电针对局灶性脑缺血再灌注大鼠脑细胞凋亡及神经功能恢复的影响

Objective To investigate the influence of acpuncture on free calcium in rat brain cells after focal cerebral ischemia reperfusion.Methods 145 adult male SD rats were randomly divided into control group,simple ischemia reperfusion group and acupuncture with ischemia reperfusion group.The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established by the modified Longa occlusion method. ①The part of free calcium in rat brain cells,focal cevebral ischemia model of rats were made by thread locking up the blood vessel for 15 min.30 min later after reperfusion, the Baihui and Shuigou Point in Du meridian were acupunctured electrically 30 min.After 3h, 6h and 12h, the rat was killed and its brain cells were made into single cell suspension,marked by Fluo-3/AM.The fluorescence optical density was recorded by laser confocal microscopy.②The part of nerve functional reconstruction, focal cevebral ischemia model of rats were made by thread locking up the blood vessel for 12 hours.30 min later after reperfusion, the Baihui and Shuigou Point in Du meridian were acupunctured electrically 30 min.After 7 d, 14 d,30 d,60 d and 90 d, the rat was forced to detect it's strength of the dog.Results ①Free calcium in rats of acupuncture therapy group(6h:10.96±1.18;2h:20.9±4.37) was significantly less than that in control group in 6 h and 12 h after reperfusion (6 h: 16.87 ± 3.56,12 h: 34.10 ±1.06)(P<0.05).②The dog in rats of acupuncture therapy group was significantly more than that in control group in 7 d, 14 d after reperfusion (P＜ 0.05 ).No difference of the dog was detected in 30 d ,60 d and 90 d after reperfusion between the two groups.Conclusion Acupunture could decreases the concentration of free calcium and the expression of Caspase-3 mRNA in rat brain cells after focal cerebral ischemia reperfusion, and it can facilitate the recovery of nerve function.     

针刺对缺血再灌注大鼠脑细胞凋亡的保护作用及机制

Objective To investigate the protective effect and mechanisms of acupuncture on the mem-brane potential in mitochondria of the brain cells injury induced by ischemia and reperfusion. Method The mid-dle cerebral artery occlusion/repeffusion (MCAO/R) rat model was established by the modified Longa occlusion method. The Baihui and Shuigou Point in Du meridian was acupunctured electrically 30 min. After reperfusion 24 h and 48 h, mitochondrial membrane potential, apoptosis and mitochondria ultrastrocture of brain cells were detected by the method of Rhodamin123 fluorescence staining,TUNEL in situ labeling and electron microscopy respectively after repeffusion 24 h and 48 h. Results The fluorescence intensity of Rhodamin123 was 961.27±34.22 and 1231.23±121.54 after focal cerebral ischemic reperfusion 24 h and 48 h respectively,this result was increased to compare with sham-operated group. The fluorescence intensity of Rhodamin 123 in group of using the intervention of acupuncture was 808.44±56.23 and 954.25±35.11 after operation 24 h and 48 h respectively ,this values de-creased significantly to compare with the group of ischemic reperfusion. The amount of apoptosis cells after cerebral ischemia reperfusion 24 h and 48 h were more than the sham group. The amount of apoptosis cells after acupuncture was less than the cerebral ischemic reperfusion group. The mitochondrial membrane shown intumesee,disaggrega-tion, endocrista collapsing and toxic granulation increased after ischemie reperfusion and this non-specificity change of mitochondrion alleviated by using acupuncture. Conclusion Acupuncture stabilized the permeability of mitochon-drial membrane, reduced the dissipation of mitochondrial transmembrane potential, decreased the amount of apopto-sis of brain cells of ischemic reperfusion and increased the toleration of brain against the ischemic and anoxie inju-ry. Conclusion Acupuncture could stabilized the permeability of mitochondria membrane. It could decreased the dissipation of cytochondriome transmembrane potential and the amount of apoptosis of brain cells. It could increased the toleration of brain against the ischemic and anoxic injury.     

Effect of Batroxobin on Neuronal Apoptosis During Focal Cerebral Ischemia and Reperfusion in Rats

We have found that Batroxobin plays a protactive role in ischemic brain injury,which attracted us toinvestigate the effect of Batroxobin on apoptosis of neurons during cerebral ischemia and reperfusion.The apoptotic cells in ischemic rat brains at different reperfusion intervals were tested with method ofTdT-mediated dUTP-DIG nick end labeling (TUNEL) and the effect of Batroxobin on the apoptosis ofneurons was studied in left middle cerebral artery (LMCA) occlusion and reperfusion in rat models(n=18).The results showed that few scattered apoptosis cells were observed in right cerebralhemispheres after LMCA occlusion and reperfusion,and that a lot of apoptosis cells were found in leftischemic cortex and caudoputamen at 12h reperfusion,and they reached peak at 24h～48h reperfusion.However,in the rats pretreated with Batroxobin,the number of apoptosis cells in left cerebral cortexand caudoputamen reduced significantly and the neuronal damage was much milder at 24h reperfusionthan that of saline-treated rats.The results indicate that administration of Batroxobin may reduce theapoptosis of neurons induced by cerebral ischemia and reperfusion and afford significantcerebroprotection in the model of focal cerebral ischemia and reperfusion.     

PROTECTIVE EFFECT OF RADIX SALVIAE MILTIORRHIZAE ON APOPTOSIS OF NEURONSDUREVG FOCAL CEREBRAL ISCHEMIA AND REPERFUSION INJURY

We have found that Radix Salviae Miltiorrhizae (RSM) plays a protective role in ischemic brain injury,which attracted us to investigate the effect of RSM on apoptosis of neurons during cerebral ischemia and reperfusion. The apoptotic cells in ischemic brains at different reperfusion intervals were tested with the method of TdT-mediated dUTP-DIG nick end labeling (TUNEL), and the effect of RSM on the apoptosis of neurons was studied in left middle cerebral artery (LMCA) occlusion in rat models (n=18). The results showed that few scattered apoptotic cells were observed in right cerebral hemisphere after LMCA occlusion and reperfusion, and that a lot of apoptotic cells were found in left ischemic cerebral cortex and caudoputamen at 12 h reperfusion, and they reached peak at 24-48h reperfusion. However, in rats pretreated with RSM, the number of apoptotic cells in left cortex and caudoputamen reduced significantly and the neuronal damage was much milder at 24h reperfusion as compared with those of saline-treated rats. From this study, we conclude that administration of RSM can reduce the apoptosis of neurons induced by cerebral ischemia and reperfusion and afford significant cerebroprotection in the model of focal cerebral ischemia and reperfusion.     

THE EFFECT OF RADIX SALVIAE MILTIORRHIZAE(RSM)ON SUBSTANCEP IN CEREBRAL ISCHEMIA—ANIMAL EXPERIMENT

The levels of substance P(SP)in rat brains were assayed in 64 rats.Bilateral common carotid ar-tery ligation was done in 49 rats.Half an hour before ligation,25 rats were given 10 g/kg ofRSM;24 rats were given the same volume of normal saline as controls.Sham operation was donein 15 rats.Half an hour and 3 hours after cerebral ischemia,the rats were quickly decapitated.SP concentration was assayed in the cerebral cortex,caudate nucleus and brain stem.Insaline-treated animals,the SP level of caudate nucleus at 3-hour group was significantly de-creased as compared with the 0.5-hour group and sham-operated group respectively.No signifi-cances were found among RSM-treated groups and sham-operated groups.The SP levels wereshown:brain stemcerebral cortex.The preliminary results suggest that SPmay be involved in the pathophysiologic procedures of cerebral ischemia and RSM mayattenuate the dysfunction of SP during cerebral ischemia.     

EFFECTS OF PHYCOCYANIN ON EXPRESSION OF CytC mRNA AND CASPASE-3 mRNA AFTER FOCAL CEREBRAL ISCHEMIA／REPERFUSION IN RATS

Objective To study the effects of phycocyanin on the expression of Cytochrome C (CytC) genes and Caspase-3 genes after focal cerebral ischemia/reperfusion in rats. Methods A rat middle cerebral artery occlusion (MCAO)/reperfusion model was produced using the intraluminal filament method. The rats were divided into three groups: sham operation group, model control group and phycocyanin group. After MCAO, the neurobehavioral testing of all rats was made. The infarction area was evaluated with the method of 2,3,7-triphenylt-etrazolium chloride (TTC) staining. The expression of CytC mRNA and Caspase-3 mRNA were determined by in situ hybridization. Results In the sham operation group and the model control group, there was only a few CytCpositive cells were seen in the normal cerebral tissue. In the model control group, the upregulation of CytC mRNA began 6h after ischemia, reached a maximum at 12h (cortex)-24h (striatum) , then subsided gradually, but still in high level. In the phycocyanin group, CytC-positive cells were also mainly in cortex and striatum, but the number of the cells was significantly lower than the number of the model control group. The time-phase pattern of CytC mRNA in the phycocyanin group was similar to the pattern of the model control group. In the sham operation group and the model control group, there was only a few Caspase-3-positive cells were seen in the normal cerebral tissue. In the model control group, the upregulation of Caspase-3 mRNA began 6h after ischemia, reached a maximum at 24h and subsided at 48h, but still in high level. In the phycocyanin group, Caspase-3-positive cells were also mainly in the penumbral area, but the number of the cells were significantly lower than the number of the model control group. The time-phase pattern of Caspase-3 mRNA in the phycocyanin group was similar to the pattern of the model control group. Conclusion The over-expression of CytC mRNA and Caspase-3 mRNA might play a key role in ischemic cerebral injury after MCAO. Phycocyanin could inhibit the over-expression of CytC mRNA and Caspase-3 mRNA in the cerebral cortex, and might play an important role in the protection of ischemic neurons.     

Changes of c-fos,malondialdehyde and lactate in brain tissue after global cerebral ischemia under different brain temperatures

Summary： Under global cerebral ischemia, the effect of different brain temperature on cerebral ischemic injury was studied. Male Sprague-Dawley rats were divided into normothermic （37-38℃） ischemia, mild hypothermic （31 32℃） ischemia, hyperthermic （41-42℃） ischemia and sham-operated groups. Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model and brain temperature was maintained at defined level for 60 min after 20omin ischemia. The expression of c-fos protein and the levels of malondialdehyde （MDA） and lactate in brain regions were detected by immunochemistry and spectrophotometrical methods, respectively. C-fos positive neurons were found in the hippocampus and cerebral cortex after cerebral ischemia reperfusion. Mild hypothermia increased the expression of c-fos protein in both areas, whereas hyperthermia decreased the expression of c-los protein in the hippocampus at 24 h reperfusion, and the cerebral cortex at 48 h reperfusion when compared to normothermic conditions. In normothermic, mild hypothermic and hyperthermic ischemia groups, the levels of MDA and lactate in brain tissue were increased at 24, 48 and 72 h reperfusion fol- lowing 20-min ischemia as compared with the sham-operated group （P〈0.01）. The levels of MDA and lactate in mild hypothermic group were significantly lower than those in normothermic group （P〈0.01）. It is suggested that brain temperature influences the translation of the immunoreactive protein product of c-fos after global cerebral ischemia, and MDA and lactate are also affected by hypothermia and hyperthermia.     

Attenuation of Brain Inflammatory Response after Focal Cerebral Ischemia/Reperfusion with Xuesaitong Injection(血塞通注射液) in Rats

Objective： To investigate the neuro-protective effect of Xuesaitong Injection （血塞通注射液, XST） on brain inflammatory response after transient focal cerebral ischemia/reperfusion in rats. Methods： Focal cerebral ischemia/reperfusion models of male rats were induced by transient occlusion for 2 h of middle cerebral artery （MCA） which was followed by 24 h reperfusion. XST was administered through intraperitoneal injection of 25 mg/kg or 50 mg/kg at 4 h after the onset of ischemia. After reperfusion for 24 h, the neurological function score was evaluated, the brain edema was detected with dry-wet weight method, the myeloperoxidase （MPO） activity and the expression of intercellular adhesion molecule-1 （IOAM-l） of ischemic cerebral cortex and caudate putamen was determined by spectrophotometry and immunohistochemistry respectively. Results： XST not only lowered neurological function score at the dose of 50 mg/kg, but reduced brain edema and inhibited MPO activity and IOAM-1 expression as compared with the ischemia/reperfusion model group （P〈0.01）. Conclusion： XST has a definite effect on inhibiting the expression of IOAM-1 and neutrophil infiltration in rats with cerebral ischemia/reperfusion when treatment started at 4 h after ischemia onset, and also attenuates inflammation in the infarcted cerebral area.     

Inhibitory Effect of Allopurinol on the Generation of Free Radicals in Reperfused Rat Brain

Free radicals in ischemic and reperfused rat brain were measured by electron spinresonance(ESR) spectrometer.The inhibitory effect of allopurinol on the free radical genera-tion in reperfused rat brain was observed.The experimental results revealed that the free radi-cal content of the brain in the ischemia group was markedly higher than that in the control group(P<0.01),that in the reperfusion group was markedly higher than that in the ischemia group(P<0.01)and that in the allopurinol group was markedly lower than that in the reperfusiongroup(P<0.01).These results suggest that free radicals increase.greatly after cerebralischemia-reperfusion and that allopurinol plays a certain inhibitory role in the free radical genera-tion in the reperfused rat brain.     

The effect of hyperglycemia on blood brain barrier of rats with focal cerebral ischemia/reperfusion injury

Objective： To determine whether hyperglycemia could aggravate the microvascular damage in ischemic stroke. Methods： Hyperglycemia model was made by injection of streptozocin through subcutaneous injection in wistar rats. Using the suture model, the rats were subjected to 3h of focal ischemia and different times of reperfusion, including 6,12,24,48,96h and 7d. TIC dyeing was used to Show the infarction area of rats. The infarctive volume of rats were calculated by computer imaging analysis system;Matrix metalloproteinase （MMP-2） and （MMP-9）were detected by immunohistochemistly and in situ hybridization histochemistly in Wistar rats. Results： The infarctive volume was siginificantly larger in hyperglycemic rats than that of nonhyperglycemic rats. The level of MMP-2, MMP-9 expression in the group of hyperglycemic rats was higher than that of nonhyperglycemic rats. Conclusion： Hyperglycemia aggravated the injury of focal ischmia-repeffusion in wistar rats and the higher expression of MMP-2,MMP-9 might be one of the mechanism in aggravation of focal ischemia/repeffusion injury.     

人白介素-10在NIH3T3细胞中的表达及生物学效应的初步观察

目的观察IL-10的免疫抑制作用.方法将构建好的pLXHIL-10载体,经包装细胞PA317包装后感染小鼠成纤维细胞株NIH3T3,用ELISA和原位杂交的方法检测hIL-10的表达,并对其生物学活性进行初步观察.结果检测到感染细胞中有hIL-10 mRNA的表达,感染细胞培养上清中存在hIL-10,证实感染细胞培养上清对混合淋巴细胞培养反应有明显的抑制作用.结论hIL-10可抑制混合淋巴细胞反应.     

Foxp3、CD3在非小细胞肺癌组织中的表达及意义

目的探讨Foxp3、CD3在非小细胞肺癌（NSCLC）组织中的表达及其与肺癌临床病理特征和预后的关系。方法选取62例NSCLC患者术后的肺癌组织标本（NSCLC组）及27例正常肺组织（距离肿块边缘5cm以上正常肺组织或另一叶肺组织）作为正常对照组。采用免疫组化方法分别检测两组组织中Foxp3、CD3表达的表达情况,并分析其与肺癌临床病理特征（年龄、性别、组织学类型、肿瘤大小、分化程度、pTNM分期、淋巴结转移与否）的关系。结果NSCLC组Foxp3、CD3阳性细胞数均显著多于正常对照组（P〈0．01）。两者显著相关（P〈001）。Foxp3阳性表达仅在不同分化程度间存在明显差异,低分化者Foxp3阳性细胞浸润量高于高、中分化者（P〈0．05）;CD3阳性表达仅在不同术后pTNM分期间存在明显差异,ⅢB期、Ⅳ期NSCLC组织中CD3阳性细胞数明显减少（P〈0.05）。Foxp3表达与患者术后生存时间无关（P〉0.05）,CD3阳性细胞数与NSCLC患者术后生存时间呈线性正相关（P〈0．01）,Foxp3阳性细胞数与CD3阳性细胞数比值（Foxp3／CD3）与患者生存时间呈线性负相关（P〈0.05）。pTNM分期、CD3阳性细胞绝对数有统计学意义,可作为NSCLC患者的独立预后因素。结论NSCLC组织中CD3阳性细胞数低、Foxp3／CD3大者T细胞肿瘤免疫作用抑制,影响患者术后生存时间。联合检测术后肺癌组织中Foxp3、CD3,并结合患者术后病理分期可以更好地评估NSCLC患者手术预后。     

小鼠不同细胞间组蛋白修饰变化对MafA基因转录表达的影响

目的通过比较不同细胞类型之间MafA基因转录起始区的组蛋白修饰差异,探讨组蛋白修饰对MafA基因转录表达的作用。方法采用染色质免疫共沉淀-实时定量PCR法检测小鼠胰岛素瘤β细胞(NIT-1)、NIH小鼠成纤维细胞(NIH3T3)及小鼠胚胎干细胞(mES)三者中的MafA和MLH1基因转录起始区组蛋白修饰(H3K4m3、H3K9m3和H3乙酰化)的状况。同时采用实时定量RT-PCR检测上述三种细胞各基因mRNA表达水平。分析基因的H3K4m3、H3K9m3和H3乙酰化修饰与基因表达之间的相互关系。结果 (1)以mES细胞为参照,NIT-1细胞MafA基因的转录起始区的H3K4m3修饰水平明显增高(P<0.05),H3K9m3修饰水平明显降低(P<0.05);NIH 3T3细胞MafA基因的转录起始区的H3K9m3修饰水平明显增高(P<0.05),H3K4m3修饰水平明显降低(P<0.05);(2)MafA基因的仅在NIT-1细胞表达,其表达与H3K4m3修饰存在直线相关(相关系数0.995);与H3K9m3修饰存在直线负相关(相关系数-0.751);(3)管家基因MLH1的表达与所检测组蛋白修饰无相关性。结论 H3K9m3与H3K4m3修饰能相互协调,共同调控MafA基因的表达,对胚胎干细胞向β细胞分化具有重要的意义。     

硫化氢对脂肪细胞前体增殖的影响

目的：探讨硫化氢对小鼠3T3-L1前脂肪细胞增殖的影响。方法：培养3T3-L1前脂肪细胞,分为对照组、低剂量组和高剂量组（硫化氢浓度分别为0、10、25、50和100μmol/L）,采用四甲基偶氮唑盐比色（MTT）法检测硫化氢对3T3-L1前脂肪细胞活性的影响;用BRDU法检测细胞的增殖;结果：与对照组相比,H2S在较高浓度时,对细胞有一定的毒性作用（P〈0.05）。结论：硫化氢抑制3T3-L1前脂肪细胞的增殖。     

硒对3T3-L1前脂肪细胞增殖和分化的影响

目的:研究硒对3T3-L1前脂肪细胞增殖和分化的影响。方法:培养3T3-L1前脂肪细胞,分为对照组、低硒组、中硒组和高硒组(亚硒酸钠浓度分别为0、0.01、0.1和1μmol/L),采用四甲基偶氮唑盐比色(MTT)法检测硒对3T3-L1前脂肪细胞增殖的影响,利用油红O染色法测定硒对3T3-L1前脂肪细胞分化的影响。结果:在增殖实验中,与对照组相比,各处理组的OD值降低,并具有剂量依赖性(P<0.05);在分化实验中,各处理组的OD值与对照组差异无统计学意义(P>0.05)。结论:硒能抑制3T3-L1前脂肪细胞的增殖,而对其分化无影响。     

梓醇与小檗碱及其配伍对胰岛素抵抗3T3-L1脂肪细胞的影响

目的观察梓醇与小檗碱及其配伍对地塞米松诱导的胰岛素抵抗3T3-L1脂肪细胞葡萄糖消耗、转运及这一过程中过氧化物体增殖物激活受体(PPAR-γ)mRNA表达的影响。方法采用地塞米松诱导胰岛素抵抗细胞模型,分别给予罗格列酮、小檗碱、梓醇、梓醇 小檗碱进行干预,以葡萄糖氧化酶法检测培养液中葡萄糖消耗量,以2-脱氧-[3H]-D-葡萄糖摄入法观察葡萄糖的转运率,以RT-PCR检测PPAR-γmRNA的表达。结果含或不含10nmol/L胰岛素的条件下,梓醇、小檗碱及其配伍组胰岛素抵抗脂肪细胞的葡萄糖消耗量和转运率较模型组明显改善(P<0.05、0.01),配伍组效应优于梓醇、小檗碱单药组(P<0.05、0.01);且小檗碱组及配伍组PPAR-γmR-NA的表达降低(P<0.05、0.01)。结论梓醇、小檗碱均能增加葡萄糖消耗和转运,改善胰岛素抵抗,其作用不依赖胰岛素的存在,且小檗碱及两药配伍还能下调脂肪细胞PPAR-γmRNA的表达水平,提示梓醇、小檗碱改善胰岛素抵抗的作用机制可能与罗格列酮不同。     

RNAi沉默STAT3基因对人卵巢癌SKOV3细胞裸鼠移植瘤生长的抑制作用

目的：研究pSilencer1.0-U6-siRNA-STAT3重组质粒对人卵巢癌SKOV3细胞裸鼠皮下移植瘤生长的抑制作用,阐明RNA干涉技术在卵巢癌生物治疗领域中的作用。方法：应用人卵巢癌细胞系SKOV3对15例裸鼠建立人卵巢癌皮下移植瘤模型,随机分为3组：对照组（生理盐水）、空质粒对照组及重组质粒组（pSilencer1.0-U6-siRNA-STAT3）。应用电子穿孔仪将质粒转入裸鼠移植瘤内,观察重组质粒注射后第7、14、21和28天各组裸鼠皮下移植瘤体积变化。利用Western blotting检测重组质粒对STAT3蛋白表达的影响,采用HE及TUNEL染色方法观察肿瘤组织形态变化及细胞凋亡情况。结果：重组质粒瘤内注射对SKOV3裸鼠皮下移植瘤的生长有明显的抑制作用,与2个对照组比较,重组质粒组裸鼠移植瘤生长速度明显减慢（P<0.01）。重组质粒组瘤体内STAT3及CyclinD1、VEGF、survivin、c-myc蛋白表达明显低于2个对照组（P<0.01）。HE染色显示,重组质粒组肿瘤细胞出现大片细胞坏死,2个对照组肿瘤细胞以正常形态居多。TUNEL染色显示,重组质粒组癌组织有大量细胞凋亡,2个对照组几乎均为TUNEL阴性反应细胞。结论：pSilencer1.0-U6-siRNA-STAT3重组质粒能明显抑制人卵巢癌细胞SKOV3裸鼠皮下移植瘤的生长。     

RNA干扰GPC3基因表达影响肝癌细胞Huh-7凋亡的机制研究

目的观察RNA干扰GPC3基因表达对人肝癌细胞Huh-7凋亡的影响,并探讨其初步机制。方法设计并合成靶向GPC3的特异性siRNA片段,通过脂质体转染法瞬时转染肝癌细胞Huh-7,48h后验证siRNA的干扰效率;Annexin V/PI染色法观察GPC3基因沉默对细胞凋亡的影响;通过Western blot和定量PCR来检测caspase3的蛋白水平和核酸水平表达。结果设计合成的GPC3siRNA转染后,能够有效抑制Huh-7细胞中GPC3的表达;流式细胞仪检测结果显示,GPC3干扰组细胞在转染后48、72h的凋亡率显著高于空白对照组(P<0.01),96h后两组凋亡率差异无统计学意义(P>0.05)。GPC3干扰组的caspase3在转染后48、72h的表达高于未转染组,96h后两组间caspase3水平差异无统计学意义(P>0.05)。结论 siRNA静默GPC3基因能促进肝癌细胞凋亡,其机理可能与上调caspase3有关。进而推测,GPC3基因可能成为肝癌靶向治疗的一个新靶点。     

不同葡萄糖浓度对3T3-L1细胞脂联素mRNA表达的影响

目的探讨不同葡萄糖浓度对3T3-L1细胞脂联素mRNA表达的影响.方法以β-actin为内对照,用半定量RT-PCR法检测不同浓度葡萄糖培养下3T3-L1细胞脂联素mRNA表达.结果培养液中的葡萄糖浓度在5.5～25.0mmol/L范围中,脂联素mRNA表达改变均无统计学意义(均P＞0.05).结论培养液中的短时间葡萄糖浓度变化不是影响3T3-L1细胞脂联素mRNA表达的主要因素.