| Injury of peripheral nerve is very common. Many kinds of trauma (such ascompression, traction, tear and cut off) and other factors (such as ischemia and tumor)can cause partial or complete injury of nervous system, which will result in functionloss of nerve and other neurological diseases. With the development of technology, thetreatment of peripheral nerve injury has significantly been improved. However, themorpgologic and functional regeneration are still seldom achieved. Slow rates of nerveregeneration and functional recovery as well as the atrophy, degeneration anddysfunction of target organs are still the difficulty in clinics. Combined with theprinciple of peripheral nerve tissue engineering, induced pluripotent stem cells derivedneural crest stem cells (iPSCs-NCSCs) were used as the source of seed cells in thisstudy. Low intensity ultrasound stimulation (LIUS) was used to enhance the rate ofperipheral nerve regeneration and functional recovery. In this thesis, the effects of LIUSon the viability, proliferation and differentiation (into neurons and Schwann cells (SCs))of iPSCs-NCSCs cultured in induction media were emphatically investigated. The mainworks and results are as follows:①Identify multi-lineages differentiation potential of iPSCs-NCSCs and therealization of LIUS at cellular level.Multilineage differentiation potential of iPSCs-NCSCs was investigated withosteogenic, chondrogenic, adipogenic and neural induced medium. The results showedthat iPSCs-NCSCs had the ability to differentiate into osteoblast, chondrocytes, adipocyte,neurons and SCs with specific induced media.In this study, LIUS equipment (US10, purchased from Cosmogamma Corporation,Italy) was used to realize ultrasound stimulation with fixed frequency (1MHz), dutycycle (20%), pulse repetition rate (100Hz) and treatment time (10min). Then theeffects of LIUS on cell viability, proliferation and differentiation of iPSCs-NCSCs wereinvestigated by changing the ultrasound intensity.②Effects of LIUS on cell viability and proliferation of iPSCs-NCSCs.The viability and proliferation of iPSCs-NCSCs treated by LIUS were evaluated byMTS assay and Cell-Light EdU DNA cell proliferation kit. The MTS results showedthat LIUS with intensity of300and500mW/cm~2could significantly enhance the cellviability of iPSCs-NCSCs by1.12(P<0.05) and1.19(P<0.01) times at day2, respectively. But when the intensity was raised to1500mW/cm, the inhibiting effect ofLIUS on the cell viability was significant (P<0.05). However, the viability ofiPSCs-NCSCs was not affected by LIUS after4days’ treatment (P>0.05).The EdU results showed that500mW/cm~2LIUS treatment for2days couldsignificantly increase the average number of EdU~+cells under the field of view from20.1±1.8to30.7±7.4(P<0.05), but significantly reduced the average number to12.1±5.1(P<0.05) after stimulated with1500mW/cm~2LIUS compared with the controlgroup. Proliferation rate of iPSCs-NCSCs was significantly increased from19.24±2.11%to23.59±1.17%(P<0.05) by500mW/cm~2LIUS. The proliferation rate ofiPSCs-NCSCs has not been obviously changed after treated by1500mW/cm~2LIUS. Onthe other hand, the average number of EdU~+cells under the field of view was increasedfrom4.2±1.5to13.9±5.6(P<0.05) and the proliferation rate of cells was increased from3.12±0.90%to7.87±2.23%(P<0.01) by500mW/cm~2LIUS for4days. Nevertheless,there was no significant difference between the control group and the group stimulatedwith1500mW/cm~2LIUS (P>0.05).③Effects of LIUS on iPSCs-NCSCs differentiation into neurons and SCs.Effects of LIUS on the neuron-and SCs-related genes and proteins (Tuj1, NFM,S100β and GFAP) expression were investigated by real-time quantitative PCR andimmunofluorescent staining, respectively. The results of quantitative real-time RT-PCRshowed that mRNA expression of NFM was significantly enhanced by1.22(P<0.01)and1.41(P<0.001) times after treated by300and500mW/cm~2LIUS for4dayscompared to control group, respectively. Moreover, mRNA expression of S100β wassignificantly enhanced about2.05times (P<0.001) and1.79times (P<0.05) by the sameLIUS, respectively. The mRNA expression of Tuj1and GFAP was enhancedsignificantly about1.36times (P<0.05) and2.1times (P<0.05) after treated by500mW/cm~2LIUS for4days compared to control group. After treated by500mW/cm~2LIUS for7days, the mRNA expression of NFM, S100β and GFAP except Tuj1werealso enhanced.The results of immunocytochemistry (ICC) showed that500mW/cm~2LIUS couldsignificantly enhance the fluorescence intensity of markers of NFM, Tuj1, S100β andGFAP in iPSCs-NCSCs.In summary,500mW/cm~2LIUS could not only enhance cell viability andproliferation, but also facilitate the iPSCs-NCSCs differentiation into neurons and SCs.Results of this study have important theoretical and clinical significance in exploring efficient methods for peripheral nerve repair. |
PDF Full Text Request