Recovery Effects Of The Combination Safflower Yellow By Intraperitoneal Injection And Transplantation MSCs On Spinal Cord Injury In Rats

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.