炎症因子TGF β1、PDGF-BB在肝组织的表达,正常组、模型对照组、低姜黄组、中姜黄组、高姜黄组、小柴胡汤组、复方鳖甲组肝组织TGF阳性面积百分比分别为7.56%±2.18%、29.25%±7.84%、13.54%±4.15%、21.82%±6.64%、20.06%±7.14%、13.78%±4.35%、12.75%±3.98%,P<0.05;PDGF-BB阳性百分比分别为1.68%±0.41%、11.70%±2.28%、3.65%±0.76%、5.24%±1.04%、6.37%±1.12%、4.16%±0.61%、3.38%±0.56%,P<0.05.上述作用以低姜黄组效果明显.结论 护肝解纤汤可改善大鼠肝纤维化的病理改变,降低Ⅰ型胶原、Ⅲ型胶原合成、减少PDGF-BB,TGF β1表达可能是其相关作用机制.     

螺内酯对肝纤维化大鼠Ⅰ/Ⅲ型胶原蛋白表达的影响

目的 探讨螺内酯对实验性大鼠肝纤维化Ⅰ/Ⅲ型胶原蛋白表达的影响,初步揭示螺内酯对大鼠肝纤维化的预防作用及可能的机制.方法雄性SD大鼠81只,随机分为正常对照组(10只)、肝纤维化模型对照组(45只)及螺内酯组(26只).采用复合因素造模,螺内酯从造模开始每日1mL 100mg@kg-1灌胃,共7wk.免疫组化染色及图像分析检测Ⅰ/Ⅲ型胶原含量.结果螺内酯组与肝纤维化组胶原面积比Ⅰ型胶原分别为:2.84±0.86,5.41±2.08,Ⅲ型胶原分别为:2.95±0.82,5.35±2.30.螺内酯组Ⅰ/Ⅲ型胶原增生程度明显降低(P＜0.01).结论螺内酯可使肝组织Ⅰ/Ⅲ型胶原沉积明显减轻,提示螺内酯可明显预防肝纤维化的形成,ALD在肝纤维化的发病中有一定意义.     

大鼠肝星状细胞转化生长因子β3/β1 mRNA比值与Ⅰ型胶原合成的关系

转化生长因子β1(TGF β 1)是激活肝星状细胞(HSC)并促进其胶原合成的最重要因子.TGF β 3被认为具有抗组织纤维化的功能.Carrington等[1]研究提示:TGF β 1与TGF β 3的比值是纤维化发生程度的关键因素.本实验研究大鼠HSC中TGF β 3/TGF β 1 mRNA比值的变化及与Ⅰ型胶原合成的关系,阐明TGF β 3是拮抗TGF β 1的重要因子,为TGF β 3对肝纤维化的防治作用提供实验依据.     

蕲艾提取液对免疫性肝纤维化大鼠Ⅰ、Ⅲ型胶原及基质金属蛋白酶抑制因子-1表达的影响

目的:研究蕲艾提取液对免疫性肝纤维化大鼠Ⅰ、Ⅲ型胶原及TIMP-1表达的影响.方法:Wistar大鼠60只随机分为正常对照组、模型组和高、中、低剂量蕲艾提取液治疗组,采用异种血清腹腔内注射构建肝纤维化大鼠模型,肝组织切片HE染色后,光镜下观察大鼠肝组织纤维化程度;SABC型免疫组化染色法观察肝组织Ⅰ、Ⅲ型胶原及TIMP-1含量变化.结果:蕲艾提取液各治疗组与模型组相比,肝纤维化程度显著减轻;肝纤维化模型组及蕲艾提取液治疗组Ⅰ、Ⅲ型胶原含量及TIMP-1水平显著高于正常对照组;蕲艾提取液治疗组Ⅰ、Ⅲ型胶原及TIMP-1水平又显著低于肝纤维化模型组.结论:蕲艾提取液能够显著降低免疫性肝纤维化大鼠Ⅰ、Ⅲ型胶原及TIMP-1表达.     

β-榄香烯对实验性肝纤维化大鼠TGF-β1、α-SMA、Col-Ⅰ表达的影响

目的观察β-榄香烯对四氯化碳肝纤维化大鼠TGF-β1、α-SMA、Col-Ⅰ表达的影响.方法采用CCl4皮下注射诱导Wistar ♂大鼠肝纤维化模型,用β-榄香烯0.1 mL/100 g剂量每天腹腔注射8 wk后,用苏木精-伊红染色(HE)和胶原纤维(Masson)染色观察大鼠肝脏病理变化,酶动力法检测肝功能,SP免疫组化法检测肝组织中α-肌动蛋白(α-SMA)、转化生长因子β1(TGF-β1)、Ⅰ型胶原(Col-Ⅰ)表达的变化,样本碱水解法检测肝组织中羟脯氨酸(HYP)的含量.结果8 wk后,正常组、模型组、对照组及治疗组肝组织胶原纤维面积百分比分别为1.22%±0.24%,7.47%±0.81%,5.57%±0.78%,4.33%±0.48%,治疗组与模型组、对照组相比均有显著差异(P＜0.01),并且治疗组肝组织纤维化程度分级较模型组逐渐好转,胶原纤维所占面积显著缩小;在肝组织中测得的Col-Ⅰ阳性面积比分别为3.022%±0.553%,9.998%±1.431%,7.554%±0.914%,4.587%±1.008%,治疗组与模型组、对照组相比均有显著差异(P＜0.01).α-SMA和TGF-β1在治疗组和模型组肝组织中的表达也有显著差异(3.172%±0.542% vs 5.605%±1.315%,P＜0.01;2.868%±0.554% vs 5.653%±0.9%,P＜0.01).结论β-榄香烯对四氯化碳肝纤维化大鼠具有拮抗作用,主要是通过抑制肝星状细胞激活,降低TGF-β1,α-SMA在肝组织中的表达,减少细胞外基质在肝脏中的沉积,从而延缓肝纤维化的进程.     

外源性尿激酶抗肝纤维化的作用机制

目的 探讨外源性尿激酶抗大鼠肝纤维化的作用机制.方法 采用复合致病因子复制肝纤维化大鼠模型.大鼠随机分为对照组(正常饮食)、肝纤维化组(复合致病因子饲养6周)和尿激酶预防组(复合致病因子+尿激酶饲养6周).检测并比较各组大鼠血透明质酸含量、肝组织内羟脯氨酸、α-平滑肌肌动蛋白(α-SMA)、金属蛋白酶组织抑制因子-1(TIMP-1)、尿激酶型纤溶酶原激活物(μPA).尿激酶型纤溶酶原激活物抑制剂-1(PAI-1)、转化生长因子β1(TGF β 1)、Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达量以及PAI-1 mRNA和TGF β 1 mRNA的相对表达量.多组间用单因素方差分析q检验,两组间比较采用t检验,多组间等级资料比较采用Kruskal Wallis H检验,多样本间两两比较采用扩展的t检验. 结果血浆ALT、AST、总胆红素、透明质酸和肝组织内羟脯氨酸含量在尿激酶预防组大鼠分别为(46.66±6.30)U/L、(126.26±31.65)U/L、(31.11±4.20)μmol/L、(109.70±18.81)μg/L和(0.98±0.09)mg/g,较肝纤维化组的(101.57±11.97)U/L、(205.89±56.26)U/L、(67.75±2.75)μmol/L、(184.43±32.36)μg/L和(1.65±0.16)mg/g均明显降低(q值分别为3.3801～20.0061,P值均<0.01).尿激酶预防组α-SMA、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、TIMP-1、PAI-1及TGF β 1蛋白相对表达量分别为299.27±37.36、210.05±27.17、192.94±24.48、213.70±32.21、204.25±17.92和205.97±23.81,较肝纤维化组的418.83±30.21、323.77±21.53、302.37±31.43、376.63±25.19、313.53±26.67和327.42±36.75均明显减少,而uPA蛋白表达增加.尿激酶预防组PAI-1 mRNA,TGF β 1 mRNA表达减少,肝纤维化程度明显减轻. 结论 预防性使用外源性尿激酶可减轻肝损伤,降低血浆转氨酶、胆红素,减少肝星状细胞活化,降低TIMP-1、PAI-1蛋白表达,增加uPA蛋白表达,加速组织损伤修复,延缓肝纤维化的发生.     

Blockage of transforming growth factor β receptors prevents progression of pig serum-induced rat liver fibrosis

AIM: To test the hypothesis that introduction of antisense TβR Ⅰ and TβR Ⅱ eukaryotic expressing plasmids into a rat model of immunologically induced liver fibrosis might block the action of TGF-β1 and halt the progression of liver fibrosis.METHODS: RT-Nest-PCR and gene recombination techniques were used to construct rat antisense TβR Ⅰ and TβR Ⅱ recombinant plasmids which could be expressed in eukaryotic cells. The recombinant plasmids and empty vector (pcDNA3) were encapsulated by glycosyl-poly-Llysine and then transducted into rats of pig serum-induced liver fibrosis model. Expression of exogenously transfected gene was assessed by Northern blot, and hepatic expressions of TβR Ⅰ and TβR Ⅱ were evaluated by RTPCR and Western blot. We also performed ELISA for serum TGF-β1, hydroxyproline of hepatic tissues, immunohistochemistry for collagen types Ⅰ and Ⅲ, and VG staining for pathological study of the liver tissues.RESULTS: The exogenous antisense TβR Ⅰ and TβR Ⅱ plasmids could be well expressed in vivo, and block mRNA and protein expression of TβR Ⅰ and TβR Ⅱ in the fibrotic liver at the level of mRNA respectively. These exogenous plasmid expressions reduced the level of TGF-β1(antisense TβR Ⅰ group 23.998±3.045 ng/mL, antisense TβR Ⅱ group 23.156±3.131 ng/mL, disease control group 32.960±3.789 ng/mL; F=38.19, 36.73, P＜0.01). Compared with disease control group, the contents of hepatic hydroxyproline (antisense TβR Ⅰ group 0.169±0.015 mg/g liver, antisense TβR Ⅱ group 0.167±0.009 mg/g liver,disease control group 0.296±0.026 mg/g liver; F=14.39,15.48, P＜0.01) and the deposition of collagen types Ⅰ and Ⅲ decreased in the two antisense treatment groups (antisense TβR Ⅰ group, collagen type Ⅰ 669.90±50.67,collagen type Ⅲ 657.29±49.48; antisense TβR Ⅱ group,collagen type Ⅰ 650.26±51.51, collagen type Ⅲ 661.58±55.28;disease control group, collagen type Ⅰ 1209.44±116.60,collagen type Ⅲ 1175.14±121.44; F=15.48 to 74.89, P＜0.01).Their expression also improved the pathologic classification of liver fibrosis models (compared with disease control group, x2=17.14, 17.24, P＜0.01). No difference was found in the level of TGF-β1, the contents of hepatic hydroxyproline and collagen types Ⅰ and Ⅲ and pathologic grade between pcDNA3 control group and disease control group or between the two antisense treatment groups (F =0.11 to 1.06, x2=0.13 to 0.16, P＞0.05).CONCLUSION: Antisense TβR Ⅰ and TβR Ⅱ recombinant plasmids have certain reverse effects on liver fibrosis and can be used as possible candidates for gene therapy.     

白细胞介素9促进日本血吸虫感染小鼠肝星状细胞激活

目的 探讨小鼠感染日本血吸虫后,白细胞介素-9(interleukin-9, IL-9)在小鼠肝星状细胞活化中的作用。方法采用肝脏原位灌注消化结合密度梯度离心法,分离日本血吸虫感染7周后小鼠原代肝星状细胞（hepatic stellate cells, HSCs）并进行体外培养。将HSCs分为PBS对照组和IL-9刺激组（浓度20 ng/mL）。分别于刺激后48、72 h收集细胞,采用免疫印迹试验检测HSCs中α平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）、Ⅰ型胶原蛋白（typeⅠcollagen,ColⅠ）和Ⅲ型胶原蛋白（typeⅢcollagen, ColⅢ）表达水平。结果 经20 ng/mL IL-9刺激48 h后,IL-9刺激组HSCs中α-SMA [(0.87±0.02)vs.(0.69±0.01);t=17.39,P <0.01]、ColⅠ[（0.74±0.02）vs.(0.65±0.01);t=9.56,P <0.01]、ColⅢ蛋白[（0.94±0.04）vs.(0.75±0.03);t=6.15,P <0.01]表达水平...     

姜黄素对大鼠肠纤维化的作用及其机制研究

目的 探讨姜黄素在三硝基苯磺酸(TNBS)诱导的大鼠结肠纤维化中的抗纤维化作用和机制.方法 SD大鼠40只随机分组,模型组(10只)、治疗组(10只)和对照组(10只)分别于第1、8、15、22和29天予TNBS 10 mg、15 mg、20 mg、25 mg和30 mg灌肠,另取10只大鼠给予50%乙醇灌肠,作为阴性对照(正常组).从实验周期第1天起,治疗组大鼠每日予姜黄素30 mg/kg腹腔注射,对照组每日予0.9%NaCl腹腔注射,模型组和正常组不予处理.采用HE染色及Masson胶原三色染色观察大鼠结肠组织损伤和纤维化变化,采用ELISA法检测结肠黏膜中Th1/Th2型细胞因子IL-2、TNF-α、IL-4、IL-17的含量,采用荧光定量PCR法检测结肠黏膜中肠纤维化相关细胞因子如转化生长因子(TGF)-β1、结缔组织生长因子(CTGF)、Smad3、胶原Ⅰ、ⅢmRNA的表达.结果 模型组与对照组大鼠结肠组织大体损伤评分[(6.14±1.07)分,(6.17±1.47)分]及组织损伤评分[(8.42±1.40)分,(8.17±1.47)分]、胶原面积比(36.59%±4.07%,37.18%±4.05%)较正常组[分别为(2.13±0.64)分,(2.25±1.28)分和25.43%±5.39%]明显升高(均P＜0.05).模型组与对照组大鼠黏膜组织中IL-2[(378.25±29.90)ng/L,(410.06±64.74)ng/L]、TNF-α[(87.11±23.85)ng/L,(100.41±12.59)ng/L)]、IL-17[(47.80±5.62)ng/L,(41.45±2.12)ng/L]含量及TGF-β1(4.71%±2.71%,4.12%±3.01%)、CTGF(10.33%±6.99%,11.46%±4.72%)、Smad3(9.35%±7.32%,10.11%±3.80%)、胶原Ⅰ(1.52%±1.11%,1.57%±1.35%)、胶原Ⅲ(3.04%±1.33%,3.03%±3.53%)mRNA表达量较正常组[分别为(179.74±20.73)ng/L,(35.47±7.13)ng/L,(14.48±7.52)ng/L和0.90%±1.13%,0.53%±0.47%,0.62%±0.44%,0.16%±0.09%,0.18%±0.10%]均明显升高(均P＜0.05).而治疗组大鼠结肠组织大体损伤评分[(4.00±1.07)分]及组织损伤评分[(5.13±1.46)分]、胶原面积比(30.01%±7.56%),IL-2[(223.91±28.04)ng/L]、TNF-α[(44.19±4.77)ng/L]、IL-17[(14.89±4.31)ng/L]含量和TGF-β1(0.85%±0.76%)、CTGF(1.56%±1.13%)、Smad3(3.62%±3.03%)、胶原Ⅰ(0.40%±0.31%)、胶原Ⅲ(0.60%±1.02%)mRNA表达量较模型组及对照组明显降低(P＜0.05),与正常组无明显差异(P＞0.05).结论 姜黄素可通过降低细胞因子的过度表达,减轻大鼠结肠炎症,从而抑制过度"损伤-修复"所致的组织纤维化.

Abstract：

Objective To investigate the anti-fibrotic effects of curcumin in trinitrobenzene sulphonic acid (TNBS) induced intestinal fibrosis in rats and its mechanism. Methods Forty SD rats were randomly divided into model group, treatment group, control group and normal group with 10each. Except the normal group, the other three groups were given 10, 15, 20, 25 and 30 mg of TNBS enema on the 1st, 8 th, 15th, 22nd and 29th days,respectively. The rats in treatment group were intraperitonealy injected with 30 mg/kg of curcumin daily. Control group was injected with 0. 9%NaCl solution and normal group received an equal volume of 50% ethanol enema without any treatment. The damage and fibrosis of colon were detected with HE staining and Masson collagen staining, respectively. The contents of interleukin (IL) -2, tumor necrosis factor (TNF) -α, IL-4 and IL-17 in colon were measured by enzyme-link immunosorbent analysis (ELISA). The expressions of intestinal fibrosis related cytokines such as transforming growth factor (TGF) -β1, connective tissue growth factor (CTGF), Smad3, collagen Ⅰ and collagen Ⅲ mRNA were determined by FQ-PCR.Results The macroscopic and micrpscopic colonic damage scores and collagen area were significantly higher in model group (6.14 ± 1.07, 8. 42 ± 1.40 and 36. 59% ± 4.07%, respectively) and control group (6.17 ± 1.47, 8. 17 ±1.47 and 37.18 %±4.05 %, respectively) than those in normal group (2.13±0.64, 2.25±1.28 and 25.43%±5.39% ,respectively)(P＜0.05). Contents of IL2, TNF-α, IL-17, as well as expressions of intestinal fibrosis related cytokines including TGF-β1, CTGF,Smad3, collagen Ⅰ and Ⅲ mRNA were also higher in model group [(378. 25±29. 90) ng/L,(87.11±23.85) ng/L, (47.80±5.62) ng/L, 4.71%±2.71%,10.33%±6.99%,9.35%±7.32%,1.52% ± 1.11% and 3.04% ±1.33%, respectively] and control group [(410. 06 ± 64.74) ng/L,(100.41±12.59) ng/L, (41.45±2. 12) ng/L, 4. 12%±3.01%,11.46%±4.72%,10. 11%±3.80%,1. 57% ± 1. 35% and 3. 03% ± 3. 53%, respectively] in comparision with normal group [(179.74±20. 73) ng/L, (35. 47±7. 13) ng/L, (14. 48±7. 52) ng/L and 0. 90%± 1. 13%,0.53%±0.47%, 0. 62%±0. 44%, 0. 16%±0. 09% and 0. 18%±0. 10%, respectively] (P＜0.05). While in treatment group, the macroscopic (4.00 ± 1.07 ) and micrpscopic (5. 13 ± 1.46)colonic damage scores, collagen area (30.01%±7.56%), contents of IL-2 [(223.91±28.04) ng/L],TNF-α [(44.19±4. 77) ng/L] and IL-17 [(14.89±4. 31) ng/L], expressions of TGF-β1 (0.85%±0.76%), CTGF (1.56%±1.13%), Smad3 (3.62%±3.03%), collagen Ⅰ (0.40%±0.31%) and Ⅲ (0.60 % ± 1.02 % ) mRNA were much lower than those in model group and control group (P＜0.05 ), but similar to those in normal group (P＞ 0.05 ). Conclusions Curcumin can inhibit intestinal fibrosis caused by excessive "wound-healing" reaction via reducing the overexpression of cytokines in colonic mucosa and attenuating the inflammation of colon.     

血小板衍生生长因子和Ⅰ型胶原在饮水砷暴露小鼠肝组织中的表达

目的 探讨血小板衍生生长因子(PDGF)和Ⅰ型胶原(Col-Ⅰ)在饮水砷暴露小鼠肝组织中的表达及其意义.方法 50只昆明种雄性小鼠被分成对照组、iAs3+组、iAs5+组.分别饮用自来水,亚砷酸钠溶液(NaAsO2,含砷离子300 mg/L),砷酸钠溶液(Na2HAsO4·7H2O,含砷离子300 mg/L).10个月后处死小鼠,使用全自动生化分析仪检查肝功能,包括血清丙氨酸转氨酶(ALT)、天冬氨酸转氧酶(AST)、球蛋白(GLB);Masson染色肝组织,检测单位视场内的纤维面积;免疫组化(SABC)法检测肝组织中PDGF和Col-Ⅰ的表达.结果 ①对照组、iAs3+组、iAs5+组,血清ALT水平分别为(36.67±3.51)、(61.46±13.85)、(43.31±4.21)U/L,组间比较差异有统计学意义(F=6.56,P<0.05),iAs3+组明显高于对照组(P<0.05);血清AST水平分别为(135.00±20.42)、(510.86±59.01)、(258.93±22.40)U/L,组间比较差异有统计学意义(F=83.33,P<0.05),iAs3+、iAs5+组均明显高于对照组(P<0.05),iA3+组明显高于iAs5+组(P<0.05);血清GLB水平分别为(20.86±0.61)、(26.94±3.73)、(24.59±5.27)g/L,组间比较差异无统计学意义(F=2.80,P>0.05).②光镜下染砷组小鼠肝组织出现明显的细胞变性、坏死、再生,汇管区炎细胞浸润,纤维增生.对照组(0.069±0.013)、iAs3+组(0.192±0.108)和iAs5+组(0.143±0.122)单位视场内的纤维面积组间比较,差异有统计学意义(F=1.91,P<0.05),iAs3+组明显高于对照组(P<0.05).③对照组、iAs3+组、iAs5+组PDGF、Col-Ⅰ光密度值分别为202.788±7.462、174.382±7.706、177.644±7.811,200.11±7.46、176.47±10.20、177.38±7.95,组间比较差异均有统计学意义(F值分别为102.91、78.51,P均<0.05),与对照组比较,iAg3+、iAs5+组PDGF、Col-Ⅰ光密度值明显降低(P<0.05).表明肝组织内PDGF、Col-Ⅰ表达明显增多.且PDGF在汇管区、间隔细胞、窦旁细胞内均有表达,而Col-Ⅰ表达广泛分布于血管及胆管周围,并自门管区向肝实质内延伸形成纤维间隔.结论 长期饮水砷暴露,可导致慢性肝脏损伤、肝纤维化形成.PDGF、Col-Ⅰ在砷敛小鼠肝纤维化形成中起重要作用.     

非酒精性脂肪性肝病患者血清瘦素和脂联素与胰岛素抵抗的关系

目的 探讨非酒精性脂肪性肝病(NAFLD)患者血清瘦素、脂联素的变化及其与胰岛素抵抗的关系. 方法 选取NAFLD患者60例,同期门诊体检健康者60名为对照组,ELJSA法测定血清瘦素、脂联素水平,并检测体质量指数、腰臀比、甘油三酯、总胆固醇、高密度脂蛋白胆固醇(HDL-C)、空腹血糖、ALT、AST、γ-谷氨酰转肽酶(GGT),稳态模型评估的胰岛素抵抗指数(HOMA-IR).采用SPSS10.0软件包进行统计学分析,计量资料差异性比较用方差分析和t检验,多因素相关性用Spearman分析和Logistic回归分析. 结果 血清瘦素、脂联素水平NAFLD组分别为(12.37±1.99)μg/L和(12.69±2.83)mg/L,对照组分别为(5.20±1.03)μg/L和(22.83±4.61)mg/L,t值分别为24.661和14.516,P值均<0.01;HOMA-IR,NAFLD组为4.86±0.63,对照组为1.91±0.41,t值为30.451,P<0.01.Logistic多因素回归分析显示瘦素与腰臀围之比、HOMA-IR、空腹血糖呈独立正相关,β值分别为8.175、0.974和0.564,P值均<0.01;脂联素与HOMA-IR、体质量指数呈独立负相关,β值分别为-0.495和-0.314,P值均<0.01.结论 NAFLD患者血清瘦素、脂联素的变化与胰岛素抵抗有关.     

Extraction and purification of TGFβ and its effect on the induction of apoptosis of hepatocytes

AIM To extract and purify the transforming growth factor β (TGF β), and to demonstrate its biological activity in vivo and induction of apoptosis of hepatocytes in vitro. METHODS TGF β was isolated from fresh bovine platelets by acid/ethanol extraction method and purified with ion exchange and gel chromatography. The extracted TGF β was injected subcutaneously to mice, and its biological activity in vivo was observed 72 hfs post-injection by HE staining. The morphological changes were observed by HE staining and the occurrence of apoptosis was detected by TUNEL method after the human normal hepatic cell line QZG was treated with 8 μg@L 1 TGFβ for 12 hrs in vitro. RESULTS The molecular mass 25 ku TGF β protein was successfully extracted. It was able to induce localized granulation tissue formation in vivo. TGF β-treated hepatocytes showed obvious apoptotic morphological changes, including the pyknosis and dense-stained nuclei and cytoplasm, the fragmentary, annular or crescent nuclei, and the "bubbling" cytoplasm. Moreover, its apoptotic rate was significantly higher than that of the control group (P＜0.05). CONCLUSION Biological active TGF β protein is extracted and purified successfully from bovine platelets, and it is able to induce the apoptosis of hepatocytes.     

Transforming growth factor beta can be a parameter of aggressiveness of pT1 colorectal cancer

AIM: To evaluate the significance of transforming growth factor beta (TGF β) expression, in correlation with histopathological parameters, at the front of invasion in T1 colorectal cancer (CRC) and presence of metastases. METHODS: TGF p immunohistochemical expression was studied in 34 specimens of colorectal adenocarcinomas (pT1). A three-step avidin-biotinylated immuno-peroxidase (ABCu-NCL) staining technique was performed on 4-μm paraffin-embedded tissue sections with a monoclonal antibody to TGF β (Novocastra, NCL-TGFB, clone TGFB 17, dilution 1:40). RESULTS: Seventeen (50%) out of 34 lesions were positive for TGF p expression. The TGF β-positive rate in patients with vascular invasion was significantly higher than in those without vascular invasion (11/14 cases, P<0.01, P= 0.005). The TGF p-positive rate was observed in 91.7% of patients with presence of tumor budding at the front of invasion (11/12 cases, P<0.01, P= 0.0003). A statistically significant correlation was found between the presence of lymph node metastases and positive expression of TGF β (14/16 cases, P<0.01, P= 0.0001). We also observed that the TGF β-positive rates in groups with distant and non-distant metastases were 92.8% and 20% respectively, and a significant correlation between TGF β expression and distant metastasis was shown (P<0.01, P= 0.00003). CONCLUSION: The evaluation of TGF β expression of protein in association with histological parameters can be used as a parameter of the aggressiveness of pT1 CRC.     

金钗石斛多糖对肝纤维化大鼠TGF-β1、α-SMA、Ⅰ型胶原、Ⅲ型胶原表达的影响

目的 探讨金钗石斛多糖(DNP)对肝纤维化(HF)大鼠肝组织转化生长因子(TGF)-β1、α-平滑肌肌动蛋白(α-SMA)、Ⅰ、Ⅲ型胶原表达的影响.方法 SD大鼠随机分为正常组,模型组,秋水仙碱组(0.2 mg/kg),扶正化瘀组(0.415 g/kg),DNP低、中、高剂量组(5、10、20 g/kg),采用50％四...