摘要
目的探讨同种异体骨髓基质细胞(MSCs)移植对SD大鼠缺血肢体治疗机制和规律,为MSCs移植治疗肢体缺血提供理论基础。方法体外全骨髓培养法培养雄性SD大鼠MSCs至三代。60只SD大鼠经结扎腹主动脉及双侧腹壁阴部动脉造成双下肢缺血模型,随机分为对照组(n=30)及移植组(n=30),造模后3天度过反应期后移植组右下肢腓肠肌及股二头肌内分5点注射移植含1×106个MSCs细胞悬液0.5ml,对照组同法注射生理盐水0.5ml。按移植后1周、2周、4周、6周、8周时两组再随机分为5个亚组(n=6),分别取动物麻醉后穿刺双侧髂静脉血行血氧分压测定,处死动物取左前肢,左后、右后肢骨骼肌标本4%多聚甲醛固定,制切片。行Y染色体性别决定区(SRY)原位杂交,血管内皮生长因子(VEGF)-mRNA原位杂交、HE染色,计数VEGF-mRNA阳性细胞数和毛细血管数。对照组仅作右后肢的相关检测。结果 SRY:移植组右后肢、左后肢、骨骼肌间间隙内有阳性细胞构成血管样结构,有的在血管周围,未见到呈阳性的骨骼肌细胞。左前肢的骨骼肌周围筋膜内偶尔可见到阳性细胞。对照组无阳性细胞发现。VEGF-mRNA原位杂交:对照组和移植组右后肢、左后肢VEGF-mRNA呈进行性升高(P<0.001),而左前肢则无明显升高(P=0.694)。在第1周时对照组与移植组相比无统计学差异(P=0.079),随后移植组明显高于对照组(P<0.001)。静脉血氧分压:对照组及移植组髂静脉血氧分压呈进行性升高(P<0.001),移植组右后肢髂静脉氧分压较左后肢及对照组明显高(P<0.05)。毛细血管计数:对照组及移植组右后肢、左后肢的毛细血管计数呈进行性增高(P<0.05),而左前肢则无统计学意义(P=0.055),移植组中右后肢毛细血管计数明显高于左后肢及对照组(P<0.05)。HE染色:未见明显组织细胞核异常分裂改变。结论移植的MSCs可以在同种异体动物体内长期生存、分化,促进缺血肢体的VEGF-mRNA的表达和毛细血管的成长。
Objective To investigate the mechanisms and rules on allogeneic bone marrow stromal cells(MSCs) transplantation to treat hind limb ischemic in Sprague-Dawley(SD) rats. Methods MSCs of male SD rat were cultured to third generation in vitro by whole bone marrow culture method. Ligatured abdominal aorta and bilateral abdominal wall pudendal artery to induce lower limbs ischemia in 60 SD rats. The rats divided into control group(n=30) and transplanting group(n=30) randomly.Three days after the operation, 0.5ml cell suspension including 1×106 MSCs implanted intramuscularly into the gastrocnemius and biceps femoris of the right lower limb at five sites in transplanting group, the control group with physiological saline 0.5ml injection at same positions. Animals were randomly divided into 5 subgroups(n=6) at 1, 2, 4, 6 and 8 week posttransplantation. Rats in the subgroup of transplanting group were respectively collected venous blood in bilateral iliac vein for blood gas analysis via a laparotomy under anesthesia then killed and taken left upper limb's, left and right hind limb's skeletal muscle specimens, made slice, checking SRY and vascular endothelial growth factor(VEGF)-mRNA in situ hybridization, HE staining, counting the number of VEGF-mRNA-positive cells and the capillary number. Control group, only the right hind limb for the correlation detection. Results SRY in situ hybridization: the positive cells were found at skeletal muscle gap of the right and left hind legs in rats of transplanting group, some cells participating the blood vessel-like structure and some near the blood vessels, without positive skeletal muscle cells. Positive cells can be seen occasionally in the skeletal muscle of the left forelimb. No positive cells were found in the control group. VEGF-mRNA in situ hybridization: the positive cells of VEGF-mRNA was progressively increased in the right and left hind limbs(P<0.001) both in transplanting and control group. There was no significant difference between the two groups(P=0.079) in the first week, soon afterwards the count in transplanting group was significantly higher(P<0.001). Venous blood oxygen partial pressure: in the control group and transplanting group venous oxygen blood partial pressure was progressively increased(P<0.001), venous blood oxygen partial pressure of the right hind limb was significantly higher compared with that of left hind limb of transplanting group and the control group(P<0.05). Capillary count: the capillary count of the right and left hind limbs was progressive increase in the control and transplanting groups(P<0.05), while the left forelimb had no statistical significance(P=0.055). The right hind limb capillary count in transplanting group was significantly higher than that in the left hind limb and higher than that in the control group too(P<0.05). HE staining: no significant change in the organization split abnormal nuclei. Conclusion Transplanted allogeneic MSCs can survive and differentiate in animal body for a long time, promote VEGF-mRNA expression and increase capillaries in ischemic limbs, which can be used as a new method of treatment for limb ischemia with relative safety.
出处
《中国血管外科杂志（电子版）》
2014年第2期88-92,共5页
Chinese Journal of Vascular Surgery(Electronic Version)
基金
新疆维吾尔自治区自然科学基金(2010211A48)
