钼对培养心肌细胞的双向性影响

黄嘌呤-黄嘌呤氧化酶系统(X-XOD)可诱发培养心肌细胞的自由基损伤。鉴于钼是黄嘌呤氧化酶(XOD)的重要组分,而且钼对于心肌细胞有保护作用还是有损伤作用的报道不尽一致,本实验拟以心肌细胞动作电位与自发性搏动为指标,探讨钼对心肌细胞机能影响的规律。 实验方法取新生昆明种小鼠与Wistar大鼠的乳鼠;胎龄4～6个月,水囊引产的人胚心尖,分别经0.1％胰蛋白酶分离心肌细胞,然后将其置入聚苯乙烯培养瓶,在36.5℃、5％CO_2和95％空气的孵箱内培养。常规培养基由80％199及20％小牛血清组成。小鼠心肌细     

白藜三醇对黄嘌呤/黄嘌呤氧化酶所致平滑机细胞增殖的干预作用

目的探讨红葡萄酒 (RW)是否对血管SMC增殖具有抑制作用。方法以培养幼兔主动脉平滑肌细胞为研究对象 ,分别给予不同浓度黄嘌呤/黄嘌呤氧化酶和/或白藜三醇 ,采用免疫组织化学、3H -TdR掺入试验和MTT法技术检测不同处理对平滑肌细胞的增殖作用。结果RES呈剂量依赖性拮抗X/XO对VSMC的促增生效应 ;终浓度50umol/L的RES对X200umol/L/XO200u/L所致促VSMC的增殖效应即有抑制作用(P<0.05) ;终浓度200umol/L的RES对X200umol/L/XO200u/L所致促VSMC的增殖效应则具有极显著的抑制作用 (P<0.001)。结论红葡萄酒中的有效成份白藜三醇可拮抗黄嘌呤/黄嘌呤氧化酶所致平滑肌细胞的作用 ,这可能有助于减轻或防治氧化损伤对血管平滑肌细胞增殖的影响。     

APPL1减轻LPS诱导的心肌细胞损伤的实验研究

目的:探讨APPL1对脂多糖(LPS)诱导的心肌细胞损伤的影响。方法:用LPS处理心肌细胞,RT-qPCR和Western blot检测细胞中APPL1的表达变化。用过表达APPL1重组慢病毒载体转染心肌细胞,经LPS处理后,RT-qPCR和Western blot检测过表达效果。MTT测定心肌细胞活力,流式细胞术测定心肌细胞凋亡变化,Western blot测定心肌细胞中活化的caspase-3蛋白水平,用硫代巴比妥酸法检测心肌细胞中丙二醛(MDA)含量,用2,4-二硝基苯肼显色法检测心肌细胞培养液中乳酸脱氢酶(LDH)水平,用黄嘌呤氧化酶法检测心肌细胞中超氧化物歧化酶(SOD)活性,5,5’-二硫代双(2-硝基苯甲酸)比色法测定心肌细胞中谷胱甘肽过氧化物酶(GSH-Px)活性,DCFH-DA法检测心肌细胞中活性氧(ROS)水平。结果:与对照心肌细胞比较,LPS处理后心肌细胞中APPL1的mRNA和蛋白水平均显著降低(P0.05)。与单纯LPS处理的心肌细胞比较,过表达APPL1重组慢病毒载体可以显著提高LPS条件下心肌细胞中APPL1 mRNA和蛋白水平(P0.05)。LPS处理后的心肌细胞活力降低,细胞凋亡率和细胞中活化的caspase-3蛋白水平升高,细胞中MDA水平升高,培养液中LDH水平也升高,细胞SOD活性、GSH-Px活性降低,ROS水平升高,(P0.05)。过表达APPL1后的心肌细胞经LPS诱导后,细胞活力升高,细胞凋亡率及细胞中活化的Caspase-3蛋白水平降低,MDA水平降低,培养液中LDH水平降低,细胞中SOD活性、GSH-Px活性升高,ROS水平降低,(P0.05)。结论:过表达APPL1能够降低LPS诱导的心肌细胞氧化损伤,减少细胞凋亡,具有减轻LPS诱导的心肌细胞损伤的作用。     

NIV毒素和硒对软骨细胞IL-1β、TNF-α分泌的影响

目的:通过测定NIV毒素和硒作用下软骨细胞IL-1β、TNF-α含量的变化,从细胞因子角度探讨大骨节病发病机制。方法:在体外单层和立体培养的人胚软骨细胞中加入NIV毒素和硒,构建软骨损伤模型,收集软骨组织、细胞和细胞培养液,用光学显微镜观察体外构建组织的形态;用分光光度法测定软骨细胞DNA含量;用酶联免疫吸附方法(ELISA)检测细胞培养液中IL-1β、TNF-α的水平。结果:形态学观察发现NIV毒素能抑制软骨细胞生长,局部出现点片状坏死;毒素加硒后,上述损伤变化减弱。NIV毒素作用下软骨细胞DNA的合成受到抑制,但培养液中IL-1β、TNF-α含量显著升高,毒素加硒与毒素组变化趋势一致,但数值略下降。结论:NIV毒素能诱导软骨细胞IL-1β与TNF-α的分泌,这可能是造成软骨细胞损伤的原因之一。     

硫化氢通过调控瘦素保护心肌细胞对抗高糖引起的损伤

目的探讨瘦素(leptin)在高糖损伤心肌细胞中的作用及硫化氢(H2S)是否通过调控瘦素保护心肌细胞对抗高糖引起的损伤。方法应用细胞计数盒(CCK-8)检测细胞存活率;Hoechst 33258核染色检测凋亡细胞形态及数量的改变,双氯荧光素(DCFH-DA)染色荧光显微镜照相测定胞内活性氧(ROS)水平;罗丹明123(Rh123)染色及荧光显微镜照相测定线粒体膜电位(MMP);Western blot法检测瘦素蛋白的表达。结果 35 mmol/L葡萄糖(高糖)作用H9c2心肌细胞9h可明显地促进瘦素表达,并引起心肌细胞损伤,表现为细胞存活率降低、凋亡细胞数量和ROS生成增多及线粒体膜电位(MMP)丢失。硫化氢钠(NaHS,为H2S的供体)预处理能抑制高糖对心肌细胞瘦素表达的上调作用。NaHS预处理或瘦素拮抗剂均能保护H9c2心肌细胞对抗高糖引起的上述损伤。结论瘦素参与高糖引起的心肌细胞损伤。外源性H2S可通过抑制瘦素表达保护心肌细胞对抗高糖引起的损伤。     

锌对培养的心肌细胞氧自由基损伤效应的观察

通过向培养基中加入黄嘌呤和黄嘌呤氧化酶造成心肌细胞的氧自由基损伤,观察了微量元素Zn的抗氧化损伤作用。实验分为①对照组:MEM 20%小牛血清;②XOD组:对照组加黄嘌呤0.42mM和黄嘌呤氧化酶5.3nM;     

黄芪皂甙抗犬心肌自由基损伤作用

在黄嘌呤－黄嘌呤氧化酶制造的自由基损伤模型上，用引导培养心肌细胞动作电位和离体心脏灌流的方法，发现黄嘌呤－黄嘌呤氧化酶使动作电位的各参数减少，使离体心功能显著下降，而黄芪皂甙可使上述改变向正常方向转化，因此，黄芪皂甙具有抗自由基损伤作用。     

黄嘌呤氧化酶在激素诱导成骨细胞凋亡过程中的作用及机制

目的探究黄嘌呤氧化酶在糖皮质激素诱导小鼠胚胎成骨细胞前体细胞(MC3T3-E1)细胞凋亡过程中的作用及机制。方法将细胞分为空白组、模型组、5 mol/L组、10 mol/L组和15 mol/L组,接种完成后培养24 h,确认细胞贴壁后换液。空白组加入10%FBS培养基2 mL,模型组加入1 10-6 mol/L DEX培养基2 mL, 5 mol/L组加入5 mol/L别嘌醇DEX培养基2 mL,10 mol/L组加入10 mol/L别嘌醇DEX培养基2 mL,15 mol/L组加入15 mol/L别嘌醇DEX培养基2 mL。培养72 h后检测Caspase-3、STAT-1、Bax、Bcl-2等蛋白表达情况,检测细胞凋亡情况以及活性氧簇含量。检测细胞内黄嘌呤氧化酶活性、丙二醛(MDA)含量及线粒体膜电位。结果模型组中Caspase-3、STAT-1和Bax蛋白表达量明显升高,Bcl-2蛋白表达量降低,细胞凋亡比例增高,细胞内活性氧簇含量增高,细胞内黄嘌呤氧化酶活性、丙二醛含量增高,而线粒体膜电位降低,与空白组、5 mol/L组、10mol/L组和15 mol/L组相比,差异有统计学意义(P 0.05)。结论黄嘌呤氧化酶通过产生过量的活性氧簇从而导致成骨细胞氧化应激损伤,通过激活STAT-1和Bax蛋白来诱导成骨细胞凋亡,同时诱导线粒体膜电位稳态崩溃触发内源性线粒体凋亡途径。别嘌醇可以较好地抑制黄嘌呤氧化酶的活性,减少活性氧簇的产生,通过减少Caspase-3、STAT-1和Bax蛋白表达,稳定线粒体膜电位,从而减少成骨细胞凋亡。     

在培养的心肌细胞中锌的抗氧自由基损伤作用的观察

本文通过向培养基中加入黄嘌呤和黄(?)呤氧化酶系统成分造成培养的心肌细胞受氧自由基伤害,以电生理的改变和BaCL_2引起心肌细胞停搏的闭浓度为指标,观察了微量元素Zn的抗氧化损伤作用。结果示:XOD组与对照组比较,动作电位APA、OS、MDP、TP、Vmax及APD_(50)各电参数明显减小。BaCl_2引起心肌细胞停搏的阈浓度亦减低。而加Zn组与对照组比较,以上结果除TP值偏低P<0.05外,其它参数则无显著性改变。加Zn组与XOD组相比,动作电位各电参数明显增大,BaCl_2引起心肌细胞停搏的阈浓度亦增高。提示Zn对培养的心肌细胞氧自由基所致的损伤具有保护作用。     

麝香保心丸通过抑制p38 MAPK和NF-κB信号传导通路表达对抗高糖诱导的心肌细胞损伤

目的探讨麝香保心丸(SBP)是否通过抑制p38丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和核因子kappa B(nuclear factor kappa B,NF-κB)通路保护H9c2心肌细胞对抗高浓度葡萄糖(高糖,HG)引起的损伤。方法应用35 mmol/L的HG处理H9c2心肌细胞24 h,建立HG诱导的心肌细胞损伤模型;细胞计数试剂盒8测定细胞存活率;Hoechst 33258核染色荧光显微镜照相法测定细胞凋亡;双氯荧光素(2’,7’-dichlorfluorescein-diacetate,DCFH-DA)染色/荧光显微镜照相法测定胞内活性氧(ROS)水平;罗丹明123(Rh123)染色法测定线粒体膜电位(MMP);Western blot法测定p38MAPK和NF-κB p65蛋白表达水平。结果 HG处理H9c2心肌细胞24 h能引起细胞明显的损伤,使细胞存活率降低,凋亡细胞数量和细胞内ROS生成增多,MMP丢失;HG能增加p38 MAPK和NF-κB p65磷酸化水平;SBP预处理能明显抑制HG上调p38 MAPK和NF-κB p65磷酸化水平这一作用;SBP、p38MAPK通路抑制剂SB203580和NF-κB通路抑制剂PDTC均能阻断HG对心肌细胞的上述损伤作用,包括细胞毒性、凋亡、ROS生成增多及MMP丢失等。结论麝香保心丸(SBP)可通过抑制p38 MAPK和NF-κB信号传导通路保护H9c2心肌细胞对抗高糖(HG)引起的损伤。     

Protective effect of the antioxidants selenium, vitamin E, and superoxide dismutase on cultured human endothelial cells (ECs) injured by lipid peroxidation

Lipid peroxidation was induced in cultured human ECs with cumene hydroxide and the protective effect of antioxidants (selenium, vitamin E and SOD) on endothelial cell injury by lipid peroxidation was investigated. The results showed that after administration of antioxidants (Se, Vit E and SOD), concentration of LPO in ECs induced by cumene hydroperoxide was reduced markedly while 6-Keto-PGF1 alpha content in the medium was increased and cell injury was alleviated. These results indicate that through the protective effect on ECs against lipid peroxidation, antioxidants (Se, Vit E and SOD) might play an important role in retarding the process of atherogenesis.     

高速投射物压力波作用下内皮细胞磷酸肌醇信号通路的变化

目的:探讨投射物压力波作用于血管内皮细胞时,细胞内磷脂酰肌醇代谢与细胞损伤的关系。方法:以枪弹压力波作用培养细胞为实验模型,测定压力波作用后细胞内三磷酸肌醇(IP₃)、游离钙[Ca²⁺]i、蛋白激酶C(PKC)以及培养液中乳酸脱氢酶(LDH)活性,并以特异阻断磷脂酰肌醇代谢后重复检测上述参数的变化。结果:压力波可导致细胞内(IP₃)、[Ca²⁺]i、以及PKC活性明显升高,且与培养液中LDH活性升高一致。阻断磷脂酰肌醇代谢后可部分抑制上述生化参数的变化。结论:枪弹压力波可激活血管内皮细胞磷酸肌醇代谢,进而导致细胞损伤、LDH泄漏。     

大鼠大脑皮质神经前体细胞的分离培养及其增殖分化鉴定

目的:体外分离和培养大鼠大脑皮质神经前体细胞并进行增殖和分化鉴定。方法:分离2周龄大鼠皮质神经前体细胞,在含EGF、bFGF和GDNF的NSC培养基中进行体外培养,使用免疫细胞荧光染色技术对细胞的分化特性进行鉴定。结果:EGF、bFGF和GDNF可促进神经前体细胞的增殖及神经球的克隆形成,并获得了Nestin阳性的神经前体细胞,其可分化为分别表达β-Ⅲtubulin、GFAP和GalC的阳性细胞。结论:体外分离和培养的大脑皮质神经前体细胞,在含EGF、bFGF和GDNF的NSC培养基中培养,具有增殖分化的能力,有望应用于神经系统疾病的细胞移植治疗。     

依达拉奉对骨髓基质干细胞氧化损伤的调节作用

背景：既往依达拉奉作为抗氧化剂对神经细胞氧化损伤的保护作用已得到证实,但其对骨髓基质干细胞氧化损伤的保护作用未见明确报道及深入研究。 目的：观察依达拉奉对骨髓基质干细胞在氧化损伤中的调节作用。 方法：通过冲洗髓腔的方法提取新西兰大耳白兔的长骨骨髓,然后应用密度梯度离心联合贴壁筛选的方法体外培养获得骨髓基质干细胞。实验将第3代骨髓基质干细胞分为5组：空白组仅加入体积分数为10%胎牛血清、1%的双抗的低糖DMEM培养液;地塞米松组加入含有1×10-7 mol/L地塞米松的细胞培养液,不含有依达拉奉;50,100,300 mg/L依达拉奉组分别加入1×10-7 mol/L的地塞米松和质量浓度为50,100,300 mg/L的依达拉奉,培养后分别用四甲基偶氮唑蓝法、流式细胞仪法检测细胞增殖水平及细胞周期。 结果与结论：依达拉奉组较空白组及地塞米松组细胞增殖水平明显增强,依达拉奉对骨髓基质干细胞起到了保护作用,当依达拉奉质量浓度为50 mg/L时即发挥作用(P < 0.05),并且与依达拉奉的质量浓度有一定的量效关系,当依达拉奉质量浓度为100 mg/L时,其保护作用明显提高(P < 0.01),但随着质量浓度的增加,这种保护作用没有进一步增加,反而稍有下降。结果表明：高浓度地塞米松可以使骨髓基质干细胞受到氧化损伤,依达拉奉可以通过抗氧化作用保护骨髓基质干细胞免受氧化损伤。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Injury to cultured endothelial cells by thrombin-stimulated platelets

In vivo, stimulated platelets may injure the endothelium. We have used cultured endothelial cells to assess endothelial cell damage caused by platelet stimulation with thrombin. Endothelial cells were cultured from umbilical veins and semiconfluent cultures were labeled with Na2 51CrO4. Twenty four hours later washed human platelets (final concentration 200,000 platelets/microliters) and thrombin (final concentration 4 units/ml) were added to the medium and the culture dish was shaken for 15 minutes. The percentage of cells detached from the culture dish and the percentage of 51Cr lost from the endothelial cells into the ambient fluid during the shaking were determined and used as indicators of cell injury. Increased percentages of loosened cells and 51Cr in the ambient fluid were observed with platelet suspension and thrombin compared to controls with neither platelet suspension nor thrombin and controls with either platelet suspension or thrombin. The platelet-free supernatant obtained after reaction of the platelets with thrombin also increased the percentage of loosened cells, but it did not increase the percentage of 51Cr in the ambient fluid to a significant degree. Thrombin alone caused a moderate loss of 51Cr, but no increased loosening of cells. Treatment of the platelets with acetylsalicylic acid prior to the experiment depressed the detachment effect of thrombin-stimulated platelets, but did not alter the effect on the release of 51Cr into the ambient fluid. Scanning and transmission electron microscopy of cultured endothelial cells exposed to thrombin-stimulated platelets confirmed the presence of loosening and injury to the endothelial cells. Thus, platelet stimulation with thrombin had at least two effects on the cultured endothelial cells: a loosening effect caused by material released from the platelets; an injury effect which, in order to reach its maximum, required the presence of stimulated platelets.     

Sister chromatid exchange analysis in lung and peripheral blood lymphocytes of mice exposed to methyl isocyanate by inhalation

Mice were exposed to 1, 3, or 6 ppm methyl isocyanate (MIC) for 6 hr/day for four consecutive days. Lung cells and peripheral blood lymphocytes (PBLs) were removed and cultured for analysis of sister chromatid exchange (SCE) and cell cycle kinetics. MIC caused a small but significant increase in SCE frequency of cultured lung cells from mice exposed to 1, 3, or 6 ppm MIC. MIC did not significantly increase SCE levels in PBLs of mice exposed to concentrations as high as 6 ppm. In cultured PBLs, MIC had a stimulatory effect on cell cycling rates as measured by the replicative index, and it caused a significant reduction in mononuclear leucocyte counts and the mitotic indices.     

柚皮甙干预鼠颅顶骨破骨细胞的形成及功能

背景：柚皮甙能诱导骨形态发生蛋白2的基因表达,促进成骨细胞系的增殖和分化。体外细胞实验提示柚皮甙有抑制破骨细胞的形成及抗骨质疏松的作用。 目的：建立含有柚皮甙的鼠颅顶骨培养模型,观察不同剂量下柚皮甙对破骨细胞增殖分化的影响。 方法：实验选取出生4 d的SD乳鼠颅顶骨,在分别含有0,1,10,100 mg/L 的柚皮甙的培养基中培养,于培养的第1,3,7,10天测定柚皮甙对鼠颅顶骨中破骨细胞标志酶抗酒石酸酸性磷酸酶阳性细胞数及培养基中钙离子浓度的影响。 结果与结论：第1天各组间抗酒石酸酸性磷酸酶阳性细胞数及培养基中钙离子浓度无明显变化,第3,7天各组间抗酒石酸酸性磷酸酶阳性细胞数随柚皮甙质量浓度增加而减少,而培养基中钙离子浓度变化明显增加,到第10天这种变化趋势最为明显。说明柚皮甙能影响鼠颅顶骨中抗酒石酸酸性磷酸酶阳性细胞数量及其功能,并且这种作用成明显的时间剂量相关性。提示柚皮甙不仅能促进成骨细胞的增殖,同时也能减少破骨细胞的分化或加速其凋亡。     

Evidence that X-irradiation and 4-hydroperoxycyclophosphamide affects different lymphocytes that respond to specific antigen in vitro

The present study examined the effect of pulse treatment with the in vitro active synthetic derivative of cyclophosphamide (CY), 4-hydroperoxycyclophosphamide (4-HPCY), and exposure to X-irradiation on the in vitro Concanavalin A (ConA), lipopolysaccharide (LPS), and antigen-specific blastogenic responses of in vivo-primed lymph node cells. Primed lymph node cells from CY-pretreated, aggregated (A) human IgG-complete Freund's adjuvant (AHGG-CFA)-immunized mice were untreated, exposed to various doses of irradiation, or pulse treated with different concentrations of 4-HPCY before being cultured in medium alone or in medium containing HGG, ConA, or LPS. The results show that HGG-responding and LPS-responding cells exhibited similar dose-inactivation profiles following exposure to irradiation or pulse treatment with 4-HPCY. More than 75% of reactivity was eliminated by exposure to 100 rads or pulse treatment with 20 microM 4-HPCY. In contrast to preculture pulse treatment with 4-HPCY, however, when primed lymph node cells were cultured in medium containing 4-HPCY (culture treatment) LPS-responding cells were shown to be more sensitive to inactivation than HGG-responding cells. The data further show that the effect of low-dose irradiation and of culture treatment with 4-HPCY on the HGG-specific response of primed lymph node cells was additive, suggesting that these agents inactivate different cell subtypes that contribute to the HGG-specific response in vitro.     

Effect of macrophage-derived growth factor on the cell cycle kinetics of cultured arterial smooth muscle cells

Effects of macrophage-derived growth factor on the progression of the cell cycle of cultured aortic smooth muscle cells were investigated by using rabbit peritoneal macrophage conditioned-medium (M phi-CM) as the source of the growth factors. DNA content of individual SMC in the cell population which had been exposed either to platelet-poor plasma serum (PPPS) or to M phi-CM was also measured by flow cytometry and microspectrophotometry, as well as by phase contrast microscopy. The results showed that the cells quiescent in PPPS medium had a high population not only in G0/G1 but also in G2 phase. This indicated that the cell cycle process was blocked both in G0/G1 and G2 phase. The cell cycle distribution of SMCs exposed to M phi-CM for 48 hours was very similar to that cultured in 10% fetal calf serum medium. It is suggested that M phi-CM can promote the cell growth-arrested in ppps medium passing through G0/G1 into S phase as well as through G2 into M phase.     

Engineering Bone Formation from Human Dental Pulp- and Periodontal Ligament-Derived Cells

A robust method for inducing bone formation from cultured dental mesenchymal cells has not been established. In this study, a method for generating bone tissue in vivo from cultured human dental pulp- and periodontal ligament-derived cells (DPCs and PDLCs, respectively) was designed using exogenous bone morphogenetic protein 2 (BMP2). DPCs and PDLCs showed enhanced alkaline phosphatase (ALP) activity and calcified nodule formation in medium containing dexamethasone, β-glycerophosphate, and ascorbic acid (osteogenic medium). However, the addition of recombinant human bone morphogenetic protein 2 (rhBMP2) to osteogenic medium remarkably increased ALP activity and in vitro calcification above the increases observed with osteogenic medium alone. rhBMP2 also significantly upregulated the expression of osteocalcin, osteopontin, and dentin matrix protein 1 mRNA in both cell types cultured in osteogenic medium. Finally, we detected prominent bone-like tissue formation in vivo when cells had been exposed to rhBMP2 in osteogenic medium. In contrast, treatments with osteogenic medium or rhBMP2 alone could not induce abundant mineralized tissue formation. We propose here that treatment with rhBMP2 in osteogenic medium can make dental mesenchymal tissues a highly useful source of cells for bone tissue engineering. In addition, both DPCs and PDLCs showed similar and remarkable osteo-inducibility.