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Therapy of rat denervated gastrocnemius muscles damage using human cord blood mononuclear cells
- LIU Zilin, LI Dong, SHI Qing, LI Cong, HUANG Jinxian, ZHU Huasu, JU Xiuli
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Journal of Shandong University (Health Sciences). 2019, 57(2):
61-69.
doi:10.6040/j.issn.1671-7554.0.2018.1442
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Objective To explore the effect of human cord blood mononuclear cells(CB-MNCs)on relieving the gastrocnemius muscle atrophy and promoting the damage repair in the isolated sciatic nerve rats after intramuscular administration. Methods CB-MNCs were isolated from healthy term infants and cultured in vitro. Thirty juvenile Wistar rats weighting about 200 g were randomized into three groups: CB-MNCs intramuscular administration group(Therapy group,n=10), saline intramuscular administration group(NS group,n=10)and healthy group(Normal group,n=10). The animal model of denervated gastrocnemius muscles were formed by cutting both sides of the sciatic nerve of rats 山 东 大 学 学 报 (医 学 版)57卷2期 -刘子琳,等.人脐血单个核细胞对大鼠腓肠肌失神经损伤的治疗作用 \=-which resulted in the nerve disconnection about 1 cm in the first two groups. One million of CB-MNCs were injected into the gastrocnemius muscles of rats in d7, d14, and d21 after despairing the nerves of Therapy group. The rats in Normal and NS groups were only injected saline with equal volume at the same time. Hind limb movements and gangrenous area ratios of rats were observed. The both sides of gastrocnemius muscles of each rat were weighed to measure wet weight ratios and HE staining was taken to determine cross-sectional area of muscle fiber at the end of the forth week. Peripheral blood serum and bilateral gastrocnemius muscles were taken to measure creatine kinase(CK), catalase(CAT), malondialdehyde(MDA), and superoxide dismutase(SOD)levels. The mRNA expressions of Bax, B-cell lymphoma-2(Bcl-2), Caspase-3, vascular endothelial growth factor-α(VEGF-α), tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), α-actin and Dystrophin were tested by qRT-PCR. The expressions of α-actin, Dystrophin and VEGF-α protein were detected by Western blotting. The expression of Dystrophin in cells was also observed by immunohistochemistry. Results The gangrenous area ratio of the Therapy group was lower than that of the NS group(P=0.002)and the wet weight of the gastrocnemius muscle in the Therapy group was higher than that in the NS group(P<0.001). The HE staining results showed that the muscle cross-sectional areas of the Normal group, NS group and Therapy group were respective(12 452.0±202.8),(6 287.0±142.2), and(8 193.0±115.5)μm2 with a statistical difference(P<0.001). Compared with the NS group, the CK and MDA contents of the serum and muscles in the Therapy group were significantly decreased, and the SOD and CAT contents were significantly increased(all P<0.001). In contrast to the NS group, the mRNA levels of Caspase-3, Bax and TNF-α decreased while the mRNA levels of Bcl-2, IL-10, α-actin, Dystrophin and VEGF-α increased in the Therapy group(all P<0.05). Compared with the NS group, the expressions of α-actin, Dystrophin and VEGF-α protein in the Therapy group were increased(all P<0.05). IHC analysis showed that the expression of Dystrophin was increased in both NS and Therapy groups, but the expression of Dystrophin in the Therapy group was significantly higher than that in the NS group(all P<0.05). Conclusion Injecting CB-MNCs into the paralyzed gastrocnemius can reduce the proportion of apoptotic cells, promote the wound healing, prevent muscle atrophy, and improve muscle functions.
