TGF-β1和Collagen-Ⅲ在牦牛感染细粒棘球蚴肝纤维化中的表达

研究转化生长因子β1(TGF-β1)、Ⅲ型胶原(Collagen-Ⅲ)在牦牛感染细粒棘球蚴导致肝纤维化中的表达及其意义。收集西藏地区40例细粒棘球蚴感染的牦牛肝组织和血清样本,同时收集未感染的肝组织和血清进行对照。采用苏木素-伊红(HE)染色法和天狼猩红染色法观察牦牛肝脏病理变化和肝纤维化程度;酶联免疫吸附试验(ELISA)和免疫组化法(IHA)分别检测肝组织和血清中TGF-β1、Collagen-Ⅲ的表达水平及分布;RT-PCR检测肝细胞内TGF-β1和Collagen-Ⅲ基因mRNA的相对表达水平。HE染色和天狼猩红染色结果显示,与未感染细粒棘球蚴病牦牛肝组织相比,感染棘球蚴周围的肝组织肝界板细胞破坏,呈虫蚀状,感染肝组织外围纤维化,细粒棘球蚴寄生的牦牛肝脏组织引起组织增生,纤维化;ELISA结果显示感染细粒棘球蚴牦牛血清样中细胞因子TGF-β1、Collagen-Ⅲ的含量明显超过未感染组(P<0.05),且免疫组化的结果显示它们的表达主要存在于细胞基质中;RT-PCR显示细粒棘球蚴感染肝组织中TGF-β1、Collagen-Ⅲ的mRNA相对表达量超出未感染组(P<0.05)。结果表明,细粒棘球蚴感染期间TGF-β1、Collagen-Ⅲ在细粒棘球蚴感染引起的肝纤维化中起重要作用。

Effects of gentiana scabra bage on expression of hepatic type Ⅰ,Ⅲ collagen proteins in Paragonimus skrjabini rats with liver fibrosis

Objective:To exploit- the effects of gentiana scabra bage on the expression of hepatic collagen proteins in Paragonimus skrjabini rats with liver fibrosis.Methods:Immunohistochemical technique was used to observe the changes of content of hepatic type Ⅰ,Ⅲ collagen proteins in Paragonimus skrjabini rats with Liver fibrosis before and after the gentiana scabra bage treatmeat.Results:Comparing with the model group,changes of hepatic tvpe Ⅰ and type Ⅲ collagen proteins in gentiana scabra bage treated group were significantly weakened.Conclusions:Gentiana scabra bage treatment can reduce the content of hepatic type Ⅲ and type Ⅰ collagen protein significantly in Paragonimus skrjabini rats with liver fibrosis,thereby,playing the role against hepatic fibrosis.

基于上皮细胞-间充质细胞转分化(EMT)理论的艾灸配合化纤Ⅳ号方对实验大鼠Collagen Type Ⅲ和PDGF干预作用实验研究

目的:基于上皮细胞-间充质细胞转分化(EMT)学说观察化纤Ⅳ号方、艾灸以及二者相配合治疗肺纤维化大鼠Collagen TypeⅢ(Ⅲ-C)和PDGF的变化,探讨其治疗效应及生物学机制。方法:将鼠龄约为6周的SD大鼠随机分为空白组、模型组、化纤Ⅳ号方组、艾灸组、化纤Ⅳ号方与艾灸配合治疗组(简称为"灸药组"),治疗30 d后处死观察其肺组织病理改变,并检测其Collagen TypeⅢ、PDGF的基因和蛋白表达情况。结果:实时荧光定量结果显示:与空白组相比,各组Ⅲ-C和PDGF m RNA表达增高(P<0.05)。与模型组相比,各组的Ⅲ-C和PDGF m RNA表达有明显降低(P<0.01)。而各组中,灸药组疗效最明显,Ⅲ-C和PDGF的表达最低。蛋白免疫印迹法检测结果显示:与模型组相比各组的Ⅲ-C蛋白表达有差异。结论:1艾灸、化纤Ⅳ号方均可减轻博莱霉素诱导肺纤维化大鼠的肺纤维化程度。2艾灸配合化纤Ⅳ号方可减轻博莱霉素诱导肺纤维化大鼠的肺纤维化程度,且其效果优于单用艾灸或单用化纤Ⅳ号方。3艾灸、化纤Ⅳ号方及其二者配合使用不同程度阻抑博莱霉素诱导肺纤维化大鼠肺纤维化进程的效应机制,可能与通过调控其EMT过程中的Ⅲ-C和PDGF表达环节紧密相关。

绝经后盆底组织TGF-β_1和CollagenⅠ、Ⅲ表达与SUI的关系

目的:探讨绝经后压力性尿失禁(SUI)患者盆底支持结构转化生长因子β1(TGF-β1)、胶原蛋白Ⅰ、Ⅲ(CollagenⅠ、Ⅲ)与SUI的关系。方法:采用免疫组化方法,测定30例压力性尿失禁患者(SUI组)、28例盆底器官膨出(POP)患者(POP组)阴道前壁组织中TGF-β1及Collagen阳性率,并选择同期30例非卵巢功能性肿瘤和宫颈病变患者作为对照(对照组)。结果:SUI组、POP组及对照组患者阴道前壁组织中,TGF-β1表达阳性率分别为20.00%、14.30%、93.33%,SUI组与对照组比较,差异有极显著性(P<0.01);SUI组与POP组比较,差异无显著性(P>0.05)。3组患者阴道前壁组织中胶原Ⅰ含量的表达以辉度值为标准,在SUI组、POP组及对照组分别为98.62、98.15和100.03。SUI组与对照组比较,差异有极显著性(P<0.01);POP组与对照组比较,差异有极显著性(P<0.01);SUI组与POP组比较,差异无显著性(P>0.05)。3组患者阴道前壁组织中胶原Ⅲ含量的表达以辉度值为标准,在SUI组、POP组及对照组分别为98.46、98.23和100.12。SUI组与对照组比较,差异有极显著性(P<0.01);POP组与对照组比较,差异有极显著性(P<0.01);SUI组与POP组比较,差异无显著性(P>0.05)。结论:绝经后SUI患者盆底支持结构的退行性病变与组织中的转化生长因子β1减少及胶原蛋白含量水平低下有关。

女性压力性尿失禁患者盆底组织MMP-3和Collagen Ⅲ的研究

目的探讨女性压力性尿失禁(SUI)患者盆底支持结构中MMP-3、胶原蛋白Ⅲ与SUI发生的关系。方法采用免疫组化方法,测定30例压力性尿失禁患者(SUI组)MMP-3,胶原蛋白Ⅲ,并选择同期20例妇科良性病变而行子宫切除术者作为对照(对照组),于手术中取患者阴道前壁,采用免疫组织化学方法观察MMP-3、CollagenⅢ的表达情况。结果SUI组阴道前壁组织中MMP-3表达明显高于对照组(P<0.05),Collagen Ⅲ的表达明显低于对照组(P<0.05)。结论SUI患者阴道前壁组织中胶原代谢活跃、胶原分解增加可能是SUI的发病机制之一。

微针导入重组Ⅲ型人源化胶原蛋白在皮肤屏障功能修复中的应用效果

目的探究微针导入重组Ⅲ型人源化胶原蛋白治疗因皮肤炎性衰老导致的皮肤屏障功能下降的有效性及安全性。方法选取2020年6月至2021年5月在本院接受面部皮肤治疗的30例患者作为研究对象,采用随机数字表法将其分为对照组(重组Ⅲ型人源化胶原蛋白)与治疗组(微针导入重组Ⅲ型人源化胶原蛋白),各15例。治疗后定期对患者进行随访,分别进行主观疗效评价及客观疗效评价。结果治疗后4、8、12周,治疗组的满意度显著高于对照组(P<0.05)。治疗后,治疗组的皮肤皱纹、纹理、红区、毛孔、弹性、皮肤经皮失水及皮肤含水量改善明显(P<0.05);对照组治疗后油脂改善明显(P<0.05)。治疗后12周,治疗组的总症状评分显著低于对照组(P<0.05)。结论微针导入重组Ⅲ型人源化胶原蛋白对皮肤修复有较好的疗效,可增强皮肤屏障功能,为改善皮肤屏障功能、治疗因皮肤屏障受损引起的炎性衰老提供了新选择。

MMP-9、CollagenⅢ在胎膜组织中的表达及意义

目的探讨基质金属蛋白酶-9(MMP-9)、Collagen III在胎膜组织中的表达及其与胎膜早破之间的关系。方法应用免疫组化分别检测20例足月胎膜早破(tPROM)、20例未足月胎膜早破(pPROM)、10例早产和10例正常足月分娩患者胎膜组织标本中MMP-9、Collagen III的表达,分析其相关性及其与胎膜早破的关系。结果胎膜早破组胎膜组织中,MMP-9的阳性表达率为90%,高于对照组(P<0.01)。胎膜早破组CLO-Ⅲ的阳性表达率为30%,低于对照组P<0.05),MMP-9的表达与Collagen III的表达呈负相关(r=-0.612,P<0.05)。结论 MMP-9的高表达及Collagen III的减少可能与胎膜早破的发生有关。

葡甘露聚糖凝胶对宫颈炎大鼠CollagenⅠ、CollagenⅢ表达的影响

目的:探讨葡甘露聚糖凝胶对宫颈炎大鼠宫颈组织中Ⅰ型胶原蛋白(CollagenⅠ)、Ⅲ型胶原蛋白(CollagenⅢ)的调节作用。方法:采用苯酚胶浆进行阴道内注射建立宫颈炎模型,造模结束后进行局部给药治疗,将18只雌性SD大鼠造模后分为模型组、葡甘露聚糖凝胶高、中、低剂量组(1.2 g·kg-1、0.6 g·kg-1、0.3 g·kg-1)、复方甲硝唑阴道栓组(0.4 g·kg-1)、凝胶基质组各3只,另外3只作为空白组。阴道内灌注给药7天后,RT-PCR和Western blot法检测大鼠宫颈组织中CollagenⅠ、CollagenⅢmRNA及蛋白的表达。结果:RT-PCR法结果显示与空白组比较,模型组大鼠宫颈组织中CollagenⅠ、CollagenⅢmRNA的表达均明显降低(P﹤0.01);与模型组比较,葡甘露聚糖凝胶给药组大鼠宫颈组织中CollagenⅠ、CollagenⅢ的表达有不同程度的提高(P﹤0.01或P﹤0.05)。Western blotting法检测宫颈组织中CollagenⅠ、CollagenⅢ蛋白的表达:与空白组比较,模型组大鼠宫颈组织中CollagenⅠ、CollagenⅢ蛋白的表达均有所降低(P﹤0.01);与模型组比较,葡甘露聚糖凝胶给药组大鼠宫颈组织中CollagenⅠ、CollagenⅢ蛋白的表达有不同程度的提高(P﹤0.01或P﹤0.05)。结论:葡甘露聚糖凝胶对宫颈炎大鼠宫颈组织中CollagenⅠ、CollagenⅢ表达具有促进作用,从而促进宫颈黏膜的组织修复。

Serum hyaluronic acid, procollagen type Ⅲ and Ⅳ in histological diagnosis of liver fibrosis

OBJECTIVE: To assess the significance of serum hyaluronic acid (HA), proeollagen type Ⅲ (PCⅢ), collagen type Ⅳ (CⅣ) in the histological diagnosis of liver fibrosis. METHODS: The concentrations of serum HA, PCⅢ, CⅣ in 253 patients with chronic liver diseases were measured by radioimmunoassay. Liver biopsies were performed in all patients at the same time. The liver was pathologically evaluated by a pathologist according to a scoring system. Combined with the results of liver pathological diagnosis, the accuracy of serum HA, PCⅢ, CⅣ in diagnosing patients with hepatic fibrosis (staging≥S_2) or cirrhosis (S_4) was assessed using the receiver operating curve (ROC). RESULTS: The cutoff values of serum HA, PCⅢ and CⅣ for identifying patients with hepatic fibrosis (≥S_2) or cirrhosis (S_4) were determined. The cutoff values of serum HA, PCⅢ and CⅣ for detecting patients with fibrosis (stage≥S_2) were 90μg/L, 90μg/L, 75μg/L, respectively; their sensitivity (Se) was 80.4%, 82%, 63.1%; their specificity (Spe) was 70.2%, 60.8%, 83.8%; their positive predictive values (PPV) were 86.7%, 83.5%, 90.4%; their negative predictive values (NPV) were 59.8%, 58.4%, 48.4%, respectively. The cutoff values for detecting patients with liver cirrhosis were 210μg/L for HA, 96.2% for Se, 85.3% for Spe, 65.4% for PPV, 98.8% for NPV; 150μg/L for PCⅢ, 76.4% for Se, 68.7% for Spe, 40.4% for PPV, 91.3% for NPV; 90μg/L for CⅣ, 80% for Se, 75.8% for Spe, 47.8% for PPV, 93.2% for NPV, respectively. CONCLUSIONS: Serum HA, PCⅢ and CⅣ can be determined for an accurate diagnosis of hepatic fibrosis in various stages. HA is the best for screening liver cirrhosis.

Dynamic changes of activator protein 1 and collagen I expression in the sclera of myopia guinea pigs

AIM: To investigate the dynamic changes of activator protein 1(AP1) and collagen I expression in the sclera of form-deprivation myopic model in guinea pigs. METHODS: A form-deprivation myopic model in guinea pigs were established with the left eye covered for 2 to 6 wk(FDM group). Normal control group(n=25) were untreated. Changes in refractive power and axial length(AL) were measured and recorded at different time points. Expressions of AP1 and collagen 1 of the sclera were measured with Western blotting and reverse transcription-polymerase chain reaction(RT-PCR). The relationship between AP1 and collagen I levels was analyzed. RESULTS: After 0, 2, 4, 6 wk, and 4/-1 wk of form-deprivation, the diopter in the FDM group was gradually changed(2.08±0.31,-1.23±0.68,-4.17±0.58,-7.07±0.55, and-2.67±0.59 D, respectively, P<0.05), and the AL was gradually increased(5.90±0.38, 6.62±0.37, 7.30±0.35, 7.99±0.31, and 6.97±0.32 mm, respectively, P<0.05). With the prolongation of covered time, the protein expressions of AP1 and collagen I in the FDM group were gradually down-regulated(all P<0.05);the mRNA expressions of them were also gradually down-regulated(all P<0.05);and there was positive correlation between them. The control group had no obvious change in each index(all P>0.05). CONCLUSION: AP1 may be an important transcription factor involved in the regulation of collagen I synthesis and degradation during myopic scleral remodeling.

Regulation of activin receptor-interacting protein 2 expression in mouse hepatoma Hepa1-6 cells and its relationship with collagen type Ⅳ

AIM： To investigate the regulation of activin receptor-interacting protein 2 （ARIP2） expression and its possible relationships with collagen type Ⅳ （collagen Ⅳ） in mouse hepatoma cell line Hepal-6 cells. METHODS： The ARIP2 mRNA expression kinetics in Hepal-6 cells was detected by RT-PCR, and its regulation factors were analyzed by treatment with signal transduction activators such as phorbol 12-myristate 13-acetate （PMA）, forskolin and A23187. After pcDNA3- ARIP2 was transfected into Hepal-6 cells, the effects of ARIP2 overexpression on activin type Ⅱ receptor （ActRⅡ） and collagen Ⅳ expression were evaluated. RESULTS： The expression levels of ARIP2 mRNA in Hapel-6 cells were elevated in time-dependent manner 12 h after treatment with activin A and endotoxin LPS, but not changed evidently in the early stage of stimulation （2 or 4 h）. The ARIP2 mRNA expression was increased after stimulated with signal transduction activators such as PMA and forskolin in Hepal-6 cells, whereas decreased after treatment with A23187 （25.3% ± 5.7% vs 48.1% ± 3.6%, P 〈 0.01）. ARIP2 overexpression could remarkably suppress the expression of ActRⅡA mRNA in dose-dependent manner, but has no effect on ActRⅡB in Hepal-6 cells induced by activin A. Furthermore, we have found that overexpression of ARIP2 could inhibit collagen Ⅳ mRNA and protein expressions induced by activin A in Hapel-6 cells. CONCLUSION： These findings suggest that ARIP2 expression can be influenced by various factors. ARIP2 may participate in the negative feedback regulation of signal transduction in the late stage by affecting the expression of ActRIIA and play an important role in regulation of development of liver fibrosis induced by activin.

An enriched environment increases the expression of fibronectin type Ⅲ domain-containing protein 5 and brain-derived neurotrophic factor in the cerebral cortex of the ischemic mouse brain

Many studies have shown that fibronectin type III domain-containing protein 5(FDNC5) and brain-derived neurotrophic factor(BDNF) play vital roles in plasticity after brain injury. An enriched environment refers to an environment that provides animals with multi-sensory stimulation and movement opportunities. An enriched environment has been shown to promote the regeneration of nerve cells, synapses, and blood vessels in the animal brain after cerebral ischemia;however, the exact mechanisms have not been clarified. This study aimed to determine whether an enriched environment could improve neurobehavioral functions after the experimental inducement of cerebral ischemia and whether neurobehavioral outcomes were associated with the expression of FDNC5 and BDNF. This study established ischemic mouse models using permanent middle cerebral artery occlusion(pMCAO) on the left side. On postoperative day 1, the mice were randomly assigned to either enriched environment or standard housing condition groups. Mice in the standard housing condition group were housed and fed under standard conditions. Mice in the enriched environment group were housed in a large cage, containing various toys, and fed with a standard diet. Sham-operated mice received the same procedure, but without artery occlusion, and were housed and fed under standard conditions. On postoperative days 7 and 14, a beam-walking test was used to assess coordination, balance, and spatial learning. On postoperative days 16–20, a Morris water maze test was used to assess spatial learning and memory. On postoperative day 15, the expression levels of FDNC5 and BDNF proteins in the ipsilateral cerebral cortex were analyzed by western blot assay. The results showed that compared with the standard housing condition group, the motor balance and coordination functions(based on beam-walking test scores 7 and 14 days after operation), spatial learning abilities(based on the spatial learning scores from the Morris water maze test 16–19 days after operation), and memory abilities(based on the memory scores of the Morris water maze test 20 days after operation) of the enriched environment group improved significantly. In addition, the expression levels of FDNC5 and BDNF proteins in the ipsilateral cerebral cortex increased in the enriched environment group compared with those in the standard housing condition group. Furthermore, the Pearson correlation coefficient showed that neurobehavioral functions were positively associated with the expression levels of FDNC5 and BDNF(r = 0.587 and r = 0.840, respectively). These findings suggest that an enriched environment upregulates FDNC5 protein expression in the ipsilateral cerebral cortex after cerebral ischemia, which then activates BDNF protein expression, improving neurological function. BDNF protein expression was positively correlated with improved neurological function. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Fudan University, China(approval Nos. 20160858 A232, 20160860 A234) on February 24, 2016.

温经汤加味对EM肾虚血瘀证大鼠局部微环境MMP-9、TNF-α、Collagen Ⅰ、Collagen Ⅲ的影响及其意义

目的探讨子宫内膜异位症(Endometosis,EM)肾虚血瘀证大鼠异位病灶局部MMP-9,TNF-α,CollagenⅠ、CollagenⅢ表达水平,以及温经汤加味对其干预作用及机制。方法SPF级雌性未孕健康SD大鼠设为空白组;构建肾虚血瘀证大鼠模型,造模成功后,设为假手术组(仅开腹);其余大鼠以自体内膜移植法构建EM肾虚血瘀证模型,造模成功后的大鼠分为模型组(不用药物干预)、阳性药(达那唑)组、温经汤加味组(低、中、高3种剂量)。阳性药对照组给予达那唑63 mg·kg^(-1)连续4周灌胃,中药低、中、高剂量组分别以5 g·kg^(-1)、10 g·kg^(-1)、20 g·kg^(-1)连续4周灌胃。取各组大鼠子宫内膜组织观察组织病理变化;免疫组化法(IHC)检测各组大鼠子宫内膜组织MMP-9,TNF-α的蛋白表达;蛋白免疫印迹法(WB)、实时荧光聚合酶链式反应法(RT-PCR)检测各组大鼠子宫内膜组织MMP-9、TNF-α、CollagenⅠ、CollagenⅢ基因表达。结果与空白组、假手术组比较,EM肾虚血瘀证模型组大鼠在位、异位内膜组织中MMP-9、TNF-α、CollagenⅠ、CollagenⅢ蛋白及其基因表达在模型组子宫内膜组织升高(P<0.01);与模型组比较,阳性药对照组、温经汤加味治疗组大鼠MMP-9、TNF-α、CollagenⅠ、CollagenⅢ蛋白及其基因表达水平均降低(P<0.01或P<0.05)。结论局部微环境免疫抑制微血管新生导致异位内膜侵袭是EM发生、发展过程中的重要病理表现,MMP-9、TNF-α、CollagenⅠ、CollagenⅢ过度表达可能促进了这一过程的发生、发展;温经汤加味通过"逆转"MMP-9、TNF-α、CollagenⅠ、CollagenⅢ等细胞因子异常升高,起到了解除局部微环境免疫抑制、阻断微血管新生的作用,在治疗EM肾虚血瘀证过程中表现出确切的疗效。

Dietary protein levels changed the hardness of muscle by acting on muscle fiber growth and the metabolism of collagen in sub-adult grass carp(Ctenopharyngodon idella)

Background:Nutrient regulation has been proven to be an effective way to improve the flesh quality in fish.As a necessary nutrient for fish growth,protein accounts for the highest proportion in the fish diet and is expensive.Although our team found that the effect of protein on the muscle hardness of grass carp was probably related to an increased collagen content,the mechanism for this effect has not been deeply explored.Moreover,few studies have explored the protein requirements of sub-adult grass crap(Ctenopharyngodon idella).Therefore,the effects of different dietary protein levels on the growth performance,nutritional value,muscle hardness,muscle fiber growth,collagen metabolism and related molecule expression in grass carp were investigated.Methods:A total of 450 healthy grass carp(721.16±1.98 g)were selected and assigned randomly to six experimen-tal groups with three replicates each(n=25/replicate),and were fed six diets with 15.91%,19.39%,22.10%,25.59%,28.53%and 31.42%protein for 60 d.Results:Appropriate levels of dietary protein increased the feed intake,percentage weight gain,specific growth rate,body composition,unsaturated fatty acid content in muscle,partial free amino acid content in muscle,and muscle hardness of grass carp.These protein levels also increased the muscle fiber density,the frequency of new muscle fibers,the contents of collagen and IGF-1,and the enzyme activities of prolyl 4-hydroxylases and lysyloxidase,and decreased the activity of matrix metalloproteinase-2.At the molecular level,the optimal dietary protein increased col-lagen type Iα1(Colα1),Colα2,PI3K,Akt,S6K1,La ribonucleoprotein domain family member 6a(LARP6a),TGF-β1,Smad2,Smad4,Smad3,tissue inhibitor of metalloproteinase-2,MyoD,Myf5,MyoG and MyHC relative mRNA levels.The levels of the myostatin-1 and myostatin-2 genes were downregulated,and the protein expression levels of p-Smad2,Smad2,Smad4,p-Akt,Akt,LARP6 and Smad3 were increased.Conclusions:The appropriate levels of dietary protein promoted the growth of sub-adult grass carp and improved muscle hardness by promoting the growth of muscle fibers,improving collagen synthesis and depressing collagen degradation.In addition,the dietary protein requirements of sub-adult grass carp were 26.21%and 24.85%according to the quadratic regression analysis of growth performance(SGR)and the muscle hardness(collagen content),respectively.

胰岛素对糖尿病大鼠模型的心肌病理改变及TGF-β1、collagenⅢ表达的影响

目的探讨胰岛素对糖尿病大鼠模型的心肌病理改变及TGF-β1、胶原蛋白Ⅲ(collagenⅢ)表达的影响。方法将30只SD大鼠分别分为A、B、C组,每组10只,A组为对照组,B、C两组腹腔注射链脲佐菌素建立糖尿病大鼠模型,A、B组给予1 mL生理盐水腹腔注射,C组腹部皮下注射胰岛素。三组大鼠造模5周后处死,检测三组大鼠的血糖和血脂的变化,观察大鼠心肌的变化,TGF-β1、collagenⅢ的表达情况。结果①血糖、血脂改善:B、C两组大鼠的三酰甘油(TG)、总胆固醇(TC)均显著高于A组(P<0.05)。C组的血糖、TG、TC、低密度脂蛋白胆固醇(LDL-C)显著低于B组(P<0.05),高密度脂蛋白胆固醇(HDL-C)显著高于B组(P<0.05)。②心肌病理学改变:A组大鼠心肌细胞结构正常;B组大鼠心肌细胞排列紊乱,局灶性心肌纤维溶解,可见变性、坏死;C组大鼠心肌细胞排列比较完好,无明显的溶解断裂,无变性坏死;③因子、蛋白表达:B、C两组大鼠心肌组织中的TGF-β1、collagenⅢ表达显著高于A组(P<0.05)。但C组中的TGF-β1、collagenⅢ表达显著低于B组(P<0.05)。结论胰岛素可调节糖尿病大鼠的糖、脂质代谢紊乱,降低的心肌病变程度,其机制可能与其降低了心肌组织中TGF-β1及collagenⅢ的表达有关。

CaMKⅡ抑制剂抑制AngⅡ或电场刺激诱导的心肌成纤维细胞TNF-α、TGF-β_1及collagenⅠ、Ⅲ的表达

目的:观察钙-钙调素依赖性蛋白激酶(CaMK)Ⅱ在血管紧张素Ⅱ(AngⅡ)或电场刺激(EFS)诱导的大鼠心肌成纤维细胞增殖、分泌细胞因子及胶原酶表达中的作用及其机制。方法:培养新生1-3 d乳鼠心肌成纤维细胞(3代),分为正常对照组(control)、0.1μmol/L AngⅡ组、0.1μmol/L AngⅡ+0.5μmol/L CaMKⅡ抑制剂KN92组、0.1μmol/L AngⅡ+0.5μmol/L CaMKⅡ抑制剂KN93组、0.1μmol/L AngⅡ+0.5μmol/L CaMKⅡ抑制剂AIP组;10 V1.0 Hz EFS组、10 V 1.0 Hz EFS+0.5μmol/L KN92组、10 V 1.0 Hz EFS+0.5μmol/L KN93组、10 V1.0 Hz EFS+0.5μmol/L AIP组、10 V1.0 Hz EFS+0.1μmol/L AngⅡ组。MTT法测定心肌成纤维细胞增殖;ELISA法测定细胞因子(TGF-β1,TNF-α)分泌;RT-PCR检测TGF-β1、TNF-α、collagenⅠ、ⅢmRNA水平。结果:CaMKⅡ抑制剂(0.5μmol/L KN93,0.5μmol/L AIP)能预防AngⅡ或EFS诱导的心肌成纤维细胞增殖;CaMKⅡ抑制剂(0.5μmol/L KN93,0.5μmol/L AIP)可预防AngⅡ或EFS引起的细胞培养上清液TGF-β1、TNF-α含量及相应mRNA表达增加。CaMKⅡ抑制剂(0.5μmol/L AIP,1.0μmol/L AIP)预防0.1μmol/L AngⅡ引起的collagenⅠ、Ⅲ表达增加。结论:抑制CaMKⅡ对AngⅡ或EFS诱导的心肌成纤维细胞增殖具有预防作用,其机制可能与CaMKⅡ抑制剂抑制TGF-β1、TNF-α以及胶原的表达有关。

Molecular Cloning and Bioinformatics Analysis of TypeⅢSecretion System Effector Protein Va1686 Gene of Vibrio alginolyticus

In this study, Va 1686 gene was cloned from Vibrio alginolyticus . The total length of the gene is 1 164 bp, and it could encode 387 amino acids. The physicochemical properties, protein structure, genetic evolutionary relationship and antigenic characteristics of the effect protein Va1686 of V. alginolyticus HY9901 type Ⅲ secretion system were studied and analyzed by bioinformatics methods and tools. The results showed that Va1686 is a stable hydrophilic and acidic protein without a transmembrane region and a signal peptide, and secondary structure to α-helix. The evolutionary analysis showed that V. alginolyticus HY9901 and V. harveyi were clustered together, which indicated that the genetic relationship between the two species was the closest. Va1686 contains a Fic superfamily conserved domain associated with cell division. Bioinformatics analysis showed that the B-cell preponderant epitopes of Va1686 might be localized in the regions of 48-49, 82-85, 125-126, 150-153, 185-186, 236-237 and so on. The 3D structure model of Va1686 subunit was simulated by SWISS-MODEL software and it was found that the vopS of V. parahaemolyticus was similar and the similarity was 89.46%. In this study, the feasibility of Va1686 as a common antigen of Vibrio was verified from the perspective of bioinformatics, which laid the foundation for the next step in vaccine development.

Expression and role of specificity protein 1 and collagenⅠin recurrent pterygial tissues

AIM:To investigate the expression profiles of the transcription factor specificity protein 1(Sp1)and collagenⅠin recurrent pterygial tissues.What is more,to compare the changes of Sp1 and collagen I among primary pterygial tissue,recurrent pterygial tissue and conjunctival tissue.METHODS:In the prospective study,we collected the pterygial tissues of 40 patients who underwent resection of primary pterygial tissue and recurrent pterygial tissue,and the conjunctival tissues of 10 patients with enucleation due to trauma.The relative expression levels of Sp1 and collagen I were analyzed by reverse transcription quantitative-polymerase chain reaction and Western blot.Paired t-test was performed to compare the Sp1 and collagen I of recurrent pterygial tissues,as well as the primary pterygial tissues and conjunctival tissues.In further,Pearson’s hypothesis testing of correlation coefficients was used to compare the correlations of Sp1 and Collagen I.RESULTS:The content of Sp1 and collagen I m RNA and protein was significantly greater in recurrent pterygial tissue than that was in primary and conjunctival tissue(P<0.05).There was a positive correlation between the m RNA and protein levels of Sp1 and collagen I in recurrent pterygial tissues(protein:r=0.913,P<0.05;m RNA:r=0.945,P<0.05).CONCLUSION:Sp1 and collagen I are expressed in normal conjunctival,primary,and recurrent pterygial tissues,but expression is significantly greater in the latter.Sp1 and collagen I may be involved in the regulation of the development of recurrent pterygium.

Collagen Ⅲ和Smad2在犬裁剪去粘膜回肠输尿管中的表达

目的探讨1年期犬裁剪去粘膜回肠输尿管中Collagen Ⅲ和Smad2的表达,评估其纤维化。方法用5只比格犬参照前期实验方法,建立裁剪去粘膜回肠代输尿管模型,术后饲养至1年,造影检查与在体观察后切取回肠输尿管;用正常回肠远段部分组织为对照;采用组织病理、免疫组织化学、Western blot和Real-time PCR技术,检测Collagen Ⅲ和Smad2在样本中的表达,比较两组的差异。结果术后模型犬4只持续存活,1只肠梗阻死亡,裁剪去粘膜回肠输尿管显影较好、未见明确狭窄与梗阻;肉眼及镜下观察未发现其与对照组间存在明显差异,管壁各层无显著纤维增生与胶原沉积增加;Collagen Ⅲ和Smad2在两组中均呈阴性表达,其中Smad2实验组低于对照组(P<0.05);Collagen Ⅲ和Smad2在蛋白和基因水平的表达,两组无显著差异(P>0.05)。结论犬裁剪去粘膜回肠输尿管1年仍能保持较好形态结构,Collagen Ⅲ和Smad2表达无显著增加,管壁无明确纤维化改变,具有长期稳定性。

Collagen type Ⅲ is an important linking molecule between generated renal tubules and an artificial polyester interstitium--Reticulin as a linker between generated tubules and the interstitium

在再生医学领域,已经有很多人考虑在将来用干/祖细胞治疗急慢性肾功能衰竭。但是,要使这种治疗方案行之有效,需要了解关于患病肾脏肾小管发育的细胞生物学信息。与干/祖细胞治疗急慢性肾功能衰竭相关尚待明晰的细胞生物学问题包括:①干/祖细胞的整合。②干/祖细胞的定向分化类型。③新形成肾小管的空间构成。为了更好的了解这项技术的相关机制,文章应用了先进的培养技术在人工非细胞外基质材料界面条件下构建肾小管。将新生兔肾来源的干/祖细胞用多层聚酯纤维网覆盖,放置在灌注培养器中,用含1×10-7mol/L醛固酮的新鲜合成的在DMEM基础上改良的IMDM培养基诱导培养13d。扫描电镜下,在大豆凝集素、银染和抗Ⅲ型胶原或层粘连蛋白γ1单克隆抗体标记的全标本包埋爬片或冰冻切片上观察肾小管的生长情况。扫描电镜观察结果显示,新生成的肾小管完全被基底膜覆盖,纤维网状板有大量的纤维与生成的肾小管基底面和周围人工聚酯材料相互交联。抗Ⅲ型胶原染色和银染结果表明,在新生成的肾小管的基底膜和邻近的人工聚酯材料间有大量的纤维交织,在发育成熟的肾小管中有与新生成肾小管相同排列形式的Ⅲ型胶原纤维。以上实验结果说明,Ⅲ胶原是联系新生成的肾小管基底面和人工基质聚酯纤维的分子纽带。