自体血清培养人骨髓间充质干细胞及其向神经细胞分化

目的:目前多采用胎牛血清培养骨髓间充质干细胞,实验应用自体血清培养骨髓间充质干细胞并诱导其向神细胞分化,拟验证其可行性及并分析其可能的机制。方法:实验于2005-10/2006-08在同济医学院附属同济医院神经外科实验室完成。培养骨髓间充质干细胞所用骨髓来源为本院需行骨髓穿刺的健康成人(共3例,年龄26～44岁),本人及家属知情同意。梯度离心法分离成人骨髓间充质干细胞,并利用自体血清进行体外培养扩增。主要观察指标:采用相差显微镜观察细胞形态变化;透射电镜观察骨髓间充质干细胞的超微结构;流式细胞术鉴定细胞表面标记物;3～5代细胞利用生长因子和N2添加剂进行诱导分化,采用RT-PCR检测Netin和神经元特异性烯醇化酶mRNA在诱导前后的表达。结果:①细胞形态变化:利用自体血清培养的骨髓间充质干细胞主要呈长或宽扁的梭形,在体外传代扩增速度较快。②超微结构:电镜下细胞表面可见细胞核较大,不规则,核质比较小,胞质中有丰富的细胞器,可见缝隙连接。③细胞表面标记物鉴定:多数细胞为CD44、CD105阳性,CD34、CD45阴性。④Netin和神经元特异性烯醇化酶mRNA的表达:诱导后部分细胞呈神经元样改变,Nestin mRNA表达水平于3d达高峰,随后减少,神经元特异性烯醇化酶mRNA于5～7d达高峰,并可持续数日。结论:应用自体血清可成功培养骨髓间充质干细胞,并在体外条件下定向诱导分化为神经元样细胞。     

嗅鞘细胞再移植治疗肌萎缩侧索硬化症1例

目的:探讨嗅鞘细胞再移植对肌萎缩侧索硬化症患者的治疗价值。方法:①患者资料:选择2005-05北京市虹天济神经科学研究院收治的肌萎缩侧索硬化症患者1例,男性白种人,42岁,西班牙籍,四肢无力伴肌萎缩进行性加重2年,分别于2005-05-27日和2006-10-11日给予嗅鞘细胞脑内移植。治疗方案患者知情同意。②移植方法:取4个月流产胚胎(孕妇及其家属均知情同意)的嗅球,消化成单个嗅鞘细胞后培养2周,细胞浓度约2×1010L-1。第2次移植术前进行供、受体细胞HLA配型。根据两次术前MRI图片,选取双额放射冠锥体束走行部位为注射细胞靶点,局麻下采用立体定向技术注射胚嗅鞘细胞100万个/侧。术后口服他克莫司胶囊1mg,2次/d,共42d。③功能评估:电子邮件随访5个月,采用国际统一的肌萎缩侧索硬化功能评分标准进行延髓和呼吸功能、上肢功能、下肢功能、其他功能评定,功能从完全丧失至正常为0～4分,总分40分。结果:①患者在围手术期及随访期内均未发现任何不良事件及副反应。②术前5个月内,肌萎缩侧索硬化功能评分由33分迅速降至17分。③第1次术后2周检查,患者肢体运动功能改善,双上肢抬举好转,书写较术前清晰,站立行走时平衡能力改善,翻身及穿衣好转,肌萎缩侧索硬化功能评分由17分增加至24分。出院后半年患者病情保持稳定。2005-10,双下肢力量较前稍下降,且坐位控制平衡能力下降。2005-12患者腕部及手部无力开始缓慢加重。至术后17个月肌萎缩侧索硬化功能评分为18分。④第2次术后1周检查,双上肢、腹部和双下肢力量有所增加,但肌萎缩侧索硬化功能评分仍为18分。再次出院后5个月随访,分数增加至20分。结论:嗅鞘细胞移植能改善肌萎缩侧索硬化症患者神经功能障碍的症状,再次治疗仍然有效。     

Accuracy and precision of calibrated arterial pulse contour analysis in patients with subarachnoid hemorrhage requiring high-dose vasopressor therapy: a prospective observational clinical trial

Introduction

Calibrated arterial pulse contour analysis has become an established method for the continuous monitoring of cardiac output (PCCO). However, data on its validity in hemodynamically instable patients beyond the setting of cardiac surgery are scarce. We performed the present study to assess the validity and precision of PCCO-measurements using the PiCCO™-device compared to transpulmonary thermodilution derived cardiac output (TPCO) as the reference technique in neurosurgical patients requiring high-dose vasopressor-therapy.

Methods

A total of 20 patients (16 females and 4 males) were included in this prospective observational clinical trial. All of them suffered from subarachnoid hemorrhage (Hunt&Hess grade I-V) due to rupture of a cerebral arterial aneurysm and underwent high-dose vasopressor therapy for the prevention/treatment of delayed cerebral ischemia (DCI). Simultaneous CO measurements by bolus TPCO and PCCO were obtained at baseline as well as 2 h, 6 h, 12 h, 24 h, 48 h and 72 h after inclusion.

Results

PCCO- and TPCO-measurements were obtained at baseline as well as 2 h, 6 h, 12 h, 24 h, 48 h and 72 h after inclusion. Patients received vasoactive support with (mean ± standard deviation, SD) 0.57 ± 0.49 μg · kg^-1 · min^-1 norepinephrine resulting in a mean arterial pressure of 103 ± 13 mmHg and a systemic vascular resistance of 943 ± 248 dyn · s · cm^-5. 136 CO-data pairs were analyzed. TPCO ranged from 5.2 to 14.3 l · min^-1 (mean ± SD 8.5 ± 2.0 l · min^-1) and PCCO ranged from 5.0 to 14.4 l · min^-1 (mean ± SD 8.6 ± 2.0 l · min^-1). Bias and limits of agreement (1.96 SD of the bias) were −0.03 ± 0.82 l · min^-1 and 1.62 l · min^-1, resulting in an overall percentage error of 18.8%. The precision of PCCO-measurements was 17.8%. Insufficient trending ability was indicated by concordance rates of 74% (exclusion zone of 15% (1.29 l · min^-1)) and 67% (without exclusion zone), as well as by polar plot analysis.

Conclusions

In neurosurgical patients requiring extensive vasoactive support, CO values obtained by calibrated PCCO showed clinically and statistically acceptable agreement with TPCO-measurements, but the results from concordance and polar plot analysis indicate an unreliable trending ability.     

2-(E)-取代苯亚甲基环戊酮及其Mannich碱盐酸盐类化合物的合成和抗炎、抗癌活性研究

本文设计,合成了2-(E)-取代苯亚甲基环戊酮(Ⅰ)类化合物18个及其Mannich碱盐酸盐(Ⅱ)类化合物11个,其中22个为新化合物。初步药理结果显示:对于角叉菜胶诱发的大鼠足趾水肿,Ⅰ_4,Ⅰ_(12)及Ⅰ_(13)口服有较显著的抑制作用,水溶性Ⅱ类化合物皮下注射有极强的抑制作用,其中Ⅱ_3于50,25和12.5 mg/kg剂量下抑制率分别为95.8%,70.3%和44.2%,它与布洛芬效力(25 mg/bg抑制率72.9%)相当.Ⅱ类化合物体外对L1210细胞及体内对荷艾氏腹水癌小鼠均有一定抗癌活性。     

鼠胶质瘤模型在紫杉醇-聚乳酸纳米缓释纤维片抗胶质瘤中的应用

目的:紫杉醇由于水溶性低及全身毒性的问题而限制了其抗癌的临床应用,为克服这些缺陷,应用紫杉醇-聚乳酸纳米缓释膜片对鼠脑胶质瘤进行了局部缓释化疗,观察其对人脑胶质瘤细胞系C6抑瘤作用。方法:实验于2005-05/2006-03在中科院长春应用化学研究所高分子与物理国家重点实验室和吉林大学基础医学院实验室完成。Wistar雌性大鼠40只,在立体定向条件下选取大鼠脑右侧尾状核区为靶点,接种2×105个处于对数生长期的C6细胞,1周后经MRI检查后证实32只大鼠种植成功,将其随机分成2组各16只:①实验组:于接种C6脑胶质瘤细胞后第7天将紫杉醇-聚乳酸纳米缓释膜片(由中科院长春应用化学研究所徐秀玲博士惠赠)植入瘤细胞接种部位。②对照组:手术,但不植入膜片。观察两组一般情况及死亡时间;采用复查MRI检测及苏木精-伊红染色组织病理和免疫组织化学染色观察肿瘤生长情况。结果:①接种1周后MRI检查,脑内形成实体瘤,成瘤率为80%,无远隔转移;苏木精-伊红组织病理证实是胶质瘤,GFAP免疫组化染色阳性。②实验组平均生存期长于对照组[(56.5±18.7),(17.6±1.6)d,P<0.05];实验组肿瘤平均直径小于对照组(P<0.05)。结论:紫杉醇-聚乳酸纳米缓释膜片进行局部化疗可有效抑制脑胶质瘤细胞的生长。