转化生长因子-β在骨髓间充质干细胞治疗实验性自身免疫性重症肌无力机制中的作用

目的探讨转化生长因子-β(TGF-β)在骨髓间充质干细胞(BMSCs)治疗实验性自身免疫性重症肌无力(EAMG)机制中的作用。方法分离培养健康Lewis大鼠BMSCs,并进行大量体外扩增;以大鼠来源的乙酰胆碱受体(R-AChR)2次免疫Lewis大鼠,建立EAMG模型;第2次免疫的同时,经尾静脉移植BMSCs,1×107个/只,依据Lennon评分标准,进行体重测量和临床体征评定。并通过体外实验进一步探讨TGF-β在治疗EAMG过程中的具体机制。结果BMSCs移植明显缓解了EAMG的临床症状,临床评分及体重变化差异均有统计学意义,表明治疗有效;体外实验结果显示,BMSCs能通过TGF-β的分泌影响AChR特异性Th17/Treg细胞亚群的分布及其相关因子的分泌,以anti-TGF-β抗体封闭后,这种调节作用在一定程度上被抑制。结论BMSCs通过细胞因子TGF-β,能在一定程度上调节AChR特异性Th17/Treg细胞亚群的平衡,从而起到治疗EAMG的作用。     

骨髓间充质干细胞对大鼠脑胶质瘤C6细胞系分化的影响

目的探讨骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs)对大鼠脑胶质瘤C6细胞系分化的影响及其相关机制。方法用Transwell小室共培养BMSCs与C6细胞,采用细胞计数法检测C6细胞增殖水平;流式细胞术检测C6细胞的细胞周期;免疫荧光与免疫组化法分别检测波形蛋白(vimentin)和胶质纤维酸性蛋白(Glia lfibrillary acidic protein,GFAP)的表达;生化法检测谷氨酰胺合成酶(Glutamine synthetase,GS)的活性。结果 BMSCs对C6细胞的增殖具有抑制作用;与BMSCs进行双层培养72h的C6细胞出现G0/G1期细胞周期阻滞,双层培养组的C6细胞GFAP表达明显增强,同时vimentin表达减弱,并且这两种分化标志物在细胞内的分布及排列发生了改变;双层培养组C6细胞内GS活性升高。结论 BMSCs能诱导C6细胞向成熟星形胶质细胞分化,可能与BMSCs分泌的某些细胞因子有关。     

阻断IFN-β改善脂多糖所致T淋巴细胞增殖抑制的实验研究

目的：探究细胞因子在脂多糖所致脓毒症T淋巴细胞免疫抑制中的作用。方法：以革兰阴性细菌成分脂多糖诱导脓毒症T淋巴细胞免疫抑制,采用流式细胞分析检测对照组和模型组树突状细胞的细胞因子白介素-1α(IL-1α)、IL-6、肿瘤坏死因子-α(TNF-α)、IL-10、转化生长因子-β(TGF-β)、干扰素-α(IFN-α)、IFN-β的表达情况。然后分别阻断树突状细胞的细胞因子进行混合淋巴细胞共培养实验,采用Brd U细胞增殖检测试剂盒检测阻断后T淋巴细胞的免疫增殖抑制的逆转情况。结果：与对照组相比,模型组树突状细胞的细胞因子IL-1α、IL-6、TNF-α、TGF-β、IFN-β阳性百分比显著升高,IL-10、IFN-α阳性百分比无统计学差异。阻断细胞因子后,与模型组相比,αIFN-β+模型组T淋巴细胞增殖反应显著升高。结论：脂多糖所致脓毒症过程中树突状细胞的细胞因子IL-1α、IL-6和TNF-α表达上调可能参与促炎反应过程。细胞因子IFN-β表达上调可能参与淋巴细胞凋亡的免疫抑制过程。阻断IFN-β可改善脂多糖诱导的脓毒症T淋巴细胞增殖抑制现象。     

血脂康对新生大鼠心肌成纤维细胞增殖及胶原合成的影响

目的观察血脂康对血管紧张素Ⅱ(AngⅡ)诱导的新生大鼠心肌成纤维细胞(CFs)增殖及胶原合成的影响,并探讨可能的分子机制。方法采用胰酶消化法分离培养Sprague-Dawley大鼠心肌成纤维细胞,建立AngⅡ诱导CFs增殖的模型,以MTT比色法和流式细胞仪检测细胞周期分析法观察血脂康对CFs数目和细胞周期的影响。AngⅡ及不同浓度血脂康作用48 h后,用天狼星红染色法检测培养上清中胶原的含量;ELISA法检测细胞培养上清液中TGF-β1蛋白表达;RT-PCR法检测胶原和TGF-β1 mRNA表达。结果AngⅡ对CFs增殖有明显促进作用,血脂康可明显抑制AngⅡ诱导的CFs增殖,且呈剂量依赖性;血脂康可增加G0/G1期细胞百分率,降低S、G2/M期细胞百分率,降低胶原含量、TGF-β1蛋白及胶原和TGF-β1 mRNA的表达。结论血脂康能抑制AngⅡ诱导的CFs增殖和胶原的产生,其作用可能是通过抑制TGF-β1表达实现的。     

RNA干扰LKB1基因对前列腺癌细胞转化生长因子-β1表达的影响

目的探讨沉默肝激酶B1/丝氨酸-苏氨酸激酶11(liver kinase B1/serine-threonine kinase 11,LKB1/STK11),基因对前列腺癌细胞株RWPE-1中转化生长因子-β1(transforming growth factor-β1,TGF-β1)的调控作用。方法构建3个sh RNA LKB1重组载体,转染至高表达LKB1的前列腺癌细胞株RWPE-1中,RT-PCR、Western blot法分别检测LKB1及TGF-β1 m RNA及蛋白表达水平;ELISA法检测细胞培养液上清中TGF-β1的分泌水平。结果构建获得的3种sh RNA LKB1表达载体,均能抑制LKB1基因的表达,其中以LKB1-sh RNA1沉默效果最强,其对LKB1 m RNA水平的抑制率为74.20%,对蛋白水平的抑制率为71.66%;转染重组质粒LKB1-sh RNA1后,TGF-β1的m RNA、蛋白表达及分泌水平均明显增加(P﹤0.05)。结论 LKB1基因沉默后,TGF-β1的表达明显增高,LKB1参与调节前列腺癌细胞中TGF-β1的表达,提示LKB1基因可作为研究前列腺癌发生发展的分子机制的新靶点。     

神经干细胞对去血清诱导PC12细胞凋亡的作用

肾上腺嗜铬细胞瘤细胞(PC12 cells)去血清后,其凋亡与多巴胺能神经元的凋亡有许多共同之处,今通过研究神经干细胞(NSCs)对去血清诱导的PC12细胞凋亡的作用,进一步为NSCs移植治疗神经系统疾病提供相应的实验和理论依据.将正常培养的PC12细胞与NSCs以不同的方式进行去血清共培养,观察PC12细胞的形态,检测PC12细胞的活性,计算PC12细胞的存活率,同时检测培养基中胶质细胞源神经营养因子(GDNF)的浓度,分析不同培养方式下NSCs对去血清诱导凋亡的PC12细胞的作用以及NSCs与去血清诱导凋亡的PC12细胞共培养后,其分泌GDNF的能力.结果表明:①去血清诱导PC12细胞凋亡呈时间依赖性,去血清72 h后,PC12细胞存活的比率为44.25%;②NSCs培养基对去血清诱导凋亡的PC12细胞没有明显的保护作用;③NSCs培养上清及NSCs对去血清诱导凋亡的PC12细胞具有保护作用;④NSCs与去血清诱导凋亡的PC12细胞共培养后,分泌GDNF的能力增强.     

肝癌细胞上清液对成纤维细胞表达α-平滑肌肌动蛋白、环氧合酶-2和肝细胞生长因子的影响

目的观察小鼠肝癌细胞上清液对成纤维细胞增殖及表达α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、环氧合酶-2(cyclooxygenase-2,COX-2)、肝细胞生长因子(hepatocyte growth factor,HGF)的影响。方法制备小鼠成纤维细胞,并分别用普通培养液、小鼠肝癌细胞上清液的条件培养液及含转化生长因子-β1(transforming growth factor-β1,TGF-β1)的培养液培养成纤维细胞。采用MTT法检测3种培养液培养的成纤维细胞的增殖活力;RT-PCR和免疫组织化学法检测3种培养液培养的成纤维细胞中α-SMA、COX-2、HGF基因mRNA的转录水平及蛋白的表达情况。结果条件培养液及含TGF-β1的培养液对成纤维细胞的增殖均有促进作用,但条件培养液的促进作用更强。普通培养液培养的成纤维细胞在第5天时α-SMA基因mRNA的转录水平最高,含TGF-β1的培养液及条件培养液培养的成纤维细胞在第3天时α-SMA基因mRNA的转录水平最高,且明显高于普通培养液在第5天时的转录水平,第3天以后转录水平稳定;普通培养液培养的成纤维细胞中无COX-2和HGF转录;条件培养液及含TGF-β1的培养液培养的成纤维细胞在第5天时COX-2和HGF基因mRNA的转录水平最高,第5天后无明显下降。不同培养液培养的成纤维细胞中α-SMA蛋白在胞浆、胞膜均有不同程度的表达;普通培养液培养的成纤维细胞中COX-2和HGF蛋白不表达,而含TGF-β1的培养液及条件培养液培养的成纤维细胞中COX-2和HGF蛋白为阳性表达,表达部位均为胞浆。结论小鼠肝癌细胞上清液能有效、稳定地将成纤维细胞激活为稳定表达COX-2和HGF的肌成纤维细胞,其有望成为在细胞移植中促进移植细胞存活及血管重建的细胞。     

姜黄素对转化生长因子-β1诱导的乳腺癌MDA-MB-231细胞基质金属蛋白酶-9表达及其侵袭能力的影响

目的研究姜黄素对转化生长因子-β1(Transforming growth factor-beta 1,TGF-β1)诱导的人乳腺癌MDA-MB-231细胞基质金属蛋白酶-9(Matrix metallopeptidase-9,MMP-9)表达及其侵袭能力的影响,探讨姜黄素在防治TGF-β1诱导的乳腺癌侵袭转移中可能的作用机制。方法采用CCK-8法检测姜黄素对MDA-MB-231细胞的细胞毒性作用。将MDA-MB-231细胞分为阴性对照组(无血清RPMI1640培养基/DMSO)、TGF-β1处理组(无血清RPMI1640培养基/DMSO+10 ng/ml TGF-β1),姜黄素+TGF-β1处理组(无血清RPMI1640培养基+5、7.5、10μmol/L姜黄素+10 ng/ml TGF-β1)。处理24 h后,采用侵袭小室试验观察细胞的侵袭能力;处理不同时间后,采用Western blot法检测细胞MMP-9、p-Smad2、p-ERK1/2以及p-p38的表达,明胶酶谱法检测各组细胞上清液中MMP-9的活性变化;以ERK特异性抑制剂PD98059、p38特异性抑制剂SB202580、姜黄素和/或TGF-β1处理细胞48 h,采用Western blot和明胶酶谱法检测MMP-9的表达。结果低浓度姜黄素(≤10μmol/L)对MDA-MB-231细胞无明显的细胞毒性作用;姜黄素呈剂量依赖性明显减少了TGF-β1诱导的细胞穿膜数量(P<0.001);姜黄素可显著抑制TGF-β1诱导的MDA-MB-231细胞MMP-9、p-Smad2、p-ERK1/2及p-p38蛋白的表达,且呈浓度、时间依赖性(P<0.05);姜黄素随浓度的增加,对TGF-β1诱导的MDA-MB-231细胞MMP-9活性的抑制作用明显增强(P<0.05);PD98059抑制MMP-9蛋白表达及活性的作用与姜黄素相似,而SB202580对MMP-9无明显影响。结论姜黄素可能通过TGF-β/Smad、TGF-β/ERK信号通路下调TGF-β1诱导的MMP-9的表达及其活性,从而抑制肿瘤的侵袭能力。     

过表达脑红蛋白对水溶性β-淀粉样蛋白片段1-42诱导SH-SY5Y细胞线粒体损伤的保护作用及其机制

目的观察过表达脑红蛋白(neuroglobin,NGB)对水溶性β-淀粉样蛋白片段1-42(Amyloid-beta 1-42,Aβ_(1-42))诱导SH-SY5Y细胞线粒体损伤的保护作用,并探讨其机制。方法体外培养SH-SY5Y细胞,经Aβ_(1-42)预处理后转染质粒p EGFP-NGB及空载体p EGFP-N1,同时设空白对照组(未转染)。MTT法、流式细胞术及JC-1染色法分别检测过表达NGB对Aβ_(1-42)诱导损伤的SH-SY5Y细胞存活率、细胞凋亡及线粒体膜电位的影响,采用相应试剂盒检测细胞内半胱氨酸蛋白酶3(caspase 3)、caspase 9、ATP及细胞色素C(cytochrome C,cyto C)氧化酶活性;Western blot法检测细胞中cyto C、凋亡蛋白酶激活因子-1(apoptosis protease activating factor-1,Apaf-1)、caspase 3及caspase 9蛋白的表达水平。结果过表达NGB可显著提高Aβ_(1-42)诱导损伤SH-SY5Y细胞的存活率(P0.001)及细胞中cyto C氧化酶、ATP的活性(P0.01);可显著抑制损伤细胞的凋亡(P0.05)、线粒体膜电位的降低(P0.001)及细胞中caspase 3、caspase 9的活性(P0.01);可明显降低损伤细胞内cyto C、Apaf-1、caspase 3及caspase 9蛋白的表达水平(P0.05)。结论 NGB可显著抑制Aβ_(1-42)诱导的SH-SY5Y细胞损伤,促进其增殖,其机制可能与NGB恢复线粒体功能及抑制cyto C触发细胞凋亡密切相关。     

大鼠骨髓间充质干细胞向肝样细胞的定向诱导分化

目的 探讨肝细胞生长因子(Hepatocyte growth factor,HGF)和碱性成纤维细胞生长因子(Basic fibroblast growthfactor,bFGF)诱导大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BM-MSCs)分化为肝样细胞的可行性。方法取SD大鼠股骨骨髓,直接贴壁法分离纯化BM-MSCs,并体外传代,流式细胞术和成骨诱导对其进行鉴定。取第3代BM-MSCs,分为2组:实验组用HGF(20 ng/ml)和bFGF(10 ng/ml)进行诱导,阴性对照组不加诱导剂,倒置显微镜下观察细胞形态变化;RT-PCR法检测诱导后细胞甲胎蛋白(Alpha fetoprotein,AFP)和白蛋白(Albumin,ALB)基因mRNA的转录水平;免疫细胞化学染色法检测诱导后细胞的AFP和ALB蛋白的表达。结果第3代BM-MSCs表型标志和功能特性均符合MSCs的特点。BM-MSCs经HGF和bFGF诱导后呈肝样细胞形态。实验组细胞可检测出AFP和ALB基因mRNA的表达。实验组细胞诱导后第7天,AFP蛋白开始表达,第14天时表达降低,第21天时不表达;ALB于诱导后第14天出现表达,并随诱导时间的延长表达逐渐增加。结论 HGF和bFGF具有体外诱导BM-MSCs向肝样细胞分化的作用。     

Adult Renal Stem/Progenitor Cells Can Modulate T Regulatory Cells and Double Negative T Cells

Adult Renal Stem/Progenitor Cells (ARPCs) have been recently identified in the human kidney and several studies show their active role in kidney repair processes during acute or chronic injury. However, little is known about their immunomodulatory properties and their capacity to regulate specific T cell subpopulations. We co-cultured ARPCs activated by triggering Toll-Like Receptor 2 (TLR2) with human peripheral blood mononuclear cells for 5 days and 15 days and studied their immunomodulatory capacity on T cell subpopulations. We found that activated-ARPCs were able to decrease T cell proliferation but did not affect CD8⁺ and CD4⁺ T cells. Instead, Tregs and CD3⁺ CD4^- CD8^- double-negative (DN) T cells decreased after 5 days and increased after 15 days of co-culture. In addition, we found that PAI1, MCP1, GM-CSF, and CXCL1 were significantly expressed by TLR2-activated ARPCs alone and were up-regulated in T cells co-cultured with activated ARPCs. The exogenous cocktail of cytokines was able to reproduce the immunomodulatory effects of the co-culture with activated ARPCs. These data showed that ARPCs can regulate immune response by inducing Tregs and DN T cells cell modulation, which are involved in the balance between immune tolerance and autoimmunity.     

Notch2胞内段基因过表达对K562细胞增殖的影响及其机制

目的探讨外源性Notch2胞内段基因过表达对慢性粒细胞白血病(Chronic myeloid leukemia,CML)细胞株K562增殖的影响及其机制。方法将携带Notch2胞内段(ICN2)基因的质粒转染K562细胞,MTT法检测细胞的增殖;流式细胞仪检测细胞的细胞周期分布;RT-PCR检测Notch2基因全长mRNA的转录,Western blot检测Notch2蛋白的表达;RT-PCR检测Notch通路下游靶基因Hes1、Hey1及细胞增殖、凋亡相关基因numb、Bcl-2、NF-κB和TGF-β1的mRNA转录水平。结果与未转染组相比,转染组K562细胞数量减少,增殖显著受抑(P<0.01);转染48 h后的K562细胞G1期细胞比例显著增多(P<0.01),S期细胞比例显著减少(P<0.01);Notch2基因mRNA及下游靶基因Hes1和Hey1 mRNA转录水平均明显增强(P<0.01),Notch2蛋白表达量增加(P<0.01);numb基因mRNA转录水平无变化;Bcl-2表达下调,NF-κB和TGF-β1表达上调。结论Notch2胞内段基因过表达可抑制K562细胞增殖,其机制可能是通过上调TGF-β1和NF-κB基因表达及下调Bcl-2基因表达,将细胞阻滞于G1期来实现的。     

Nonylphenol Induces Apoptosis through ROS/JNK Signaling in a Spermatogonia Cell Line

Nonylphenol (NP) is an endocrine-disruptor chemical that negatively affects reproductive health. Testes exposure to NP results in testicular structure disruption and a reduction in testicular size and testosterone levels. However, the effects of NP on spermatogonia in testes have not been fully elucidated. In this study, the molecular mechanisms of NP in GC-1 spermatogonia (spg) cells were investigated. We found that cell viability significantly decreased and apoptosis increased in a dose-dependent manner when GC-1 spg cells were exposed to NP. Furthermore, the expression levels of the pro-apoptotic proteins increased, whereas anti-apoptosis markers decreased in NP-exposed GC-1 spg cells. We also found that NP increased reactive oxygen species (ROS) generation, suggesting that ROS-induced activation of the MAPK signaling pathway is the molecular mechanism of NP-induced apoptosis in GC-1 spg cells. Thus, NP could induce c-Jun phosphorylation; dose-dependent expression of JNK, MKK4, p53, and p38; and the subsequent inhibition of ERK1/2 and MEK1/2 phosphorylation. The genes involved in apoptosis and JNK signaling were also upregulated in GC-1 spg cells treated with NP compared to those in the controls. Our findings suggest that NP induces apoptosis through ROS/JNK signaling in GC-1 spg cells.     

NGF/TRKA Promotes ADAM17-Dependent Cleavage of P75 in Ovarian Cells: Elucidating a Pro-Tumoral Mechanism

Nerve growth factor (NGF) and its high-affinity receptor TRKA are overexpressed in epithelial ovarian cancer (EOC) displaying a crucial role in the disease progression. Otherwise, NGF interacts with its low-affinity receptor P75, activating pro-apoptotic pathways. In neurons, P75 could be cleaved by metalloproteinases (α and γ-secretases), leading to a decrease in P75 signaling. Therefore, this study aimed to evaluate whether the shedding of P75 occurs in EOC cells and whether NGF/TRKA could promote the cleavage of the P75 receptor. The immunodetection of the α-secretase, ADAM17, TRKA, P75, and P75 fragments was assessed by immunohisto/cytochemistry and Western blot in biopsies and ovarian cell lines. The TRKA and secretases’ inhibition was performed using specific inhibitors. The results show that P75 immunodetection decreased during EOC progression and was negatively correlated with the presence of TRKA in EOC biopsies. NGF/TRKA increases ADAM17 levels and the fragments of P75 in ovarian cells. This effect is abolished when cells are previously treated with ADAM17, γ-secretase, and TRKA inhibitors. These results indicate that NGF/TRKA promotes the shedding of P75, involving the activation of secretases such as ADAM17. Since ADAM17 has been proposed as a screening marker for early detection of EOC, our results contribute to understanding better the role of ADAM17 and NGF/TRKA in EOC pathogenesis, which includes the NGF/TRKA-mediated cleavage of P75.     

Less Expression of Prohibitin Is Associated with Increased Paired Box 2 (PAX2) in Renal Interstitial Fibrosis Rats

Prohibitin (PHB) and paired box 2 (PAX2) are associated with the development of renal interstitial fibrosis (RIF). This study was performed to investigate whether or not the PHB could regulate the PAX2 gene expression in unilateral ureteral obstruction (UUO) in rats. Eighty Wistar male rats were randomly divided into two groups: sham operation group (SHO) and model group subjected to unilateral ureteral obstruction (GU), n = 40, respectively. The model was established by left ureteral ligation. Renal tissues were collected at 14-day and 28-day after surgery. RIF index, protein expression of PHB, PAX2, transforming growth factor-βl (TGF-β1), α-smooth muscle actin (α-SMA), collagen-IV (Col-IV), fibronectin (FN) or cleaved Caspase-3, and cell apoptosis index in renal interstitium, and mRNA expressions of PHB, PAX2 and TGF-β1 in renal tissue were detected. When compared with those in SHO group, expression of PHB (mRNA and protein) was significantly reduced, and expressions of PAX2 and TGF-β1 (protein and mRNA) were markedly increased in the GU group (each p < 0.01). Protein expressions of α-SMA, Col-IV, FN and cleaved Caspase-3, and RIF index or cell apoptosis index in the GU group were markedly increased when compared with those in the SHO group (each p < 0.01). The protein expression of PHB was negatively correlated with protein expression of PAX2, TGF-β1, α-SMA, Col-IV, FN or cleaved Caspase-3, and RIF index or cell apoptosis index (all p < 0.01). In conclusion, less expression of PHB is associated with increased PAX2 gene expression and RIF index in UUO rats, suggesting that increasing the PHB expression is a potential therapeutic target for prevention of RIF.