ObjectiveTo evaluate the most efficient method for transfection of human umbilical cord mesenchymal stem cells (HUMCSs) in vivo. MethodsHUCMSCs were isolated from human umbilical cord and cultured, which were labelled by PKH26 and lentivirus-GFP, then were observed by using a fluorescence microscope. Sixty SD rats were randomly divided into PKH26 transfection group and lentivirus-GFP transfection group. The right hepatic lobe of rat was resected, then the transfected stem cells were injected into portal vein. The rats were sacrificed on day 3, 8, and 13 after transfection. The liver specimens were observed by using a fluorescence microscope. Flow cytometry was used to evaluate the percentage of transfected stem cells and the apoptotic stem cells. ResultsThe third generation of HUCMSCs labelled by PKH26 and lentivirus-GFP were spindle shaped. PKH26 red dye was evenly distributed in the cell membrane of HUCMSCs and could be clearly labelled. The HUCMSCs labelled by lentivirus-GFP were green fluorescence under the fluorescence microscope, and it was clear and stable. The HUCMSCs were clear and could be clearly distinguished on day 3 after transfection by two methods in vivo. As the time went by, red was faded and blurred, then was gradually disappeared on day 13 after transfection in the HUCMSCs stansfected by PKH26; but the color in the HUCMSCs stansfected by lentivirus-GFP were clear at all the time points. The transfection rate of the lentivirus-GFP was significantly higher that that of the PKH26 (P < 0.05), the rate of apoptotic stem cells had no significant differences at all the time points between these two groups (P > 0.05). ConclusionLentivirus-GFP transfection is a higher efficient method for stem cell labelling in vivo, it could be used to observe transplantation cells for a long time in future.
ObjectiveTo investigate the regularity of myelin degeneration and regeneration and the difference of axonal density between tibial nerve and common peroneal nerve after sciatic nerve injury repair in rhesue monkey. MethodsNine adult rhesue monkeys (male or female, weighing 3.5-4.5 kg) were selected to establish the model of rat sciatic nerve transaction injury. The tibial nerve and common peroneal nerve of 5 mm in length were harvested at 5 mm from injury site as controls in 3 monkeys; the distal tibial nerve and common peroneal nerve were repaired with 9-0 suture immediately in the other 6 monkeys. And the gross observation and neural electrophysiological examination were performed at 3 and 8 weeks after repair respectively. Then, distal tibial nerve and common peroneal nerve at anastomotic site were harvested to observe the myelin sheath changes, and to calculate the number of axon counts and axonal density by staining with Luxol Fast Blue. ResultsAtrophy of the lower limb muscle and various degrees of plantar ulcer were observed. Gross observation showed nerve enlargement at anastomosis site, the peripheral connective tissue hyperplasia, and obvious adhesion. The compound muscle action potential (CMAP) of tibial nerve and common peroneal nerve could not be detected at 3 weeks; the CMAP amplitude of common peroneal nerve was less than that of the tibial nerve at 8 weeks. Different degrees of axonal degeneration was shown in the tibial nerve and common peroneal nerve, especially in the common peroneal nerve. The average axonal density of common peroneal nerve was lower than that of tibial nerve at 3 weeks (13.2% vs. 44.5%) and at 8 weeks (10.3% vs. 35.3%) after repair. ConclusionThe regeneration of tibial nerve is better and faster than that of common peroneal nerve, and gastrocnemius muscle CMAP recovers quicker, and amplitude is higher, which is the reason of better recovery of tibial nerve.