静脉注射骨髓间充质干细胞在缺血鼠脑中的分布与转化  

Distribution and transformation of intravenous administration of mesenchymal stem cells in the brain of rats after cerebral ischemia

作　　者：李高华[1] 张朝东[2] 王翠琴[1] 

机构地区：[1]抚顺矿业集团总医院神经内科,辽宁省抚顺市113008 [2]中国医科大学附属第一医院神经内科,辽宁省沈阳市110001

出　　处：《中国组织工程研究与临床康复》2007年第3期490-493,I0004,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:观察经静脉输入骨髓源性骨髓间充质干细胞在缺血鼠脑中的分布与转化情况。方法:实验于2003-07/2004-08在中国医科大学完成。①取两周龄Wistar胎鼠1只制备骨髓间充质干细胞,将传至2,3代的细胞于移植前3d加入溴脱氧核苷尿嘧啶进行标记备用。②选取健康成年Wistar大鼠22只,随机数字表法分为缺血再灌注模型组12只、假手术组10只。缺血再灌注模型组大鼠建立大脑中动脉闭塞模型,假手术组手术步骤相同,但不阻塞大脑中动脉。术后大鼠提尾右前肢屈曲内收、向右侧转圈或向右侧倾倒为造模成功,缺血再灌注模型组12只大鼠均符合标准。③造模24h后,取2,3代经BrdU标记的骨髓间充质干细胞,消化离心后分别取3×106个细胞(总量大约1mL)静脉输入两组每只鼠尾。④造模后14d,两组大鼠断头后以前囟为中心前后1mm处取脑组织制作标本切片,苏木精-伊红染色。应用BrdU标记骨髓间充质干细胞,计数两组同一部位的10个冠状切片的所有BrdU反应阳性细胞。应用免疫荧光与免疫酶法双重染色,计数BrdU阳性细胞表达神经元特异性烯醇酶、神经胶质原纤维酸性蛋白的比例。结果:实验选取22只大鼠均进入结果分析。①神经元凋亡检测结果:缺血再灌注模型组大鼠缺血区脑组织均发现了暗红色神经元,神经细胞间隙增宽。假手术组均未检测到以上现象。②骨髓间充质干细胞BrdU免疫组化染色结果:骨髓间充质干细胞的胞核为圆形或椭圆形,暗棕色,胞浆少且形态不规则,BrdU标记出现在大脑中动脉闭塞范围的供体细胞胞核中。大多数BrdU标记的骨髓间充质干细胞定位于缺血中心和边缘区,平均阳性细胞数为(15002.75±4972.64);少量分布在对侧半球皮层,平均阳性细胞数为(999.58±655.41)。假手术组脑组织内未发现BrdU阳性细胞。③骨髓间充质干细胞双重免疫组化染色结果:缺血区BrdU阳性细胞表达AIM： To observe the distribution and differentiation of intravenous administration of meshenchymal stem cells （MSCs） in the brains of rats after cerebral ischemia. METHODS： The experiment was conducted in China Medical University from July 2003 to August 2004. （1）MSCs were prepared from one Wistar fetus rat of two weeks old and the second and third generation MSCs were prelabeled with bromodeoxyuddine （BrdU） 3 days before transplantation. （2）Twenty-two healthy adult Wistar rats were selected and randomly divided into ischemic reperfusion group （n =12） and sham operation group （n =10）. The rats in the ischemic reperfusion group were made into middle cerebral artery occlusion （MCAO） models; the rats in the sham operation group were also given the same operation procedure but the middle cerebral artery was not occluded. If the right limbs appeared flection and adduction, dextrad version or latedversion after lifted tail of the rats, the models were successfully established. All 12 rats in the ischemic reperfusion group met the criteria. （3）At 24 hour after MCAO, the second and third MSCs prelabeled with bromodeoxyuridine were selected, and 3×10^6 cells （about 1 mL） were colleted and intravenously infused into the tail vein of all rats after digestion and centrifugalization. （4）Fourteen days after modeling, the cerebral tissues of the rats corresponding to coronal coordinates for bregma 1 mm were taken to prepare the sections, which were stained with hematoxylin and eosin. MSCs were labeled with BrdU, so numbers of BrdU-reactive cells were measured in 10 coronal sections in the same original brain tissue. Immunofluorescence and immunoenzymic method double staining were used to measure the ratio of neuron specific enolase and glial flbrillary acidic protein expressed by BrdU positive cells. RESULTS： All 22 rats were involved in the result analysis. （1）Results of neuron apoptosis： Madder red neurons were detected in the ischemic region of rats in the ischemic reperfu

关 键 词：骨髓细胞 间质干细胞 移植 脑缺血 

分 类 号：R394.2[医药卫生—医学遗传学]