干扰硫氧还蛋白-1对H_2O_2诱导PC12细胞超氧化损伤的保护作用

目的探讨硫氧还蛋白-1(sulfiredoxin-1,Srxn-1)对H2O2诱导的PC12细胞超氧化损伤的保护作用及其可能机制。方法在Lipofectamine 2000的介导下,分别将pLVT574、pLVT575和pLVT576干扰质粒转染PC12细胞,同时设空白对照组(正常培养细胞)及阴性对照组(转染pLVT4质粒),普通显微镜和倒置荧光显微镜下观察转染效率,并采用RT-PCR和Western blot法分别检测转染细胞中Srxn-1基因mRNA转录和蛋白表达的水平。采用不同终浓度(50、100、150、200、250、300μmol/L)的H2O2诱导PC12细胞,建立超氧化细胞损伤模型,MTT法检测Srxn-1对细胞存活率的影响,并采用超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA)试剂盒检测细胞SOD活性及MDA含量。结果镜下观察可见,转染后细胞死亡数较多,胞体缩小;有绿色荧光,转染效率达50%。si-Srxn-1-575组细胞中Srxn-1基因mRNA转录及蛋白表达水平较空白对照组明显下降(P0.05)。终浓度为200μmol/L的H2O2处理12 h后,PC12细胞的存活率为66%;H2O2+si-Srxn-1-575组的细胞存活率(49.3%)明显低于空白对照组(65.2%)(P0.05)。H2O2+si-Srxn-1-575组细胞中SOD活性[(10.319±1.362)U/mg pro]明显低于H2O2+空白对照组[(15.7±1.138)U/mg pro],MDA含量[(5.507±0.297)nmol/mg pro]明显高于H2O2+空白对照组[(3.41±0.281)nmol/mg pro](P均0.05)。结论 Srxn-1对H2O2损伤的PC12细胞具有保护作用,其机制可能与其发挥内源性抗氧化作用有关。     

牛毛水解物对PC12细胞氧化损伤的抑制

通过考察H₂O₂浓度和作用时间对细胞活力的影响,确定PC12细胞氧化损伤模型的建立条件;通过细胞活力及形态分析,测定牛毛水解物对H₂O₂致PC12细胞氧化损伤的抑制作用。结果表明,H₂O₂浓度0.15 mmol/L、作用时间2 h为建立PC12细胞氧化损伤模型的理想条件;不同质量浓度的牛毛水解物 (10、20、40、60、80 mg/L)可有效抑制H₂O₂对PC12细胞的氧化损伤,使细胞活力达到58.79%、67.53%、76.89%、85.96%、87.58%,也能在一定程度上修复氧化损伤的细胞,使其活力恢复至53.69%、57.18%、62.56%、65.68%、67.23%,还能有效地维持细胞原有的形态特征。     

嗜酸乳杆菌上清液制备及其对PC12细胞增殖的影响

本实验首先对嗜酸乳杆菌进行相应的处理,使其复苏和活化,在进行生长曲线测定的过程中,发现其生长28h左右达到稳定期.之后将以28h为培养时间的L.a.s按不同体积分数作用于PC12细胞,荧光染色和CCK-8细胞增殖检测结果表明,PC12细胞能够正常生长,研究发现加入L.a.s体积分数为6％时,PC12细胞的存活率提高至对...     

川牛膝多糖对H2O2诱导PC12细胞氧化损伤保护作用的研究

目的:探讨川牛膝多糖组分对H_2O_2诱导PC12细胞产生的氧化和凋亡作用,并对其机制进行深入研究。方法:通过H_2O_2诱导PC12细胞,建立神经氧化损伤模型,通过MTT法测定PC12细胞存活率,丙二醛(MDA)、活性氧(ROS)和超氧化物歧化酶(SOD)检测氧化作用,Hoechst 33342法染色观察川牛膝多糖组分对PC12细胞遭受H_2O_2损伤凋亡状态特征。结果:浓度为100μmol·L~(-1) H_2O_2时,PC12细胞产生明显损伤,为最佳造模浓度;川牛膝多糖组分能够减少H_2O_2诱导PC12的损伤程度,减少MDA含量、降低ROS的活性、提高SOD的活性,并减轻细胞凋亡作用。结论:川牛膝多糖能够减少H_2O_2对PC12细胞的氧化作用,增强细胞保护作用。     

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.     

蛋白酶体抑制剂诱导的PC12细胞帕金森病模型中血红素加氧酶-1的差异表达

目的探讨蛋白酶体抑制剂(Proteasome inhibitor,PSI)诱导的PC12细胞帕金森病(Parkinson disease,PD)模型中血红素加氧酶-1(Hemeoxygenase-1,HO-1)的差异表达,为深入研究PD的发病机制提供理论依据。方法取培养的PC12细胞,加入终浓度为10μmol/L的PSI,建立PSI诱导的PC12细胞模型,以加入终浓度为10μmol/L的二甲基亚砜(DMSO)为对照组。经HE、AO&EB及α-SYN染色进行鉴定;PSI作用48h后提取蛋白,应用荧光差异凝胶电泳(DIGE)系统获得差异蛋白点,运用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOFProMS)鉴定差异蛋白。结果模型组与对照组比较,PSI作用48h可见细胞内嗜酸性类Lewy小体形成及细胞凋亡,凋亡率达(24.74±4.55)%。模型组与对照组比较,HO-1表达量显著增加。结论在泛素-蛋白酶体系统(Ubiquitin-proteasome system,UPS)功能障碍诱发PD过程中,氧化应激反应发挥着一定的作用。     

槲皮素对皮质酮损伤的pc12细胞的保护作用

利用pc12细胞的抗抑郁损伤模型,研究了槲皮素的抗抑郁作用.槲皮素、氯丙咪嗪与0.1 mmol·L-1皮质酮共孵pc12细胞48 h,用MTT比色法判断细胞损伤程度.结果表明,高浓度皮质酮处理pc12细胞后,细胞存活率比正常细胞降低;当给予槲皮素或氯丙咪嗪后,细胞存活率升高,表明细胞损伤减少或接近正常.与经典抗抑郁剂氯丙咪嗪的作用效果一样,槲皮素对皮质酮损伤的pc12细胞有明显的保护作用,有抗抑郁作用.     

糖基化终末产物对大鼠肾小球系膜细胞尾加压素Ⅱ及G蛋白偶联受体mRNA表达的影响

目的观察不同浓度糖基化终末产物(Advanced glycation end products,AGEs)及AGEs作用不同时间对大鼠肾小球系膜细胞尾加压素Ⅱ(UrotensinⅡ,UⅡ)及G蛋白偶联受体(G-protein-couple receptor,GPR14)mRNA表达的影响。方法制备AGE-BSA,体外培养大鼠肾小球系膜细胞(Mesangial Cells,MC),加入不同浓度的AGE-BSA(终浓度分别为0、25、50、100和200mg/L),37℃孵育24h;加入100mg/L AGE-BSA,分别培养0、2、8、16和24h,以不含葡萄糖的BSA作为对照。收集细胞,采用RT-PCR检测各组MC UⅡ及GPR14mRNA的表达。结果 AGE-BSA各组MC UⅡ及GPR14mRNA的表达量均随AGEs浓度的增加而增加,50、100和200mg/L与0mg/L组比较,差异有统计学意义(P<0.05);100mg/L AGE-BSA各组MC UⅡ及GPR14mRNA的表达量随着作用时间的延长而增加,作用8、16、24h组与0h组比较,差异有统计学意义(P<0.05)。BSA组MC UⅡ及GPR14mRNA的表达量无明显增加(P>0.05)。结论 AGEs能上调大鼠MC UⅡ及GPR14mRNA的表达。     

培养条件对HBsAg转基因人参细胞的生长及表达量的影响

目的研究培养条件对HBsAg转基因人参细胞生长及HBsAg表达量的影响。方法通过单因子实验,分别考察碳源、氮源、有机成分对HBsAg转基因人参细胞生长和HBsAg表达量的影响,并通过不同组合实验,探索氮源浓度、有机成分和起始pH的最佳组合。结果在3%蔗糖、10mmol/L氮源、0·25%乳清蛋白和起始pH6·2的情况下,细胞生长量略有增加,HBsAg的表达量增加1倍。结论通过改善培养条件,可以提高HBsAg转基因人参细胞的生长速度和抗原表达量。     

Inhibitory Effects of Saururus chinensis Extract on Receptor for Advanced Glycation End-Products-Dependent Inflammation and Diabetes-Induced Dysregulation of Vasodilation

Advanced glycation end-products (AGEs) and the receptor for AGEs (RAGE) are implicated in inflammatory reactions and vascular complications in diabetes. Signaling pathways downstream of RAGE are involved in NF-κB activation. In this study, we examined whether ethanol extracts of Saururus chinensis (Lour.) Baill. (SE) could affect RAGE signaling and vascular relaxation in streptozotocin (STZ)-induced diabetic rats. Treatment with SE inhibited AGEs-modified bovine serum albumin (AGEs-BSA)-elicited activation of NF-κB and could compete with AGEs-BSA binding to RAGE in a dose-dependent manner. Tumor necrosis factor-α (TNF-α) secretion induced by lipopolysaccharide (LPS)—a RAGE ligand—was also reduced by SE treatment in wild-type Ager^+/+ mice as well as in cultured peritoneal macrophages from Ager^+/+ mice but not in Ager^−/− mice. SE administration significantly ameliorated diabetes-related dysregulation of acetylcholine-mediated vascular relaxation in STZ-induced diabetic rats. These results suggest that SE would inhibit RAGE signaling and would be useful for the improvement of vascular endothelial dysfunction in diabetes.     

The Role of Advanced Glycation End Products and Its Soluble Receptor in Kidney Diseases

Patients with chronic kidney disease (CKD) are more prone to oxidative stress and chronic inflammation, which may lead to an increase in the synthesis of advanced glycation end products (AGEs). Because AGEs are mostly removed by healthy kidneys, AGE accumulation is a result of both increased production and decreased kidney clearance. On the other hand, AGEs may potentially hasten decreasing kidney function in CKD patients, and are independently related to all-cause mortality. They are one of the non-traditional risk factors that play a significant role in the underlying processes that lead to excessive cardiovascular disease in CKD patients. When AGEs interact with their cell-bound receptor (RAGE), cell dysfunction is initiated by activating nuclear factor kappa-B (NF-κB), increasing the production and release of inflammatory cytokines. Alterations in the AGE-RAGE system have been related to the development of several chronic kidney diseases. Soluble RAGE (sRAGE) is a decoy receptor that suppresses membrane-bound RAGE activation and AGE-RAGE-related toxicity. sRAGE, and more specifically, the AGE/sRAGE ratio, may be promising tools for predicting the prognosis of kidney diseases. In the present review, we discuss the potential role of AGEs and sRAGE as biomarkers in different kidney pathologies.     

Soluble Receptor for Advanced Glycation End Products (sRAGE) Is a Sensitive Biomarker in Human Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive condition with an unmet need for early diagnosis, better monitoring, and risk stratification. The receptor for advanced glycation end products (RAGE) is activated in response to hypoxia and vascular injury, and is associated with inflammation, cell proliferation and migration in PAH. For the adult cohort, we recruited 120 patients with PAH, 83 with idiopathic PAH (IPAH) and 37 with connective tissue disease-associated PAH (CTD-PAH), and 48 controls, and determined potential plasma biomarkers by enzyme-linked immunoassay. The established heart failure marker NTproBNP and IL-6 plasma levels were several-fold higher in both adult IPAH and CTD-PAH patients versus controls. Plasma soluble RAGE (sRAGE) was elevated in IPAH patients (3044 ± 215.2 pg/mL) and was even higher in CTD-PAH patients (3332 ± 321.6 pg/mL) versus controls (1766 ± 121.9 pg/mL; p < 0.01). All three markers were increased in WHO functional class II+III PAH versus controls (p < 0.001). Receiver-operating characteristic analysis revealed that sRAGE has diagnostic accuracy comparable to prognostic NTproBNP, and even outperforms NTproBNP in the distinction of PAH FC I from controls. Lung tissue RAGE expression was increased in IPAH versus controls (mRNA) and was located predominantly in the PA intima, media, and inflammatory cells in the perivascular space (immunohistochemistry). In the pediatric cohort, plasma sRAGE concentrations were higher than in adults, but were similar in PH (n = 10) and non-PH controls (n = 10). Taken together, in the largest adult sRAGE PAH study to date, we identify plasma sRAGE as a sensitive and accurate PAH biomarker with better performance than NTproBNP in the distinction of mild PAH from controls.     

Advanced Glycation End-Products Induce Apoptosis of Vascular Smooth Muscle Cells: A Mechanism for Vascular Calcification

Vascular calcification, especially medial artery calcification, is associated with cardiovascular death in patients with diabetes mellitus and chronic kidney disease (CKD). To determine the underlying mechanism of vascular calcification, we have demonstrated in our previous report that advanced glycation end-products (AGEs) stimulated calcium deposition in vascular smooth muscle cells (VSMCs) through excessive oxidative stress and phenotypic transition into osteoblastic cells. Since AGEs can induce apoptosis, in this study we investigated its role on VSMC apoptosis, focusing mainly on the underlying mechanisms. A rat VSMC line (A7r5) was cultured, and treated with glycolaldehyde-derived AGE-bovine serum albumin (AGE3-BSA). Apoptotic cells were identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. To quantify apoptosis, an enzyme-linked immunosorbent assay (ELISA) for histone-complexed DNA fragments was employed. Real-time PCR was performed to determine the mRNA levels. Treatment of A7r5 cells with AGE3-BSA from 100 µg/mL concentration markedly increased apoptosis, which was suppressed by Nox inhibitors. AGE3-BSA significantly increased the mRNA expression of NAD(P)H oxidase components including Nox4 and p22^phox, and these findings were confirmed by protein levels using immunofluorescence. Dihydroethidisum assay showed that compared with cBSA, AGE3-BSA increased reactive oxygen species level in A7r5 cells. Furthermore, AGE3-induced apoptosis was significantly inhibited by siRNA-mediated knockdown of Nox4 or p22^phox. Double knockdown of Nox4 and p22^phox showed a similar inhibitory effect on apoptosis as single gene silencing. Thus, our results demonstrated that NAD(P)H oxidase-derived oxidative stress are involved in AGEs-induced apoptosis of VSMCs. These findings might be important to understand the pathogenesis of vascular calcification in diabetes and CKD.     

Association between Toll-Like Receptor 4 Expression and Neural Stem Cell Proliferation in the Hippocampus Following Traumatic Brain Injury in Mice

Whether or how neural stem cells (NSCs) respond to toll-like receptor 4 (TLR4) in an inflammatory environment caused by traumatic brain injury (TBI) has not been understood. In the present study, association between TLR4 expression and NSCs proliferation in the hippocampus was investigated in a mouse model of TBI using controlled cortical impact (CCI). Hippocampal proliferating cells were labeled with the thymidine analog 5-bromo-2-deoxyuridine (BrdU). In order to identify NSCs, the proliferating cells were further co-labeled with BrdU/sex determination region of Y chromosome related high mobility group box gene 2 (SOX2). Morphological observation on the expression of BrdU, SOX2, and TLR4 in the hippocampus was performed by inmmunofluorescence (IF). Relative quantification of TLR4 expression at the protein and mRNA level was performed using Western blotting and real-time polymerase chain reaction (PCR). It was observed that BrdU⁺/SOX2⁺ cells accounted for 95.80% ± 7.91% among BrdU⁺ cells; several BrdU⁺ cells and SOX2⁺ cells in the hippocampus were also TLR4-positive post injury, and that BrdU⁺ cell numbers, together with TLR4 expression at either protein or mRNA level, increased significantly in TBI mice over 1, 3, 7, 14, and 21 days survivals and changed in a similar temporal pattern with a peak at 3 day post-injury. These results indicate that hippocampal proliferating cells (suggestive of NSCs) expressed TLR4, and that there was a potential association between increased expression of TLR4 and the proliferation of NSCs post TBI. It is concluded that hippocampal TLR4 may play a potential role in endogenous neurogenesis after TBI.     

KDRn3蛋白在人脐静脉血管内皮细胞中的表达及对其增殖的抑制作用

目的观察KDRn3蛋白在人脐静脉血管内皮细胞中的表达及对其增殖的抑制作用。方法将质粒pEGFP-N1/KDRn3扩增并鉴定后,以脂质体介导转染人脐静脉血管内皮细胞,培养一定时间后,在荧光显微镜下观察绿色荧光蛋白的表达,ELISA检测细胞培养上清中KDRn3的含量,MTT法检测KDRn3蛋白对人脐静脉血管内皮细胞增殖的影响。结果转染后48h,在荧光显微镜下可见pEGFP-N1/KDRn3转染组人脐静脉血管内皮细胞发出绿色荧光,72h发出绿色荧光的细胞增多,强度增强;转染后24、48和72h,细胞培养上清中KDRn3含量与转染前比较均提高,且差异有显著意义;转染后48和72h,pEGFP-N1/KDRn3转染组与对照组比较,细胞增殖有明显的抑制,且差异有显著意义。结论KDRn3蛋白可在人脐静脉血管内皮细胞中表达,并能抑制其增殖。     

CXCR4在乳腺癌中的表达及乌斯他丁对其表达的影响

目的探讨趋化因子受体CXCR4在乳腺癌中的表达及其与肿瘤转移的相关性,以及乌斯他丁(UTI)对其表达的影响。方法采用流式细胞术和RT-PCR法检测22例乳腺癌患者的癌组织、癌旁组织、正常组织以及乳腺癌细胞MDA-MB-231中CXCR4在蛋白和mRNA水平的表达情况,以及UTI对MDA-MB-231细胞CXCR4表达的影响。结果在乳腺癌组织和MDA-MB-231细胞中,CXCR4在蛋白和mRNA水平的表达均显著高于癌旁组织和正常组织,且CXCR4的表达与乳腺癌的转移密切相关;UTI能下调CXCR4的表达,经UTI作用后,MDA-MB-231细胞的趋化活性降低。结论CXCR4在乳腺癌中高表达,在乳腺癌的转移中起重要作用,下调乳腺癌细胞CXCR4的表达水平可减少或抑制其转移。     

Survivin-siRNA真核表达载体的构建及其对胃癌SGC-7901细胞增殖和凋亡的影响

目的构建Survivin-siRNA真核表达载体,并探讨其对胃癌SGC-7901细胞增殖和凋亡的影响。方法化学合成4条能转录出siRNA的模板DNA,各75个碱基,退火形成2条双链DNA,双酶切后插入pSUPER.basic载体。将阳性重组质粒转染SGC-7901细胞,进行细胞计数,并用MTT法检测细胞的增殖活性;半定量RT-PCR检测细胞Survivin基因mRNA的转录水平;流式细胞术检测细胞周期的变化。结果PCR和酶切鉴定表明Survivin-siRNA真核表达载体构建正确,其能下调SGC-7901细胞Survivin基因mRNA的转录水平,抑制SGC-7901细胞生长和增殖,并促进细胞凋亡,使G0/G1和亚G1期细胞增多,S期细胞减少。结论已成功构建了Survivin-siRNA真核表达载体,其能下调SGC-7901细胞中Survivin基因mRNA的转录水平,使细胞增殖减弱,凋亡增加,为RNA干扰技术应用于胃癌的基因治疗提供了一定的实验依据。     

三七总皂甙对大鼠肺动脉血管平滑肌细胞增殖和凋亡的影响及其机制

目的探讨三七总皂甙(Panax notoginseng saponins,PNS)对大鼠肺动脉血管平滑肌细胞(Pulmonary artery smoothmuscle cell,PASMC)增殖和凋亡的影响及其机制。方法将PASMC分为5组:对照组(不给药)、bFGF组(8 ku/L)、bFGF+低、中、高剂量PNS组(bFGF 8 ku/L+300、450、600μg/L PNS),给药24 h后,采用MTT法检测PASMC的增殖活力;流式细胞仪检测细胞周期分布和细胞凋亡率;Westernblot检测细胞中胱天蛋白酶-3(caspase-3)蛋白的表达。结果 PNS干预可抑制PASMC增殖,阻遏bFGF诱导的PASMC进入S期,提高滞留在G0/G1期细胞的比例,并使PASMC凋亡率和caspase-3蛋白表达上调,PASMC凋亡率达(18.70%±0.33%)～(28.40%±0.79%),是bFGF诱导组的10～14倍。结论 PNS能显著抑制bFGF诱导的PASMC增殖,促进其凋亡,其机制可能与其调控细胞周期转换、激活caspase-3蛋白的表达有关。