可溶性载体复合成肌细胞移植对胫前肌深度冻伤的修复作用研究

EFFECTS OF IMPLANTED MYOBLASTS WITH SOLUBLE CARRIERS ON SEVERELY-CRYODAMAGED TIBIALIS ANTERIOR MUSCLES

目的探讨利用可溶性载体能否提高成肌细胞移植对胫前肌深度冻伤的修复效率。方法采用酶消化法分离出新生SD大鼠的骨骼肌成肌细胞,经纯化并传代培养,取第3代细胞用BrdU标记。取84只5月龄SD大鼠,建立胫前肌深度冻伤模型,随机分为4组,分别植入含0.1%透明质酸钠的F12培养基为载体的成肌细胞(A1组),不含透明质酸钠的F12培养基为载体的成肌细胞(A2组),含0.1%透明质酸钠的F12培养基载体溶液(B1组)及不作任何处理作对照组(B2组)。分别于术后2、5、9d,2、4、8、12周各处死大鼠6只,通过免疫组织化学和Mallory三色染色观察冻伤组织的修复情况,通过图像分析比较第2天各组细胞存留数和第8周各组胶原纤维面积比。结果BrdU免疫组织化学染色显示,各时间点A1组移植细胞在受体的存留数明显高于A2组,成肌细胞的迁移范围更大,细胞分布更均匀,细胞的分化潜能未受到破坏,移植效率得到提高。B组各时间点未见蓝染的细胞核。Mallory三色染色法显示A1、A2及B1组冻伤组织修复过程中的纤维化明显受到抑制。抑制作用在A1组最明显,其次是A2组。图像分析提示术后第2天A1组移植细胞存留数明显大于A2组(P<0.05),术后第8周各组胶原纤维面积A1组最少,A2组其次,B1组较多,B2组最多(P<0.05)。结论成肌细胞移植对冻伤的肌肉组织有明显的修复作用,以0.1%透明质酸钠溶液为载体可提高成肌细胞移植效率。

Objective To investigate whether the implanted myoblasts with the soluble carriers can improve the repairing efficiency for the severely-cryodamaged tihialis anterior muscles. Methods The skeletal myoblasts were isolated from the newborn SD rats by the use of the enzyme digestion. They were purified and serially subcultivated ; the subcultivated myoblasts of the 3rd generation were marked with BrdU. The severely-cryodamaged tibialis anterior muscle models were established from 84 SD rats aged 5 months. They were randomly divided into 4 groups, including Group A1 （the implanted myoblasts with the carriers-F12 containing 0. 1% sodium hyaluronate）, Group A2 （the implanted myoblasts, with the carriers-F12 that did not contain 0.1% sodium hyaluronate）, Group B1 （the implanted carrier solution containing 0.1% sodium hyaluronate, but with no myoblasts）, and Group B2 （with no carrier solution or myoblasts）. Six rats were killed at the following time points： at 2, 5 and 9 days, and 2, 4, 8 and 12 weeks after operation; the immunohistochemical and the Mallory staining studies were performed for an evaluation on the repairing efficiency for the severely-cryodamaged tibialis anterior muscles. By the imaging analysis, the number of the survived cells in each group was compared at 2 days, and the area ratio of the collagen fiber in each group was also compared at 8 weeks. Results The BrdU immunohistochemical staining showed that the number of the remaining implanted cells was significantly greater in Groups A1 than in Group A2, the migrating area of the myoblasts was greater, the distribution of the cells was more uniform, the cell differentiating potential was undestroyed, the repairing efficiency for the severely-cryodamaged tibialis anterior muscles was significantly improved. There was no blue-stained nucleus at each time point in Group B. The Mallory staining showed that the fibrous degeneration in the tissue repairing process was significantly inhibited in Groups A1, A2 and B1 ; the inhibition was most obvious in Group A1, and next in Group A2. The imaging analysis indicated that at 2 days after operation, the number of the survived cells was significantly greater in Group A1 than in Group A2 （P〈0.05）. At 8 weeks after operation, the collagen fiber was the least in Group A1, less in Group A2, more in Group B1, and the most in Group B2 （P〈0. 05）. Conclusion The implanted myoblasts can significantly improve the repairing efficiency for the severely-cryodamaged muscle tissues, and the implanted carrier solution containing 0.1% sodium hyaluronate can improve the implanting efficiency for the myoblasts.