摘要
目的:观察脊髓全横断SD大鼠模型骨髓CD34+细胞移植术后,脊髓功能恢复情况。方法:实验于2004-11/2005-07在昆明医学院神经科学研究所完成。①采用Percoll细胞分离液离心+贴壁分离法对绿色荧光蛋白转基因小鼠骨髓细胞进行粗筛后,收集骨髓悬浮细胞进行体外培养,密切观察细胞生长情况。②收集培养至第3周的悬浮细胞,采用免疫细胞化学法进行CD34单克隆抗体鉴定。③选健康雌性SD大鼠30只,采用随机数字法分为移植组和对照组,每组15只(1d,1,2,4和8周各3只)。将经CD34单克隆抗体鉴定后的CD34+细胞移植入脊髓全横断大鼠模型脊髓横断处尾侧。④术后密切观察大鼠后肢运动功能恢复情况,分别在术后1d,2,4和8周行BBB评分检测大鼠后肢运动功能的恢复。⑤同时行冰冻切片于荧光显微镜下观察绿色荧光细胞的分布情况,并用免疫组织化学法检测CD34+细胞的存活。结果:对照组大鼠第22天死亡1只,进入结果分析29只。①免疫荧光镜下见,培养至两三周的骨髓悬浮细胞中,圆形绿色荧光细胞可达90%以上。②免疫细胞化学染色结果显示,培养至两三周后的骨髓悬浮细胞多数呈CD34阳性,阳性率达(74.6±1.7)%。③两组大鼠BBB评分结果显示,移植组大鼠后肢自主运动功能的恢复明显优于对照组。④移植后2,4和8周时移植组脊髓横断处头尾两侧均可见绿色荧光细胞,且多分布于灰质中,散在或聚集成片;免疫组织化学法可见移植组脊髓横断处头尾两侧切片中均有CD34+细胞散在。结论:移植骨髓CD34+细胞可在脊髓全横断大鼠脊髓中存活并迁移,其大鼠后肢自主运动功能得到部分恢复。
AIM： To observe the functional recovery of spinal cord after transplanting CD34^＋ cells into the completely transected spinal cord of rats.
METHODS： The experiment was carried out in the Institute of Neuroscience, Kunming Medical College between November 2004 and July 2005. ①The marrow cells from transgenetic green fluorescent protein mice were sieved roughly by centrifugation and adherence method with Pereoll separating fluid to collect the marrow suspended cells for in vitro culture, and the growing state of cells was observed. ②The suspended cells were collected after 3 weeks of culture for anti-CD34 monoclonal antibodies identification by immunocytochemistry. ③Thirty healthy male SD rats were divided into transplantation group and control group by random number method with 15 rats in each group （respective 3 rats at the 1^st day, 1^st, 2nd, 4^th and 8^th weeks）. The CD34^＋ cells after identified by anti-CD34 monoclonal antibodies were transferred into the caudal of transaction of rat model. ④ The motor functional recovery of hind limbs was observed： Respectively at the 1^st day, 1^st, 2^nd, 4^th and 8^th weeks, the locomotor functions of the rats were detected by BBB score. ⑤Through sank and frozen, the spinal cord was sectioned. Then the green fluorescence cells were observed with the fluorescence microscope and the survivorship of CD34^＋ cells was detected by immunohistochemistry staining.
RESULTS： One rat in the control group died at the 22^nd day, finally a total of 29 rats entered the result analysis. ① More than 90% of round green fluorescent cells in marrow suspended cells cultured after 2 to 3 weeks were observed by immuno-fluoroscope. ② The result of immunohistochemistry staining suggested that most of the suspended cells cultured for 2 to 3 weeks showed CD34-positive with positive rate of （74.6±1.7）%. ③Compared with thecontrol group, the locomotors functional recovery of the transplantation group, which evaluated by BBB score, was obviously better. ④At the end of the 2^nd, 4^th and 8^th weeks, diffused or accumulated green fluorescence cells could be found in gray matter of the rostral and caudal part around the transected sites of the transplantation group, where scattered CD34^＋ cells were also observed by immunohistochemistry staining. CONCLUSION： The survival and immigration phenomenon can be observed after CD34^＋ cells from bone marrow transplanted into the completely transected spinal cord, and partly locomotors functional recovery of hind limbs also present.
出处
《中国临床康复》
CSCD
北大核心
2006年第21期30-33,i0002,共5页
Chinese Journal of Clinical Rehabilitation
