三七总皂苷对大鼠脑缺血再灌注后脑内NGF和bFGF表达的影响

目的:观察三七总皂苷(PNS)对局灶性脑缺血再灌注后脑组织神经生长因子(NGF)、碱性成纤维细胞生长因子(bFGF)蛋白表达的影响.方法:采用线栓法建立大鼠大脑中动脉栓塞局灶性脑缺血再灌注模型.实验动物随机分为假手术组、脑缺血再灌注模型组、模型 PNS治疗组和模型 尼莫地平治疗组.用免疫组织化学方法检测脑内皮质、海马等区域NGF和bFGF蛋白表达.结果:缺血2h再灌注46h后,脑内海马和皮质区的NGF表达降低,PNS能显著上调海马、皮质区及丘脑区域NGF的表达.缺血2h再灌注46h后,bFGF的表达各脑区无明显差异;但PNS能显著上调缺血再灌注损伤后胼胝体区域内bFGF的表达.结论:局灶性脑缺血再灌注后,PNS能上调缺血脑组织内NGF和bFGF表达,尤其是促进了NGF的表达,这可能是PNS对脑缺血后损伤神经元的保护机制之一.     

大鼠脑缺血/再灌注后bFGF和GAP-43的表达与神经再生

目的:观察大鼠脑缺血/再灌注后不同时间段碱性成纤维生长因子(bFGF)和生长相关蛋白43(GAP-43)表达与神经元再生的变化,探讨其与神经再生的有关机制。方法:建立大鼠大脑中动脉阻塞模型(MCAO),并分为缺血再灌注3 d、7 d、14 d和28 d四组(n=6)。以神经损伤严重程度评分(NSS),运动评分测试(SMT)评估神经功能缺损程度,Nissl和TUNEL染色法观察不同时段缺血区周边组织神经元存活和凋亡情况,应用蛋白免疫印迹法和免疫荧光双标法检测缺血/再灌注后不同时段缺血区周边组织bFGF和GAP-43的表达水平和神经元再生的变化情况。结果:大鼠脑缺血/再灌注后3 d,大鼠出现了明显的神经功能缺损及运动功能障碍,缺血区周边组织神经元凋亡亦达到高峰,同时bFGF和GAP-43表达增强,7 d达到高峰,以后逐渐减弱,缺血周边组织可见散在的新生神经元,持续到28 d。结论:大鼠脑缺血/再灌注后内源性bFGF和GAP-43表达水平增加,可能与其神经修复和再生有关。     

三七总皂苷对脑缺血再灌注大鼠神经血管单元超微结构的影响

为了观察脑缺血再灌注(cerebral ischemia reperfusion, CIR)大鼠缺血灶周边脑组织不同时间点神经血管单元(neurovascular unit, NVU)超微结构变化,研究三七总皂苷(Panax notoginseng saponins, PNS)对脑缺血再灌注大鼠脑组织NVU超微结构的影响,本研究采用改良Zea Longa法制作局灶性大脑中动脉闭塞(MCAO)模型,缺血2 h后再灌注;采用Longa法评分标准检测各组大鼠术后4 h神经功能评分,随之各组进行干预,分别在缺血再灌注后24 h、72 h、7 d、3周进行神经功能评分和透射电镜下观察各组大鼠缺血灶周边脑组织的NVU超微结构变化。研究结果表明,干预前即术后4 h治疗组和对照组神经功能评分比较无明显差异;PNS干预后治疗组大鼠神经功能评分逐渐改善,缺血再灌注后24 h与对照组比较,差异无统计学意义(p>0.05),再灌注72 h、7 d、3周的大鼠神经缺损评分与同时间点对照组相比差异具有统计学意义(p<0.05)。电镜观察发现再灌注24 h、72 h、7 d、3周治疗组大鼠脑组织NVU超微结构的病理形态损伤均较同时间点对照组明显减轻。本研究结论认为,PNS通过整合促进脑缺血后NVU的神经元、胶质细胞和微血管的修复,改善神经功能缺损症状,对脑缺血具有保护作用。     

促红细胞生成素对大鼠局灶性脑缺血再灌注神经元的保护作用及P53蛋白的表达

目的:探讨促红细胞生成素(Epo)对大鼠局灶性脑缺血再灌注神经细胞的保护作用.方法:60只SD大鼠随机分为缺血再灌注Epo治疗组(又分为高剂量A组、低剂量B组)、缺血再灌注组(C组)及假手术组(D组),采用大脑中动脉线栓法制备大鼠局灶性脑缺血再灌注模型.参考Longa的5分制法在大鼠麻醉清醒后进行评分,TTC染色法观察线栓侧的梗死体积,并检测脑组织含水量的变化,HE染色法观察脑缺血再灌注后脑组织的病理变化,TUNEL法观察神经细胞凋亡情况,western blot法观察p53蛋白的表达变化.结果:对照组比较,大鼠脑缺血再灌注后出现不同程度的脑梗死,24h后缺血中心区及周围区均可见到p53蛋白表达.缺血再灌注6h内给予Epo可显著改善大鼠神经功能评分,减少梗死体积及脑组织含水量,减轻病理学变化及神经细胞凋亡.结论:Epo通过调控神经细胞凋亡、改善缺血再灌注损伤而发挥脑保护作用,P53蛋白参与缺血再灌注后神经细胞凋亡机制.     

Ephrin-B2促进大鼠局灶脑缺血再灌注后VEGF表达及血管新生

目的:探讨Ephrin-B2对大鼠脑缺血再灌注后脑组织中血管新生的调节作用及其可能的机制。方法:雄性SD大鼠随机分为正常组及、缺血再灌注组及Ephrin-B2干预组,后两组再分为4天、7天、14天、28天亚组;线栓法制备局灶性大脑中动脉缺血再灌注模型;改良神经功能评分(modified neurological severity scores mNSS)评分法对各时间点模型进行评分;Western blot及荧光定量PCR检测缺血脑组织中血管内皮生长因子(Vascular Endothelial Growth Factor VEGF)的表达;以免疫荧光双标法定位VEGF表达的细胞类型;以CD31+BrdU计数缺血半暗带中新生微血管密度(microvessel densityMVD)。结果:Ephrin-B2干预组与缺血再灌注组各时间点亚组比较,新生微血管密度测定计数较缺血再灌注组均显著增加(P0.05),神经功能评分均显著降低(P0.05),VEGF mRNA水平及蛋白表达水平均显著增加(P0.05),VEGF主要表达于CD31阳性的血管内皮细胞。结论:Ephrin-B2通过上调VEGF的表达促进脑缺血再灌注后缺血半暗带血管新生,从而促进神经功能缺失的修复。     

加味温胆汤对MCAO模型大鼠神经功能缺失及血管新生的影响

目的:通过观察加味温胆汤对脑缺血再灌注大鼠神经功能缺损评分、血管新生和肝细胞生长因子HGF蛋白表达的影响,研究加味温胆汤的神经保护机制。方法:采用改良Longa法建立大鼠脑缺血再灌注模型,与补阳还五汤作对照,观察加味温胆汤对大鼠神经功能缺损程度评分的影响,并用免疫组织化学法检测缺血脑组织微血管密度及HGF蛋白表达。结果:加味温胆汤能明显改善脑缺血再灌注大鼠神经缺损症状,促进血管新生及大鼠脑组织HGF蛋白表达,与补阳还五汤比较有显著差异。结论:加味温胆汤可明显改善大鼠脑缺血再灌注后神经功能,促进缺血脑组织血管新生,其机制可能与其促进HGF表达有关。     

人参皂甙Rg1对脑缺血再灌注大鼠脑组织Bax和Bcl-2蛋白表达的影响

目的探讨人参皂甙Rg1对脑缺血再灌注大鼠脑组织Bcl-2和Bax表达的影响及其意义。方法分别给大鼠腹腔注射人参皂甙Rg1 10、20、40 mg/kg,采用大脑中动脉闭塞方法建立脑缺血再灌注模型,观察大鼠脑缺血再灌注后不同时间段（2 h、24 h）神经功能缺损评分;应用免疫组化法检测脑组织缺血再灌注24h后Bcl-2、Bax的表达。结果人参皂甙Rg1组大鼠脑缺血后各时间点神经功能缺损评分显著低于单纯缺血再灌注组（P〈0.05）;与单纯缺血再灌注组相比,人参皂甙Rg1各组Bcl-2表达显著增高,Bax表达显著降低,Bcl-2/Bax比值显著上调（P〈0.05）。结论人参皂甙Rg1防治大鼠脑缺血再灌注损伤的机制可能与促进脑组织Bcl-2表达、抑制Bax表达有关,且以高剂量效果较好。     

大鼠脑缺血再灌注后血清中血管内皮生长因子与神经元凋亡的研究

目的:通过检测SD大鼠脑缺血再灌注模型血清中血管内皮生长因子(VEGF)与神经元凋亡动态表达变化的关系,以探讨两者之间的相关性。方法:将40只大鼠随机分为8组:对照组、假手术组和脑缺血30min再灌注12h组、1d组、3d组、5d组、7d组、及14d组,每组5只。采用ELISA双抗夹心法检测大鼠血清中血管内皮生长因子、原位细胞凋亡TUNEL法检测脑组织中的凋亡神经细胞数。结果:再灌注12h、1d、3d、5d、7d及14d大鼠血清VEGF表达和凋亡神经元百分比的变化均为负相关性(均为P<0.05)。结论:在脑缺血再灌注大鼠模型中,缺血诱导使VEGF的表达发生变化,VEGF通过直接或间接的途径抑制神经元凋亡。     

三七总皂苷对大鼠脑缺血再灌注损伤后脑组织的凋亡抑制作用

目的:探讨三七总皂苷(PNS)对大鼠脑缺血再灌注损伤后大脑皮层细胞的凋亡抑制作用.方法:采用大脑中动脉栓塞再通法建立脑缺血再灌注模型,将大鼠随机分为假手术组、缺血再灌注组和三七总皂苷治疗组;根据再灌注时间不同分为再灌注10h、12 h、24h组,缺血时间为90 min.大鼠脑缺血再灌注10h、12h和24h不同时间点进行神经功能评分,采用原位末端标记法检测神经细胞凋亡情况,同时用免疫组化法检测抑制凋亡蛋白XIAP和促凋亡蛋白Smac阳性细胞数.结果:缺血再灌注组神经细胞凋亡数明显增加,XIAP蛋白的表达呈先高后低的变化(P＜0.05),Smac蛋白的表达明显上升(P＜0.05);PNS治疗组能明显减少脑皮层组织神经细胞凋亡数(P＜0.05),增加XIAP蛋白表达(P＜0.05),减少Smac蛋白表达(P＜0.05).结论:PNS可能通过促进抑制凋亡蛋白XIAP的表达和抑制促凋亡蛋白Smac的表达,减少脑组织缺血再灌注损伤后的神经细胞凋亡,进而对再灌注后脑组织具有抑制脑细胞凋亡的作用.     

孕酮对大鼠局灶性脑缺血/再灌注损伤的神经保护作用及其机制

目的:观察孕酮(PROG)对大鼠局灶性脑缺血/再灌注损伤后的神经保护作用,并探讨其作用机制。方法:120只雄性SD大鼠随机分为:假手术组、大脑中动脉栓塞(MCAO)组和PROG+MCAO组(n=40)。线栓法建立大鼠右侧MCAO模型,PROG+MCAO组于建模型前30 min按8 mg/kg腹腔内注射PROG。大鼠脑缺血2 h再灌注0、24、48、72 h,通过Longa评分标准进行神经功能缺陷评分;实时荧光定量聚合酶链反应技术检测大鼠脑组织中双孔道结构域钾离子通道3(TASK3)mRNA的表达。结果:PROG(8 mg/kg)可显著降低大鼠脑缺血2 h再灌注24、48、72 h时的神经功能缺陷评分(P0.05)。与假手术组相比,MCAO组再灌注各时间点脑组织TASK3 mRNA的表达均显著降低(P0.05);与MCAO组相比,PROG+MCAO组再灌注各时间点大鼠脑组织中TASK3 mRNA的表达均显著增多(P0.05)。结论:PROG可改善局灶性脑缺血/再灌注损伤后大鼠神经功能缺陷症状,其作用机制可能与上调脑组织中TASK3 mRNA的表达有关。     

Mesenchymal stem cell therapy of brain ischemic stroke in rats

Mesenchymal stem cell (MSCs)-based therapy is a promising attempt to improve the recovery after stroke. Our experiments were carried out on inbred Wistar-Kyoto rats. MSCs were isolated, expanded in culture, and labeled with vital fluorescent dye PKH-26. Animals were subjected to middle cerebral artery occlusion (MCAO). After three days, MCAO 5 × 10⁶ isolated MSCs were injected into the tail vein of the experimental rats. The control animal group received PBS injections (negative control). Therapy results were evaluated by the following parameters: behavioral and neurological testing, the inured brain areas, damaged brain structures, neuron state, and vessel quantity in the region close to with necrosis zone. It was shown that control animals (PBS injection) did not return to their initial behavioral and neurological state within 6 weeks, while the experimental animals (MSCs injection), within 2–3 weeks after MCAO, had parameters like intact rats. The size of the damaged region in the control group was larger than in the experimental group by a factor of approximately 1.3. The damage in MSC-treated rats was limited to the neocortex; caudate nucleus, capsula externa and piriform cortex remained uninjured. The small vessel quantity in the “border” regions was twice as high as compared to the control group and approximately equal to the number of vessels in an intact brain. For the first time, we demonstrated that the vessel quantity in the neocortex and caudate nucleus of the contralateral hemisphere after MSC transplantation was twice as high as in control rats. It is concluded that the MSC transplantation exerts a beneficial influence upon the brain tissue reparation after stroke.     

移植途径对大鼠骨髓间充质干细胞归巢及肝功能恢复的影响

目的 探讨移植途径对骨髓间充质干细胞（MSCs）归巢及促进肝切除大鼠肝再生的影响.方法 建立肝切除大鼠模型,随机分为3 组,即肝切除对照组、尾静脉移植组和门静脉移植组.移植组分别经尾静脉和门静脉注射DAPI 标记的MSCs 约1.5×106/ 只,分别于第3 天和第9 天后采血清检测肝功能,第9 天处死大鼠取肝脏标本,并通过荧光显微镜观察两种移植途径对MSCs 向肝脏迁移的影响.结果 门静脉移植组（18.1 ± 3.4）个细胞/100 倍视野到肝脏归巢及定植的 MSCs 多于尾静脉移植组（7.6 ± 2.0）个细胞/100 倍视野,差异有统计学意义（P 〈 0.01）.术后第9 天各组大鼠肝功能均有好转,丙氨酸氨基转移酶（ALT）及天冬氨酸氨基转移酶（AST）3 组之间对比差异无统计学意义（F = 2.822,1.046,P = 0.057,0.365,P 〉 0.05）;但两移植组与单纯肝切除组比较血浆白蛋白（ALB）均有明显升高,差异具有统计学意义（F = 6.259,P = 0.006）;尾静脉移植组与门静脉移植组两移植组之间相比,差异无统计学意义（P 〉 0.05）.结论 移植途径对 MSCs 归巢、定植到肝脏有一定影响,门静脉途径优于外周静脉,MSCs 移植对肝大部切除大鼠肝功能恢复具有促进作用.     

高压氧预处理对大鼠局灶性脑缺血再灌注损伤的保护作用

目的:研究高压氧预处理对大鼠脑缺血再灌注损伤的保护作用。方法:36只SD大鼠随机分为假手术组、模型组及高压氧预处理组,每组12只。高压氧预处理组大鼠在造模前5天给予高压氧预处理。采用线栓法建立大鼠脑缺血再灌注模型,观察高压氧预处理对脑缺血再灌注损伤大鼠神经功能缺损评分、脑梗死面积的影响,检测大鼠缺血脑组织COX-2 mRNA和蛋白的表达以及IL-1β、TNF-α、MDA的含量。结果:高压氧预处理可明显改善脑缺血再灌注大鼠神经功能缺损评分,减少脑梗死面积,降低COX-2m RNA和蛋白表达量,抑制IL-1β、TNF-α的表达,降低MDA水平。结论:高压氧预处理对大鼠脑缺血再灌注损伤具有明显的保护作用,其机制可能与抑制IL-1β、TNF-α、COX-2的表达以及减弱脂质过氧化反应有关。     

bFGF对脑缺血再灌注大鼠海马及顶叶皮质神经元的保护作用

目的本实验应用大脑中动脉栓塞（MCAO）模型,观察bFGF对脑缺血再灌注损伤后海马及顶叶皮质中Wnt通路抑制因子Dickkopf-1（DKK-1）和Wnt通路中pCatenin的表达作用的影响,以探讨Wnt通路对缺血性脑损伤的作用机制,为临床治疗缺血性脑血管病提供实验依据。方法应用线栓法制作大鼠局灶性脑缺血再灌注模型,大脑中动脉阻塞1h再灌注损伤24h,采用免疫组织化学SABC法及RT-PCR法检测海马及顶叶皮质CA1区神经元β-Catenin和DKK-1mRNA的表达。结果正常sham组,大鼠海马组织DKK-1 mRNA表达较少,β-Catenin阳性产物在细胞质内有所表达;I／R组,DKK-1 mRNA表达明显增多,β-Catenin在胞质内表达明显减少;bFGF组,大鼠海马组织DKK-1 mRNA表达较I／R组明显减少,而海马细胞质内β-Catenin表达较I／R组明显增加。结论bFGF抑制缺血神经元凋亡,参与DKK-1 mRNA和β-Catenin的调节,对缺血神经元有保护作用。     

骨髓间质干细胞移植对大鼠脑缺血再灌注凋亡蛋白Caspase-3的影响

目的研究静脉移植骨髓间充质干细胞（MSCs）对脑缺血再灌注模型大鼠神经功能及凋亡相关蛋白caspase-3的影响。方法体外培养及扩增MSCs后,用绿色荧光染料羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）标记,通过静脉途径移植给大脑中动脉缺血2 h再灌注的SD大鼠,按不同时间点取材,荧光显微镜观察BMSCs在脑内的分布,免疫组织化学染色及RT-PCR检测大鼠脑内caspase-3蛋白表达情况。结果移植组在移植后第6天神经功能明显好于对照组（P〈0.05）。移植组移植后3、12、24、48、72 h caspase 3免疫组化阳性目标面密度分别为（1.34±0.31）%、（3.98±0.67）%、（5.58±0.92）%、（4.65±0.69）%、（3.51±0.63）%,对照组分别为（2.09±0.19）%、（5.23±0.30）%、（6.89±0.57）%、（5.93±0.56）%、（4.39±0.57）%,移植组和对照组比较均（P〈0.05）。6h及7 d移植组caspase 3阳性目标面密度分别为（2.81±0.35）%、（1.64±0.29）%,与对照组（3.92±0.44）%,（2.29±0.21）%比较差异显著（P〈0.01）。移植组相应时间点caspase-3的表达明显低于对照组（P〈0.05,P〈0.01）;移植组大鼠缺血侧皮层的caspase-3 mRNA相对量明显低于对照组（P〈0.01）。结论经静脉注射骨髓间充质干细胞可明显改善神经功能。其可能通过下调caspase-3表达方式对脑缺血再灌注损伤起保护作用。     

大豆异黄酮基于RhoA/ROCK2信号通路改善MCAO大鼠神经功能损伤

目的:探讨大豆异黄酮对脑缺血再灌注大鼠RhoA/ROCK2信号通路介导的氧化应激反应和神经元凋亡的影响。方法:60只SD大鼠随机分为3组,对照组、模型组、大豆异黄酮组。连续给药7天后,给药剂量200 mg/kg。应用中动脉栓塞再灌注模型致大鼠缺血损伤。24 h后评价大鼠神经功能,TTC染色检测脑梗死体积,试剂盒检测脑中氧化因子含量,免疫组化检测神经元损伤,Western Blotting检测RhoA/ROCK2相关蛋白含量。结果:与对照组比较,模型组大鼠神经功能评分降低(P0.05),脑梗死体积增加(P0.05),氧化因子含量增加(P0.05),神经元凋亡显著(P0.05),RhoA/ROCK2蛋白表达增加(P0.05)。与模型组相比,大豆异黄酮升高了大鼠神经功能评分(P0.05),减少的脑梗死体积(P0.05),降低脑中氧化因子含量(P0.05),抑制了神经元凋亡(P0.05),抑制了RhoA/ROCK2蛋白表达(P0.05)。结论:大豆异黄酮可以缓解脑缺血再灌注损伤介导的氧化应激及细胞凋亡,进而减轻神经功能障碍,其机制可能与抑制RhoA/ROCK2信号通路相关。     

Bone marrow-derived mesenchymal stem cells inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia after arterial injury in rats

We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8 × 10⁶ cells, n = 10) or vehicle (controls, n = 10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3 × 10⁶ cells, tail vein; n = 4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67 + proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.     

Mesenchymal stem cell transplantation for myocardial reparation of rat experimental heart failure

Mesenchymal stem cells (MSC) are resident pluripotent cells of bone marrow stroma. MSC have the ability to differentiate into osteoblasts, chondroblasts and adipocytes, neurons, glia and also into cardiomyocytes. The problem of MSC use in cell therapy of various diseases and in myocardial infarction therapy is widely discussed at present. The experiments were carried out on the inbred line Wistar--Kyoto rats. Myocardial experimental infarction (EI) was induced by left descending coronary artery ligation. MSC were isolated from bone marrow, cultivated in vitro and injected into the tail vein on the day of experimental infarction operation. It was shown that the structure of injured myocardium in experimental group significantly differed from that in control group. MSC transplantation led to inflammatory process acceleration and to increased angiogenesis in the damaged myocardium; also, live cardiomyocyte layers were detected in the scar. As a result, ventricular dilatation and overload of the border zone of infarct region decreased, no features of infarction relapse were shown in the border zone.     

Mesenchymal stem cells transplantation influences upon dynamics of morphological changes in rat brain after stroke

Study of dynamic morphological changes if the brain after ischemic stroke is an important phase of pre-clinical trial of mesenchymal stem cell (MSC) therapy for this widespread disease. Experoments were carried out in inbred Wistar-Kyoto rats. MSCs were isolated, expanded in culture and labeled with vital fluorescent dye PKH26. Animals were subjected to middle cerebral artery occlusion (MCAO) followed by injection of 5 x 10(6) rat MSCs into the tail vein on the day of MCAO. Control group of animals received PBS injection (negative control). Animals were sacrificed in 1, 2, 3 and 5 days and in 1, 2, 4 and 6 weeks after operation. MSCs were revealed in the brain on the third day transplantation. They distributed around brain vessels in both the ipsilateral and contralateral hemispheres. This pattern of distribution remained unchanged during 6 weeks of observation. It was demonstrated that inflammation process and scar formation in the experimental group progressed 25-30 % faster than in the control group. MSC transplantation stimulated endogenous stem cell proliferation on the subependimal zone of lateral ventricles (subventrecular zone). What is more, MSC injection showed neuroprotective effect: almost all penumbra neurons in animals treated with cell therapy retained their normal structure, whereas in animals of control group penumbra neurons died or had signs of serious damage.