Objective To obtain highly purified and large amount of Schwann cells (SCs) by improved primary culture method, to investigate the biocompatibility of small intestinal submucosa (SIS) and SCs, and to make SIS load nerve growth factor (NGF) through co-culture with SCs. Methods Sciatic nerves were isolated from 2-3 days old Sprague Dawley rats and digested with collagenase II and trypsin. SCs were purified by differential adhesion method for 20 minutes and treated with G418 for 48 hours. Then the fibroblasts were further removed by reducing fetal bovine serum to 2.5% in H-DMEM. MTT assay was used to test the proliferation of SCs and the growth curve of SCs was drawn. The purity of SCs was calculated by immunofluorescence staining for S-100. SIS and SCs at passage 3 were co-cultured in vitro. And then the adhesion, proliferation, and differentiation of SCs were investigated by optical microscope and scanning electron microscope (SEM). The NGF content by SCs was also evaluated at 1, 2, 3, 4, 5, and 7 days by ELISA. SCs were removed from SIS by repeated freeze thawing after 3, 5, 7, 10, 13, and 15 days of co-culture. The NGF content in modified SIS was tested by ELISA. Results The purity of SCs was more than 98%. MTT assay showed that the SCs entered the logarithmic growth phase on the 3rd day, and reached the plateau phase on the 7th day. SCs well adhered to the surface of SIS by HE staining and SEM; SCs were fusiform in shape with obvious prominence and the protein granules secreted on cellular surface were also observed. Furthermore, ELISA measurement revealed that, co-culture with SIS, SCs secreted NGF prosperously without significant difference when compared with the control group (P gt; 0.05). The NGF content increased with increasing time. The concentration of NGF released from SIS which were cultured with SCs for 10 days was (414.29 ± 20.87) pg/cm2, while in simple SIS was (4.92 ± 2.06) pg/cm2, showing significant difference (P lt; 0.05). Conclusion A large number of highly purified SCs can be obtained by digestion with collagenase II and trypsin in combination with 20-minute differential adhesion and selection by G418. SIS possesses good biocompatibility with SCs, providing the basis for further study in vivo to fabricate the artificial nerve conduit.
Objective To investigate the targeted combination and anti-inflammatory effects of anti-intercellular adhesion molecule 1 (ICAM-1) targeted perfluorooctylbromide (PFOB) particles on myocardial ischemia-reperfusion injury in rat model. Methods Seventy-six adult Sprague Dawley rats (male or female, weighing 250-300 g) were selected for experiment. The models of myocardial ischemia-reperfusion injury were established by ligating the left anterior descending coronary artery for 30 minutes in 30 rats. The expression of ICAM-1 protein was detected by immunohistochemistry staining at 6 hours after reperfusion, and the normal myocardium of 10 rats were harvested as control; then the content of interleukin 8 (IL-8) in serum was tested every 6 hours from 6 hours to 48 hours after reperfusion. The other 36 rats were randomly divided into 6 groups (n=6): ischemia-reperfusion injury model/targeted PFOB particles group (group A), ischemia-reperfusion injury model/untargeted PFOB group (group B), normal control/targeted PFOB particles group (group C), normal control/untargeted PFOB particles group (group D), ischemia-reperfusion injury model/normal saline group (group E), and sham operation group (group F). The ischemia-reperfusion injury models were established in groups A, B, and E; while a thread crossed under the coronary artery, which was not ligated after open-chest in group F. After 6 hours of reperfusion, 1 mL of corresponding PFOB particles was injected through juglar vein in groups A, B, C, and D, while 1 mL of nomal saline was injected in group E. Ultrasonography was performed in groups A, B, C, and D before and after injection. The targeted combination was tested by fluorescence microscope. The content of IL-8 was tested after 6 and 24 hours of reperfusion by liquid chip technology in groups A, B, E, and F. Results After 6 hours of reperfusion, the expression of ICAM-1 protein significantly increased in the anterior septum and left ventricular anterior wall of the rat model. The content of IL-8 rised markedly from 6 hours after reperfusion, and reached the peak at 24 hours. Ultrasonography observation showed no specific acoustic enhancement after injection of PFOB particles in groups A, B, C, and D. Targeted combination was observed in the anterior septum and left ventricular anterior wall in group A, but no targeted combination in groups B, C, and D. There was no significant difference in the content of IL-8 among groups A, B, and E after 6 hours of reperfusion (P gt; 0.05), but the content in groups A, B, and E was significantly higher than that in group F (P lt; 0.05). After 24 hours of reperfusion, no sigificant difference was found in the content of IL-8 between groups A and B (P gt; 0.05), but the content of IL-8 in groups A and B were significantly lower than that in group E (P lt; 0.05). Conclusion Anti-ICAM-1 targeted PFOB particles can target to bind and pretect injured myocardium of rat by its anti-inflammation effects.