| Part I: Culture,purification,detection of rat bone marrow mesenchymal stem cells(MSCs)Objective: To prepare the bone marrow mesenchymal stem cells in treatment of rats with hemisection injury,we isolated and culturd BMSCs from rat tibia, observed their morphology and identified the surface markers.Methods: The mononuclear cells were collected from the femur in infant SD rats under sterile conditions; MSCs were isolated and purified from the mononuclear cells by sticking wall method, and then were confirmed by flow cytometric analysis of CD29,CD90, CD34 and CD45 after three passenges.Results: It could been seen that there were many long fusiform or polygonal cells adherent after original generation were incubated for 24 hours. The in primary cells reached 80% to 90%, and integration into the film with a swirl array after 5 to 7 days, and then inoculate according to the ratio of 1:2 after digestion. The MSCs start adhered and spread 4 hours later and can reach 80% of the fusion after 3 days. The flow cytometer(FCM)showed that the cells from rat bone marrow were positive in CD90 and CD29, and were negative in CD45 and CD34.Conclusion: Rat bone marrow mesenchymal stem cells were isolated and cultured successfully.The second part: The changes of macrophage subsets after spinal hemisection injury in ratsObjective: To investigate the changes of macrophage subsets in different time points after SCI by constructing a rat model of semi transection injury.Method: 1. The model of left-T9 hemisection spinal cord injury in rat. 2. 27 health six week old male SD rats were randomly assigned to two groups: 1) Sham operation group; 2.) Control group; 3) The histological evaluation was performed at 3 days, 12 days,28 day post injury respectively(M1 : anti-iNOS, M2 : anti-CD206).Results: 1) Three rats were eliminated because of incomplete half transaction according to the exclusion criteria, and finally there were 24 rats left.2) The M1 macrophage marker iNOS began express within 3 days after injury,reached a peak at 12 days post injury, and then decreased and until 28 days with low level expression.3) M2 macrophage marker CD206 was significantly higher at 3 days after the injury,and then decreased until 12 days.Conclusion: The expression of M2 in the spinal cord is short; however, the expressio n of M1 is longer than that of M2. The 12 days post injury can be used as the time point for the study of the regulation of macrophages in the rat model of semi transection injury.The third part: Labeling of mesenchymal stem cells and loading on scaffoldObjective: To observe the cell adhesion, fixation and distribution of bone marrow mesenchymal stem cells on the collagen scaffold by fluorescence labeling and scaffold.Methods: 1) The bone marrow mesenchymal stem cells were labeled with 8μmol/L CM- Dil, and then use flow cytometry to evaluate the labeling rate, CCK-8 assay for the detection of labeled cells proliferation. 2) In vitro, the cell suspensions of bone marrow mesenchymastemal stem cells with certain concentration were prepared by cell counting,and then were seeded on the collagen scaffold. 3) The blank collagen scaffold and scaffold and stem cell complex were observed by using SEM.Results: 1) The bone marrow mesenchymal stem cells labeled with CM-Dil showed red fluorescence. The cells were spindle shaped and maintained good morphology. The labeling rate of CM-Dil was over 98%, and the proliferative activity of CM-Dil labeled stem cells was not affected.2) BMSCs could be seen in the center and edge of collagen scaffold with relatively homogeneous distribution after 12 days co-culture3). The results of electron microscopy scanning showed that the collagen scaffold can provide a three-dimensional cell adhesion with appropriate porosity and nano-grade linear fiber.Conclusion: The collagen scaffold can be used as a carrier for bone marrow mesenchymal stem cells to adhere.The fourth part. The mesenchymal stem cells can promote the repair of spinal hemisection injury in rats by the regulation of macrophage subsets.Objective: To analyze and evaluate the effect of MSCs on macrophage subtypes and recovery of nerve function by using collagen scaffold stem cell complex to repair the spinal hemisection injury in rats.Method: 1) T9 left spinal cord hemisection injury model in rats were established: 99 health six week old male SD rats were randomly divided into four groups: 1. Sham operation group(n = 20); 2. Control group(n = 32); 3. CS transplantation group(n = 26); 4.CS/MSC transplantation group(n = 21).2) The rat mortality was recorded within 21 days after surgery; The 21-point Basso-Beattie-Bresnahan(BBB) scale was performed at 1 week intervals after SCI for 8weeks.3) The histological evaluation was performed at 12 days, 4 and 8 weeks post injury.For immonohistochemistry, the spinal cord sections were incubated with primary antibody:anti-CD68, anti-iNOS, cd206, anti-NF, anti-GFAP,Tunel.Results:1. There were eight rats eliminated with incomplete left hemisection(4 from control group, 2 from CS group, 2 from CS/MSC group), and 91 rats finally were included.2. The mortality rate of CS/MSC group was lower than that of CS group and Control group, the recovery of left hind limb function was faster than other groups. At 8 week, the BBB score of CS/MSC group was significantly higher than that of CS group and control group.3. H&E staining showed that the cavity of the CS/MSC group was significantly smaller than that of the CS group and the Control group after 12 days and 8 weeks, whilethe cavity of the Control group was smaller than that of the CS group at eight weeks post SCI significantly.4. Immonohistochemistry results showed that expression of CD68, iNOS, Tunel,GFAP in CS/MSC group were less than those in control group and CS group, while NF-200 and CD206 expression increased in group at 12 days.Conclusion: The combined application of collagen scaffolds and bone marrow mesenchymal stem cells can achieve significant therapeutic effect. Bone marrow mesenchymal stem cells can modulate macrophage subsets after spinal cord hemisection injury by reducing macrophage infiltration and promoting macrophage to M2 polarization,reducing holes, apoptosis and scar,promoting nerve regeneration. |
PDF Full Text Request