Study On Induce Nscs To Differentiate Into Oligodendrocytes By 1,25(oh)2d3 And Construct Long Segment Tissue Engineered Nerve

The first part: Study on induce NSCs to differentiate into oligodendrocytes by 1,25（OH）₂D₃Objective: Culture and purify neural stem cells（NSCs）of SD rats and induce NSCs to differentiate into a large number of oligodendrocytes in a short time.Explore the experimental method to obtain a high concentration and purity of oligodendrocytes.Observe and analysis the biological characteristics of oligodendrocytes.Methods: The bilateral cerebral cortex of neonatal SD rats（48 h）was cut after anesthesia,excluding the meninges,blood vessels and other tissues.The cerebral cortex was cut into minced,repeated pipetting after filtration with DMEM/F12 medium.The cell density was adjusted to 5×10⁵ cells/m L,added EGF（20 ug/m L）,b FGF（10 ug/m L）,B27（20 ul/m L）according to the proportion.NSCs cultured for third generations were used for cell identification.DMEM containing 10%FBS was used as the basic medium,and the neurospheres were centrifuged and cultured.The next day,the 1×10^-6 mol/L 1,25（OH）₂D₃,an inductor for NSCs,was added to the basic medium and cultured for 7 days.After a short time of 0.0125% trypsin digestion,cells suspension were collected and centrifuged.The supernatant were moved away and the cells were suspended again by DMFM containing 10% FBS for culture.Trypan blue was used to calculate total cells.The next day,the 1×10^-6 mol/L 1,25（OH）₂D₃ was added to the basic medium again and cultured for 7 days.The follow culture steps were same with the induction for the first time.The total cells were calculated by trypan blue staining.The 12 cover-slips without digestion and the other 12 cover-slips with 0.0125% trypsin digestion about 1 min were divided into two groups of GC and GFAP separately,detected by immunohistochemistry at the first and second induction time.In other words,it was identified into A,B,C,D four groups: group A for the first time by direct identification;group B for the first time after digestion identification;group C for the second time by direct identification;group D for the second time induced after digestion identification.The obtained oligodendrocytes were cryopreserved by conventional methods and observed the biological characteristics after resuscitation.Results: NSCs were suspension growth in the serum free culture medium.At the 7 culture days,dozens of NSCs begun to aggregate and they could form large neurospheres.The results of immunocytochemistry showed that there were a large number of Nestin positive cells on the surface of neurospheres,and the range of staining was similar to the cells morphology.After differentiation of 24 hours,the neurospheres were able to produce more projections,and the neurites between the adjacent neurospheres were formed mesh structures.After culture for 7 days,more NF-200 positive cells and some GFAP positive cells were formed,which the range of staining was basically the same with the cells.For the first induction time,1,25（OH）₂D₃ was added into the medium,and the neurospheres formed into more protrusions at the periphery.At the same time,it could also be seen that single cell adherent differentiation,generally grew 1² projections.After differentiation for 3 days,glial cells were adherent growth at the bottle,and some cells had 1² or 3⁴ projections.After differentiation for 7 days,the neurospheres were fully differentiated.And the bottom of the bottle was overspread with cells,which with 1² protrusions and other 3⁴ protrusions.The second induction time,1,25（OH）₂D₃ was added into the medium and cultured about 7 days.The cells were met with cells at the bottom of the bottle.There was mainly 1² protruding cells,and some 3⁴ protruding cells.Trypan blue staining to calculate the total differentiation cells showed that the first induction of differentiation cells were（1.51±0.08）×106,and the second time of the total cells were（1.34±0.12）×106.There were significant differences between the two groups（P<0.05）.Immunocytochemical staining showed that oligodendrocytes were 1² projections.The GC positive cells rate of group A was（64.90±3.38）% and GFAP positive cells rate was （32.30±2.90）%;GC positive cells rate of group B was（4.70±1.33）% and GFAP positive cells rate was（31.10±2.42）%.The GC positive cells rate of group C was（90.10±2.72）% and GFAP positive cells rate was（10.00±1.88）%.The GC positive cells rate of group D was（6.90±1.91）% and GFAP positive cells rate was（9.90±1.66）%.The differences between the group A and the group C were statistically significant（P<0.05）and no significant differences between the group A and the group B of GFAP positive cells（P>0.05）.Cryopreservation of different periods of oligodendrocytes,the survival rate was up to 50⁶0%,continued to culture cells well and cell morphology and growth conditions was closed to the pre frozen.Conclusions: 1,25（OH）₂D₃ two-induced method combined with conditioned medium can induce neural stem cells to differentiate into oligodendrocyte,further purified cells with a low concentration trypsin digestion method.So it can obtain high purity and high concentration of oligodendrocytes.The second part: Study on the construction of long segment tissue engineered nerve by oligodendrocyte derived from neural stem cells and acellular nerve graftsObjective: Through the modified chemical method to prepare acellular nerve grafts（ANGs）of human tibial nerve,which reserve space structure and low immunogenicity.Oligodendrocytes derived from NSCs of SD rats combine with ANGs from human tibial nerve to construct tissue engineered nerve,researching the biological characteristics of tissue engineered nerve,exploring the suitable conditions of ideal tissue engineered nerve construction.Methods: The ANGs of tibial nerve were prepared by a modified chemical method,and the 32 parts of the human tibial nerve were taken.The length of each segment was about 30 mm.（1）Removed the tibial nerve stem surfaces loose fibrous tissue until hard smooth epineurium.（2）The 0.05 mol/L Tris-Hcl（PH7.4）and protease inhibitor were added to the 50 ml centrifuge tube,vibrated at constant temperature（4℃）for 24 hours.（3）The liquid in the centrifugal tube was replaced with 3% Triton X-100 phosphate buffer,vibrated at constant temperature（4℃）for 24 hours.They were bathed in double distilled water for 12 hours and then washed for 3 times.（4）The original liquid in the centrifuge tube was replaced by 4% sodium deoxycholate,oscillated at a room temperature for 24 hours and then washed for 3 times by distilled water.（5）Repeat 3⁴ steps for 1 time.The prepared ANGs were saved in PBS liquid in 4℃.The prepared ANGs and human tibial nerve were examined by H-E staining,transmission electron microscopy and immunohistochemistry to evaluate the effect of ANGs.Tissue engineered nerves were randomly separated into A,B,C,D,E five groups,each group of 5 nerves,each segment length of about 30 mm.Group A: the injection cells density was 5×10⁵ cells/ml and the medium cells density was 5×10⁵ cells/ml;group B: the injection cells density was 1× 106 cells/ml and the medium cells density was 5×10⁵ cells/ml;group C: the injection cells density was 1×107 cells/ml and the medium cells density was 5×10⁵ cells/ml;group D: no injection cells and medium cells density was 5×10⁵ cells/ml;group E: the injection cells density was 1×106 cells/ml,no medium cells.Cells and ANGs were cultured together for 14 days.The adhesion of the cells in the scaffolds was examined at 7 days and 14 days respectively.At 7 days,the midpoint of each tissue engineered nerve was cut off,obtained the 15 mm tissue engineered nerve to immunohistochemistry.GC positive cells were counted by IPP 6.0 software.Each slice was randomly selected for ten visual fields,which the cells were counted respectively,and the average value of the GC positive cells was considered as the target number.The remaining 15 mm tissue engineered nerves continued to culture.At 14 days,the detection method was same with the 7 days’ method and the statistical analysis was through SSPS 19.0 software.Fifteen SD rats were divided into three groups randomly,subcutaneous embedding after skin disinfection and anesthesia.There was embedded in human tibial nerve,tissue engineered nerve and allograft nerve of SD rat respectively.After continued to feed for 7 days,HE staining for the embedded nerve was used to detect lymphocyte infiltration.Results: Longitudinal slice HE staining showed the ANGs have visible neat pipeline structure,without the cells component;the myelin sheath and cells disappeared in the cross section.Transmission electron microscopy showed that there were no myelin and cell structures,with an irregular circular cavity in the middle,which formed by the nerve fiber matrix and the basement membrane.Immunohistochemical staining showed that the laminin staining was positive and uniform distribution on the longitudinal section of ANGs,while the S-100 dyeing showed negative.At 7 days,immunohistochemical of tissue engineered nerve showed that there were a large number of GC positive cells on the longitudinal section and the cells distributed along the injection part to the distal end,and there were more GC positive cells in the injection site.The number of GC positive cells in groups B and C were more than that in groups A,D and E,and there were statistically differences between them（P<0.05）and no meaningful differences between groups B and C（P>0.05）.At 14 days,tissue engineered nerve showed a large number of GC positive cells,and the cells distribution was more uniform that the 7 days culture.The injected cells were migrated distribution to the distal part and longitudinal distribution of cells more uniform.The number of GC positive cells in groups B and C was more than that in groups A,D and E,and the difference was statistically significant（P<0.05）while no noteworthy differences between groups B and C（P>0.05）.The number of GC positive cells of 14 days in culture increased than the number of 7 days in culture,but no noteworthy differences between then（P>0.05）.Under the light microscope,the number of lymphocytes in the other two groups was less than that of the tibial nerve group.There were no significant differences between tissue engineered nerve and allograft nerve（P>0.05）.Conclusions: Oligodendrocyte derived from NSCs combined with ANGs to build the long segment tissue engineered nerve,which with three-dimensional fiber pipeline structure like normal nerve,low immunogenicity,good cell adhesion and biocompatibility.The tissue engineered nerve might be an effective substitute for nerve graft.