ABSTRACT
BACKGROUND: Current research on the differentiation of mesenchymal stem cells into endothelial cells mainly focuses on the induction of cytokines or co-culture of two types of cells. Research has been not yet reported on heat shock-treated endothelial cells inducing the differentiation of mesenchymal stem cells into endothelial cells. OBJECTIVE: To investigate the ability of heat shock-treated endothelial cells to induce bone marrow mesenchymal stem cells to differentiate into vascular endothelial cells, and to explore the angiogenic capacity of induced bone marrow mesenchymal stem cells. METHODS: Human bone marrow mesenchymal stem cells were co-cultured with heat shock-treated human umbilical vein endothelial cells in a non-contact manner. The expression of CD31, CD144, VEGFR2 and vWF in the bone marrow mesenchymal stem cells was detected by flow cytometry at 14 days after co-culture. The immunofluorescence of the cells was further verified. In vivo angiogenesis test was detected using hematoxylin-eosin staining in nude mice undergoing subcutaneous transplantation of induced and non-induced bone marrow mesenchymal stem cells. Matrigel angiogenesis test was applied to observe the angiogenic ability of induced and non-induced bone marrow mesenchymal stem cells. RESULTS AND CONCLUSION: After coculture, bone marrow mesenchymal stem cells had paving-stone like structure. Flow cytometry and cellular immunofluorescence results showed an increase in the expression of VEGFR2, vWF, CD31 and CD144 after co-culture. The hematoxylin-eosin staining of the graft showed that the induced bone marrow mesenchymal stem cells arranged in a regular manner and had an angiogenic tendency. The Matrigel angiogenesis test showed that the angiogenic ability of bone marrow mesenchymal stem cells was increased after induction compared with the control group. These findings indicate that co-culture with heat shock-treated human umbilical vein endothelial cells can promote the differentiation of mesenchymal stem cells into endothelial cells, and the specific phenotype of endothelial cells is obviously transformed, which has a certain tendency of angiogenesis.
ABSTRACT
BACKGROUND: miRNA-136-5 p plays a crucial regulatory role in pathological changes, inflammatory response and regeneration after spinal cord injury. OBJECTIVE: To investigate the effect of miRNA-136-5 p on the expression of cytokines in serum and NF-κB protein in spinal cord in rats with spinal cord injury and to explore the molecular mechanism. METHODS: Thirty-six male Sprague-Dawley rats, of SPF grade were provided by Laboratory Animal Center of Guangxi Medical University. The lentiviral vector system was prepared and transfected into spinal cord injured rats. Thirty-six rat models of spinal cord injury were established by modified Allen's method. Basso Beattie Bresnahan scores were performed. Rats were randomly divided into normal control, modeling (LV-ctrl plus spinal cord injury), overexpression (spinal cord injury plus LV-miRNA-136-5 p), and inhibition (spinal cord injury plus LV-sponge) groups (n=9/group). Seven days before surgery and the day of surgery, the overexpression and inhibition groups were continuously injected with the lentivirus suspension into the injured area, and the normal control and modeling groups were injected with the same amount of normal saline. Three rats were sacrificed at 1, 3 and 7 days, and blood and spinal cord tissues were taken. The levels of interleukin-1β, interleukion-6 and interferon-α in rat serum were determined by ELISA. The expression of NF-κB protein was detected by western blot assay and double immunofluorescence. RESULTS AND CONCLUSION: (1) There was no significant difference in preoperative Basso Beattie Bresnahan scores (P＞ 0.05). In the modeling group, the rats showed prone walking, vary degrees of urinary retention, and spinal shock, with complete loss of function of both hind limbs and muscle strength of 0. (2) Compared with the normal control group, the levels of inflammatory factors in the other groups were increased significantly (P ＜ 0.05). The expression levels of inflammatory factors were highest in the overexpression group, followed by modeling group, and lowest in the inhibition group. (3) Results of western blot assay and double immunofluorescence showed that the expression level of NF-κB protein in the modeling, overexpression and inhibition groups was significantly higher than that in the normal control group (P ＜ 0.05), and the level was highest in the overexpression group. (4) In summary, miRNA-136-5 p can affect inflammatory factors and NF-κB in rats with acute spinal cord injury.