The Experimental Study On Effect Of MSC And Autologous PRP On Peripheral Nerve Regeneration

Part IThe approach to isolate and culture bone mesenchymal stem cells of rabbitObjective To explore the approach to isolate and culture mesenchymal stem cells (MSCs) in vitro and identify it.Methods MSCs were isolated from the bone marrow of the newborn rabbit by density gradient centrifugation. Light and electron microscopy were used to study the morphologic features, growth status. Immunocytochemistry technique was employed to examine the cell surface antigens. Observe the proliferation status of 1^st, 3^rd, 5^th , 8^th, 10^th generation and draw growth curve of cells.Results MSCs that we have obtained was very purified and the shape was whirlpool. The cells of 1^st, 3^rd, 5^th generation proliferated more rapidly than those of 8^th, 10^th generation. The cells that we have obtained express the CD44 and CD90 while CD34 are negative.Conclusion The cells that we have obtained were only composed of MSC, being of the property of stem cell. The cells of 1^st, 3^rd, 5^th generation is fit to the experiment of later stage. Part IIThe effect of platelet-rich plasma on proliferation of mesenchymal stem cellsObjective To explore the effect of platelet-rich plasma (PRP) on proliferation of MSCs and to be hope to find a ideal way to multiply the MSCs.Methods We have extracted PRP by the method of Appel. MSCs were cultured with different concentration of PRP(PRP-A, PRP-B, PRP-C and control group). Growth curve of MSCs was recorded and cytoactive was detected by methyl thiazolyl tetrazolium(MTT).Results Comparing to the control group, MSCs proliferation of PRP interfering groups(PRP-A, PRP-B, PRP-C group) were promoted. The effect of highest dose of PRP(PRP-A group) is most outstanding.Conclusion It was demonstrated that PRP could promote MSCs to proliferate and the effect is to be dose- dependence.Part IIIMesenchymal Stem Cells differentiate into Schwann cells in vitro studyObjective To explore the possibilities of Mesenchymal Stem Cells(MSCs) differentiating to Schwann Cells and examine secretion function of the cells.Methods β -ME and RA were added to induce the 3rd generation MSCs and the cell were cultured by PRP. Schwann cells marker S-100 was used to discriminate the MSC properties by fluorescent staining. And measure NGF contents of culture fluid at 4^th, 7^th, 9^th and 11^th by ELISA. The levels of NGF mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR). Results MSCs changed morphologically into cells resembling Schwann cells after induction in vitro and express S-100. The NGF contents (pg/ml) of induction group were 254 ± 25,372 ± 31,485 ± 29, 534±32 at 4^th, 7^th, 9^th and 11^th day, obviously exceed to the content of simple induction group and control group (P<0. 01) . RT-PCR manifest that the NGF mRNA expression union induction group is brightest and simple induction group is brighter than control group.Conclusion These results suggested that MSCs may differentiate into schwann cells in vitro and excrete NGF. The PRP could raise efficiency of induction.Part IVThe experimental study on effect of MSCs and autologous PRP on peripheral nerve regenerationObjective To observe the effect of mesenchymal stem cell(MSC) and platelet-rich plasma(PRP) on regeneration of peripheral nerve.Methods Thirty-two rabbits were divided randomly into 4 group, 8 in each. NGF group, PRP group, MSC group and union group. Epineurorrhaphy was done(NGF group and PRP group) or sleeve joint by silicone conduit(MSC group and union group) after transaction of the left sciatic nerve. The silicone conduit containing MSC(MSC group and union group) and/or injection autologous PRP(PRP group), and compare with NGF the group(injection NGF). The examination index include morphological examination, wet muscle weight and motor nerve conduction velocity(MNCV).Results All index in union group were superior to those in the NGF group, PRP group and MSC group. All index in MSC group and MSC group were superior to those in NGF group.Conclusion It was demonstrated that both PRP and MSC could enhance strongly the peripheral nerve regeneration and the effect was superior to NGF. MSC united PRP the effect is more excellent.