【作者】 李安军；

【导师】 施永彦；

【作者基本信息】 湖北中医药大学 ， 中西医结合临床， 2010， 硕士

【摘要】 目的：脊髓损伤是一种常见病、多发病,可以造成损伤平面以下不可逆的感觉、运动及括约肌功能障碍,临床治疗效果不理想,一直是医学领域的热点和难点。本实验应用对脊髓损伤具有保护作用的红花黄素腹腔注射联合MSCs移植治疗大鼠脊髓损伤模型,探讨红花黄素腹腔注射和MSCs移植对损伤脊髓修复作用的机制,为提高脊髓损伤临床治疗效果寻找一条可行的途径。方法：贴壁分离法分离纯化大鼠MSCs,观察原代及传代MSCs的形态特点、生长特性,探讨其作为种子细胞的可行性并用流式细胞分析技术鉴定MSCs表面蛋白表达。携带GFP基因的腺病毒感染MSCs,荧光显微镜观察MSCs转染情况并计算转染效率,观察GFP-MSCs形态特点、生长特性,研究GFP标记对MSCs生物学性状的影响。55只成年S-D大鼠,雌雄各半,根据处理方法的不同,随机分为5组：A组(红花黄素腹腔注射+MSCs移植组)、B组(生理盐水腹腔注射+MSCs移植组)、C组(红花黄素腹腔注射+PBS组)和D组(生理盐水腹腔注射+PBS组),以上每组各10只；E组为补充组,15只。改良Allen法T7平面制作脊髓损伤模型。造模成功后,A组和B组损伤局部即时直接移植标记后的MSCs (GFP-MSCs) 6×106/60μl,C组和D组注射同等体积PBS。A组和C组术后按40mg/kg的剂量腹腔注射红花黄素,B组和D组注射同等剂量的生理盐水,每日1次,共用2w。实验过程中,各组的死亡动物后从E组中随机选取补充,重新开始计算模型动物的喂养处理时间,以保证各组实验动物数目的相同。术后1w、2w、3w和4w时采用BBB评分法进行后肢功能评分后心脏灌注取材,制作冰冻切片,荧光显微镜观察移植的MSCs; HE染色观察组织形态学变化；免疫组化检测GFAP、NeuN和NGF的表达。结果：1、贴壁分离法分离纯化的贴壁细胞形态、生长特性与MSCs一致,流式细胞分析技术检测细胞表面蛋白的表达：CD45阳性率14.47%,CD44阳性率89.26%,CD29阳性率93.27%提示获得的细胞是MSCs;2、携带GFP基因的腺病毒感染MSCs后,荧光显微镜观察证实MSCs的GFP标记成功,腺病毒的转染效率为(93%±2%),转染后MSCs的形态学、生长特性无明显变化；3、荧光显微镜下,GFP-MSCs移植后各个时间点1w、2w、3w和4w时的冰冻组织切片均可观察到GFP-MSCs发出的明亮的绿色荧光,证明移植的MSCs可以在大鼠损伤的脊髓中存活；4、术后2w-4W,红花黄素腹腔注射或MSCs移植的单独应用均可提高大鼠BBB评分的得分,二者联用作用更加明显,差异有显著性(P<0.05),术后1w时,各组间评分的差异性不显著(P>0.05)；5、术后1w、2w、3w和4w各个时间点冰冻组织切片HE染色显示红花黄素腹腔注射或MSCs移植的单独应用均可改善神经组织病变情况,减少空腔的形成,其改善程度和BBB评分得分情况一致,二者联用病变作用更加明显；6、免疫组化检测显示各组GFAP、NeuN和NGF表达均随着术后时间的延长而增加,相同时间点同一指标A组、B组和C组的表达水平明显高于D组,差异有显著性(P<0.05),其中A组的表达水平又高于B组和C组,差异有显著性(P<0.05),B组的表达水平高于C组,差异有显著性(P<0.05)。结论：1、贴壁分离法是一种较好的MSCs分离方法,所获贴壁细胞生长迅速,活力良好,流式细胞分析技术证实为MSCs,可用作组织工程的种子细胞；2、应用携带GFP基因的腺病毒感染MSCs,GFP转染效率高,对MSCs的生物学特性没有明显影响,标记后的MSCs能够在大鼠的脊髓中成活、显示,GFP是一种较好的MSCs标记方法；3、红花黄素腹腔注射或MSCs移植单独应用能够提高NGF的分泌,促使干细胞向神经细胞和神经胶质细胞方向分化,有利于受损神经组织的修复,二者联用具有协同作用。更多还原

【Abstract】 Objective:Spinal cord injury (SCI) is very frequent in our society, and often results in loss of voluntary motor and sensory function below the lesion site, and causes the dysfunction of urination and defecation, which decreases the life quality seriously. The treatment of SCI remains to be a puzzling and challenging issue in medical science. There are some reports that safflower yellow can promote the neural function to resume, has close relationship with repairing of the nervous system. In this experience, we study the recovery effects of the combination safflower yellow by intraperitoneal injection and transplantation MSCs on spinal cord injury in Rats, discuss the mechanisms of safflower yellow by intraperitoneal injection and transplantation MSCs on the SCI model, and examine whether the combination of these protocols is more effective than a single protocol.Methods:MSCs were collected from S-D rat bone marrow with adherence separation method, expanded in vitro. The morphology was observed and CD44, CD29 and CD45 of P3 MSCs were detected with flow cytometry analysis. GFP genes were transfected into MSCs with adenovirus vector (Adv-GFP), GFP positive MSCs (GFP-MSCs) were observed with fluorescent microscopy, the morphology and the growth curve were also observed in order to study biocharacteristics. By the striking of the spinal cord at the level of T7 with Allen’s device, acute spinal cord injury was induced in anesthetized rats. Fifty-five SCI model rats were randomly divided into 5 groups:group A (safflower yellow +MSCs), B (saline+MSCs), C (safflower yellow+PBS), D (saline +PBS) and E (supplementary group). There were 10 rats in every group except group E which has 15 rats. The labeled MSCs (GFP-MSCs) in a dose of 6×106/60μl were injected into SCI space in group A and B, the identical volume of PBS in group C and D. A dose of saff lower yellow (40mg/kg) was administered by intraperitoneal injection at post-lesion from 1d tol4d in group A and C and the identical volume of saline in group B and D. At 1w、2w、3w 4w after SCI, Neurological functional changes was evaluated by another man who was proficient in the regulations with open field Basso, Beattie, Bresnahan (BBB)locomotors rating scale. At the same time of post-operation, cardiac perfusion by Para-formaldehyde was performed; the injury spinal cord was made into frozen section. histomorphology change was observed by HE staining, the survival of MSCs by fluorescence microscopy and the expressions of GFAP、NeuN and NGF through immunohistochemical staining technique and image analysis. During the experiment, if any rat died, another one would be selected from group E instead of it and calculate feeding time from beginning, which in order to make sure the same number in each group.Results:(1) It was believed that the harvested cells was identical to MSCs, because the morphology and biocharacteri-stics were similar to MSCs’and flow cytometry analysis showed the expression of CD44, CD45 and CD29 were 89.26%,14.47%and 93.27%.(2) After GFP genes were transfected into MSCs, Nearly (93±2)%of cells expressed GFP genes, which be confirmed Adv-GFP can efficient transfect MSCs and make GFP mass expression in MSCs. The morphology and biocharacteristics of GFP-MSCs were found no difference.(3) GFP positive MSCs were observed on frozen section by fluorescent microscopy at 1w、2w、3w 4w after GFP-MSCs transplantation, which be conformed MSCs can survive in the injured spinal cord of rats.(4) From the second week to the fourth week postoperatively, the BBB score of in the group B and C were higher than those in the group D, those in the group B and C was compared with those in the group D, there was significant difference (P<0.05), the BBB score of in the group A was higher than those in the group B, C and D, there was significant difference (P<0.05), while there was no obvious difference among in every group (P>0.05) at the first week postoperatively.(5) The application of either safflower yellow intra-peritoneal injection or MSCs transplantation can improve histomorphology of nerve tissue at every time point postoperatively. Improvement effects of the combination safflower yellow by intraperitoneal injection and transplantation MSCs was significant better than either safflower yellow intraperitoneal injection or MSCs transplantation. The degree of improvement was parallel to the BBB score.(6) Immunohistochemistry staining results showed that the expression of GFAP、NeuN and NGF was consistently increased in all of groups. At identical time point the expression of GFAP、NeuN and NGF was higher in the group A, B and C than those in the group D, there was significant difference (P<0.05); Those in the group A than those in the group B and those group C, and those in the group B than those in the group C, there was significant difference (P<0.05).Conclusions:(1) Adherence separation method might be an ideal method for isolation and purified MSCs from bone marrow, because the harvested adherence cells proliferated fast, and flow cytometry analysis showed the cells were identical to MSCs. all those results shows MSCs are suitable for seeding cells in tissue engineering.(2) MSCs which be transfected GFP gene carry bright green fluorescence, which be observed in injured spinal cord of rats, and have no impact on biological function of MSCs, so Adv-GFP transfected MSCs is an ideal method for GFP labeled of MSCs, with its characteristics of high accuracy, high ratio of the labeled and stable expression.(3) The application of either safflower yellow intra-peritoneal injection or MSCs transplantation can increase expression of NGF, enhance ratio of the differentiation into neurons and neuroglial cells, and facilitate recovery of injured nerve tissue on SCI model in rats. The combination safflower yellow by intraperitoneal injection and transplantation MSCs may result in better effect. It provides the better theoretical basis for more extensive study application.更多还原