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BACKGROUND: Hyperbaric oxygen therapy and inhibition of Nogo-A/NgR-RhoA/ROCK signaling pathways are conducive to the proliferation and differentiation of transplanted cells. OBJECTIVE: To explore the effects of hyperbaric oxygen plus rat bone marrow mesenchymal stem cells transplantation carrying miRNA silencing NgR gene on repair of rat spinal cord injury. METHODS: Rat bone marrow mesenchymal stem cells were transfected using constructed miRNA recombinant plasmid targeting NgR. The expression levels of NgR were detected by real-time quantitative PCR and western blot assay after transfection. The 15 of 80 Sprague-Dawley rats were randomly selected as sham group and the remaining 65 rats were used to construct models of spinal cord injury; and 60 successful models were randomly divided into spinal cord injury group, hyperbaric oxygen group, rat bone marrow mesenchymal stem cells group and rat bone marrow mesenchymal stem cells plus hyperbaric oxygen group. The rat bone marrow mesenchymal stem cells group and hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group were injected with 5 μL rat bone marrow mesenchymal stem cells suspension (5×106 cells) after 24 hours, respectively, and the spinal cord injury and hyperbaric oxygen groups were injected with PBS of the same volume. The hyperbaric oxygen and hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group received continuous treatment for 7 days. The hind limb motor function was evaluated by Basso Beattie Bresnahan score before and after the modeling, once a week, for 4 consecutive weeks. After 4 weeks of modeling, somatosensory evoked potential testing was performed to detect the recovery of neural conduction. The histopathological damage, apoptosis of cells and axon regeneration in spinal cord tissues was observed by hematoxylin-eosin staining, TUNEL staining and fluorescent gold retrograde tracing, respectively. After hyperbaric oxygen therapy, the mRNA relative expression levels of Nogo-A, NgR, RhoA, ROCK-1, Lingo-1 and p75NTR were detected by real-time quantitative PCR around the injured region. RESULTS AND CONCLUSION: (1) An obvious decrease was found in NgR expression levels after rat bone marrow mesenchymal stem cells transfection. (2) Basso Beattie Bresnahan score of 2 to 4 weeks after modeling: hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group > rat bone marrow mesenchymal stem cells group and hyperbaric oxygen group > spinal cord injury group (P < 0.05). (3) The latency of somatosensory evoked potential was shortened and the amplitude of somatosensory evoked potential was increased in hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group compared with other treatment groups (P < 0.05). There was no significant difference in latency and amplitude of somatosensory evoked potential between the rat bone marrow mesenchymal stem cells group and the hyperbaric oxygen group (P > 0.05). (4) Compared with the spinal cord injury group, the number of neurons increased; the number of axons increased; the regeneration effect of neurons was obvious and the apoptosis was reduced in the hyperbaric oxygen group and rat bone marrow mesenchymal stem cells group. The repair effect was better in the hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group than that in other two treatment groups. (5) The mRNA relative expression of Nogo-A/NgR/RhoA-ROCK signaling pathway was significantly inhibited in hyperbaric oxygen plus rat bone marrow mesenchymal stem cells group, rat bone marrow mesenchymal stem cells group and hyperbaric oxygen group compared with the spinal cord injury group, but there was no significant difference between hyperbaric oxygen group and rat bone marrow mesenchymal stem cells group (P > 0.05). (6) In summary, hyperbaric oxygen combined with NgR gene silencing rat bone marrow mesenchymal stem cells transplantation can inhibit the gene expression of Nogo-A/NgR/RhoA-ROCK signaling pathway, promote axon regeneration and improve the electrophysiological function and locomotor function of rats. [ABSTRACT FROM AUTHOR]

Objective To prepare the new NGR-targeted shRNA-loaded liposome, and investigate its suppressing effect on orthotopic C6 glioma in rats. Methods (1) Liposome(lipo), the NGR-targeted liposome (lipo-NGR) and shBirc5-loaded lipo-NGR were prepared successively, and all kinds of products were characterized accordingly; (2) The targeting property of shBirc5-lipo-NGR was researched by immunohistochemical method in orthotopic C6 glioma rats model; (3) The tumor growth in treated rats was monitored longitudinally with MRI T2, and the survival time of each rat was recorded simultaneously. Results (1) We successfully prepared shBirc5-lipo-NGR whose size and zeta potential were (186.2±74.07)nm and (10.9±3.76)mV; (2) The Birc5 protein was significantly decreased in shBirc5-lipo-NGR group, compared with shBirc5-lipo group, shControl-lipo-NGR group and control group; (3) The control group exhibited an average tumor volume and growth rate 4.7 times greater than that in the shBirc5-lipo-NGR group on the 12th, 17th and 22th day(P < 0.05). The median survival times of the shBirc5-lipo-NGR, shBirc5-lipo, shControl-lipo-NGR and control groups were 30, 24, 22, 21 days, respectively. Conclusion We successfully prepare the new NGR-targeted shRNA-loaded liposome, and demonstrate its targeting property, obvious inhibitory effect on tumor growth and extending the survival time of orthotopic C6 glioma-bearing rats.

BACKGROUND: Mild hypothermia can effectively ease secondary brain and spinal cord injuries, which has a definite protective effect on the central nervous system. Meanwhile, mild hypothermia is conducive to the proliferation, activation and growth of transplanted cells by improving the microenvironment of the injured spinal cord. OBJECTIVE: To investigate whether mild hypothermia intervention can regulate the proliferation and apoptosis of neural stem cells through the RhoA/ROCK pathway. METHODS: The neural stem cell injury model was prepared and randomly divided into two groups: normothermia group and mild hypothermia group. The mild hypothermia group was treated with mild hypothermia [(32.0±0.5) ℃] for 4 hours. Expression of RhoA, RHOCK, Nogo-A and NgR in neural stem cells was detected by RT-PCR and western blot assay. RhoA/RHOCK positive cells were observed by fluorescence microscope. The intracellular Ca2+ concentration in neurons was measured by laser scanning confocal microscopy. RESULTS AND CONCLUSION: The expression of RhoA, RHOCK, Nogo-A and NgR in neural stem cells at mRNA and protein levels was significantly lower in mild hypothermia group than in the normothermia group (P < 0.05). The intracellular Ca2+ concentration in the neural stem cells was lower in the mild hypothermia group than in the normothermia group (P < 0.05). The number of RhoA/RHOCK positive cells in the brain tissue of rats was significantly lower in the mild hypothermia group than in the in the mild hypothermia group (P < 0.05). To conclude, mild hypothermia regulates the proliferation and apoptosis of neural stem cells by inhibiting the RhoA/ROCK pathway. [ABSTRACT FROM AUTHOR]