| Part I：Culture and identification of rat adipose derived stem cells and bone marrowmesenchymal stem cells.Objective: Rat bone marrow mesenchymal stem cells (rBMSCs) and adipose derived stemcells (rADSCs) were cultured in vitro. Cell morphology was observed. Surface markersand osteogenic, adipogenic, neural induction of differentiation capacity was assessed,which provided adequate cells for follow-up experiments.Methods: rBMSCs were isolated and cultured using the repeated adherent culture method.rADSCs were isolated and cultured using I-type collagenase digestion and the adherentculture method. CD29, CD34and CD90surface markers of rBMSCs were identified byflow cytometry (FCM). CD29, CD34, CD45and CD90surface markers of rADSCs wereidentified. They were performed the osteogenic, adipogenic, neural induction ofdifferentiation. The osteogenic were identified by alkaline phosphatase and Von Cassastaining. The adipogenic were identified by oil red staining. The neural differentiation wasidentified by NSE and MAP2.Results: rBMSCs were isolated and cultured using the repeated adherent culture method.rADSCs were isolated and cultured using I-type collagenase digestion and the adherentculture method. These cells proliferated well and their morphology was more uniform.Surface marker analysis showed that the rBMSCs were positive for CD90and CD29，negative for CD34. The rADSCs were positive for CD90and CD29，negative for CD34and CD45. After induction of differentiation, rBMSCs and rADSCs appear as themorphology of bone cells, fat cells and nerve cells. Osteoblasts was confirmed by alkalinephosphatases and Von Cossa staining, fat cells confirmed by oil red staining, and nervecells was proved by NSE and MAP2. After the cultivation of the cells in three generations cells proliferated well.Conclusion: After3passages, relative purified rBMSCs can obtained by repeated adherentculture method and relative purified rADSCs can obtained by I-type collagenase digestionand the adherent culture method. In vitro，the rBMSCs and rADSCs can be induced, whichindicates that the two have multipotent differentiation capacity.Part II：The influence of adipose derived stem cells and bone mesenchymal stem cellstransplantation to the neurocyte apoptosis of injured rat spinal cord．Objective: To compare rBMSCs and rADSCs transplant treatment of spinal cord injury andto investigate potential anti-apoptotic mechanisms.Methods: A total of8SD rats aged6-week-old were used for rBMSCs and rADSCsharvest.96adult SD rats, according to the surgical approach and graft were randomlydivided into6(A~F) group (n=16), each group was divided into two groups inaccordance with the executed time, each subgroup of8. Group A1, the PBS sham surgerygroup, day14post; Group A2, the PBS sham surgery group, day28post; Group B1, thePBS sham surgery group, day14post; Group B2, the PBS sham surgery group, day28post; Group C1, rBMSCs sham surgery14days group; Group C2, the rBMSCs28dayssham operation group; Group D1, rBMSCs surgery14days group; Group D2, rBMSCs,surgery28days group; Group E1, rADSCs sham surgery14days group; Group E2,rADSCs, sham operation28days group; Group F1, rADSCs surgery14days group; GroupF2, rADSCs surgery28days group. Model groups are modified Allen methodmanufacturing the rat thoracic spinal cord injury model in sham group only bite segmentspinous process and lamina in addition to the corresponding section, non-intervention inthe spinal cord. Seven days after the modeling, the level of (L4/L5clearance) in the lumbarsubarachnoid space to the group was injected with the corresponding graft. Then observedthe hind limb motor function of all animals，and evaluated by BBB scale．Rats wereanesthetized at day14，28postoperatively according to the timing point of group．Every8rat spinal cord segments containing the injured sites or counterparts in each group wereharvested after perfused with normal saline followed paraformaldehyde intracardially．Spinal cords were postfixed in neutral formalin and were paraffin-embeddedand sectioned． Hematoxylin and eosin staining were performed for histologicalobservation． Neurocyte apoptosis was examined by the terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining.Results: Before surgery, all animals’ BBB scale score was21(the maximum value).Postoperative anesthesia, the animals of control group (A1, A2, C1, C2, E1, E2) reached21BBB scale score. Surgery group (B1, B2, D1, D2, F1, F2) rats after modeling the first3days,7days BBB score lower than the sham operation group was statistically significant(P <0.05). rBMSCs after transplantation, rBMSCs surgery group (E1, E2) BBB score ofrats continued to increase, higher than the PBS operation group (B1, B2) rats (P <0.05).But it still lower than rBMSCs in sham operation group (C1, C2). There was a significantdifference (P <0.05). After rADSCs transplantation in the treatment, the rADSCs surgerygroup (F1, F2) The BBB score continued to increase, higher than the PBS operation group(B1, B2) rats (P <0.05), but still lower than the rADSCs the sham operation group (D1,D2), with significant difference (P <0.05). rBMSCs surgery group (E1, E2) and rADSCssurgery group (F1, F2), The BBB score was no significant difference (P>0.05). Nosignificant change was in the PBS sham operation group (A1, A2) the BBB rating. Fromthe first seven days of the transplant recipients, rBMSCs operation group and rADSCsoperated rats BBB score were higher than the corresponding sham operation group, weresignificantly different (P <0.05). Spinal cord tissue sections were stained with HE results:28days, the sham operation group (A1, A2, C1, C2, of E1, E2),14days aftertransplantation, spinal cord tissue sections were normal. Each surgical group (B1, B2, D1,D2, F1, F2), spinal cord tissue sections showed cavity formation, the PBS operation groupcavity formation becomes apparent. Transplantation14days after PBS operation group(B1), the number of TUNEL staining cells than rBMSCs surgery group (D1) and rADSCssurgery group (F1), there is a significant difference (P <0.05). While rBMSCs surgerygroup (D1) and rADSCs operation group (F1) were higher than the corresponding shamgroup, a significant difference (P <0.05).28days after transplantation, groups group TUNEL-positive cells were reduced, PBS operation group (B2) is still higher than othergroups, but there was no significant difference (P>0.05). Conclusion: The transplantrBMSCs and rADSCs can improve spinal cord injury rat model of hindlimb motor function,and can reduce apoptosis after spinal cord injury. Those findings suggest that rBMSCs andrADSCs transplantation has potential anti-apoptotic effects in the treatment of spinal cordinjury. |
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