乙酰左旋肉碱预处理对低氧低糖诱导PC12细胞凋亡的影响

目的 评价乙酰左旋肉碱预处理对低氧低糖诱导PC12细胞凋亡的影响.方法 PC12细胞接种于96孔板中,采用随机数字表法,将细胞随机分为5组(n=6):对照组(C组)、细胞损伤组(Ⅰ组)和不同浓度乙酰左旋肉碱预处理组(A1-3组).C组加入含葡萄糖4.5 g/L的DMEM溶液孵育3h;Ⅰ组加入含葡萄糖0.5 g/L和Na2SO4 3 mmol/L的DMEM溶液孵育3h;A1-3组分别加入0.2、0.4和0.6 mmol/L乙酰左旋肉碱孵育24h,然后加入含葡萄糖0.5 g/L和Na2S2O4 3mmol/L的DMEM溶液孵育3h.采用MTT法检测细胞活力,采用TUNEL法检测细胞凋亡率,测定细胞SOD和ATP酶的活性和MDA含量.结果 与C组比较,Ⅰ组细胞活力降低,细胞凋亡率升高,SOD和ATP酶的活性降低,MDA含量增加(P＜0.05),A1-3组细胞活力、细胞凋亡率、SOD和ATP酶的活性和MDA含量差异无统计学意义(P＞0.05);与Ⅰ组比较,A1-3组细胞活力升高,细胞凋亡率降低,SOD和ATP酶的活性升高,MDA含量下降(P＜ 0.05);A1-3组间细胞活力、细胞凋亡率、SOD和ATP酶的活性和MDA含量比较差异无统计学意义(P＞0.05).结论 乙酰左旋肉碱预处理可通过抑制细胞凋亡,减轻低氧低糖诱导PC12细胞损伤.     

ROS/GADD153蛋白在血管紧张素Ⅱ诱导心肌细胞凋亡中的作用

目的 探讨活性氧簇(ROS)/GADD153蛋白在血管紧张素Ⅱ(AngⅡ)诱导心肌细胞凋亡中的作用.方法 体外培养乳鼠心肌细胞,随机分为9组(n=5),Ⅰ组(C组)常规心肌细胞培养,不予其他处理;Ⅱ组(AngⅡ6组)给予100 nmol/L AngⅡ,孵育6 h;Ⅲ组(AnsⅡ12组)给予100 nmol/L AngⅡ,孵育12 h;Ⅳ组(ArtsⅡ24组)给予100 nmol/L Ang Ⅱ,孵育24 h;V组[N-乙酰-L半胱氨酸(NAC)+AngⅡ组]预先给予抗氧化剂NAC 5 mmol/L,2 h后给予100 nmol/L Ang Ⅱ,孵育24 h;Ⅵ组(NAC组)仅给予NAC 5 mmol/L,孵育24 h;Ⅶ组(anti-ODN组)GADD153反义寡核苷酸10 μmol/L转染24 h后,加入100nmoFL AngⅡ,孵育24 h;Ⅷ组(mis-ODN组)GADDl53错义寡核苷酸10 μmol/L转染24 h后,加入100nmol/L Ang Ⅱ,孵育24 h;Ⅸ组(脂质体对照组)加入等容量转染试剂,孵育24 h.检测细胞活力、细胞内ROS产量、细胞凋亡率及GADD153蛋白表达水平.结果 与C组相比,AngⅡ6组、AngⅡ12组、AngⅡ24组细胞活力降低,细胞凋亡率、ROS产量及GADD153蛋白表达升高,且呈时间依赖性(P＜0.05);与AngⅡ24组比较,NAC+AngⅡ组和anti-ODN组细胞活力升高,细胞凋亡率、ROS产量及GADD153蛋白表达降低(P＜0.05).结论 AugⅡ通过增加ROS产量,诱导GADD153蛋白表达上调,从而导致心肌细胞凋亡的发生.     

柠檬酸钠对胃癌细胞增殖和凋亡的影响及其机制

目的 探讨柠檬酸盐对胃癌细胞的影响及其机制.方法 将胃癌细胞株BGC-823和SGC-7901各分成对照组、5mmol/L组、10 mmol/L组,分别用0、5、10 mmol/L柠檬酸钠处理.24、48h后,荧光显微镜下观察细胞增殖变化、进行活细胞计数、计算细胞增殖抑制率；应用4’,6-二.脒基-2-苯基吲哚(DAPI)细胞核染色和流式细胞仪检测细胞凋亡；24h后蛋白免疫印迹法(Western blot)检测凋亡相关蛋白表达.结果 5、10 mmol/L组活细胞计数均低于对照组,细胞增殖抑制率均高于对照组(P＜0.05)；DAPI染色显示凋亡细胞核变化特点.流式细胞仪凋亡检测显示,BGC-823/SGC-7901对照组、5、10 mmol/L组,24h后凋亡率分别为(10.68±1.20)％/(11.86±0.20)％,(46.36±0.60)％/(50.72±1.90)％,(58.41±3.50)％/(59.92±2.60)％；48 h后分别为(8.20±1.60)％/(4.99±0.90)％,(76.34±2.70)％/(82.92±2.80)％,(87.18±3.10)％/(95.65±1.30)％.5、10 mmol/L组与对照组比较细胞凋亡率增高(P＜0.05),并有浓度和时间依赖性.Western blot法显示两株细胞中多聚腺苷二磷酸核糖聚合酶(PARP)、活化Caspase-9(cleaved Caspase-9)、活化Caspase-3(cleaved Caspase-3)表达增高,髓细胞白血病基因-1(Mcl-1)表达降低,在SGC-7901中突变型p53( Mtp53)表达降低.结论 柠檬酸钠能有效抑制胃癌细胞增殖,并经线粒体通路诱导凋亡,其作用随浓度和作用时间增加而增强.其机制可能与Mcl-1表达降低有关.     

琥珀酸对体外培养的人成纤维细胞功能的影响

目的　探讨琥珀酸对人成纤维细胞损伤的机制及其在脆弱类杆菌感染中的作用。方法　分别以 5、10、2 0、30mmol L(pH 5.5 )的琥珀酸刺激体外培养的人成纤维细胞 ,2 4h后检测细胞活力、细胞凋亡率、培养上清中的胶原合成量及caspase 3的活性。以等渗盐水刺激的该细胞作为对照组 ,观察刺激前、后各指标的变化。　结果　随着琥珀酸浓度的增高 ,成纤维细胞增殖速度减慢 ,胶原合成量下降 ,细胞凋亡率及caspase 3活性上升。当琥珀酸浓度为 10 30mmol L时 ,caspase 3的活性为 (0 .5 70 7± 0 .0 345 )(0.5 90 4± 0.0 16 8)U,明显高于对照组 (P <0.0 5);当琥珀酸浓度为 2 0、30mmol L时 ,细胞活力及胶原合成量明显低于对照组 ,凋亡率则较之明显增高 (P <0.0 5)。　结论　琥珀酸能抑制成纤维细胞增殖及胶原合成 ,促进细胞凋亡。创面感染时 ,细菌可通过代谢产生琥珀酸抑制创面愈合 ,使感染持续存在。     

依托咪酯预处理对HL-60细胞凋亡相关蛋白表达的影响

目的 探讨依托咪酯预处理对HL-60细胞procaspase-3、caspase-9 p35和caspase-8 p20表达的影响.方法 体外培养HL-60细胞,选择指数生长期的细胞,随机分为3组(n=3):对照组(C组)、依托咪酯组(E组)和依托咪酯预处理组(EP组).C组不给予任何处理;E组加入依托咪酯标准品500 μmol/L孵育24 h;EP组先加入依托咪酯标准品1μmol/L孵育1 h,冲洗后于培养基中培养4 h,再加入500 μmol/L依托咪酯孵育24 h.采用Western blot法测定procaspase-3、caspase-9 p35和caspase-8 p20的表达水平.结果 与C组比较,E组和EP组HL-60细胞procaspase-3表达下调,caspase-8 p20和caspase-9 p35表达上调(P＜0.05);与E组比较,EP组HL-60细胞procaspase-3表达上调,caspase-8 p20 和caspase-9 p35表达下调(P＜0.05).结论 依托咪酯预处理可抑制依托咪酯导致的procaspase-3表达下调和caspase-8 p20、caspase-9 p35表达上调,从而抑制依托咪酯诱发的HL-60细胞凋亡.     

T-型钙通道在利多卡因致神经母细胞瘤SH-SY5Y细胞损伤中的作用

目的 探讨T-型钙通道在利多卡因致神经母细胞瘤SH-SY5Y细胞损伤中的作用.方法 SH-SY5Y细胞离体培养后,采用随机数字表法,将SH-SY5Y细胞随机分为4组,每组66孔,正常对照组(C组):SH-SY5Y细胞继续培养24 h;米贝地尔+SH-SY5Y细胞组(M组):培养液中加入T-型钙通道阻断剂米贝地尔,终浓度5 μmol/L,孵育24 h;利多卡因+SH-SY5Y细胞组(L组):培养液中加入利多卡因,终浓度10 mmol/L,孵育24 h;米贝地尔+利多卡因+SH-SYSY细胞组(ML组):培养液中加入米贝地尔和利多卡因,终浓度分别为5 μmol/L、10 mmol/L,孵育24 h.于开始培养或孵育时(T0)、培养或孵育1、6、12和24 h时测定SH-SY5Y细胞活力和凋亡率,孵育24 h时观察SH-SY5Y细胞形态学.结果 与C组比较,L组和ML组SH-SY5Y细胞活力降低,凋亡率升高(P＜0.05),M组差异无统计学意义(P＞0.05);与M组比较,L组和ML组SH-SY5Y细胞活力降低,凋亡率升高(P＜0.05);与L组比较,ML组SH-SYSY细胞活力升高,凋亡率降低(P＜0.05).L组SH-SY5Y细胞形态学发生改变,ML组SH-SY5Y细胞形态学改变较L组减轻.结论 T-型钙通道参与了利多卡因导致的神经细胞损伤.

Abstract：

Objective To investigate the role of T-type calcium channel in lidocaine-induced neuronal cytotoxicity . Methods SH-SYSY cell line was a gift from cell biology laboratory of our medical university. The cells were cultured in DMEM liquid culture medium at 37℃ in incubator filled with 5% CO2 , and randomly divided into 4 groups ( n = 66 each) : control group (group C)and M, L and ML groups were exposed to 5 μmol/L mibefradil (a T-type calcium channel blocker), 10 mmol/L lidocaine and 5 μmoL/L mibefradil + 10 mmol/L lidocaine for 24 h. Cell morphology was examined by electronic microscopy at 24 h of drug exposure. Cell viability (by MTT) and neuronal apoptosis (by flow cytometry) were detected immediately before and at 1, 6, 12 and 24 h of exposure to mibefradil or/and lidocaine.Results In C and M groups, the cells demonstrated dendritic protrusions, enlarged nerve processes and dense lattice. After being exposed to lidocaine for 24 h, the dendritic protrusions disappeared,the cells decreased in size, shrinked and became round; the cell viability was significantly decreased while the neuronal apoptosis increased. The lidocaine-induced changes were significantly attenuated by co-incubation with mibefradil. ConclusionT-type calcium channel is involved in lidocaine-induced neuronal cytotoxicity.     

p38丝裂原活化蛋白激酶在罗哌卡因致SH-SY5Y细胞凋亡中的作用

目的 评价p38丝裂原活化蛋白激酶(p38MAPK)在罗哌卡因致SH-SY5Y细胞凋亡中的作用,以探讨罗哌卡因诱发神经毒性的机制.方法 采用随机数字表法,将SH-SY5Y细胞随机分为4组(n=18):正常对照组(C组)、10 μmol/L p38MAPK特异性抑制剂SB203580组(SB组)、3 mmol/L罗哌卡因组(R组)、10 μmol/L SB203580+3 mmol/L罗哌卡因组(SB+R组).C组在细胞培养液中继续培养；SB组在含10 μmol/L SB203580培养液中孵育；R组在含3 mmol/L罗哌卡因的培养液中孵育；SB+R组在含10 μmol/L SB203580的培养液中孵育30 min后,用含3mmol/L罗哌卡因的培养液继续孵育.各组细胞培养或罗哌卡因孵育4 h后采用流式细胞仪检测细胞内活性氧(ROS)水平和细胞凋亡率,采用Western blot法检测p38MAPK和磷酸化p38MAPK(p-p38MAPK)的表达,采用MTT法检测细胞活力.结果 与C组比较,R组和SB+R组ROS水平升高,p-p38MAPK表达上调,细胞活力降低,细胞凋亡率升高(P＜0.01),SB组上述指标差异无统计学意义(P＞0.05)；与R组比较,SB组p-p38MAPK表达下调,细胞活力升高,细胞凋亡率降低(P＜0.01).四组p38MAPK表达水平差异无统计学意义(P＞0.05).结论 罗哌卡因致SH-SY5Y细胞凋亡作用的机制部分与p38MAPK的激活有关.     

氯胺酮对谷氨酸引起神经元样嗜铬细胞瘤细胞株凋亡的影响

目的 观察氯胺酮对谷氨酸引起神经元样嗜铬细胞瘤(PC12)细胞株凋亡的影响。方法PC12细胞株分别以2×103/孔和1×105/ml密度接种于96孔细胞培养板和60 mm细胞培养皿中,置于培养基,培养基中加10 nmol/L神经生长因子(7S-NGF),96孔细胞培养板和60 mm细胞培养皿的细胞均随机分为五组,A组暴露于20 mmol/L谷氨酸中,B组暴露于20 mmol/L谷氨酸 0.1 mmol/L氯胺酮(氯胺酮比谷氨酸提前1 min 加入)中,C组暴露于20 mmol/L 谷氨酸 1.0 mmol/L氯胺酮中,D组暴露于20 mmol/L 谷氨酸 100 μmol/L D-2-氨基-5-膦酸基戊酸(D-AP5)(细胞与D-AP5提前孵育2h)中,E组暴露于等容积的不含7S-NGF的新鲜培养液中,采用MTT法测定细胞培养板中PC细胞的细胞活力,采用死端比色TUNEL系统细胞检测培养皿中PC12细胞株的凋亡率。结果 A组、B组、C组和D组细胞活力分别为:37％±6％、65％±7％、99％±10％、90％±22％,与A组比较,B组、C组、D组细胞活力均升高(P<0.05或0.01)。A组、C组、D组、E组凋亡细胞率分别为66％±10％、20％±6％、22±7％、3.2％±1.8％,与A组比较,C组、D组、E组细胞凋亡率明显降低(P<0.01)。结论 氯胺酮通过抑制谷氨酸引起的神经元样PC12细胞株凋亡而发挥神经保护作用。     

牛磺酸减轻碘海醇诱导的人肾小管上皮细胞损伤

目的 探讨牛磺酸能否减轻碘海醇导致的HK-2细胞（人近端肾小管上皮细胞）损伤及作用机制。 方法 不同碘浓度（25、50、100、125 gI/L）的碘海醇作用HK-2细胞6 h;碘浓度为100 gI/L的碘海醇作用HK-2细胞不同时间（2 h、4 h、6 h）,观察细胞损伤程度。选用碘海醇（100 gI/L、6 h）作为刺激组,用不同浓度牛磺酸（3、12、24 mmol/L）共孵育进行干预。细胞增殖-毒性试剂盒（CCK-8）测定细胞活力。Hoechst33342染色、荧光显微镜下观察细胞凋亡形态。流式AnnexinⅤ-FITC-PI双染和半胱氨酸天冬氨酸蛋白酶（caspase-3）活性比色法观察细胞凋亡。Western印迹检测Bcl-2、Bax的表达。流式DCFH-DA细胞内标记法测定细胞内活性氧（ROS）。 结果 碘海醇呈碘浓度和时间依赖性诱导HK-2细胞活力下降、凋亡增加（P ＜ 0.05）。碘海醇（100 gI/L、6 h）导致HK-2细胞ROS升高（P ＜ 0.05）。牛磺酸呈浓度依赖性增加HK-2细胞活力,减轻凋亡。牛磺酸（3、12、24 mmol/L）干预组与碘海醇刺激组比较,细胞活力分别为（88.0±1.00）%、（91.33±0.58）%、（95.67±1.52）%比（76.67±1.53）%（均P ＜ 0.05）;细胞凋亡率分别为（8.84±1.75）%、（7.86±1.82）%、（6.30±1.50）%比（11.98±0.39）%（均P ＜ 0.05）;caspase-3的活性分别为（1.33±0.10）、（1.27±0.0）、（1.10±0.04）比（1.42±0.1）（均P ＜ 0.05）。牛磺酸上调HK-2细胞Bcl-2表达,降低了细胞内ROS的升高（均P ＜ 0.05）。 结论 碘海醇可致HK-2细胞凋亡增加。氧化应激参与了HK-2细胞损伤。牛磺酸通过减轻细胞内氧化应激、促进Bcl-2表达上调,减轻碘海醇诱导的HK-2细胞凋亡和损伤。     

吗啡对胃癌细胞生物学性状的影响

目的 观察吗啡对人胃癌MGC-803细胞生物学性状的影响.方法 将吗啡加入细胞培养液中使吗啡浓度分别稀释至0.1、10、1000 μmol/L,分别与胃癌MGC-803细胞一同孵育,应用噻唑蓝(MTT)比色法绘制12、24、36、48、60、72h的细胞生长曲线并计算细胞增殖率,7d后应用克隆形成实验检测MGC-803细胞生长增殖的变化;应用流式细胞仪检测MGC-803细胞周期和细胞凋亡的变化,用透射电子显微镜观察MGC-803细胞超微结构的变化.结果 吗啡使胃癌细胞生长缓慢,0.1 μmol/L吗啡组、10 μmol/L吗啡组、1000 μmol/L吗啡组细胞增殖率分别为对照组的(81.89±6.44)%、(78.52±4.68)%和(78.53±5.68)%(P＜0.05),3组细胞的克隆形成率分别为(0.76±0.18)%、(0.72±0.17)%和(0.56±0.18)%,低于对照组的克隆形成率(1.19±0.29)%(P＜0.05);与对照组比较,各吗啡组细胞周期G2/M期比例上升,G0/G1期和S期比例下降(P＜0.05);0.1μmol/L吗啡组、10 μmol/L吗啡组、1000 μmol/L吗啡组细胞凋亡率分别为(17.20±2.95)%、(19.13±3.86)%、(24.38 ±4.94)%,明显高于对照组的凋亡率(11.40±2.86)%(P＜0.05);透射电镜显示吗啡使胃癌细胞出现明显的凋亡表现.结论 吗啡抑制胃癌MGC-803细胞的生长增殖,促进胃癌细胞的凋亡.     

Transient hypocalcemic reperfusion does not improve postischemic recovery in the rat heart after preservation with St. Thomas' Hospital cardioplegic solution.

We used the isolated perfused working rat heart to investigate the effects of transient hypocalcemic reperfusion after cardioplegic arrest with the St. Thomas' Hospital cardioplegic solution and 25 minutes of global normothermic (37 degrees C) ischemia. Hearts were reperfused (Langendorff mode) transiently (20 minutes) with solutions containing various concentrations of calcium; this was followed by 30 minutes of reperfusion with standard (1.4 mmol/L, the physiologic concentration) calcium buffer (10 minutes in the Langendorff mode and 20 minutes in the working mode). Recovery of cardiac output in control hearts (calcium concentration 1.4 mmol/L throughout) was 51.7% +/- 4.6%; in hearts transiently reperfused with hypocalcemic buffer (0.25, 0.5, 0.75, or 1.0 mmol/L) the recoveries of cardiac output were 49.3% +/- 6.4%, 52.2% +/- 7.2%, 58.7% +/- 3.2%, and 47.2 +/- 4.7%, respectively (all not significant), whereas recovery was only 14.7% +/- 2.8% (p less than 0.05) in hearts transiently reperfused with calcium 0.1 mmol/L. Creatine kinase leakage was significantly (p less than 0.05) greater in the group reperfused with calcium 0.1 mmol/L, but it did not vary significantly between the other groups. Tissue high-energy phosphate content was similar and in the normal range in all groups except for the group reperfused with calcium 0.1 mmol/L. In further experiments, the duration of hypocalcemic (0.5 mmol/L) reperfusion was varied (0, 5, 10, 15, 20, or 30 minutes). No significant differences in recovery of cardiac output were observed (58.2% +/- 5.0%, 52.3% +/- 5.7%, 52.0% +/- 8.2%, 61.2% +/- 5.0%, 62.2% +/- 4.3%, and 66.2% +/- 3.2%, respectively). In additional studies, the standard calcium concentration (1.4 mmol/L) used before and after ischemia was replaced by hypercalcemic solution (2.5 mmol/L). Despite this, transient (10 minutes) hypocalcemic (0.5 mmol/L) reperfusion did not improve recovery. Finally, studies were undertaken with a longer duration of ischemia (40 minutes), and although recovery of cardiac output in the hypocalcemic group (0.5 mmol/L for 10 minutes) tended to be higher than in the control group (29.7% +/- 4.8% versus 18.5% +/- 4.9%, respectively), statistical significance was not achieved. We conclude that in these studies transient hypocalcemic reperfusion did not afford any additional protection over and above that afforded by cardioplegia alone.     

3-甲基腺嘌呤对大鼠内皮祖细胞增殖、凋亡及细胞周期的影响

目的 观察3-甲基腺嘌呤(3-methlyadenine,3-MA)对大鼠骨髓源性内皮祖细胞(endothelial progenitor cells,EPCs)增殖、凋亡及细胞周期的影响.方法 密度梯度法取大鼠骨髓EPCs,体外诱导分化并鉴定.实验分对照及不同浓度(1.25、2.5、5、10 mmol/L)3-MA组.四氮唑蓝(methyl thiazolyl tetrazolium,MTT)法测细胞增殖,流式细胞仪测凋亡率及细胞周期.结果 (1)5 mmol/L组促EPCs增殖;10 mmol/L组抑制EPCs增殖,差异有统计学意义(P＜0.05).其余两组对EPCs增殖无明显影响,差异无统计学意义(P＞0.05).(2)10 mmol/L组促EPCs凋亡,差异有统计学意义(P＜0.05).其余各组对EPCs凋亡率无明显影响,与对照组比较差异无统计学意义(P＞0.05).(3)5、10 mmol/L组影响细胞周期,5 mmoL/L组G0/G1期细胞减少、S和G2/M期增加;10 mmol/L组发生S期阻滞,G2/M细胞减少;差异有统计学意义(P＜0.05).结论 3-MA对EPCs增殖及凋亡的影响与其浓度有关,当浓度为10 mmol/L时抑制增殖并促进凋亡.

Abstract：

Objective To investigate the effect of proliferation,apoptosis and cell cycle of 3-MA on rat endothelial progenitor cells. Methods Bone marrow-derived mononuclear cells were isolated from rat bone marrow by ficoll. There were five groups. The control group and four 3-MA concentration groups: 1. 25 mmol/L,2. 5 mmol/L,5 mmol/L, 10 mmol/L. MTT was used to measure the proliferation of endothelial progenitor cells. Flow cytometry ( FCM) was used to detect cell apoptosis and cell cycle. Results (1)5 mmol/L 3-MA promotes proliferation of endothelial progenitor cells, while 10 mmol/L 3-MA inhibits the proliferation of endothelial progenitor cells (P ＜ 0. 05). (2) 10 mmol/L 3-MA promotes apoptosis of endothelial progenitor cells, compared with the control, the difference was significant ( P ＜ 0. 05 ). (3) 3-MA at the concentration of 5 mmol/L reduces cells at G0/G1 phase and increases S and G2/M phase cells; 10 mmol/L 3-MA induces endothelial progenitor cells blockade at S phase, G2/M phase cells decreased, compared with the control, the difference was significant (P ＜ 0. 05). Conclusions 5 mmol/L 3-MA promotes the proliferation of endothelial progenitor cells. 10 mmol/L 3-MA inhibits the proliferation and promotes apoptosis of endothelial progenitor cells.     

1，25（OH）2VD3诱导小鼠胚胎干细胞向成骨细胞分化的研究

目的 探讨1,25(OH)2VD3在诱导小鼠胚胎干细胞(embryonic stem cells,ESCs)向成骨细胞分化过程中的重要作用.方法 取2 d龄昆明小白鼠颅盖骨进行成骨细胞分离培养;参照并改进zur Nieden等的方法制备拟胚体(embryoid bodies,Ebs).将Ebs根据培养液不同分为4组:A组:不含白血病抑制因子Ebs培养液;B组:Ebs培养液中添加50μg/mL维生素C、50 mmol/L β-磷酸甘油;C组:培养液同B组,另每孔接种5×104个第3代成骨细胞;D组与C组相同,另添加4 × 10-9 mol/L 1,25(OH)2VD3.检测ALP活性,实时定量PCR检测骨钙素mRNA的表达,茜素红S染色进行矿化骨结节计数.结果 Ebs贴壁后前5 d,各组ALP表达未发生明显变化,5 d后ALP表达逐渐增加,其中D组在第20天表达水平最高.光镜下可见轮廓清晰大小不等的红色结节.茜素红S染色测得A、B、C、D组骨结节数分别为(20±8)、(18±5)、(31±1)、(50±1)个:实时定量PCR测得A、B、C、D组骨钙素mRNA分别为10.18±1.17、20.29±1.03、18.84±4.07、32.15±5.23:C、D组与A、B组比较差异均有统计学意义(P＜0.05),A、B组间比较差异无统计学意义(P＞0.05),C、D组间比较差异有统计学意义(P＜0.05).结论 在浓度为4 × 10-9 mol/L 1,25(OH)2VD3和维生素C、β-磷酸甘油共同作用下,有效促进了ESCs源性成骨细胞的产生.     

Effect of cold blood cardioplegia enriched with potassium-magnesium aspartate during coronary artery bypass grafting

AIM: The aim of this investigation is to evaluate the effect of enriched with potassium-magnesium aspartate cold-blood cardioplegia on early reperfusion injury and postoperative arrhythmias in patients with ischemic heart disease undergoing coronary artery bypass grafting (CABG), using measurements of cardiac troponin I (CTnI), hemodynamic indexes and clinical parameters. METHODS: Forty patients with three-vessel coronary artery disease (CAD) and stable angina, receiving first-time elective CABG, were randomly divided into 2 groups: patients in control group (C group n=20) received routine institutional cold blood cardioplegia (4 degrees C) concentration of Mg2+4 mmol/L, Ca2+1.2 mmol/L and K+ 24mmol/L during myocardial arrest. Patients in P group (n=20) received modified cold blood cardioplegia enriched with potassium-magnesium aspartate and maintained concentration of Mg2+10 mmol/L, Ca2+1.2 mmol/L and K+20mmol/L in the final blood cardioplegia solution. Clinical outcomes were observed during operation and postoperatively. Serial venous blood samples for CTnI were obtained before induction, after cardiopulmonary bypass (CPB), and postoperative 6, 24, and 72 hours. Hemodynamic indexes were obtained before and after bypass by the radial catheter and Swan-Ganz catheter. RESULTS: In both groups, there were no differences regarding preoperative parameters. There were no cardiac related deaths in either group. The time required to achieve cardioplegic arrest after cardioplegia administration was significantly shorter in P group (47.5+/-16.3 s) than in C group (62.5+/-17.6 s) (P<0.01). The number of patients showing a return to spontaneous rhythm after clamp off was significantly greater in P group (n=20, 100%) than in C group (n=14, 70%) (P<0.01). Eight patients in C group had atrial fibrillation (AF) compared with two patients in P group (P<0.05) in the early of postoperative period. The level of CTnI increased 6 hours and 12 hours postoperatively, and there was a significant difference between groups (P<0.05). P group also shortened the time of postoperative mechanical ventilation (P<0.05) after surgery. CONCLUSIONS: Cold blood cardioplegia enriched with potassium-magnesium aspartate is beneficial on reducing reperfusion injury.     

灵芝孢子粉对2型糖尿病大鼠附睾细胞线粒体Ca~(2+)细胞色素C的影响

目的:研究灵芝孢子粉对2型糖尿病大鼠附睾组织中细胞色素C(Cyt-C)、线粒体Ca2+的影响。方法:青春期Wistar大鼠50只,大鼠尾静脉一次性注射2%链脲佐菌素(STZ)或柠檬酸-柠檬酸钠缓冲液制备2型糖尿病大鼠模型和对照组模型。糖尿病大鼠模型随机分模型组和灵芝组,每组20只,分别给以高脂高糖饮食、高脂高糖+灵芝孢子粉[250mg/(kg.d)],对照组10只,正常饮食。10周后,取双侧附睾,检测附睾细胞线粒体Ca2+、Cyt-C含量。结果:2型糖尿病模型制备成功,模型组附睾细胞线粒体Ca2+含量[(3.279±0.502)mg/L]明显高于对照组[(2.606±0.048)mg/L,P<0.01],灵芝组[(2.693±0.196)mg/L]明显低于模型组(P<0.05),与对照组差异无显著性(P>0.05)。模型组线粒体Cyt-C含量[(3.213±1.511)μmol/L]明显少于对照组[(5.688±1.679)μmol/L,P<0.05],胞质Cyt-C含量[(2.484±0.661)μmol/L]明显高于对照组[(1.574±0.329)μmol/L,P<0.01];灵芝组线粒体Cyt-C含量[(5.258±1.560)μmol/L]高于模型组,但差异无显著性(P>0.05),胞质Cyt-C含量[(1.727±0.396μmol/L]明显低于模型组(P<0.05),与对照组差异无显著性(P>0.05)。结论:2型糖尿病大鼠附睾细胞钙稳态失衡、线粒体有损伤,附睾细胞可能存在细胞凋亡过度。灵芝孢子粉在糖尿病状态下,对附睾组织有保护作用或有对抗细胞凋亡作用。     

Maintenance of normoglycemia during cardiac surgery

We used the hyperinsulinemic normoglycemic clamp technique, i.e., infusion of insulin at a constant rate combined with dextrose titrated to clamp blood glucose at a specific level, to preserve normoglycemia during elective cardiac surgery. Ten nondiabetic and seven diabetic patients entered the clamp protocols. Perioperative glucose control was also assessed in 19 nondiabetic and 11 diabetic patients (control group) receiving a conventional insulin infusion sliding scale. In patients of the clamp group, a priming bolus of insulin (2 U) was started before the induction of anesthesia followed by infusions of insulin at 5 mU. kg(-1). min(-1) and of variable amounts of dextrose. Arterial blood glucose was measured every 5 min in the clamp group and every 20 min in the control group. Control of normoglycemia was defined as > or =95% of the glucose levels within 4.0-6.0 mmol/L. Glucose concentration was recorded before surgery, 15 min before cardiopulmonary bypass (CPB), during early and late CPB, and at sternal closure. Patients of the control group became progressively hyperglycemic during surgery (late CPB; nondiabetics, 9.0 +/- 3.2 mmol/L; diabetics, 10.1 +/- 3.6 mmol/L), whereas normoglycemia was achieved in the study group (late CPB; nondiabetics, 5.5 +/- 0.7 mmol/L; diabetics, 4.9 +/- 0.6 mmol/L; P < 0.05 versus control group). In conclusion, it seems that normal blood glucose concentration during open heart surgery can be reliably maintained in nondiabetic and diabetic patients by using the hyperinsulinemic normoglycemic clamp technique.     

Pulmonary hypertension and polymorphonuclear leukocyte elastase after esophageal cancer operations

To evaluate the role of polymorphonuclear leukocyte (PMN) elastase in pulmonary impairment occurring after operation for esophageal cancer, 10 patients were randomized preoperatively into two equal groups. One group received a placebo infusion and the other, an infusion of the PMN elastase inhibitor ulinastatin. In the placebo group, the mean plasma PMN elastase level increased from 154 +/- 23 micrograms/L preoperatively to 449 +/- 56 micrograms/L at 6 hours postoperatively (p less than 0.01), whereas the mean plasma fibronectin concentration decreased from 490 +/- 70 micrograms/mL preoperatively to 265 +/- 81 micrograms/L on postoperative day 2 (p less than 0.01). The mean pulmonary vascular resistance increased markedly from 151 +/- 24 dynes.s.cm-5.m-2 preoperatively to 284 +/- 76 dynes.s.cm-5.m-2 at 6 hours postoperatively (p less than 0.01). In the group given ulinastatin, 150,000 units every 12 hours from the start of the operation, the mean PMN elastase value at 6 hours postoperatively was lower (275 +/- 66 micrograms/L; p less than 0.01) and the fibronectin level on postoperative days 1 and 2, higher (p less than 0.05). A lower pulmonary vascular resistance was noted into day 2 (p less than 0.05). Our results suggest that PMN elastase may participate in the development of postoperative pulmonary impairment.     

全反式维甲酸对鼠胚神经干细胞增殖和分化的作用

目的 观察不同浓度的全反式维甲酸(all-trans-retinoic acid,ATRA)在鼠胚神经干细胞(neural stemcells,NSCs)增殖和分化中的作用,寻找促NSCs分化为神经元的较佳ATRA浓度. 方法 分离孕12～16 d(平均15 d)胚胎大鼠脑皮层,用无血清但含神经生长因子(20 ng/mL bFGF、20 ng/mL EGF)的培养液悬浮培养,细胞接种密度为1×106/mL,7 d传代.取第3代NSCs,分别在含0.5、1.0、5.0和10.0 μmol/L ATRA(实验组,分别为A、B、C、D组)及不含ATRA(对照组,E组)的DMEM/F12完全培养基中培养,于培养第1、2、3、4、5、6及7天倒置相差显微镜下计数形成的细胞球数;于培养后1、3、5、7及9 d采用BrdU免疫荧光染色,分析各组细胞增殖效应.另取第3代NSCs,5?S培养基调整细胞密度为1×106/mL,接种至6孔板内,分别加入0.5、1.0、5.0和10.0 μmol/L ATRA,作为各实验组(即A、B、C、D组),对照组不含ATRA(E组),于培养后3、5及7 d采用免疫荧光双标染色和流式细胞仪检测各组细胞分化为神经元的比例. 结果 培养后1、2、3、4、5、6及7 d,各实验组增殖细胞球数目接近(P＞0.05),但均明显少于对照组,且差异有统计学意义(P＜0.05);各实验组在培养后1、3、5、7、9dBrdU阳性细胞球增多,组间差异无统计学意义(P＞0.05);对照组各时间点BrdU阳性细胞球明显多于实验组,差异有统计学意义(P＜0.05).各实验组ATRA促使NSCs分化为神经元的比例明显增高,NSE阳性细胞分化率各组间差异有统计学意义(P＜0.05),其中B组最高,在培养3、5、7 d分别为29.46%±0.47%、47.25%±0.46%、66.81%±0.57%,而E组NSCs主要分化为星形胶质细胞,NSE阳性细胞分化率在培养3、5、7 d分别为11.11%±0.59%、14.10%±0.32%、15.92%±0.70%.流式细胞仪检测显示,培养后3、7 d,促神经元分化比例各实验组与对照组比较差异均有统计学意义(P＜0.05). 结论 ATRA具有显著的促NSCs分化为神经元的作用,1.0 μmol/L ATRA诱导分化效果最佳.