微管相关蛋白LC3B-Ⅱ和自噬基因Beclin1在星形细胞肿瘤中的表达及其意义

目的 探讨微管相关蛋白1轻链3B(MAP1-LC3B)和自噬相关基因Beclin1在星形细胞肿瘤中的表达及其与星形细胞肿瘤病理和临床表现的相关性及意义.方法 选择上海长征医院神经外科2006年1月至2006年12月住院手术治疗的初发性星形细胞肿瘤患者62例,其中Ⅰ级(毛细胞性星形细胞瘤)4例,Ⅱ级(星形细胞瘤)23例,Ⅲ级(间变性星形细胞瘤)12例,Ⅳ级(胶母细胞瘤)23例.免疫组织化学染色检测肿瘤标本中Beclin 1的表达,免疫印迹法检测Beclin 1和MAP1-LC3B的表达并统计Beclin 1、MAP1-LC3B的表达与星形细胞肿瘤临床病理表现的关系.结果免疫组化显示Ⅰ～Ⅳ级星形细胞肿瘤中Beclin 1的表达随肿瘤级别的升高而降低,差异有统计学意义(P＜0.05).免疫印迹检测结果显示不同病理分级、生存期患者Beclin 1的表达差异有统计学意义(P＜0.05),不同病理分级患者MAP1-LC3B-Ⅱ表达的差异有统计学意义(P＜0.05);相关性分析显示MAP1-LC3B-Ⅱ、Beclin 1在星形细胞肿瘤中的表达与病理分级呈负相关关系(r=-0.334,P=0.007;r=-0.448,P=0.000),MAP1-LC3B-Ⅱ、Beclin 1在星形细胞肿瘤中的表达与生存时间呈正相关关系(r=0.285,P=0.027;r=0.359,P=0.005).MAP1-LC3B-Ⅱ和Beclin 1的表达之间也呈正相关关系(r=0.272,P=0.035).结论 MAP1-LC3B-Ⅱ和Beclin 1在胶母细胞瘤中的表达下调,自噬活性的改变可能与星形细胞肿瘤的发生、发展相关.

Abstract：

Objective To investigate the expressions of microtubule -associated protein 1 (MAP1) light chain 3B (LC3B) and autophagy-related gene Beclin1 in astrocytic tumors, and explore their correlations with the pathological features and clinical manifestations of astrocytic tumors to further reveal their roles in tumorigenesis and development of astrocytic tumors. Methods Sixty-two specimens with different-grade astrocytic tumors, including 4 with grade Ⅰ (pilocytic astrocytoma), 23 with grade Ⅱ (astrocytoma), 12 with grade Ⅲ (anaplastic astrocytoma) and 23 with grade Ⅳ (glioblastoma multiforme), were selected in our study. Immunohistochemistry was employed to detect the expression of Beclin1; the expressions of MAP 1-LC3B and Beclin1 were detected by Western blotting.The correlations between expressions of MAP 1-LC3B and Beclin 1 and both the pathological features and clinical manifestations of astrocytic tumors were analyzed. Results Immunohistochemistry showed decreased Beclin1 expression in the astrocytic tumors following the increase of tumor grades (P＜0.05).Western blotting indicated that the expressions of Beclin1 in tumors with different grades and these patients with different life cycles were significantly different (P＜0.05) and the average optical density ratio of Beclin1 in high-grade astrocytic tumors (grade Ⅲ/Ⅳ) was obviously lower than that in low-grade astrocytic tumors (grade Ⅰ/Ⅱ, P＜0.05). The expressions of LC3B-Ⅰ showed significant differences in different-grade astrocytic tumors, and the expression of LC3B-Ⅰ of grade Ⅳ tumor was statistically lower than that of grade Ⅰ, Ⅱ and Ⅲ tumors(P＜0.05). The expressions of LC3B-Ⅱ and Beclin 1 were negatively correlated to the pathological grade of the tumors (r=-0.334, P=0.007; r=-0.448, P=0.000), but positively correlated to the survival time(r=0.285, P=0.027; r=0.359, P=0.005). The expressions of LC3B-Ⅱ and Beclin 1 had a positive correlation (r=0.272, P=0.035). Conclusion Expressions of LC3B-Ⅱ and Beclin1 are down-regulated in glioblastoma multiforme; the decrease of autophagic capacity may relate to the tumorigenesis and development of astrocytic tumors.     

Correlation of hypoxia-inducible factor-1 alpha and erythropoietin protein and mRNA to cerebral ischemic tolerance in a focal ischemia/reperfusion model using the twice suture method

BACKGROUND： Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. However, the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE： To dynamically measure erythropoietin and hypoxia-inducible factor-1α （HIF-1α） mRNA and protein expression at various times following preconditioning, and to investigate effects of erythropoietin and HIF-1α on cerebral ischemic tolerance in a model of focal ischemia/reperfusion established using the twice suture method. DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Institute of Anatomy, Medical College, Qingdao University, China from March 2006 to March 2007. MATERIALS： Rabbit anti-rat HIF-1α monoclonal antibody and biotinylated goat anti-rabbit IgG （Boster, China）, rabbit anti-rat erythropoietin monoclonal antibody （Santa Cruz Biotechnology, USA）, and one-step RT-PCR kit （Qiagen, Germany） were used in this study. METHODS： A total of 99 healthy, male, Wistar rats were randomly assigned to three groups： sham surgery （n = 9）, non-ischemic preconditioning （n = 45）, and ischemic preconditioning （n = 45）. In the ischemic preconditioning group, rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method. In the non-ischemic preconditioning group, pre-ischemia was replaced by sham surgery. Subsequently, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion, including 1-, 3-, 7-, 14-, and 21-day subgroups. The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES： HIF-la and erythropoietin protein expression was measured in the cerebral cortex, corpus striatum, and hippocampus of the ischemic hemisphere. HIF-1α and erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS： （1） Intergroup comparison： compared with the non-ischemic preconditioning group, HIF-1α protein expression significantly increased in the rat cerebral cortex, corpus striatum, and hippocampus in the ischemic hemisphere at 1,3, and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05 or P 〈 0.01）. Erythropoietin protein expression significantly increased in the cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1α and erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere, at 3 and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05）. （2） Temporal expression： HIF-1α protein expression in the rat cerebral cortex, corpus striatum, and hippocampus, as well as HIF-la mRNA expression in the frontal and parietal cortex, in the ischemic hemisphere increased at 3 days, and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group. Temporal erythropoietin protein and mRNA expression was consistent with HIF-1α protein expression. （3） Correlation： erythropoietin mRNA expression positively correlated with HIF-1α mRNA expression （r= 0.737, P 〈 0.01）. CONCLUSION： Ischemic preconditioning induced cerebral ischemic tolerance. Pre-ischemiainduced increase in endogenous HIF-1αexpression, as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance.     

N- cadherin, E- cadherin and β- catenin expressions in human brainstem gliomas

Objective To explore the expression and significance of N - cadherin, E - cadherin and β - catenin in human brainstem gliomas. Methods N - cadherin, E - cadherin and β - catenin proteins and mRNA were detected in 38 cases of brainstem gliomas using immunohistochemistry and RT - PCR Methods. Results There was no significant difference in the expression of N - cadherin between grades Ⅰ ～ Ⅱ and grade Ⅲ ～ Ⅳ ( P ＞ 0. 05) . The expression of N - cadherin mRNA in human brainstem gliomas of grade Ⅰ ～ Ⅱ was significantly lower than that in grade Ⅲ～ Ⅳ ( t = 2. 711, P ＜ 0. 05 ). There was no significant difference in the expression of E - cadherin and E - cadherin mRNA between grade Ⅰ ～ Ⅱ and grade Ⅲ～ Ⅳ ( P ＞ 0. 05 ). The expression of β - catenin in human brainstem gliomas of grade Ⅰ ～ Ⅱ was significantly lower than that in grade Ⅲ ～ Ⅳ ( P ＜ 0. 05 ). As regard to β -catenin mRNA, there was no significant difference between grade Ⅰ ～ Ⅱ and grade Ⅲ ～ Ⅳ ( P ＞ 0. 05 ). Conclusions The over -expressionof N - cadherin and β - catenin may play an important role in the invasion and malignant progression of human brainstem gliomas.     

N- cadherin, E- cadherin and β- catenin expressions in human brainstem gliomas

Objective To explore the expression and significance of N - cadherin, E - cadherin and β - catenin in human brainstem gliomas. Methods N - cadherin, E - cadherin and β - catenin proteins and mRNA were detected in 38 cases of brainstem gliomas using immunohistochemistry and RT - PCR Methods. Results There was no significant difference in the expression of N - cadherin between grades Ⅰ ～ Ⅱ and grade Ⅲ ～ Ⅳ ( P ＞ 0. 05) . The expression of N - cadherin mRNA in human brainstem gliomas of grade Ⅰ ～ Ⅱ was significantly lower than that in grade Ⅲ～ Ⅳ ( t = 2. 711, P ＜ 0. 05 ). There was no significant difference in the expression of E - cadherin and E - cadherin mRNA between grade Ⅰ ～ Ⅱ and grade Ⅲ～ Ⅳ ( P ＞ 0. 05 ). The expression of β - catenin in human brainstem gliomas of grade Ⅰ ～ Ⅱ was significantly lower than that in grade Ⅲ ～ Ⅳ ( P ＜ 0. 05 ). As regard to β -catenin mRNA, there was no significant difference between grade Ⅰ ～ Ⅱ and grade Ⅲ ～ Ⅳ ( P ＞ 0. 05 ). Conclusions The over -expressionof N - cadherin and β - catenin may play an important role in the invasion and malignant progression of human brainstem gliomas.     

hMena在胶质瘤中的表达及分布特征

目的 观察人脑胶质瘤中hMena蛋白的表达情况及分布特征,并分析其表达与病理分级的相关性. 方法 选择天津医科大学总医院神经外科自2008年8月至2009年12月间手术切除并经病理证实的胶质瘤标本65例(WHO Ⅰ级9例,Ⅱ级16例,Ⅲ级19例,Ⅳ级21例),其中6例胶质母细胞瘤(GBM,Ⅳ级)患者术中在神经导航系统引导下在肿瘤实体区、肿瘤与脑组织交界区和瘤周1.5 cm处取材,另取同期5例因脑出血行减压术患者的正常脑组织标本作为对照.应用免疫组化染色与Western blotting检测标本中hMena蛋白的表达情况和分布特征. 结果 免疫组化染色结果显示对照脑组织中无hMena阳性染色,胶质瘤hMena阳性染色定位于胞浆,不同级别胶质瘤中hMena的表达差异有统计学意义(x2=34.935,P=0.000),hMena的表达与胶质瘤的分级呈正相关关系(rs=0.682,P=0.000).Westemblotting检测发现不同病理分级的胶质瘤中,hMena的表达水平随病理级别升高而逐渐升高. 结论 hMena蛋白可能在胶质瘤恶性进展中发挥作用.

Abstract：

Objective To observe the expression and distribution characteristics of human orthology of mammalian enabled (hMena) in human glioma, and analyze the correlation of its expression with the pathological grade of the glioma. Methods Sixty-five specimens of glioma with different pathological grades were collected; among these samples, according to the WHO classification, grade Ⅰ was noted in 9, grade Ⅱ in 16, grade Ⅲ in 19 and grade Ⅳ in 21; in 6 of these patients with grade Ⅳ glioblastoma multiforme, tissues from the lesion, the junctional zone between the glioma and the normal brain tissue, and the surrounding area (with a diameter of 1.5 cm) were separated under the assistance of neuronavigation system. Another 5 normal brain tissues from patients with cerebral hemorrhage performed decompression were chosen as controls. Immunohistochemistry and Western blotting were used to detect the expression and distribution characteristics of hMena. Results The hMena expression was negative in control brain tissue, and its positive rates of gliomas with different grades were 11.11% (1/9), 25% (4/16), 84.21% (16/19) and 90.48% (19/21) in gliomas of grade Ⅰ, grade Ⅱ, grade Ⅲ and grade Ⅳ, respectively; and these staining was mainly located in cytoplasm; the hMena expression in gliomas with different grades was significantly different (x2=34.935, P=0.000). The expression rate of hMena was positively correlated with the increasing grade of WHO classification (rs=0.682, P=0.000).Western blotting indicated that, in gliomas with different pathological grades, hMena expression levels increased gradually with the increase of pathological grade. Conclusion The hMena protein might play a role in the malignant progression of glioma.     

人脑干胶质瘤N-cadherin、E-cadherin及β-catenin的表达及其意义

目的 探讨神经-钙粘素(N-cadherin)、上皮-钙粘素(E-cadherin)及相关因子β-连环蛋白(β-catenin)在高、低级别人脑干胶质瘤中的表达及其意义.方法 采用免疫组化方法和RT-PCR法检测不同级别脑干胶质瘤N-cadherin、E-cadherin及β-catenin的表达.结果 N-cadherin蛋白在高、低级别脑干胶质瘤中表达差异无统计学意义(P＞0.05),mRNA的表达在脑干胶质瘤中随着肿瘤级别的升高而增高,高、低级别间差异有统计学意义(t=2.711,P＜0.05);E-cadherin蛋白和mRNA在高、低级别脑干胶质瘤中均表达缺失或减少,组间差异无统计学意义(P＞0.05);β-catenin蛋白的表达在脑干胶质瘤中随着肿瘤级别的升高而增高,高、低级别间差异有统计学意义(P＜0.05),mRNA的表达在脑干各级别胶质瘤中差异无统计学意义(P＞0.05).结论 N-cadherin转录水平的过度表达及β-catenin蛋白水平的过度表达可能与人脑干胶质瘤的恶性进展有关,而E-cadherin转录和蛋白水平的表达下调可能与此无关.

Abstract：

Objective To explore the expression and significance of N - cadherin, E - cadherin and β - catenin in human brainstem gliomas. Methods N - cadherin, E - cadherin and β - catenin proteins and mRNA were detected in 38 cases of brainstem gliomas using immunohistochemistry and RT - PCR Methods. Results There was no significant difference in the expression of N - cadherin between grades Ⅰ ～ Ⅱ and grade Ⅲ ～ Ⅳ ( P ＞ 0. 05) . The expression of N - cadherin mRNA in human brainstem gliomas of grade Ⅰ ～ Ⅱ was significantly lower than that in grade Ⅲ～ Ⅳ ( t = 2. 711, P ＜ 0. 05 ). There was no significant difference in the expression of E - cadherin and E - cadherin mRNA between grade Ⅰ ～ Ⅱ and grade Ⅲ～ Ⅳ ( P ＞ 0. 05 ). The expression of β - catenin in human brainstem gliomas of grade Ⅰ ～ Ⅱ was significantly lower than that in grade Ⅲ ～ Ⅳ ( P ＜ 0. 05 ). As regard to β -catenin mRNA, there was no significant difference between grade Ⅰ ～ Ⅱ and grade Ⅲ ～ Ⅳ ( P ＞ 0. 05 ). Conclusions The over -expressionof N - cadherin and β - catenin may play an important role in the invasion and malignant progression of human brainstem gliomas.     

Midkine expression in 52 human meningiomas

BACKGROUND： Several studies have shown that midkine directly participates in tumor cell growth and invasion, as well as the regulation of angiogenesis. OBJECTIVE： To investigate midkine expression in meningioma tissue in relation to angiogenesis, invasion, peritumoral edema, and clinicopathology. DESIGN, TIME AND SETTING： The present clinical, case-controlled, neuropathological study was performed at the Laboratory of Molecular Organism, People＇s Hospital of Deyang City between May 2007 and April 2008. MATERIALS： Fifty-two meningioma tissues were classified by WHO tumor classification of the central nervous system, comprising 40 grade Ⅰ meningioma, five grade Ⅱ meningioma, and seven grade Ⅲ meningioma. Ten normal, human cerebral maters were selected from cerebral trauma patients. METHODS： Midkine protein expression and mean microvessel density were detected using immunohistochemical techniques. Simultaneously, all data were statistically analyzed. MAIN OUTCOME MEASURES： Midkine expression and microvessel density in meningiomas and normal cerebral maters. RESULTS： The positive midkine expression rate was 64% in the meningioma tissues. However, midkine expression was not detected in normal cerebral mater tissue. The mean microvessel density was 82.0 ± 22.7 in the meningiomas, and 25.8± 6.2 in the normal cerebral mater tissues. There was significant difference in midkine expression and mean microvessel density between meningioma tissues and human cerebral maters （P 〈 0.05）. Midkine expression positively correlated with microvessel density （r = 0.756, P 〈 0.05）. Midkine expression did not correlate to patient age, gender, or tumor size, location, and shape （P 〉 0.05）. However, it closely correlated with patient clinical condition, pathological grade, invasion, and peritumoral edema （r = 0.3785,0.741 2,0.6518, 0.614 2, P 〈 0.05）. CONCLUSION： Midkine protein was overexpressed in meningiomas and correlated to tumor angiogenesis, invasion, pefitumoral edema, and clinicopa     

WISP-2在人脑星形细胞瘤中的表达研究

目的探讨WISP-2mRNA及蛋白在人脑星形细胞瘤中的表达及意义。方法采用半定量PCR检测WISP-2mRNA在人脑星形细胞瘤组织与正常脑组织中的表达差异;随后利用免疫组化方法验证WISP-2蛋白在星形细胞瘤组织与正常脑组织之间的表达差异。结果半定量PCR检测发现,WISP-2mRNA在正常脑组织中无表达,在星形细胞瘤中相对表达量为0.677±0.445,两者比较有统计学意义(t=4.783,P0.05),在高级别组(Ⅲ-Ⅳ)与低级别组(Ⅰ-Ⅱ)星形细胞瘤中的相对表达量分别为0.924±0.438、0.478±0.344,两者比较有统计学意义(t=3.909,P0.05)。免疫组化检测发现,正常脑组织细胞中无WISP-2蛋白阳性表达(0/10),WISP-2蛋白在星形细胞瘤组织内呈高表达,阳性率为66.0%(31/47),两者比较有统计学意义(χ2=11.92,P0.01),WISP-2蛋白在高级别组(Ⅲ-Ⅳ)与低级别组(Ⅰ-Ⅱ)星形细胞瘤中的表达阳性率分别为85.7%(18/21)、50.0%(13/26),两者比较有统计学意义(χ2=6.60,P0.05).与WISP-2mRNA在星形细胞瘤组织内表达特征相一致,两者具有正相关性(r=0.63,P0.001)。结论WISP-2mRNA和蛋白在人脑星形细胞瘤组织中明显高表达,并与其恶性程度有关,可作为判断脑星形细胞瘤恶性程度的一个辅助指标。     

沉默HIF-1α基因对U87细胞增殖、侵袭及转移能力的影响

目的 观察沉默低氧诱导因子1α(HIF-1a)基因后对胶质母细胞瘤U87细胞增殖、侵袭及转移能力的影响.方法 实验分3组,干扰组(转染表达HIF-1α-shRNA的质粒)、对照干扰组(转染阴性对照shRNA序列)及未处理组.干扰组利用前期构建的HIF-1α基因的短发夹RNA(shRNA)沉默HIF-1α基因,构建HIF-1α-shRNA慢病毒表达载体,在脂质体介导下转染人胶质母细胞瘤细胞U87.采用RT-PCR和Western blotting检测HIF-1α基因干扰效率,MTT法检测细胞生长增殖能力,迁移实验检测细胞的体外迁移能力,Transwell小室模型检测细胞的体外侵袭及转移能力.结果 RT-PCR和Western blotting实验证实,与对照干扰组及未处理组比较,干扰组细胞HIF-1αmRNA表达水平明显下降,蛋白条带明显减弱.MTT细胞增殖实验显示,干扰组细胞增殖水平较其他2组明显降低,差异有统计学意义(P<0.05).Transwell小室侵袭实验中,未处理组、对照干扰组、干扰组穿膜细胞数分别为(125.2±10.8)个、(118.3±8.3)个、(60.9±5.4)个,差异有统计学意义(P<0.05).结论 HIF-1α-shRNA能有效抑制U87细胞株HIF-1α mRNA及蛋白的表达,并能抑制U87细胞的增殖、侵袭及转移能力.

Abstract：

Objective To observe the influence of silencing hypoxia-inducible factor-1α(HIF-1α)gene on the proliferation, invasion and metastasis of glioblastoma U87 cells. Methods The samples were divided into 3 groups: blank group: samples without giving any treatments, control group: cells with empty shRNA vector, and experimental group: cells with HIF-1α-shRNA transfection complex. HIF-1α gene was silenced by shRNA constructed in early time; and HIF-1α-shRNA lentivirus vector was constructed in the experimental group, and then transfected into glioblastoma U87 cells with the mediation of liposome. The interference efficiency was detected by using RT-PCR and Western blotting, and cell proliferation was measured by MTT assay; cell migration in vitro was observed by migration test, and invasion and metastasis abilities were detected by Transwell booth model. Results As compared with those in cells of the control and blank groups, the mRNA and protein expressions of HIF-1α in cells of the experimental group were significantly decreased; MTT assay showed that the cell proliferation in the experimental group was significantly lower than that in the other 2 groups (P<0.05). The number of penetrating cells of the blank group, control group and experimental group in Transwell chamber invasion assay were (125.2±10.8), (118.3±8.3), (60.9±5.4), respectively, and significant differences were noted between each 2 groups (P<0.05). Conclusion The mRNA and protein levels of HIF-1α in U87 cells are efficiently depressed by HIF-1α-shRNA, and so are the proliferation, invasion and metastasis abilities of U87 cells.     

mTOR、VEGF的表达与脑膜瘤病理分级的关系

目的 探讨哺乳动物雷帕霉素靶蛋白(mTOR)、血管内皮生长因子(VEGF)在脑膜瘤组织的表达及其与脑膜瘤病理级别之间的关系.方法 取中山大学第一附属医院神经外科自1995年1月至2010年8月手术切除的脑膜瘤标本76例,其中Ⅰ级40例,Ⅱ级24例,Ⅲ级12例.采用免疫组化SABC法检测mTOR、VEGF在脑膜瘤组织中的表达.结果 mTOR、VEGF阳性蛋白主要位于肿瘤细胞的胞浆中,为浅黄色至深黄色颗粒样物质.Ⅰ、Ⅱ、Ⅲ级脑膜瘤mTOR、VEGF的表达不同,差异均有统计学意3L(P<0.05);脑膜瘤mTOR、VEGF蛋白的表达呈正相关关系(r=0.440,P=0.0000).结论 mTOR、VEGF的表达与脑膜瘤的病理级别有关.

Abstract：

Objective To investigate the relationship between expressions of mammalian target of rapamycin (mTOR) and vascular endothelial growth factor (VEGF) and pathological grading of meningiomas. Methods Seventy-six specimens of meningiomas, performed resection in our hospital from January 1995 to August 2010, were chosen, in which, 40 were of WHO grade Ⅰ, 24 of WHO grade Ⅱ, and 12 of WHO grade Ⅲ. The expressions of mTOR and VEGF were immunohistochemically studied with SABC method. Results The protein expressions of mTOR and VEGF mainly located in cytoplasm of meningioma cells, and these proteins were granular materials colored from pale yellow to dark yellow. The expressions of mTOR and VEGF were significantly different among meningiomas of grade Ⅰ,Ⅱ and Ⅲ (P<0.05);the expressions of mTOR and VEGF had a positive correlation (r=0.440, P=0.000). Conclusion The expressions of mTOR and VEGF are positively correlated with the tumor grades.     

Operative strategy of complex internal carotid artery- posterior communicating artery aneurysms Defensive effect on perioperative nerve injury

BACKGROUND: The two problems in treating intracranial aneurysm are the vascular reconstruction and brain protection, especially for complex internal carotid artery-posterior communicating artery (ICA-PComA) aneurysms. OBJECTIVE: To analyze the anatomic features and operative technique of complex ICA-PComA aneurysms, and investigate how to better protect the brain tissue. DESIGN: A retrospective case analysis. SETTING: Department of Neurosurgery, Dalian Central Hospital. PARTICIPANTS: Totally 154 inpatients with ICA-PComA aneurysms were selected from the Department of Neurosurgery, Dalian Central Hospital from January 1998 to December 2006, including 19 cases (12.3%) of complex ICA-PComA aneurysms, 8 males and 11 females, 38–67 years of age. Informed contents for surgery and observation were obtained from all the patients or their relatives. METHODS: The clinical manifestations, including initial symptoms and Hunt&Hess grading, were observed. Corresponding strategies were selected for different types of ICA-PComA aneurysms. The patients were followed up at 3 months postoperatively. According to the results of Glasgow scoring, the curative effects were classified as good (4–5 points), bad (2–3 points) and dead (1 point). The results at discharge were taken as early results, whereas the follow-up results as late results. MAIN OUTCOME MEASURES: Clinical manifestations and curative effects of the patients. RESULTS: All the 19 patients with ICA-PComA were involved in the analysis of results. For clinical manifestations, the initial symptoms were subarachnoid hemorrhage (n =15), paralysis of oculomotor nerve (n =3), and occasional attack (n =1); The Hunt&Hess grading was grade Ⅰ in 4 cases, grade Ⅱ in 6 cases, grade Ⅲ in 6 cases, grade Ⅳ in 2 cases, and grade Ⅴ in 1 case. The curative effects were that aneurysm breakage and bleeding occurred in 6 cases perioperatively, uncomplete clipping of aneurysm in 2 cases and constriction of parent artery in 1 case. The complications were nervous and ischemic ones. The early outcome was good in 12 cases and bad in 7 cases, no one died. The late outcome was good in 17 cases and bad in 2 cases. CONCLUSION: Complex ICA-PComA aneurysm is a particular aneurysm, thus different operative strategies should be adopted according to the conditions to improve the operative outcome and reduce ischemic and nervous injuries.     

Expression of hypoxia inducible factor-1 alpha and ischemic erythropoietin tolerance in the brain of cerebral ischemic tolerance model rats

BACKGROUND: Hypoxia inducible factor-1 alpha (HIF-1 α) and erythropoietin(EPO), possessing neuroprotective effect in the cerebral ischemia, might play an important role in the formation of cerebral ischemic tolerance (IT). OBJECTIVE:To observe the neuroprotective effect of cerebral ischemic preconditioning(IPC) of rats, and the expression and mechanism of HIF-1α and target gene erythropoietin in the brain tissue following the formation of cerebral IT. DESIGN:A randomized and controlled observation. SETTING: Department of Neurology, the Affiliated Hospital of Medical College, Qingdao University. MATERIALS: Totally 84 enrolled adult healthy male Wistar rats of clean grade, weighing 250 to 300 g, were provided by the Animal Experimental Department, Tongji Medical College of Huazhong University of Science and Technology. Ready-to-use SABC reagent kit and rabbit anti-rat HIF-1α monoclonal antibody were purchased from Boshide Bioengineering Co.Ltd (Wuhan); Rabbit anti-rat EPO monoclonal antibody was purchased from Santa Cruz Company (USA). METHODS: This experiment was carried out in the Department of Anatomy, Medical College, Qingdao University during March 2005 to March 2006. ① The 84 rats were divided into 3 groups by a lot: IPC group (n =40), sham-operation group (n =40) and control group (n =4). In the IPC group, middle cerebral artery was occluded for 2 hours respectively on the 1st, 3rd, 7th, 14th and 21st days of the reperfusion following 10-minute preischemia was made using a modified middle cerebral artery second suture method from Zea-Longa. The rats were sacrificed 22 hours after reperfusion in the end of middle cerebral artery occlusion (MCAO). That was to say, after 10-minute preischemia, suture was exited to the external carotid artery and embedded subcutaneously. Middle cerebral artery was occluded again to form the second reperfusion at the set time point after reperfusion. Twenty-two hours later, rats were sacrificed; In the sham-operation group,the preischemia was substituted by sham-operation(only common carotid artery and crotch were exposed, and MCAO by suture was omitted), and the other procedures were the same as those in the IPC group. In the control group, rats were given sham-operation twice at an interval of one day, and they were sacrificed 24 hours after the second sham-operation. ② Brain tissue was taken from the rats in each group. Cerebral infarction area of each layer was measured with TTC staining, and total cerebral infarction volume (The total cerebral infarction area of each layer×interspace ) was calculated. After brain tissue was stained by haematoxylin-esoin (HE), the form of nerve cells was observed under an optical microscope, and the expressions of HIF-1α and EPO protein in the brain tissue were detected with immunohistochemical method. MAIN OUTCOME MEASURES: ①Cerebral infarction volume;②form of nerve cell; ③ the expression of HIF-1α and EPO protein in the brain tissue. RESULTS: Totally 84 rats were enrolled in the experiment. The dead rats were randomly supplied during the experiment, and finally 84 rats entered the stage of result analysis. ① Detection of cerebral infarction volume of rats in each group: Cerebral infarction volume in the IPC group was significantly smaller than that in the sham-operation group on the 1st, 3rd and 7th days after reperfusion respectively [(161.2±6.9) mm3 vs (219.9±11.2) mm3, (134.9±9.0) mm3 vs (218.6±13.0) mm3, (142.9±13.7) mm3 vs (221.3±14.2) mm3, t =8.924，10.587，7.947, P < 0.01]. ② Observation of nerve cell form of brain tissue: HE staining showed that the ischemic degree, range and cerebral edema degree of IPC group were significantly milder than those of sham-operation group. ③ The expressions of HIF-1α and EPO protein in cerebral cortex and hippocampus : The expression of HIF-1α of IPC group was significantly higher than that of sham-operation group on the 1st, 3rd and 7th days after reperfusion respectively (125.93±3.79 vs 117.65±5.60, 140.63±4.64 vs 119.33±4.26, 131.15±2.74 vs 107.60±3.89, t=2.449, 6.763，9.899，P < 0.05-0.01). The expression of EPO of IPC group was significantly higher than that of sham-operation group on the 3rd and 7th days after perfusion respectively (141.68±3.29 vs 126.33±4.51, 138.88±2.59 vs 125.58±6.18，t=5.499，3.970, P < 0.05). CONCLUSION: ① IPC can protect the never cells in rat brain and the best time to onset of cerebral IT induced by IPC is 1 to 7 days after reperfusion. ② Neuroprotective effect of cerebral IT might be related to the expression of HIF-1α and its target gene EPO.     

胶质瘤中GLUT-1与GLUT-3表达的实验研究

目的 探讨葡萄糖转运蛋白-1(GLUT-1)和葡萄糖转运蛋白-3(GLUT-3)在人脑胶质瘤中的表达及与病理分级的关系.方法 用Western blot和逆转录-聚合酶链反应(RT-PCR)的方法检测80例胶质瘤中GLUT-1和GLUT-3的表达情况.结果 GLUT-1蛋白在Ⅰ、Ⅱ、Ⅲ、Ⅳ级胶质瘤中的表达分别为0.09±0.02、0.20±0.02、0.41±0.09和0.63±0.07;GLUT-3蛋白在Ⅰ、Ⅱ、Ⅲ、Ⅳ级胶质瘤中的表达分别为0.10±0.02、0.23±0.07、0.62±0.05和0.88±0.06.各组间有显著差异(P<0.01).GLUT-1mRNA在Ⅰ、Ⅱ、Ⅲ、Ⅳ级胶质瘤中的表达分别为0.08±0.03、0.19±0.02、0.41±0.06和0.66±0.08;GLUT-3 mRNA在Ⅰ、Ⅱ、Ⅲ、Ⅳ级胶质瘤中的表达分别为0.17±0.01、0.36±0.09、0.64±0.09和0.89±0.11.各组间有显著差异(P<0.01).结论 胶质瘤中GLUT-1和GLUT-3表达随病理级别增高而增加,提示GLUT-1和GLUT-3在胶质瘤的发生过程中起重要作用.     

Influence of hypoxia-inducible factor 1-alpha on neuronal apoptosis in a rat model of hypoxia- or hypoxia-ischemia-induced brain injury

BACKGROUND： In addition to neuroprotective genes, the targeted genes of hypoxia-inducible factor 1α （HIF-1α） include pro-apoptotic genes. However, the influence of HIF-1α on neuronal apoptosis in hypoxia-ischemia remains poorly understood. OBJECTIVE： To investigate the relationship between HIF-1α expression and neuronal apoptosis in hypoxia or hypoxia-ischemia brain injury and to determine the role of HIF-1α in regulating neuronal apoptosis. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed at the Laboratory of Children Neurology of Sichuan University between May 2006 and May 2007. MATERIALS： In situ cell death detected kit was provided by Roche, USA; rabbit anti-mouse HIF-1α polyclonal antibody was purchased from Santa Cruz Biotechnologies, USA; rabbit anti-mouse cleaved caspase-3 polyclonal antibody was purchased from Chemicon, USA. METHODS： A total of 36 Sprague Dawley rats aged 10 days were randomly assigned to 3 groups： sham-surgery, hypoxia, and hypoxia-ischemia, with 12 rats per group. The rats were treated at 3 time points： 4, 8, and 24 hours, with 4 rats per time point. In the hypoxia-ischemia group, the right common carotid artery was exposed and permanently ligated through a midline cervical incision. A 2.5-hour exposure to hypoxia （8% O2/92% N2） was used to induce hypoxia-ischemia injury. In the hypoxia group, rats were exposed to hypoxia without ligation of the common carotid artery. In the sham-surgery group, the common carotid artery was exposed without ligation or hypoxia. MAIN OUTCOME MEASURES： Histopathological changes, HIF-1α and activated caspase-3 protein expression, integrated optical density of positive cells, and apoptosis-positive cells. RESULTS： Hematoxylin and eosin staining showed that neuronal degeneration and edema was most prominent at 24 hours after hypoxia-ischemia. HIF-1α protein expression was significantly upregulated at 4 hours, peaked at 8 hours, and decreased at 24 hours after hypoxia or hypoxia-ischemia. HIF-1α protein expression was significant greater in the hypoxia and hypoxia-ischemia groups compared with the sham-surgery group （P 〈 0.01）. Activated caspase-3 protein expression began to increase at 4 and 8 hours following hypoxia or hypoxia-ischemia and was significantly upregulated at 24 hours. Activated caspase-3 protein expression remained at low levels in the sham controls compared with the hypoxia and hypoxia-ischemia groups （P〈 0.01）. TUNEL staining showed that the number of apoptotic cells significantly increased at 24 hours after hypoxia or hypoxia-ischemia. In addition, HIF-1α protein expression was greater in the hypoxia group compared with the hypoxia-ischemia group at the same time point （P 〈 0.05）. However, activated caspase-3 expression and the number of TUNEL-positive cells were less in the hypoxia group compared with the hypoxia-ischemia group at the same time point （P〈 0.05）. CONCLUSION： HIF-1α played a neuroprotective role following hypoxia-ischemia brain injury.     

HER-2/neu siRNA对人胶质瘤细胞增殖及相关基因表达的影响

目的 探讨HER-2/neu特异性小干扰核糖核酸(siRNA)对高表达HER-2/neu的人胶质瘤细胞系U251MG和T98G增殖的影响及其可能机制.方法 脂质体介导HER-2/neu siRNA转染体外常规培养的U251MG和T98G细胞,同时设脂质体为对照组.转染后3 d实时定量PCR和免疫印迹实验检测HER-2/neu mRNA和蛋白的表达;四甲基偶氮唑盐(MTT)比色法检测转染后3、4d细胞增殖率的变化;免疫印迹实验检测转染后3 d细胞蛋白激酶B(AKT)、磷酸化AKT、磷酸化叉头转录因子(FOXO1)、p27、Cyclin D1蛋白的表达.结果 与脂质体组比较,HER-2/neu siRNA组U251MG、T98G细胞转染后3 d HER-2/neu mRNA和蛋白的表达均下降,转染后3、4 d细胞增殖率均下降,转染后3 d细胞磷酸化AKT和磷酸化FOXO1水平降低、p27蛋白表达增多、Cyclin D1蛋白表达减少,差异均有统计学意义(P＜0.05).结论 HER-2/neu siRNA转染人胶质瘤细胞系U251MG和T98G后明显抑制细胞增殖,可能与抑制AKT/FOXO1信号通路,调控下游基因p27、Cyclin D1蛋白的表达有关.

Abstract：

Objective To investigate the effect of HER-2/neu siRNA on proliferation of human glioma cell lines U251MG and T98G which over-express HER-2/neu, and explore its mechanism.Methods Liposome-mediated HER-2/neu siRNA was transfected into human glioma cell lines U251MG and T98G;lipofectin group was established as controls. The mRNA and protein levels of HER-2/neu were detected by real-time PCR and Western blotting 3 d after the transfection. The proliferation of glioma cells was investigated using methyl thiazolyl tetrazolium (MTT) assay 3 and 4 d after the transfection. The effects of HER-2/neu siRNA on AKT/FOXO1 pathway and protein expression of p27 and Cyclin D1 were studied using Western blotting. Results HER-2/neu mRNA and protein expressions in the transfected U251MG cells were decreased to (28.833±4.174)% and (22.167±1.955)% while those in cells of the lipofectin group were (92.067±5.698)% and (96.100±1.682)%, respectively,with significant differences (P=0.000, 0.001). HER-2/neu mRNA and protein expressions of the transfected T98G cells were decreased to (28.067 ±6.165)% and (12.433 ±8.864)% while those in the untransfected cells were (96.000 ±5.110)% and (94.333 ±3.215)%, respectively, with significant differences (P=0.001, 0.008). Three d after the transfection, the rates of proliferation in the transfected T98G and U251MG cells were (58.467±5.561)% and (63.933±5.363)%, respectively;4 d after the transfection, the rates of proliferation in the transfected T98G and U251MG cells were (57.500±4.770)% and (60.167±3.253)%, respectively;an obvious decrease was noted as compared them with cells of the lipofectin group (P=0.020, 0.023, 0.021, 0.008). Cyclin Dl expression was decreased, while p27 protein expression was up-regulated in the transfected cells as compared with those in cells of the lipofectin group (P<0.05). Moreover, the levels of phosphorylated AKT and phosphorylated FOXO1 were decreased in the transfected cells as compared with those in cells of the lipofectin group (P<0.05).Conclusion The specific siRNA targeting HER-2/neu in human glioma cell lines U251MG and T98G could inhibit the cell proliferation, which might relate to the suppression of AKT/FOXO1 pathway and the regulation of expresion of thier downstream molecules such as p27 and Cyclin D1.     

Botulinum toxin type A plus rehabilitative training for improving the motor function of the upper limbs and activities of daily life in patients with stroke and brain injury

BACKGROUND: Botulinum toxin type A (BTX-A) is mostly to be used to treat various diseases of motor disorders, whereas its effect on muscle spasm after stroke and brain injury needs further observation. OBJECTIVE: To observe the effect of BTX-A plus rehabilitative training on treating muscle spasm after stroke and brain injury. DESIGN: A randomized controlled observation. SETTINGS: Department of Rehabilitation, Department of Neurology and Department of Neurosurgery, the Second Hospital of Hebei Medical University. PARTICIPANTS: Sixty inpatients with brain injury and stroke were selected from the Department of Rehabilitation, Department of Neurology and Department of Neurosurgery, the Second Hospital of Hebei Medical University from January 2001 to August 2006. They were all confirmed by CT and MRI, and had obvious increase of spastic muscle strength in upper limbs, their Ashworth grades were grade 2 or above. The patients were randomly divided into treatment group (n =30) and control group (n =30). METHODS: ① Patients in the treatment group undertook comprehensive rehabilitative trainings, and they were administrated with domestic BTX-A, which was provided by Lanzhou Institute of Biological Products, Ministry of Health (S10970037), and the muscles of flexion spasm were selected for upper limbs, 20-25 IU for each site. ② Patients in the treatment group were assessed before injection and at 1 and 2 weeks, 1 and 3 months after injection respectively, and those in the control group were assessed at corresponding time points. The recovery of muscle spasm was assessed by modified Ashworth scale (MAS, grade 0-Ⅳ; Grade 0 for without increase of muscle strength; Grade Ⅳ for rigidity at passive flexion and extension); The recovery of motor function of the upper limbs was evaluated with Fugl-Meyer Assessment (FMA, total score was 226 points, including 100 for exercise, 14 for balance, 24 for sense, 44 for joint motion, 44 for pain and 66 for upper limb); The ADL were evaluated with Barthel index, the total score was 100 points, 60 for mild dysfunction, 60-41 for moderate dysfunction, < 40 for severe dysfunction). MAIN OUTCOME MEASURES: Changes of MAS grade, FMA scores and Barthel index before and after BTX-A injection. RESULTS: All the 60 patients with brain injury and stroke were involved in the analysis of results. ① FMA scores of upper limbs: The FMA score in the treatment group at 2 weeks after treatment was higher than that before treatment [(14.98±10.14), (13.10±9.28) points, P < 0.05], whereas there was no significant difference at corresponding time point in the control group. The FMA scores at 1 and 3 months in the treatment group [(23.36±10.69), (35.36±11.36) points] were higher than those in the control group [(20.55±10.22), (30.33±10.96) points, P < 0.01]. ② MAS grades of upper limbs: There were obviously fewer cases of grade Ⅲ in MAS at 2 weeks after treatment than before treatment in the treatment group (0, 9 cases, P < 0.05), whereas there was no obvious difference in the control group. There were obviously fewer cases of grade Ⅲ in MAS at 2 weeks and 1 month after treatment in the treatment group (0, 0 case) than the control group (5, 2 cases, P < 0.01). ③ Barthel index of upper limbs: The Barthel index at 2 weeks after treatment was higher than that before treatment in the treatment group [(30.36±22.25), (28.22±26.21) points, P < 0.05], whereas there was no significant difference in the control group. The Barthel indexes at 1 and 3 months after treatment in the treatment group were obviously higher than those in the control group [(20.55±10.22), (30.33±10.96) points, P < 0.01]. CONCLUSION: BTX-A has obvious efficacy on decreasing muscle tension after stroke and brain injury, and relieving muscle spasm; Meanwhile, the combination with rehabilitative training can effectively ameliorate the motor function of upper limbs and ADL of the patients.