杜仲叶活性部位I调控骨代谢平衡作用研究

目的 研究杜仲叶活性部位Ⅰ对人体骨代谢平衡的多靶位双重调控作用.为补肾壮骨中药临床防治骨质疏松症(OP)提供理论依据.方法 从杜仲叶5个极性不同的提取部位(Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ)中选取对骨代谢平衡调节最有效部位Ⅰ;建立体外大鼠成骨细胞堵养、肾细胞培养以及两种细胞共培养实验模型,加入高、中、低剂量杜仲叶活性部位Ⅰ培养;生物化学法测定成骨细胞早期特异性分泌的ALP活性,ABC-ELISA法测定肾细胞OPG和成骨细胞OPG与ODF的分泌量;RT-PCR技术检测OPG基因mRNA转录量.结果 杜仲叶活性部位Ⅰ促成骨作用表现剂量依赖性.对成骨细胞作用明显的剂量足10-5 g·ml-1(P＜0.001),对肾细胞作用明显的剂量足10-4 g·ml-1(P＜0.05).成骨细胞与肾细胞共培养的最佳比例是2∶1.结论 杜仲叶活性部位Ⅰ在成骨细胞、肾细胞不同靶部位对人体骨代谢平衡具有双重调控作用.     

褪黑素对H2O2诱导的成骨细胞氧化应激损伤的保护作用

摘要：目的 探讨褪黑素对H2O2诱导的成骨细胞氧化应激损伤的保护作用机制。方法 取24 h内新生SD大鼠10只，采用组织块贴壁法提取原代成骨细胞并行差速贴壁提纯细胞。细胞传至第2代时行碱性磷酸酶及茜素红染色进行鉴定。将鉴定后的成骨细胞用不同浓度的H2O2作用不同时间并行CCK8试验检测细胞增殖情况。选择合适的浓度和时间建立氧化应激损伤模型，并用褪黑素及EX527进行干预。实验分为对照组、H2O2组、褪黑素组及EX527组。分别对四组细胞行碱性磷酸酶染色、茜素红染色，检测四组细胞活性氧、丙二醛、超氧化物歧化酶含量，同时用流式细胞仪检测四组细胞凋亡率，免疫印迹法检测凋亡相关蛋白Bax、Bcl2和成骨相关蛋白BMP2、RUNX2以及SIRT1和p66SHC的表达量。结果 经鉴定，成功提取原代成骨细胞。CCK8结果显示，400 μmol/L H2O2作用4 h成骨细胞活性降至52 %，适宜用于建立氧化应激损伤模型。H2O2作用后成骨细胞活性及矿化能力降低，ROS含量、MDA含量升高，SOD活性降低，细胞凋亡率升高，伴随有SIRT1、BMP2、RUNX2、Bcl2表达降低，p66SHC和Bax表达升高。褪黑素预处理后缓解了H2O2对成骨细胞的氧化应激损伤作用，部分恢复了成骨细胞的活性及矿化能力，SIRT1抑制剂EX527能够逆转褪黑素的上述作用。结论 褪黑素通过调节SIRT1/p66SHC通路来抑制H2O2诱导的成骨细胞的氧化应激损伤并促进成骨。     

葛根素对过氧化氢致成骨细胞氧化损伤的影响

目的研究具有抗氧化作用的植物雌激素——葛根素(puerarin,Pue)对氧化应激后成骨细胞(osteoblast,OB)的影响。方法采用酶多次消化法,由出生24 h以内的Wistar大鼠头盖骨分离OB,培养、传代及诱导分化、矿化等。用0.1 mmol/L H_2O_2制造氧化应激状态模型。将细胞随机分为对照组、H_2O_2组[H'组]、"Pue(10~(-10)mol/L~10~(-3)mol/L)+H_2O_2"组["P+H'"组]和"H_2O_2+Pue(10~(-10)mol/L~10~(-3)mol/L)"组["H'+P"组]。观察不同处理组细胞的增殖、分化功能,并测定其微量丙二醛(m MDA)含量、超氧化物歧化酶(super oxide dismutase,SOD)活性、总抗氧化能力(T-AOC)等。结果与H_2O_2组相比,"P+H'"组和"H'+P"组中Pue10~(-7)mol/L~10~(-4)mol/L使细胞的增殖、分化功能,SOD活性和T-AOC均有显著升高(P0.01),而mMDA含量则显著降低(P0.01)。结论 H_2O_2过量会导致OB的氧化损伤,而葛根素在体外对H_2O_2介导OB的氧化损伤具有保护作用。     

MP联合BDNF转染对脊髓损伤细胞模型凋亡机制的作用

目的研究甲基强的松龙(MP)联合腺病毒介导脑源性神经营养因子(BDNF)转染对脊髓损伤细胞模型凋亡发生中氧化应激的影响及其作用机制。方法通过大鼠脊髓神经元细胞(rn-dsc)建立脊髓损伤细胞模型,分析MP+BDNF转染对脊髓损伤细胞模型中乳酸脱氢酶(LDH)漏出量、丙二醛(MDA)含量的影响,以及超氧化物歧化酶(SOD)活性的变化,利用流式细胞仪和MTT法分析细胞的凋亡率和存活率,Western Blot法分析细胞中Caspase 3及活化Cleaved caspase 3的表达情况。结果通过细胞外液LDH漏出量、MDA含量及SOD活性分析证实MP联合BDNF转染能够降低氧化应激对细胞的损伤;流式细胞仪、MTT法及Western Blot检测结果证实MP联合BDNF转染能够增强损伤细胞的抗凋亡作用,但是其抗氧化能力与凋亡率并不完全一致。结论 MP联合BDNF转染能够增强脊髓损伤细胞模型的抗氧化能力,并可能通过抗氧化应激反应提高模型细胞的抗凋亡能力,但是MP与BDNF抗凋亡作用还存在其他途径的参与。     

N-乙酰半胱氨酸对烟雾吸入性肺损伤氧化应激的影响

目的　研究N -乙酰半胱氨酸 (NAC)对烟雾吸入性肺损伤氧化应激及抗氧化能力的影响。　方法　采用烟雾吸入性肺损伤大鼠模型 ,检测吸入伤后肺泡灌洗液 (BALF)中白细胞数(WBC)、肺组织髓过氧化物酶 (MPO)活性、过氧化氢 (H2 O2 )含量、谷胱甘肽 (GSH)含量及总抗氧化能力(TAO)。　结果　NAC治疗可显著降低吸入伤后BALF中WBC数 ,降低肺组织MPO活性及H2 O2 含量 ,并显著提高肺组织GSH含量及TAO水平。　结论　NAC治疗可减轻吸入伤后肺脏氧化应激程度 ,提高肺脏抗氧化应激能力     

原花青素C1可有效抑制过氧化氢刺激所致的成骨细胞衰老

目的探讨原花青素C1(PCC1)对成骨细胞衰老的保护作用。方法应用不同浓度过氧化氢刺激成骨前体细胞系MC3T3后通过细胞计数试剂盒(CCK-8)检测成骨细胞活性, 选取活性抑制率约为50%的浓度用于诱导MC3T3细胞衰老。依据不同处理措施, 将细胞分为对照组、衰老组和PCC1组。正常组加入完全培养基, 衰老组加入含1 μmol/L H2O2的完全培养基, PCC1组加入含1 μmol/L H2O2和5 μmol/L PCC1的完全培养基。CCK-8用于检测成骨细胞活性。实时定量反转录聚合酶链反应(RT-qPCR)和蛋白质免疫印迹法(Western blot)用于检测经不同处理后MC3T3细胞中衰老相关基因p53、p16INK4a、p21、基质金属蛋白酶(MMP)-3、MMP-13的表达。采用β-半乳糖苷酶染色检测成骨细胞β-半乳糖苷酶阳性表达。两组之间采用t检验进行统计分析。结果 PCC1可以显著抑制过氧化氢刺激而所致的细胞活性降低, 衰老组细胞活性降低显著程度低于PCC1组[(44.54±2.38)%比(30.45±2.84)%, t=7.60, P<0.05]; RT-qPC...     

番茄红素对氧化应激后成骨细胞的影响

目的研究具有强抗氧化作用的番茄红素对氧化应激后成骨细胞的影响。方法制造氧化应激状态成骨细胞模型。将细胞随机分为4组:高、中、低浓度番茄红素组及对照组。然后用流式细胞术检测各组成骨细胞生长指数,用酶标仪检测各组成骨细胞内碱性磷酸酶的活性。在各组细胞中另加入矿化结节诱导液,观察各组成骨细胞矿化能力。结果各实验组成骨细胞较对照组生长指数高,碱性磷酸酶表达增加,形成的矿化结节多。结论番茄红素可以增加氧化应激状态成骨细胞的增殖、碱性磷酸酶的表达及矿化功能。     

人脐血间质干细胞条件培养基在受损成骨细胞中的作用与机制

目的探讨线粒体活性在人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,HUMSCs)条件培养基修复受损成骨细胞中的作用及其机制。方法提取、培养HUMSCs及制作其条件培养基,建立成骨细胞氧化损伤模型。实验分组:无血清DMEM培养基组、晚期氧化蛋白(advanced oxidation protein products,AOPPs)刺激组、常规培养基组、条件培养基组,培养1 h后用流式细胞仪以及Mito Tracker Red分析成骨细胞凋亡率、线粒体活性,并用蛋白-凝胶成像法(western-blot,WB)分析Caspase-3凋亡蛋白表达差异,从而评价HUMSCs条件培养基对遭受氧化损伤的成骨细胞的保护、修复、逆转作用,以及线粒体在其中发挥的作用及其机制。结果HUMSCs条件培养基可以增加氧化应激作用下成骨细胞内线粒体活性;HUMSCs条件培养基可降低成骨细胞凋亡率,减少Caspase-3凋亡蛋白表达。结论HUMSCs条件培养基通过缓解氧化应激(oxidative stress,OS)导致的线粒体功能与结构损伤,并下调Caspase-3凋亡蛋白的表达,抑制OB凋亡。     

剂量因素对羟基磷灰石/磷酸三钙成骨细胞相容性的影响

目的　检测不同剂量的羟基磷灰石 /磷酸三钙 (HA/ TCP)对兔成骨细胞增殖及碱性磷酸酶(AL P)活性的影响 ,明确材料的剂量因素在研究 HA/ TCP细胞相容性中的作用规律。方法　以兔成骨细胞为正常对照 ,细胞加入钛合金材料组为阴性对照 ,细胞加入聚氯乙烯材料组为阳性对照。选择三种材料剂量 ,以材料占细胞面积的比例表示 ,分别为 10 %、4 0 %和 70 %。将各组材料与兔成骨细胞复合培养 ,MTT法检测不同时间点、不同剂量材料作用下兔成骨细胞增殖的情况 ;偶氮偶联法检测不同时间点、不同剂量材料作用下兔成骨细胞表达 AL P的变化。结果　正常兔成骨细胞的生长呈现时间 -效应关系。与正常对照相比 ,10 % HA/ TCP不会对兔成骨细胞的生长产生影响 (P>0 .0 5 ) ;当 HA/ TCP剂量增大为 4 0 % ,细胞的生长速度明显变缓 (P<0 .0 5 ) ,细胞的生长仍呈现时间 -效应关系 ;70 %的 HA/ TCP使细胞生长趋于停滞 (P<0 .0 1)。 10 % HA/ TCP可造成细胞 AL P活性可逆性损伤 ,HA/ TCP浓度达 4 0 %时 ,AL P活性损伤至共培养 6天不可逆转。相对而言 ,惰性金属硬组织材料钛合金在检测三种剂量下均对细胞生长及细胞 AL P活性不会产生影响。结论　评价材料的细胞相容性应向材料接触剂量个体化、特异化的方向发展 ;除增殖指标之外 ,AL     

HO-1对醋酸铅诱导肾小管上皮细胞氧化应激的保护机制

目的:探讨血红素氧合酶-1(HO-1)对醋酸铅(LA)诱导的肾小管上皮细胞损伤的保护机制,为铅性肾病的治疗提供一定的理论依据。方法:采用醋酸铅孵育人肾小管上皮细胞48 h建立细胞损伤模型,原卟啉氯化钴(Co PP)诱导HO-1表达,自噬抑制剂(3-MA)抑制自噬,CCK-8方法检测细胞活性;流式细胞术检测细胞凋亡率; Western blot检测自噬标志蛋白LC3Ⅱ/LC3Ⅰ、p62及氧化应激相关蛋白超氧化物歧化酶(SOD2)、过氧化氢酶(CAT)、丙二醛(MDA)含量。结果:醋酸铅处理48 h后细胞活性、HO-1及自噬水平较Control组均明显下降,在补充Copp激活HO-1后细胞自噬水平上升,细胞活性明显恢复; Copp可明显改善细胞抗氧化酶CAT、SOD2表达,降低MDA,降低细胞凋亡率; 3-MA抑制自噬导致细胞CAT、SOD2表达进一步降低,MDA含量增加,细胞凋亡率升高,并且补充Copp后细胞CAT和MDA水平仅部分改善,SOD2表达未见明显恢复,提示抑制自噬减弱了HO-1的抗氧化作用。结论:HO-1可通过上调自噬减轻醋酸铅诱导的肾小管上皮细胞氧化应激损伤,减少细胞凋亡,HO-1可能可以作为治疗铅性肾病的一个干预靶点。     

Evaluation of the association of IGF2BP2 variants with type 2 diabetes in French Caucasians

OBJECTIVE—We performed a comprehensive genetic association study of common variation spanning the IGF2BP2 locus in order to replicate the association of the “confirmed” type 2 diabetes susceptibility variants rs4402960 and rs1470579 in the French Caucasian population and to further characterize the susceptibility variants at this novel locus.RESEARCH DESIGN AND METHODS—We genotyped a total of 21 tagging single nucleotide polymorphisms spanning the IGF2BP2 locus in our type 2 diabetes case-control cohort comprising 3,093 French Caucasian subjects.RESULTS—IGF2BP2 variants rs4402960 and rs1470579 were not associated with type 2 diabetes in the present study (P = 0.632 and P = 0.896, respectively). Meta-analysis of genotype data from over 34,000 subjects demonstrated that our inability to replicate rs4402960/rs1470579 was consistent with the findings from several previous genome-wide association study (GWAS) datasets that were underpowered to detect this modest association signal (odds ratio [OR] 1.14). We obtained novel evidence that rs9826022, a borderline rare variant (5% minor allele frequency) in the 3′ downstream region, was associated with type 2 diabetes (P = 0.0002; OR 1.53 [95% CI 1.22–1.91]). This result was corroborated by the meta-analysis of 10,542 genotypes from the current study and GWAS datasets using both fixed (P = 9.47 × 10⁻⁶; 1.30 [1.16–1.46]) and random effects (P = 0.001; 1.30 [1.11–1.52)] calculations.CONCLUSIONS—We were unable to replicate the confirmed rs4402960/rs1470579 susceptibility variants but found novel evidence for a rare variant in the 3′ downstream region of IGF2BP2. Further genetic and functional studies are required to identify the etiological IGF2BP2 variants.The insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) gene on chromosome 3q27 is a paralog of IGF2BP1, a known regulator of IGF2 gene expression. Genome-wide association studies (GWASs) carried out by the Finland-U.S. Investigation of NIDDM Genetics (FUSION) (1), the Wellcome Trust Case Control Consortium (WTCCC) (2), and the Diabetes Genetics Initiative (DGI) (3) groups each found modest evidence that single nucleotide polymorphisms (SNPs) in the IGF2BP2 region are associated with type 2 diabetes. The subsequent meta-analysis of primary and replication datasets from these GWASs corroborated these findings and identified two strongly correlated IGF2BP2 variants, rs1470579 and rs4402960, as “confirmed” type 2 diabetes susceptibility variants (1–3). By contrast, the French/Canadian GWAS (4) typed 10 SNPs across the IGF2BP2 locus, including rs1470579, in 1,363 subjects, but found no nominal (P < 0.05) association signals at IGF2BP2. In an attempt to replicate the IGF2BP2 association findings in the French Caucasian population in a larger study and to further characterize the susceptibility variants at this novel locus, we performed an association study of HapMap Phase II tag SNPs spanning the IGF2BP2 locus in 3,093 French Caucasian subjects.     

Role of spinal cyclooxygenase-2 and prostaglandin E2 in fentanyl-induced hyperalgesia in rats

Background

Accumulated evidence suggests that spinal cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) may be implicated in the development of opioid-induced hyperalgesia.

P2Y2 receptors and GRK2 are involved in oscillatory fluid flow induced ERK1/2 responses in chondrocytes

Mechanical loading is an important factor regulating cartilage metabolism maintained by chondrocytes. However, some of its underlying mechanisms remain poorly understood. In this study, we employed a chondrogenic cell line ATDC5 to investigate roles of P2Y₂ and GRK2 in chondrocyte mechanotransduction. We first confirmed the expression of chondrocyte markers in differentiated ATDC5 cells. We then exposed both differentiated and undifferentiated ATDC5 cells to oscillatory fluid flow, and found that differentiated ATDC5 cells responded to oscillatory fluid flow by increasing COX‐2 and aggrecan expressions. More importantly, fluid flow induced ERK1/2 response in differentiated cells was increased more than 10 times compared to those in undifferentiated cells. Furthermore, we found that P2Y₂ mRNA and protein levels in differentiated ATDC5 cells were significantly higher than those in undifferentiated cells. In contrast, GRK2 protein levels in differentiated cells were significantly lower than those in undifferentiated cells. Finally, overexpressions of P2Y₂ and GRK2 in differentiated ATDC5 cells result in a 34% increase and a 21% decrease of the ERK1/2 phosphorylation, respectively, in response to oscillatory fluid flow, suggesting important roles of P2Y₂ and GRK2 in chondrocyte mechanotransduction. © 2010 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:828–833     

c-erbB-2和cox-2在乳腺癌中的表达及其意义

应用免疫组化技术 ,检测人乳腺良恶性疾病组织中c erbB 2和cox 2蛋白的表达 ,分析其与淋巴结转移、激素受体状态的关系。 40例乳腺癌组织中c erbB 2表达阳性率为 3 5 .0 %,淋巴结阳性及激素受体阴性者表达率显著高于无淋巴结转移及受体阳性者 (均P <0 .0 5 ) ;cox 2表达阳性率为 47.5 %,激素受体阴性者表达率显著高于受体阳性者 (P <0 .0 5 ) ,但与淋巴结转移无关 (P >0 .0 5 )。 10例良性乳腺疾病及 2例正常乳腺组织中无c erbB 2表达 ,仅有 1例乳腺囊肿病cox 2表达阳性 ,c erbB 2与cox 2的表达存在显著的相关性 (P <0 .0 5 )。提示c erbB 2和cox 2与乳腺癌的发生、转移及预后均有密切关系 ,同时进行两者的免疫组化检测对评估乳腺癌的预后及选择靶向治疗对象可能有意义。     

腹腔镜胆囊切除术CO2气腹对脉搏血氧饱和度的影响

目的:观察腹腔镜胆囊切除术(laparoscop ic cholecystectomy,LC)CO2气腹对脉搏血氧饱和度的影响。方法:按照美国麻醉医师协会体格情况分级(ASA)标准,选择ASAⅠ~Ⅱ级600例患者全麻下行LC,在围手术期对脉搏血氧饱和度(SpO2)进行连续监测。结果:CO2充气后3m in SpO2明显下降(P<0.01),放气后恢复到术前水平。结论:CO2气腹对脉搏血氧饱和度存在一定程度的影响,因此CO2气腹压力不宜过大,应限制在10~12mm Hg为宜,对老年患者伴有心、肺功能不全及肥胖者更要加强麻醉管理,加强SpO2、呼气末二氧化碳分压(PETCO2)监测。     

Circulating level of alpha2-macroglobulin-beta2-microglobulin complex in hemodialysis patients

BACKGROUND: The presence of alpha2-macroglobulin (alpha2M) in amyloid tissue from patients with dialysis-related amyloidosis (DRA) was demonstrated by Argilés et al in 1989. Thereafter, the formation of the complex of beta2-microglobulin (beta2m) with alpha2m was confirmed directly by in vitro study. In Alzheimer's disease, complex formation of amyloid beta-peptide and alpha2M is considered to play an important role in the pathogenesis by modifying the degradation processes of amyloid protein. Thus, we hypothesized that the alpha2M-beta2m complex is an important factor in the pathogenesis of DRA as well. Here, we measured the circulating levels of alpha2M-beta2m complex in the maintenance hemodialysis patients and discussed about its clinical significance in DRA. METHODS: One hundred and thirty-seven hemodialysis patients and 11 prehemodialysis chronic renal failure (CRF) patients were included in this study. The affinity of purified alpha2M for beta2m was confirmed by a highly sensitive 27 MHz quartz crystal microbalance (QCM). The presence of circulating alpha2M-beta2m complex was analyzed by immunoblotting analysis. Furthermore, the serum levels of alpha2M-beta2m complex were measured by sandwich enzyme immunoassay. RESULTS: QCM analysis revealed the high affinity of alpha2M for beta2m. The presence of circulating alpha2M-beta2m complex was detected in two out of a total 11 prehemodialysis CRF patients and in 95 out of the total of 137 hemodialysis patients. None of the healthy subjects, however, were observed to present with any alpha2M-beta2m complex. Serum levels of the alpha2M-beta2m complex were correlated to the duration of hemodialysis (P= 0.043). Serum levels of the alpha2M-beta2m complex were significantly higher in patients with high DRA score than in patients with negative DRA score (P= 0.018). Moreover, serum levels of the alpha2M-beta2m complex showed significantly lower in the hemodiafiltration patients compared to the hemodialysis patients (P= 0.002) and showed a strong correlation with DRA score in hemodialysis patients excluding 11 hemodiafiltration patients (P= 0.0004). CONCLUSION: This study is the first to demonstrate the presence of circulating alpha2M-beta2m complex in hemodialysis patients. Furthermore, we observed the correlation between serum levels of alpha2M-beta2m complex and clinical characteristics of DRA. Thus we concluded that a formation of an alpha2M-beta2m complex may be implicated in DRA.