半夏提取物调节Bax、Bcl-2、Caspase-3蛋白表达诱导白血病细胞凋亡

背景:温热中药半夏拥有可人工广泛种植、易提纯等优势,其提取物可抑制慢性髓系白血病、急性髓系白血病、急性T淋巴白血病细胞增殖,但是作用机制尚不明确。目的:探讨中药半夏提取物对3种白血病细胞凋亡的作用机制。方法:慢性髓系白血病细胞株(K562)、急性髓系白血病M3细胞株(HL-60)、急性T淋巴白血病细胞株(C8166)在半夏提取物5种浓度梯度中分别作用24,48,72 h,采用CCK-8法评估3种白血病细胞增殖情况并从中筛选出有明显抑制作用的中(300 mg/L)、高(500 mg/L)质量浓度。然后进一步采用流式细胞术检测3种白血病细胞早期凋亡发生率,采用RT-PCR与Western blot检测Bax、Bcl-2、Caspase-3 mRNA及蛋白水平。结果与结论:①5种质量浓度的半夏提取物作用3种白血病细胞具有细胞增殖抑制作用,中(300 mg/L)、高(500 mg/L)质量浓度半夏提取物增殖抑制率较高,与低质量浓度组(100 mg/L)、对照组比较差异有显著性意义(P<0.01);②中、高质量浓度半夏提取物可诱导3种白血病细胞早期凋亡;③中、高质量浓度半夏提取物可以下调3种白血病细胞的Bcl-2 mRNA表达,上调Bax及Caspase-3 mRNA表达(P<0.05;P<0.01);④中、高质量浓度半夏提取物作用72 h后,K562、C8166细胞的Bcl-2蛋白条带明显减弱,而HL-60细胞无减弱变化;Bax及Caspase-3蛋白条带明显增强;⑤结果表明,半夏提取物能有效抑制髓系白血病、T淋巴细胞白血病细胞的增殖,促进其凋亡,其作用机制可能与其调节Bax/Bcl-2、Caspase-3蛋白表达有关。     

雷帕霉素联合TAT-凋亡素抑制胶质瘤的研究

目的：研究雷帕霉素(Rapamycin, Rapa)和凋亡素(TAT-apoptin, T-ap)联合应用对神经胶质瘤的治疗作用。方法用 MTT 实验比较 Rapa、 TAT-apoptin 单用和联合应用对 U87细胞增殖的影响; AO/EB 荧光染色与流式细胞仪检测对 U87细胞凋亡的影响;建立裸鼠 C6胶质瘤皮下移植模型,计算 Rapa、TAT-apoptin 单独及联合作用下的抑瘤率。结果 Rapa 与 TAT-apoptin 单独用药均能抑制 U87细胞的增殖,呈剂量依赖性,联合用药组细胞增殖率明显低于单独用药组(P ﹤0.05);联合用药组凋亡状态比单独用药更明显,呈晚期凋亡形态,细胞凋亡率显著高于单独用药组(P ﹤0.01);在体内实验中,联合用药组抑瘤率为65.5％,雷帕霉素与 TAT-凋亡素单独用药抑瘤率分别为29.59％,24.4％,前者比后者高1倍(P ﹤0.05)。结论 Rapa 与 TAT-ap 联合用药可增强对胶质瘤细胞增殖的抑制作用,促进胶质瘤细胞的凋亡,产生协同的抗肿瘤作用。     

54例非霍奇金病Bcl-2、Bax、Fas和Fas-L蛋白表达及其意义

研究凋亡相关蛋白Ｂｃｌ－２、Ｂａｘ、Ｆａｓ和Ｆａｓ－Ｌ在非霍奇金淋巴瘤中的表达及意义。方法应用免疫组化Ｓ－Ｐ法对５４例ＮＨＬ进行检测。结果：（１）Ｂｃｌ－２、Ｂａｘ、Ｆａｓ和Ｆａｓ－Ｌ阳性率分别为５０％、８３．８％、８８．９％和８８．７％。     

Bcl-2和Bax蛋白在Wistar大鼠胃粘膜的表达及其意义

Bcl-2是日前已知的抑制细胞凋亡的重要基因。Bax基因不但拮抗Bcl-2的抑制凋亡作用,而且有直接促进细胞凋亡的功能。Bax和Bcl-2是一对正负凋亡调节基因,研究其在大鼠胃粘膜的表达和分布,对研究胃粘膜的更新和发病机理有重要的意义。     

儿童喉乳头状瘤组织中Survivin及Caspase-3蛋白表达及相关性研究

目的:研究儿童喉乳头状瘤中survivin及caspase-3蛋白的表达及其相互关系.方法:应用免疫组化方法对儿童喉乳头状瘤组织、儿童声带小结组织及正常的喉黏膜组织中survivin及caspase-3的表达进行检测并进行相关性分析.结果:42例儿童喉乳头状瘤组织中survivin阳性表达率为57.14%, 明显高于儿童声带小结组(P<0.01)和正常黏膜组(P<0.01);42例喉乳头状瘤组织中caspase-3阳性表达率为26.19%, 显著高于儿童声带小结组和正常喉黏膜组 (P<0.01);Spearman相关性分析显示, 儿童喉乳头状瘤中survivin与caspase-3的表达呈显著负相关(r=-0.682, P<0.01).结论:Survivin的高表达和caspase-3的低表达可能在儿童喉乳头状瘤发生发展过程中起着重要作用.     

癫痫大鼠海马神经元Fas、FasL和Caspase-3的表达

目的观察Fas、FasL和Caspase-3在癫痫大鼠海马神经元中的表达,探讨Fas、FasL和Caspase-3表达与癫痫的关系,为癫痫的生物治疗提供实验依据。方法通过腹腔注射戊四氮建立癫痫大鼠模型,用免疫组织化学方法和图像分析技术检测Fas、FasL和Caspase-3在癫痫大鼠海马神经元中的表达。结果 Fas、FasL和Caspase-3在癫痫大鼠海马神经元中的表达水平和阳性细胞数量与正常对照组相比均明显增多(<0.05)。结论 Fas、FasL和Caspase-3在癫痫大鼠海马过表达。     

颞叶癫痫病灶内bax、fas、caspase-3蛋白的表达

目的探讨细胞凋亡调控基因bax、fas、caspase-3在颢叶癫痫患者病灶内的表达及意义。方法应用免疫组化S-P法检测24例颞叶癫痫患者手术切除病灶内凋亡相关基因bax、fas、caspase-3的表达,同时采用光镜、电镜及原位末端标记（TUNEL）方法检测神经细胞凋亡的情况。结果bax蛋白在癫痫组与对照组中均轻微表达,两者间差异无显著性（P〉0.05）;fas蛋白在对照组中无表达,在癫痫组表达明显增强（P〈0．001）;caspase-3在对照组中有轻微表达,在癫痫组表达明显增强（P〈0．01）。光镜检查及TUNEL染色均未发现凋亡的神经细胞,但电镜检查的8例标本中有3例发现少量早期凋亡征象的神经元。结论癫痫患者脑内存在神经元凋亡现象。fas、caspase-3基因在这一过程中可能发挥了重要作用。     

卵巢黏液性肿瘤caspase-3、caspase-9和Smac的表达及其意义

目的 探讨凋亡调控因子caspase-3、caspase-9和Smac在卵巢黏液性肿瘤中发生、发展过程中的作用。方法 采用免疫组化（SP法）和图像分析技术检测41例卵巢黏液性囊腺瘤、10例卵巢交界性黏液性囊腺瘤、14例卵巢黏液性囊腺癌和24例正常卵巢组织中Smac、caspase-9和caspase-3的表达水平。结果 caspase-3、caspase-9和Smac在41例黏液性囊腺瘤中的表达率分别为93%、93%和73.3%;在10例交界性黏液性囊腺瘤中的表达率分别为70%、80%和80%;在14例黏液性囊腺癌中的表达率分别为96%、96%和84%。阳性信号主要定位于细胞质,呈弥漫分布。而在正常卵巢中24例中的表达率分别为83.3%、80.9%和58.3%。caspase-3、caspase-9和Smac在黏液性卵巢肿瘤中的表达增加（P〈0.05）。结论 caspase-3、caspase-9和Smac可能会成为卵巢黏液性肿瘤治疗的新靶点。     

乳腺癌中caspase-3和caspase-9的表达及其意义

目的探讨caspase-3和caspase-9在乳腺癌发生、发展中的作用。方法采用免疫组化SP法和原位杂交(ISH)技术检测80例乳腺浸润性导管癌(invasive duct carcinoma,IDC)、23例导管内癌(ductal carcinoma in situ,DCIS)、37例增生症和9例癌旁组织中caspase-3、caspase-9 mRNA和蛋白的表达。结果 caspase-3 mRNA在各组中的表达差异无统计学意义(P>0.05)。caspase-3蛋白在IDC、DCIS、增生症中的表达均高于癌旁组织(P<0.05),在IDC、DCIS中的表达均高于增生症组(P<0.05)。caspase-9 mRNA和蛋白在IDC、DCIS、增生症中的表达均低于癌旁组织(P<0.05),IDC、DCIS中的表达均低于增生症组(P<0.05)。caspase-3、caspase-9蛋白在IDC与DCIS中的表达差异均无统计学意义(P>0.05)。caspase-3蛋白与mRNA在DCIS和增生症中的表达呈正相关(r=0.429,r=0.563,P<0.05)。caspase-9 mRNA与蛋白在各组中的表达均存在相关性(r=0.94,r=0.414,r=0.391,r=0.827,P<0.05)。caspase-3、caspase-9 mRNA和蛋白的表达与肿瘤大小等临床病理参数无相关性(P>0.05)。结论 caspase-3和caspase-9均参与乳腺导管癌的发生、发展。caspase-9蛋白表达降低,而caspase-3表达增高在乳腺癌的发生、发展过程中较常见;caspase-9表达降低可能与乳腺组织恶性转化有关。     

X染色体连锁的凋亡抑制蛋白和Caspase-3在上皮性卵巢癌中的表达及其临床意义

目的:探讨X染色体连锁的凋亡抑制蛋白(Xiap)和Caspase-3在上皮性卵巢癌中的表达变化及其临床意义.方法:采用免疫组织化学两步法检测Xiap和Caspase-3在40例上皮性卵巢癌、20例卵巢上皮性交界性肿瘤、15例卵巢良性肿瘤和15例正常卵巢组织中的表达.结果:Xiap在正常卵巢组织、良性卵巢肿瘤、交界性卵巢肿瘤组织和上皮性卵巢癌中的阳性表达率呈逐渐增高趋势,差异有统计学意义(P<0.01).Caspase-3在交界性卵巢肿瘤和上皮性卵巢癌中的阳性表达率明显低于正常卵巢组织和良件卵巢肿瘤(P<0.01).上皮性卵巢癌中Xiap的阳性表达与临床分期、组织分级和生存状况有关(P<0.01);Caspase-3的阳性表达与临床分期和生存状况有关(P<0.01).结论:XIAP、Caspase-3的表达在上皮性卵巢癌的发生、发展过程中起重要作用,可能是上皮性卵巢癌预后不良的因素.     

Pi3K-mTOR Signaling and AMOG Expression in Epilepsy-associated Glioneuronal Tumors

Gangliogliomas (GGs) and dysembryoplastic neuroepithelial tumors (DNTs) represent the most frequent type of neoplasms in pediatric medically intractable epilepsy. Several data suggest a pathogenetic relationship between GGs and other glioneuronal malformations of cortical development (MCDs), including activation of the Pi3K-mTOR signaling pathway. To further reveal these pathogenetic similarities, we investigated immunocytochemically the expression of phosphorylated (p)-PDK1, p-AKT, p-mTOR, p-4E-BP1, p-eIF4G, p-p70S6K and p-S6, the effector proteins ERM (ezrin/radixin/moesin) and the pathway regulator AMOG (adhesion molecule on glia) in both GGs and DNTs. Components of the Pi3K-mTOR signaling pathway were observed in a higher percentage of neuronal cells in GGs compared with control cortex. In DNTs, the expression of these components was low and comparable with the expression in control samples. Strong immunoreactivity for ERM was observed in GGs, but not in DNTs. Additionally, AMOG was strongly expressed within GGs (but not in DNTs) in CD34-positive precursor cells. These findings support the previously suggested pathogenic relationship between GG and MCDs concerning activation of the Pi3K-mTOR signaling pathway and suggest a different pathogenetic origin for DNTs. The strong expression of AMOG within the precursor cells of GG may represent an additional marker for the diagnostic evaluation of these glioneuronal lesions.     

Caspase-3和Bax在放射性脊髓损伤大鼠的表达

目的观察大剂量分割放疗对大鼠脊髓损伤的影响及其凋亡基因的表达。方法6MeV电子线照射,每次照射剂量为5Gy,隔日1次,总照射剂量为40Gy。照射结束后20周,取脊髓组织行HE染色与透射电镜观察脊髓损伤的形态学变化,免疫组化检测凋亡基因caspase-3与bax在脊髓组织中的表达。结果HE染色见受照的脊髓白质内神经纤维排列松散紊乱,部分灰质出现细胞肿胀、破坏、炎细胞浸润,部分神经元尼氏体消失,神经胶质细胞固缩。透射电镜表现为髓鞘的板层结构水肿明显、膨大、粘连,模糊不清,细胞器空泡变性改变,神经胶质细胞有核固缩现象。免疫组化结果显示正常大鼠脊髓神经细胞中很少有凋亡基因caspase-3与bax的表达,放疗组脊髓损伤后caspase-3与bax表达的阳性细胞明显增加(P<0.05)。结论大剂量分割放疗可引起大鼠放射性脊髓损伤,且caspase-3与bax凋亡基因在脊髓损伤中呈过表达。     

难治性癫痫二次手术41例患者脑切除标本临床病理学分析

目的分析并比较接受第二次癫痫外科手术的难治性癫痫患者脑切除标本的临床病理学特点。方法回顾性分析2008~2013年在北京市海淀医院功能神经科接受第二次手术治疗的41例难治性癫痫患者的临床资料及病理资料,并复习相关文献。结果 41例患者中男性28例,女性13例,发病年龄1~51岁(平均10.29岁),病程2~30年(平均11.91年),第二次手术距第一次手术0.5~20年(平均7.05年)。脑切除标本病理诊断为FCDⅢa者8例(19.51%),FCDⅢb者11例(26.83%),FCDⅢd者15例(36.59%),双重病理7例占17.07%(5例为FCDⅢa伴胶质瘢痕/瘢痕脑回,1例为FCDⅢa伴血管瘤,1例为FCDⅢa伴混合性少突星形细胞瘤)。术后随访0.5~5年,EngelⅠ级25例(61.0%),EngelⅡ级14例(34.1%),EngelⅢ级1例(2.45%),失访1例(2.45%)。颞叶癫痫20例(占48.78%)。结论接受第二次癫痫外科手术的难治性癫痫患者脑切除标本病理类型仅包括FCDⅢ型和双重病理。这两种类型的患者再次手术后,效果良好,EngelⅠ级和EngelⅡ级共占95.1%。获得性脑损伤(如高热惊厥、外伤、难产等和脑肿瘤切除术后)致胶质瘢痕的形成和脑功能重建的过程、颞叶及内侧结构未能完全切除、肿瘤复发及致痫灶切除范围不够,是再次行癫痫外科手术的主要原因;精准完整的术前评估可以有效地减少再次手术的风险。     

脑缺血再灌注损伤的海马神经元对Bax、Bcl-2和Caspase-3的表达

目的：凋亡调控基因在脑缺血再灌后海马神经元的表达。方法：采用免疫组织化学的方法，观察 昆明小鼠双侧颈总动脉结扎7min后不同再灌时间组(24h组、48h组、72h组、7d组、14d组)海马CAl区神经元Bax、Bcl-2和Caspase-3的活性形式CM1的免疫反应活性。结果：Bax和CM1阳性神经元数在48h组最多，与其他各组相比差异有显著性(P＜0．01)，72h组明显下降，14d组完全消失；而Bcl-2阳性神经元数在48h组增多(与24h组相比，P＜0．01)，72h组下降，7d组再次上升(与72h组相比，P＜0．01)，14d组最多(与48h组相比，P＜0．01)。在24h、48h、72h、7d组，Bax阳性神经元多于Bcl-2阳性神经元(P＜0．05)，14d组则相反。结论：Bax和caspase-3在脑缺血再灌早期表达增强，然后下降以至消失，Bcl-2于再灌后期表达增强。Bax表达上调可能与Caspase-3激活相关。     

Symmetric polymicrogyria and pachygyria associated with TUBB2B gene mutations

The purpose of the study is to explore the causative role of TUBB2B gene mutations in patients with different malformations of cortical development. We collected and evaluated clinical and MRI data of a cohort of 128 consecutive patients (61 females and 67 males) in whom brain MRI had detected a spectrum of malformations of cortical development including polymicrogyria or pachygyria, who were mutation-negative to other possible causative genes. Mutation analysis of the TUBB2B gene was performed. We identified three new TUBB2B mutations in three unrelated patients (3 out of 128; 2.3%) with a diffuse and rather symmetrical cortical abnormality, including diffuse polymicrogyria in two and bilateral regional pachygyria in one. One patient harbored a p.Asp417Asn amino-acid substitution in the C-terminal domain of the protein; one patient a p.Asn256Ser amino-acid substitution in the intermediate domain and one patient a p.Leu117Pro amino-acid substitution in the N-terminal domain. The localization of each mutation within the secondary structure of the β2-tubulin polypeptide suggests that these mutations might alter the proper functions of microtubules. The phenotypic spectrum associated with TUBB2B mutations is wider than previously reported and includes diffuse, symmetric malformations of cortical development.     

The neuroanatomy of Eml1 knockout mice,a model of subcortical heterotopia

The cerebral cortex is a highly organized structure responsible for advanced cognitive functions. Its development relies on a series of steps including neural progenitor cell proliferation, neuronal migration, axonal outgrowth and brain wiring. Disruption of these steps leads to cortical malformations, often associated with intellectual disability and epilepsy. We have generated a new resource to shed further light on subcortical heterotopia, a malformation characterized by abnormal neuronal position. We describe here the generation and characterization of a knockout (KO) mouse model for Eml1, a microtubule‐associated protein showing mutations in human ribbon‐like subcortical heterotopia. As previously reported for a spontaneous mouse mutant showing a mutation in Eml1, we observe severe cortical heterotopia in the KO. We also observe abnormal progenitor cells in early corticogenesis, likely to be the origin of the defects. EML1 KO mice on the C57BL/6N genetic background also appear to present a wider phenotype than the original mouse mutant, showing additional brain anomalies, such as corpus callosum abnormalities. We compare the anatomy of male and female mice and also study heterozygote animals. This new resource will help unravel roles for Eml1 in brain development and tissue architecture, as well as the mechanisms leading to severe subcortical heterotopia.     

Microcephalic osteodysplastic primordial dwarfism type II and pachygyria: Morphometric analysis in a 2‐year‐old girl

Microcephalic osteodysplastic primordial dwarfism (MOPD) type II is a rare disorder characterized by skeletal dysplasia, severe proportionate short stature, insulin resistance and cerebrovascular abnormalities including cerebral aneurysms and moyamoya disease. MOPD type II is caused by mutations in the pericentrin (PCNT) gene, which encodes a protein involved in centrosomes function. We report a 2 year old girl affected by MOPD type II caused by two compound heterozygous loss‐of‐function variants in PCNT gene, of which one is a novel variant (c.5304delT; p.Gly1769AlafsTer34). The patient presented atypical brain magnetic resonance imaging (MRI) findings consistent with pachygyria. This was confirmed by morphometric analysis of cortical thickness (CT) and gyrification index by comparing MRI data of the patient with a group of eight age‐matched healthy controls. The statistical analysis revealed a significant and diffuse increase of CT with an anterior‐predominant pattern and diffuse reduced gyrification (p < .05). These findings provide new evidences to the emergent concept that malformations of cortical development are complex disorders and that new genetic findings contribute to the fading of classification borders.     

The recurrent TUBB3 Gly98Ser substitution is the first described to inconsistently result in CFEOM3

Missense variants in TUBB3 have historically been associated with either congenital fibrosis of the extraocular muscles type 3 (CFEOM3) or malformations of cortical development (MCD). Until a recent report identified two amino acid substitutions in four patients that had clinical features of both disorders, pathogenic variants of TUBB3 were thought distinct to either respective disorder. Three recurrent de novo Gly71Arg TUBB3 substitutions and a single patient with a de novo Gly98Ser substitution blurred the MCD and CFEOM3 phenotypic distinctions. Here we report a second patient with a missense c.292G>A (p.Gly98Ser) substitution, but without CFEOM3, the first reported evidence that even the same TUBB3 substitution can produce a spectrum of TUBB3 syndrome phenotypes. Our patient presented with amblyopia, exotropia, optic disc pallor, and developmental delay. Neuroimaging identified hypoplasia of the corpus callosum, interdigitation of the frontal lobe gyri, and dysplasia or hypoplasia of the optic nerves, basal ganglia, brainstem, and cerebellum. This report identifies the TUBB3 Gly98Ser substitution to be recurrent but inconsistently including CFEOM3, and identifies the absence of joint contractures and the presence of optic disc abnormalities that may be genotype‐specific to the TUBB3 Gly98Ser substitution.