课题背景：课题受国家自然科学基金、广州中医药大学博士后基金、深圳市南山区科技局基金3个基金资助。课题深入探讨电针任脉承浆、气海、关元等穴位对脑缺血后神经干细胞增殖与分化与碱性成纤维细胞生长因子作用的异同。

应用要点：应用免疫荧光双标、激光共聚焦显微镜检测等技术，观察针刺任脉经穴对线栓法造成中动脉缺血模型大鼠脑缺血后神经干细胞增殖与分化的影响，解析针刺治疗脑缺血的疗效及认识针刺任脉治疗脑缺血的机制。

术语解析：碱性成纤维细胞生长因子是哺乳动物和人体组织中存在的微量蛋白质，是一种多功能细胞生长因子，对于中胚层和神经外胚层源细胞具有显著的促增殖作用，能促进神经元的存活和突起生长,对神经胶质细胞有较强的促分裂作用,对多分化潜能的神经干细胞也有促增殖作用。


摘要
目的：近来的研究认为，针刺任脉治疗脑卒中的机制在于针刺任脉可能产生与干细胞增殖分化有一些联系的生长因子。脑缺血损伤后调动内源性神经干细胞试图自我修复的途径应是多元化的,针刺及外源性生成因子的给予为其不同的途径。实验拟观察电针任脉和肌肉注射碱性成纤维细胞生长因子对脑缺血模型大鼠缺血侧脑室下区5-溴-2’-脱氧尿苷和5-溴-2’-脱氧尿苷/巢蛋白阳性细胞的表达的影响。
方法：实验于2004-09/2005-07在中山大学医学院解剖学实验室完成。①材料：选用成年健康雄性Wistar大鼠83只。将实验动物按随机抽签法分为5组：空白对照组6只、假手术组6只、模型组26只、电针任脉组23只、碱性成纤维细胞生长因子组22只。后3组又分为缺血后7，14和28 d 3个时间点进行观察。②干预：除空白对照组和假手术组外，其余大鼠采用线栓法制作局灶性脑缺血模型。再灌注后参考Longa神经病学评分标准，1~4分为造模成功。空白对照组不作任何处理；假手术组仅分离右侧颈总动脉并离断右颈外动脉，不予栓塞。电针任脉组大鼠于造模后第2天采用上海华谊医用仪器厂生产的G6805Ⅱ型电针仪针刺承浆、气海、关元3穴，针刺后加电，疏密波刺激（疏波30 Hz，密波100 Hz），强度6~15 V，以身体相应部位出现轻微颤动为准，持续时间为20 min。碱性成纤维细胞生长因子组：再灌后立即给药，肌注碱性成纤维细胞生长因子4 000 U/d，此后每天肌注碱性成纤维细胞生长因子，1次/d。模型组、假手术组大鼠于造模后第2天固定于针刺操作台上20 min，不予针刺。空白对照组大鼠不作任何处理。③观察指标：通过免疫荧光方法测定侧脑室下区5-溴-2’-脱氧尿苷和5-溴-2’-脱氧尿苷/巢蛋白阳性细胞的表达。用Olympus FV 500激光共聚焦显微镜系统，在200倍镜下，计数平均5个视野阳性细胞数。
结果：造模成功大鼠54只及空白对照组和模型组各6只进入结果分析。侧脑室5-溴-2’-脱氧尿苷阳性细胞数和5-溴-2’-脱氧尿苷/巢蛋白双标细胞：空白对照组与假手术组比较，差异无显著性意义（P > 0.05）。模型组与空白对照组相比，均有不同程度的增加，其中造模后7和14 d差异有显著性意义（P < 0.01）。电针任脉组和碱性成纤维细胞生长因子组造模后3个时间点与模型组比较，均有较大程度的增加，差异有显著性意义（P < 0.05~0.01）。碱性成纤维细胞生长因子组与电针任脉组相比,差异无显著性意义（P > 0.05）。
结论：电针任脉和肌肉注射碱性成纤维细胞生长因子均可促进局灶性缺血模型大鼠原位神经干细胞增殖，且两者作用相当。
关键词：神经干细胞；电针；任脉；碱性成纤维细胞生长因子；5-溴-2'-脱氧尿苷，5-溴-2'-脱氧尿苷/巢蛋白；脑缺血；增殖；大鼠

刁利红，于海波，皮敏，罗文舒，饶晓丹，刘远声，杨卓欣.电针任脉和肌肉注射碱性成纤维细胞生长因子对脑缺血模型大鼠侧脑室下区原位神经干细胞增殖的影响[J].中国组织工程研究与临床康复，2008，12(8):1435-1439
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1435(ps).pdf]

Effects of electroacupuncture at the Ren channel and basic fibroblast growth factor injection on in situ neural stem cell proliperation in subventricular zone of cerebral ischemic rats

Abstract

AIM：Recent studies have demonstrated that electroacupuncture at the Ren channel can treat stroke by the production of some neurodevelopment-related growth factors, which are associated with the proliferation and differentiation of stem cells. There are many approaches to regulate self-repair of endogenous neural stem cells after cerebral ischemic injury. Electroacupuncture and exogenous growth factors are two approaches of them. This study observed the effect of electroacupuncture at the Ren channel and intramuscular injection of basic fibroblast growth factor (bFGF) on the expressions of 5-bromodeoxyuridine (BrdU)-labeled and BrdU/Nestin-labeled positive cells in subventricular zone of cerebral ischemic rats.

METHODS: The experiment was performed at Laboratory of Anatomy, Medical College of Sun Yat-sen University from September 2004 to July 2005. ①Eighty-three adult healthy male Wistar rats were selected and randomly divided into blank control group (n=6), sham operation group (n=6), model group (n=26), electroacupuncture at Ren-channel group (n=23), and bFGF group (n=22). Three time points (7, 14 and 28 days after ischemia) were set in each of the latter three groups for observation. ②Except for blank group and sham operation group, the rats in the other groups were prepared into models of local cerebral ischemia by thread occlusion method. After reperfused, the rats were graded by neurological scoring and only rats ranged from 1 to 4 points were used for the experiment. Blank group was not given any treatment; in the sham-operation group, only rght common carotid artery was isolated and right external carotid artery was dissected without occlusion. On the second day after modeling, the rats in the electroacupuncture group were given acupuncture at the Chengjiang, Qihai and Guanyuan acupoints using G6805Ⅱelectroacupuncture apparatus (Huayi Medical Instrument, Shanghai) followed by sparse wave 30 Hz, dense wave 100 Hz, intensity 6 to 15 V till slight trill appeared in the corresponding position. The stimulation lasted for 20 minutes. In the bFGF group, the rats were immediately intramuscularly injected with 4 000 U/d bFGF after reperfusion, once a day. The rats in the model group and sham operation group were mounted on the acupuncture bench for 20 minutes on the second day after modeling with no acupuncture. ③The expressions of Brdu-labeled positive cells (cell proliferation) and Brdu/Nestin-labeled positive cells (neural stem cells proliferation) in the lateral subventricular zone were determined by immunofluorescence. Positive cells were counted in five visual fields using 200-fold Olympus FV 500 laser confocal microscope system.

RESULTS: Altogether 54 successfully modeled rats, 6 in blank group, and 6 in model group were included in the final analysis. There was no significant difference in BrdU and BrdU/Nestin-labeled positive cells between blank group and sham operation group (P > 0.05). BrdU and BrdU/Nestin-labeled positive cells were increased when the model group compared with normal group, and the differences were statistically significant on days 7 and 14 after modeling (P < 0.01). Compared with model group, BrdU and BrdU/Nestin-labeled positive cells were significantly increased on the 7th, 14th, and 28th days in electroacupuncture group and bFGF group (P < 0.05-0.01). But there was no significant difference between electroacupuncture group and bFGF group (P > 0.05).

CONCLUSION: The results demonstrate that both electroacupuncture at the Ren channel and injection of bFGF can equally promote multiplication of neural stem cells in rats with focal cerebral ischemia.

Diao LH, Yu HB, Pi M, Luo WS, Rao XD, Liu YS, Yang ZX.Effects of electroacupuncture at the Ren channel and basic fibroblast growth factor injection on in situ neural stem cell proliperation in subventricular zone of cerebral ischemic rats.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):1435-1439(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1435(ps).pdf]