| Objective:To investigate the mechanisms and rules on allogeneic bone marrow stromal cells (MSCs) transplantation treatment for ischemic hindlimb of SD rats to provide theoretical basis of the cell therapy for ischemic limb. Method: Whole bone marrow culture method in vitro cultured MSCs of male SD rat MSCs to three generations were made and made into cell suspension and evaluate P3 cells by immunohistochemisty. 60 SD rats ligatured the abdominal aorta and bilateral pudendal artery wall were induced ischemia of lower limbs . Randomly divided into control group (A group n = 30) and experimental group (B group n = 30).The Models passing through the reaction of the procedure after the operation three days , 0.5ml cell suspension including1×106 MSCs implanted intramuscularly into the gastrocnemius and biceps femoris of the right lower limb of the rats dividing five sites, the control group with saline injection 0.5ml. According to post-transplant week, two weeks, four weeks, six weeks, eight weeks animals were randomly divided into ten subgroups (n = 6). six rats in the subgroup of B group were respectively punctured hibateral femoral vein and aspirated venous blood for blood gas analysis via a laparotomy then killed under anesthesia and taken left upper limb, left and right hindlimb skeletal muscle specimens by 4% formaldehyde fixation, made slice , checking SRY in situ hybridization, VEGF-mRNA in situ hybridization, HE staining, counting the number of VEGF-mRNA-positive cells and the capillary number on post-transplant one week, two weeks, four weeks, six weeks, eight weeks of the timepoint.. Control group, only the right hind limb for the correlation detection. Results: P3 immunohistochemical analysis indicated CD105+ 61.9%,CD44+ 45.4%, CD29+ 16.8%,CD45+ 8.2%,CD31+ 13.7%, CD34+ 8.3%,CD11b+ 1.5% respectively.SRY in situ hybridization: In B group skeletal muscle gap of the right hind, left hind legs the positive cells participating in the blood vessel-like structure were found, some in the blood vessels around without positive skeletal muscle cells. Surrounding the skeletal muscle ?fascia of the left forelimb positive cells can be seen in the occasional. No positive cells were found in the control group. VEGF-mRNA in situ hybridization: In control group and treatment group of the right hind, left hind sample VEGF-mRNA was progressively increased (each group p = 0.000). In the first week the control group compared with treatment group, no significant difference (p = 0.065) , soon afterwards in treatment group the count was significantly higher (p = 0.000). Venous blood oxygen partial pressure:in the control group and treatment group venous oxygen blood partial pressure was progressively increased (p<0.05), the right hind limb venous blood oxygen partial pressure compared with left hind limb of treatment group and the control group was significantly higher (p <0.05 ). Capillary count: In the control group and treatment group, the capillary count of the right hind and left hind limbs sample was progressive increase (p <0.05), while the left forelimb had no statistical significance (p = 0.055), in treatment group the right hind limb capillary count was significantly higher than the left hind limb and the control group (p <0.05). HE staining: no significant change in the organization split abnormal nuclei. Conclusion: The transplantation of allogeneic MSCs can be long-term survival in animal body,and differentiation, promotion of ischemic limbs VEGF-mRNA expression and the growth of capillaries and can be used as the relative safety of a new method of treatment for limb ischemia. |
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