大鼠移植肝内胆管上皮细胞转化生长因子-β表达的变化

目的探讨肝移植术后肝内胆管上皮细胞(BEC)转化生长因子(TGF)-β的表达。方法建立大鼠原位肝移植模型,根据供肝冷保存时间而分为CP 1 h组(冷保存1 h)、CP 12 h组(冷保存12 h)、SO组(假手术组)。分别于术后1、3、7、14 d检测血清谷丙转氨酶(ALT)、总胆红素(TBIL)、碱性磷酸酶(ALP);免疫组织化学法检测BEC增殖、TGF-β表达、肌纤维母细胞(MFB)分布。结果CP12 h组术后各时相点BEC增殖率(7.0%、27.8%、23.1%、17.8%)明显高于CP 1 h组同时相点水平(2.0%、6%、2.6%、2.3%)及SO组(1.2%)水平;血清ALT(>200 IU/L)、ALP (>160 IU/L)、TBIL(>3.6μmlo/L)水平持续高于CP 1 h组(ALP<200 IU/L,ALT<160 IU/L,TBIL<3.6μmol/L);CP 12 h组术后3 d,BEC分泌TGF-β,7、14 d表达显著,伴有大量MFB聚集于BEC周围。结论供肝较长时间的冷保存严重损伤胆道,诱导BEC活跃增殖并分泌TGF-β,促进胆管周围成纤维细胞发生向MFB的表型转化,是肝移植术后胆管周围纤维化、胆道狭窄等并发症的产生的细胞学基础。     

IL-6在人胆管上皮细胞损伤修复中的作用

目的 探讨IL-6在人胆管上皮细胞(BECs)损伤修复中的作用机制.方法 IL-6按浓度分为5组:0 ng/L组,10 ng/L组,50 ng/L组,100 ng/L组,1000 ng/L组,将不同浓度的IL-6分别干预体外培养的BECs,并检测IL-6对转录激活子3(STAT3)磷酸化和三叶因子3(TFF3)表达的影响;将BECs分为未处理组、STAT3-RNAi组(细胞中转入STAT3 RNAi腺病毒)和Control-RNAi组(细胞中转入空RNAi腺病毒),检测行干扰后,IL-6对TFF3表达的影响;分别用未处理组、STAT3-RNAi组、Control-RNAi组BECs制备体外损伤模型,观察IL-6、TFF3对各组细胞的影响.两组数据间比较采用Student's t检验,多组间比较采用单因素方差或Sidak检验.结果 添加IL-6的50 ng/L组、100 ng/L组、1000 ng/L组磷酸化STAT3( p-STAT3)的表达水平分别为0.240±0.052、0.714 ±0.124、0.327± 0.069,明显强于0 ng/L组的0.033±0.011(q=5.246,17.260,7.451,P＜0.05).TFF3 mRNA及其蛋白表达水平随着IL-6浓度的增高而显著增加(q=12.045,9.889,P ＜0.05);IL-6浓度为1000 ng/L时,TFF3 mRNA及其蛋白表达有所回落.行干扰后,STAT3-RNAi组BECs TFF3蛋白表达水平为0.037±0.005,明显低于Control-RNAi组的0.267±0.038和未处理组的0.301 ±0.042(q=12.135,13.929,P＜0.05).体外损伤模型中,IL-6干预BECs 12 h后,STAT3-RNAi组BECs移行速度为(9.1±1.5)μm/h,慢于Control-RNAi组的(25.1±3.8)μm/h(q=7.737,P＜0.05);而STAT3-RNAi组中加入外源性人重组TFF31 g/L后,BECs移行速度为(39.2±4.7)μm/h,比Control-RNAi组显著提高(q=14.507,P＜0.05).结论 IL-6主要通过激活STAT3,继发上调TFF3表达,从而促进人胆管上皮细胞移行和损伤修复.     

维生素B1介导的Akt/mTOR/STAT3信号通路在谷氨酸诱导的PC12细胞损伤中的作用

[目的]研究维生素B1(thiamine,VitB1)介导的Akt/mTOR/STAT3信号通路在谷氨酸(glutamate,Glu)诱导的PC12细胞损伤中的作用.[方法]应用MTT法检测不同浓度谷氨酸对PC12细胞作用不同时间的细胞生长抑制率,筛选出合适的作用浓度与作用时间后,将细胞分为4组,分别为A组:正常对照组;B组:20 mmoL/L谷氨酸处理组;C组:20 mmol/L谷氨酸+300 μmol/L维生素B1处理组;D组:20 mmol/L谷氨酸+300 μmol/L维生素B1+ 800 nmol/L雷帕霉素(rapamycin,RAPA)处理组.应用流式细胞术观察各组作用12 h后细胞凋亡率,Western blot观察各组处理1、4、8、12 h后,p-Akt,p-mTOR,p-STAT3蛋白表达情况.[结果](1)谷氨酸对PC12细胞的生长抑制作用随作用时间和作用浓度的增加而增强;(2)A组的凋亡率为(3.42±0.79)％;C组凋亡率为(23.85±1.58)％,明显低于B组(40.20±2.54)％和D组(53.49±2.83)％(P＜0.01);(3) Western blot 检测结果表明C组各时间点p-Akt,p-mTOR,p-STAT3表达均高于A、B、D组,并且p-Akt表达量在1h时最高,p-mTOR和p-STAT3在4h时达到高峰.[结论]维生素B1激活了细胞Akt/mTOR/STAT3信号通路,该通路对谷氨酸诱导的神经细胞损伤起到了保护作用.     

基于STAT3信号通路研究IL-17在大鼠急性脊髓损伤模型中的作用研究

[目的]观察白细胞介素17通过激活STAT3信号通路在大鼠急性脊髓损伤模型中的表达。[方法]健康雄性SD大鼠75只,体重(250±25)g,分为对照组和脊髓损伤组,脊髓损伤组随机分到1、24、48和72 h四个亚组(n=15只)。改良Allen氏打击法建立脊髓损伤模型,对照组只切除椎板不损伤脊髓。术后处死前对大鼠脊髓功能进行BBB评分,HE染色观察损伤段脊髓组织形态结构,免疫组织化学检测IL-17、p-STAT3的表达变化,逆转录PCR检测IL-17 mRNA的动态变化。[结果]免疫组织化学检测显示对照组的大鼠脊髓组织中IL-17、pSTAT3表达量较少,损伤后1h明显上升,24 h到达峰值,而后缓慢下降,72 h仍高于对照组(P<0.05)。RTPCR检测显示脊髓损伤后IL-17mRNA表达趋势与免疫组化结果一致(P<0.05)。[结论]大鼠脊髓损伤后可异常激活IL-17过量表达,它可能对继发性脊髓损伤炎症反应有着重要作用。     

JAK/STAT通路在金属蛋白酶1组织抑制剂抑制肾小球系膜细胞凋亡中的作用

目的 探讨金属蛋白酶1组织抑制剂（TIMP-1）抑制大鼠肾小球系膜细胞（RMC）凋亡与Janus激酶/信号转导和转录激活子（JAK/STAT）通路的关系。 方法 用无血清培养基体外培养pcDNA3空载体、人正义、反义TIMP-1基因重组真核表达载体转染RMC。根据是否加JAK2特异性抑制剂AG490刺激24 h,将细胞分为未转染组、未转染＋AG490组、空载体组、空载体＋AG490组、正义组、正义＋AG490组、反义组和反义＋AG490组。另外设正常培养条件下的RMC作为正常对照组。应用流式细胞技术检测各组RMC的凋亡率。RT-PCR检测TIMP-1、bcl-xl、cyclin D1、p27kip1和JAK2 mRNA的表达。Western印迹检测胞质中JAK2、STAT3、STAT5及其相应磷酸化蛋白(p-JAK2、p-STAT3、p-STAT5)的表达。 结果 未转染组、正义组及反义组RMC凋亡率分别为（10.59±0.96）％、（7.08±0.43）％和（21.91±0.25）％，各组间差异有统计学意义(P < 0.05或P < 0.01)。在未加AG490的各组RMC中,bcl-xl和cyclin D1 mRNA在正义组中表达最高，反义组中最低，p27kip1 mRNA在反义组中表达最高。加入AG490后，各组细胞的凋亡率均显著增加（P < 0.01）；TIMP-1、bcl-xl和cyclin D1 mRNA表达均减少；p27kip1 mRNA表达均增加。在未加AG490的各组细胞中，p-JAK2、p-STAT3和p-STAT5在正义组中表达最高，反义组中最低。加入AG490后上述蛋白表达均减少，且正义＋AG490组最高，反义＋AG490组最低。 结论 TIMP-1表达受JAK/STAT信号通路调控，后者可通过上调前者的表达抑制RMC凋亡；TIMP-1通过JAK/STAT信号通路抑制RMC凋亡。 bcl-xl、cyclin D1和p27kip1参与了上述过程。     

NF-κB抑制剂对前列腺癌PC-3细胞STAT3核转位的影响

目的:观察IL-6刺激后的前列腺癌PC-3细胞STAT3和NF-κB的表达情况;验证NF-κB抑制剂咖啡酸苯乙基酯(CAPE)对PC-3细胞IL-6和STAT3表达的影响。方法:20 ng/ml IL-6分别作用于PC-3细胞0、5、10、20、30、45 min后,Western印迹和实时荧光定量PCR检测STAT3和NF-κB蛋白和mRNA水平的表达差异;流式细胞技术检测细胞周期。采用TNF-α或TNF-α联合CAPE作用于PC-3细胞,收集培养液上清,ELISA检测IL-6的表达;同时用Western印迹检测p-STAT3的表达。结果:IL-6刺激PC-3细胞后,p-STAT3蛋白的表达明显上调,细胞增殖指数明显增高。TNF-α作用于PC-3细胞后,培养液中IL-6的表达上调,同时p-STAT3蛋白的表达亦上调(P<0.05)。CAPE联合TNF-α作用于PC-3细胞后,培养液中IL-6的表达及p-STAT3蛋白的表达均明显低于TNF-α作用后的表达水平(P<0.05)。结论:CAPE能抑制TNF-α引起的IL-6的分泌,从而抑制IL-6引起的STAT3核转位;通过CAPE抑制NF-κB表达,继而影响STAT3等相关细胞信号传导途径,可能成为前列腺癌治疗的一条新途径。     

保存不同时间的大鼠部分肝脏移植后的肝细胞再生

目的 探讨保存不同时间的大鼠部分肝脏移植后的肝细胞再生及其可能机制.方法 采用近交系雄性Lewis大鼠为供、受者,按照实验设计分别将供肝于4℃UW液中保存1 h(冷缺血1 h组)、8 h(冷缺血8 h组)和16 h(冷缺血16 h组).然后进行原位肝移植.移植肝恢复血流前,用3-0丝线结扎供肝左侧中央叶、左外叶及尾状叶,保留右侧的肝叶.即可制成大鼠50%体积肝脏(以下简称"半肝")原位移植模型.术后观察各组移植肝的存活情况和肝细胞再生情况;采用逆转录聚合酶链反应测定肝组织中自细胞介素-6(IL-6)和肿瘤坏死因子α(TNF_n)的表达情况;采用Western印迹法检测肝组织中信号传导与转录因子-3(STAT-3)表达情况;采用免疫组织化学染色检测移植肝组织中细胞周期素DI(Cyelin D1)的表达和肝细胞摄取溴脱氧尿核苷(BrdU)情况.结果 各组手术成功率均为100%.与冷缺血1 h组相比,冷缺血8 h组和冷缺血16 h组移植肝组织中TNF-a(F=67.45,P<0.05)和IL-6(F=287.73,P<0.05)的表达明显增加.STAT-3的表达也明显增强.肝移植后24 h.冷缺血8 h组在胞浆和细胞核内均有Cyclin D1的表达.而冷缺血16 h组移植肝组织中未见明显的Cyclin D1表达.移植后24 h.冷缺血16 h组的BrdU染色阳性的肝细胞数无明显增多,而在冷缺血8 h组可见BrdU染色阳性的肝细胞明显增多(t=19.40,P<0.05).结论冷保存一定时限的大鼠部分肝脏在移植后可获得肝细胞再生,此过程可能通过TNF-α/IL,16/sTAT-3/Cyclin D1/DNA合成的途径进行调节;当冷保存时间达16 h后,肝细胞不能对肝脏再生早期信号起反应.     

Severe Preservation Injury Induces Il-6/STAT3 Activation with Lack of Cell Cycle Progression After Partial Liver Graft Transplantation

Partial liver graft transplantation is a surgical advance developed to overcome severe donor shortage. Survival of these grafts involves recovery from cold ischemia and reperfusion (CIR) injury, immediate regeneration and maintenance of function. Here we examined the outcome of partial liver grafts in comparison to whole grafts following CIR injury. Lewis rats subjected to orthotopic liver transplantation (OLT) with whole grafts preserved in Viaspan were compared to rats receiving 50% and 30% grafts. Outcome was analyzed by survival and regeneration. Transplantation was associated with 100% survival for all grafts, whereas 16 h preservation resulted in 100%, 20% and 0% survival in animals receiving whole, 50% and 30% grafts, respectively. CIR induced increased IL-6 levels in 50% and 30% grafts, and activation of STAT3. Cell cycle progression (cyclin D1) and regeneration (BrdU) was initiated in all livers preserved for 1 or 8 h, but not in partial grafts preserved for 16 h. In conclusion, partial grafts recover from CIR injury through similar molecular pathways to whole grafts. Partial grafts with severe injury fail to achieve cellular proliferation despite the early initiating signals. This failure could be attributed to the impaired ability of the parenchyma to respond to initiating signals for regeneration.     

冷保存对肝移植术后肝内胆管微循环的影响

目的探讨供肝冷保存对肝移植术后肝内胆管微循环的影响。方法实验大鼠随机分为假手术组（SO组）、供肝冷保存1h组（CP1h组）、供肝冷保存24h组（CP24h组）。采用重建肝动脉的大鼠肝移植模型。在肝移植术后的不同时间点,观察肝内胆管组织损伤程度;经肝动脉注入微球,光镜下行肝组织汇管区内微球计数;采用间接免疫荧光双染技术检测肝组织汇管区微小血管内皮细胞eNOS、El＂-1和ICAM-1的表达,并采用原位杂交技术检测其mRNA的表达。结果冷保存再灌注可引起肝内胆管结构改变,冷保存时间越长损害程度越重。冷保存再灌注可引起肝组织汇管区内微球数量增加,且时间越长微球数量增加越明显。冷保存再灌注可引起汇管区微小血管内皮细胞eNOS蛋白及mRNA表达水平降低,而ET-1和ICAM-1的蛋白及mRNA表达水平升高。结论冷保存可引起大鼠移植肝脏肝内胆管微循环及其内皮细胞功能明显改变,微循环障碍可能在肝内胆管冷保存再灌注损伤中起重要作用。     

Human amniotic fluid stem cells attenuate cholangiocyte apoptosis in a bile duct injury model of liver ductal organoids

PurposeBiliary atresia (BA) is a fibro-obliterative cholangiopathy that involves both extrahepatic and intrahepatic bile ducts in infants. Cholangiocyte apoptosis has an influence on the fibrogenesis process of bile ducts and the progression of liver fibrosis in BA. Human amniotic fluid stem cells (hAFSCs) are multipotent cells that have ability to inhibit cell apoptosis. We aimed to investigate whether hAFSCs have the potential to attenuate cholangiocyte apoptosis and injury induced fibrogenic response in our ex vivo bile duct injury model of liver ductal organoids.MethodsThe anti-apoptotic effect of hAFSCs was tested in the acetaminophen-induced injury model of neonatal mouse liver ductal organoids (AUP #42681) by using direct and indirect co-culture systems. Cell apoptosis and proliferation were evaluated by immunofluorescent staining. Expression of fibrogenic cytokines was analyzed by RT-qPCR. Data were compared using one-way ANOVA with post hoc test.ResultsIn our injury model, liver ductal organoids that were treated with hAFSCs in both direct and indirect co-culture systems had a significantly smaller number of apoptotic cholangiocytes and decreased expression of fibrogenic cytokines, transforming growth factor beta-1 (TGF-β1) and platelet-derived growth factor-BB (PDGF-BB). Moreover, hAFSCs increased cholangiocyte proliferation in injured organoids.ConclusionhAFSCs have the ability to protect the organoids from injury by decreasing cholangiocyte apoptosis and promoting cholangiocyte proliferation. This protective ability of hAFSCs leads to inhibition of the fibrogenic response in the injured organoids. hAFSCs have high therapeutic potential to attenuate liver fibrogenesis in cholangiopathic diseases such as BA.     

移植肝胆汁成分失衡与胆管损伤关系的实验研究

目的 探讨移植肝术后早期胆汁主要成分的变化规律及其在胆管损伤中的意义.方法 大鼠被随机分为对照组(A组)、供肝冷保存1h组(B组)和供肝冷保存12h组(C组),移植组均采用动脉化的大鼠肝移植胆道外引流模型.术后按1、3、5、7、10和14d共6个时相点采集标本并进行相关生化及病理学指标的检测.结果 冷保存/再灌注损伤对大鼠移植肝胆汁主要成分有显著影响.术后14 d内胆盐分泌恢复的速度明显高于磷脂分泌恢复的速度,使得在术后1～14 d的时间内,胆盐与磷脂的分泌量之比为正常水平的2～5倍,并且供肝冷保存时间呈正相关;进一步分析提示移植肝胆盐分泌量与γ-谷氨酰转肽酶、碱性磷酸酶分泌量及胆管受损程度显著相关.结论 冷保存/再灌注损伤可以导致移植肝胆汁成分失衡,在移植肝胆管损伤的发生发展中可能具有一定作用.     

Prolonged administration of secretin to normal rats increases biliary proliferation and secretin-induced ductal secretory activity

Background and aim

Cholangiocyte proliferation is coordinately regulated by a number of gastrointestinal hormones/peptides, some of which display stimulatory effects and some have inhibitory actions on cholangiocyte proliferation. Enhanced biliary proliferation [for example after bile duct ligation (BDL) and partial hepatectomy] is associated with increased expression of secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR) and Cl^–/HCO₃^– anion exchanger 2 and secretin-stimulated ductal secretion, whereas loss/damage of bile ducts [for example after acute carbon tetrachloride (CCl₄) administration] is associated with reduced secretin-stimulated ductal secretory activity. There is growing information regarding the role of gastrointestinal hormones the regulation of biliary growth. For example, while gastrin, somatostatin and serotonin inhibit bile duct hyperplasia of cholestatic rats by downregulation of cAMP signaling, secretin has been shown to stimulate the proliferation of normal mice by activation of cyclic adenosine 3'',5''-monophosphate (cAMP)-dependent signaling. However, no information exists regarding the stimulatory effects of secretin on biliary proliferation of normal rats. Thus, we evaluated the in vivo and in vitro effect of secretin on biliary proliferation, the expression of markers key of ductal secretion and secretin-stimulated ductal secretion.

Methods

Normal male rats were treated with saline or secretin (2.5 nmoles/kg BW/day by osmotic minipumps for one week). We evaluated: (I) intrahepatic bile duct mass (IBDM) in liver sections and PCNA expression in purified cholangiocytes; (II) SR and CFTR mRNA expression and secretin-stimulated cAMP levels in purified cholangiocytes; and (III) secretin-stimulated bile and bicarbonate secretion in bile fistula rats. In vitro, normal rat intrahepatic cholangiocyte lines (NRIC) were treated with BSA (basal) or secretin (100 nM) for 24 to 72 hours in the absence/presence of a PKA or a MEK inhibitor before evaluating proliferation by MTS assays.

Results

Prolonged administration of secretin to normal rats increased IBDM and PCNA expression in purified cholangiocytes compared to saline-treated normal rats. Also, secretin increased the expression of proteins (SR and CFTR) that are key in the regulating ductal secretion and enhanced secretin-stimulated cAMP levels and bile and bicarbonate secretion. In vitro, secretin increased the proliferation of NRIC, increase that was prevented by PKA and MAPK inhibitors.

Conclusions

We have demonstrated that secretin stimulates both in vivo and in vitro biliary proliferation and secretin-stimulated ductal secretory activity in normal rats. We suggest that the stimulatory effect of secretin on biliary proliferation and secretion may be important for preventing biliary dysfunction during ductopenic disorders.     

Impact of STAT1-siRNA transfection on the expressions of STAT3 and transforming growth factor β1 in human glomerulur mesangial cells induced by high glucose

Objective To observe the cell proliferation and the protein expression of STAT1,phosphorylation of STAT1 (p-STAT1), STAT3, p-STAT3 and transforming growth factor β1 (TGF-β1) in human glomerulur mesangial cells (HMCs) induced by high glucose after STAT1-siRNA transfection. Methods Three STAT1-siRNA sequences were designed and synthetized. HMCs in 6-well plate were transiently transfected with STAT1-siRNA using Lipofectamine 2000. After transfection for 48 h or 72 h, STAT1 mRNA and protein expression were detected by real-time PCR and Western blotting, respectively, to choose the effective sequence in later experiments. After transfection for 24 h and stimulated with 25 mmol/L glucose for 24 h, 48 h, 72 h, cell proliferation was measured by MTT assays, the protein expressions of STAT1, p-STAT1, STAT3 and p-STAT3 were detected by Western blotting, the expression of TGF-β1 was detected by ELISA in each group. Results High glucose could stimulate HMCs proliferation. The protein expressions of p-STAT1, p-STAT3 and TGF-β1 were increased in the group stimulated by high glucose (P＜0.05). The protein expressions of p-STAT3 and TGF-β1 were further increased in HMCs induced by high glucose after STAT1-siRNA transfection (P＜0.05). Conclusions Under high glucose conditions, JAK-STAT signal transduction pathway of HMCs can be activated, then it is far greater when HMCs are induced by high glucose after STAT1-siRNA transfection. The secretion of TGF-β1 is increased in HMCs under the state of high glucose, and it is further increased after STAT1-siRNA transfection, which is related to the kidney fibrosis.