Study On The Application Of Velvet Polypeptide In Repairing Peripheral Nerve Injury And The Epidemiological Investigation Of Peripheral Nerve Injury In Western Liaoning

Purpose:To explore the effects of Pilose Antler Polypeptides(PAP) promoting muscle derived stem cells to the class of schwann cell differentiation and its mechanism of action, Pilose Antler Polypeptides(PAP) and rat muscle derived stem cells were researched In this experiment; Muscle derived stem cells pretreated by PAP medicated serum were given microencapsulation, Combined with sodium hyaluronate of BMP and silicone tube to construct a new artificial nerve to evaluate effect of PAP and muscle derived stem cells repairing defected nerve by animal experiment.Material and method:PartⅠ: Muscle-derived stem cells of the rat were separated,cultivated and identificated. The skeletal muscle of rats were digested by enzyme. Muscle-derived stemcells were cultivated and by purified digestion of the enzyme and the improved pre-plating technique.Then, the growth conditions and morphological character of purified muscle-derived stem cells were observed through inverted microscope. And the cells were dentified with Desmin immunohistochemical staining. partⅡ: The experimental study on Pilose Antler Polypeptides(PAP) promoting Muscle-derived stem cells which were ransfected CNTF gene to differentiate into Schwann cell-likecell lineages. Ten Wistar rats were randomly divided into dose treatment groups and the control group of blank serum. Pilose Antler Polypeptides(PAP) were irrigated according to the formula.Blood was drawn from abdominal aortic under aseptic conditions and centrifuged, then the supernatant was taken, and then PAP medicated serum and blank serum were prepared; Muscle-derived stemcells were divided into Groups A, B, C, and D, and were pretreated by adding the corresponding cultures.Group A: PAP medicated serum; Group B: solution of PAP;Group C: blank serum; Group D: complete medium.After 21 days’ culture, the growing states and morphological changes of Muscle-derived stemcells were observed by inverted microscope and the proliferation of Muscle-derived stemcells were determined by MTT. The commonly used makers of Schwann cell were used in Immunohistochemistry and flow cytometry in order to evaluate the cells after induction. Part Ⅲ: Muscle-derived stem cells promote to repair the injured peripheral nerve after they were conducted by Pilose Antler Polypeptides(PAP). The muscle-derived stem cells in the PartⅠwere marked into N groups of cells,and the Schwann cell-like cell lineages in the PartⅡwere marked into M groups of cells. These transfected muscle-derived stem cells were microencapsulated. Twenty-four Wistar rabbits were taken for peripheral nerve defect mode. The rats were divided into four groups at random, with silicone tube used as nerve catheter, and sodium hyaluronate as the carrier. In the experimental group(Group A), micro capsule the cells of M groups were added, in comparison with another three groups(Group B, C and D). In group B, autologous nerves were grafted.In group D, micro capsule the cells of N groups were added.In the first, second, third, sixth, ninth and twelfth weeks after the operation, the ulcers of rats’ heels, gait and joint activities were observed in each group. In the fourth, eighth and twelfth weeks, SFI(sciatic nerve function index) was measured. In the twelfth week, the moving potential magnitude and conducting speed of sciatic nerves were measured, by calculating the number and diameter of reborn nerve fibers and using normal HE staining and S-100 immunity staining(SABC method) in order to observe nerve-regeneration condition and myelin sheath condition, as well as to observe reborn nerve axon and ultrastructure of smyelin sheath with transmission electron microscope.Results: 1、More muscle-derived stem cells could be cultivated in a short time by Improved traditional cultured method. Immunocytochemistry showed that the culturedmyoblasts were cytoplasmic strongly positive by anti-desmin and Sca-1 staining. 2 、 Pilose Antler Polypeptides(PAP) medicated serum and blank serum were successfully obtained in the experiment. Muscle-derived stem cells were pretreated by PAP medicated serum After 72 hours,under inverted phase contrast microscope: lots of the Cells in group A began to mix together and appear bipolar spindle shapes, a small amoubt of the cells appear triangle,and they connected by cellular synapse; the cells in group B appear taking off the wall and floating in the cell cultured plate, Cell shrinkage and a lot of debris were observed under the inverted phase contrast microscope. And they evolved into a long spindle, polygonal, star and other different shapes gradually;The cells in group C and group D appear long spindle and develop longly. MTT results showed that muscle-derived stem cells in different treatment groups all proliferated as time went by, reaching proliferation peak on the 4th or 5th day, and declining on the 6th day. Markers(S100, GFAP and P75) of chwann cells were expressed positively when the cells were cultivated by PAP drug-containing serum after 21 days by immunohistochemistry.The results proved that the induced cells were Schwann cell-like cell lineages. 3、In the ninth week, the reddish swelling and ulcer became better to different degrees in A, B and D groups, while the recovery of Group C was not obvious. In the twelfth week, the reddish swelling and ulcer almost disappeared in A, B and D groups, while it still existed in Group C. Also in the twelfth week, the regenerative nerves in Group A looked like normal ones; there were not scar tissues on the broken ends of transplanted nerves in Group B, but the nerves adhered slightly to surrounding tissues; in Group C, there were regenerative nerves in the tube, but they were very thin; so was Group D, but the regenerative nerves of this group were thicker than those of Group C. In the twelfth week, in regard to the SFI, the conducting velocity of nerves,the recovery rate of gastrocnemius wet weight, and the number and diameters of medullated nerve fibers, Group A was superior to Group C and D, so the differences made sense in statistics(P<0.05); while Group A was similar to Group B, so the differences made no sense in statistics(P>0.05). Observed in transmission electron microscope, the nerve fibers in Group A and B were thick and they arranged tight, myelin was thick, and the structure of axon was integrated. In comparison, Group D had fine regenerative nerves, irregular shape and thin myelin. While in Group C, regenerative nerves were the finest, nerve fibers had different sizes and arranged loosely, and the structure of axon was not integrated.Conclusions: 1.Mmuscle-derived stem cells could be isolated from the muscle conveniently through microsurgical technique and,hybrid enzyme and differential adherent culture method. 2. Pilose Antler Polypeptides(PAP) medicated serum could significantly improve the proliferation of muscle-derived stem cells,which showed that PAP had the potential to become the new drug to promote injuried nerve healing. 3.The combination of PAP and myoblasts can be applied to promote the repair of injuried nerve and be used in the researches on peripheral nerve tissue engineering.Purpose:In order to providing reference for the prevention of peripheral nerve injury, Analysis of epidemiological characteristics of jinzhou area in patients with peripheral nerve injury are used.Material and method:1410 patients with peripheral nerve injury were retrospectively analyzed in seven top-class hospitals in five cities in the west region of Liaoning province between January 2014 and December 2014. The patients were divided into five age groups: childhood(0 to 6 years of age);The young(7 to 17 years old);Youth(18 to 40 years of age);Middle-aged(41-65 years);Elderly(> 65 years of age).The statistics of injury time, age, culture level, occupation, injury locations, causes, injury parts and combined injury, whether surgery were included.The data was s analysed tatistically. Results:In one year time, 1410 patients with peripheral nerve injury were treated in ward. Male patients was 1105 and female patients was 305, the rate was 3.62:1; The incidence was high in the group of 18 to 40 years old in young adults, and it was 51.06%; There was no obvious difference in the group of primary school culture and school culture,;The injuried rate of work causes was 48.94%, the injuried rate of traffic causes was 28.72%.Different gender had different reasons, and women were mainly includeed in traffic injuries, and male patients mainly in work injuries;Conclusions:At the age of 16 to 40 years old, the risk of peripheral nerve injury was high in young adults who had not high level of culture;Cutting injury in work was the main injury factors;Weaked Consciousness and loosed Production management were the key of the prevention of peripheral nerve injury.