The Effect Of OECs And SCs On The Survival, Proliferation, And Differentition Of NSCs, BMSCs, And HSCs, As Well As The Expression Of Neutrophic Factors In Vitro

Objective:To investigate the effect of olfactory ensheathing cells (OECs) and schwann cells (SCs) derived from Sprague dawley rats on the vitality. proliferation. differentiation of neural stem cells (NSCs), bone marrow stromal cells (BMSCs) and homopoietic stem cells (HSCs), as well as the expression of neurotrophic factors. All these provide the theoretical and practical foundation of the cell-base therapy for the Central Nervous System diseases by the combination of supporting cells and stem cells.Methods:In this study, nine groups were divided as following:single NSCs culture group. NSCs/OECs co-culture group. NSCs/SCs co-culture group. single BMSCs culture group. BMSCs/OECs co-culture group. BMSCs/SCs co-culture group, single HSCs culture group. HSCs/OECs co-culture group and HSCs/SCs co-culture group. Sample harvesting, purification and subculturing of NSCs. BMSCs. HSCs. OECs and SCs were performed respectively. When above cells were cultured for the 3rd passage. immunohistochemistry was used to identify them by using specific markers, i.e. nestin. CD44. CD34. P75and S100. Subsequently, the identified OECs. SCs of the 3rd passage co-culture with NSCs, BMSCs and HSCs respectively in the Millipore embeddability culture plate for seven days. In the single cellular culture group, the Millipore embeddability culture plate was employed to continuously culture for seven days. After the period of culture finished,the active cells were observed and taken photos. The number and size of cultured cells and neurosphere was counted. MTT test was utilized to test the vitality of the stem cells in different groups. Then, the stem cells of each group were extracted and immunohistochemistry of anti-β-TubulinⅢ, NF, Neun. Gale and GFAP. The extraction of stem cells in each group was used for RT-PCR process to test the expression of neurotrophic factors and their receptors, also. Image-Pro Plus and Image J software was utilized to count the cells in different experimental groups and measure the gray scales. Finally. SPSS 13.0 software was used to deal with the data in our study.Results:1. Celluar culture and identification in vitro: immunohistochemistry showed that cultured NSCs, BMSCs, HSCs,OECs and SCs exhibit immunostaining of nestin, CD44. CD34. P75 and S100. respectively.2. Results of cellular counting:2.1 The number of BMSCs co-cultured with OECs (114±23.4) and BMSCs co-cultured with SCs (134.2±14.0) was significantly more than that of single culture group (61.4±15.0); and the number of BMSCs co-cultured with SCs was significantly more than that of BMSCs co-cultured OECs. also. There was significant difference between above two groups (p<0.05).2.2 The number of HSCs co-cultured with OECs (109.0±17.8) and HSCs co-cultured with SCs (180.5±24.5) was significantly more than that of single culture group (95.4±18.9); and the number of HSCs co-cultured with SCs was more than that of HSCs co-cultured with OECs. also. And there was significant difference between above two groups (p<0.05).3. Comparison neurosphere counting and their diameter:3.1 The number of neurosphere in NSCs co-cultured with OECs group (68.9±15.8) and NSCs co-cultured with SCs (64.7±10.7) was significantly more than that of single NSCs culture group (50.3±9.8). respectively. There was significant difference between above two groups (p<0.05). While there was no significant difference between NSCs co-cultured with OECs group and NSCs co-cultured with SCs group.3.2 The diameter of neurosphere in NSCs co-cultured with OECs group (154.1±69.3 Pixels) and NSCs co-cultured with SCs group (188.3±73.4 Pixels) was significantly bigger than that of single NSCs cultured group (109.8±50.5 Pixels); and the diameter of neurosphere in NSCs co-cultured with SCs group was significantly bigger than that of NSCs co-cultured with OECs group. also. There was significant difference between above two groups (p<0.05).4. Results of MTT test:4.1 The scale of MTT test in BMSCs co-cultured with OECs group (0.149±0.006) and BMSCs co-cultured with SC (0.188±0.007) were significantly higher than that of single BMSCs group (0.117±0.007):and BMSCs co-cultured with SCs group was significantly higher than that of BMSCs co-cultured with OECs. also. There was significant difference between the above groups (p<0.05).4.2 The scales of MTT test in HSCs co-cultured with OECs group (0.197±0.029) and HSCs co-cultured with SCs group (0.339±0.089) were significantly higher than that of the single HSCs cultured group (0.167±0.016):and HSCs co-cultured with SCs group was significantly higher than that of HSCs co-cultured with OECs. also. There was significant difference between above two groups (p<0.05).4.3 The scales of MTT test in NSCs co-cultured with OECs group (0.249±0.018) and NSCs co-cultured with SCs group (0.197±0.020) were significantly higher than that of single NSCs group (0.160±0.012). and NSCs co-cultured with OECs was significantly higher than that of NSCs co-cultured with SCs group, also. There were significant difference between above two groups (p<0.05).5. The differentiated index of immunohistochemistry:5.1 Immunohistochemical result of different groups involving BMSCs:The immunohistochemistry of differentiated index ofβ-TubulinⅢ. NF. Neun. Galc and GFAP showed that in the BMSCs co-cultured with SCs group, the percentage of immunopositive cells ofβ-TubulinⅢ(10.1±3.3%), Neun (18.3±8.9%) and NF (7.6±5.3%) were significantly higher than those of BMSCs co-cultured with OECs group (7.5±2.4%.12.3±4.3%,2.4±1.3%) and the single BMSCs culture group (6.9±3.7%.12.6±5.1%,0.0±0.0%) respectively, there were significant difference between above groups (p<0.05). In the BMSCs co-cultured with OECs group, the percentage of immunopositive cells of NF was significantly higher than this of the single BMSCs cultured group, there were significant difference between above groups (p<0.05); while as for the percentage of immunopositve neurons ofβ-TubulinⅢand Neun, there was no significant difference between above two groups (p>0.05). The immunostaining of Galc in the single BMSCs culture group, BMSCs co-cultured with OECs group and BMSCs co-cultured with SCs group all showed immunopositive. And the measurement of gray scales in three groups showed that the gray scales of the BMSCs co-cultured with SCs group (0.29±0.04) and BMSCs co-cultured with OECs (0.28±0.04) were significantly higher than that of the single BMSCs cultured group (0.26±0.04). there were significant difference between above groups (P<0.05). while between the BMSCs co-cultured with SCs group and BMSCs co-cultured OECs group, there was no significant difference (P>0.05). The immunostaining of GFAP in the single BMSCs culture group. BMSCs co-cultured with SCs group and BMSCs co-cultured with SCs group all showed immunonegative.5.2 Result of immunostaining in the groups involving HSCs:The positive percentage ofβ-Tubulin±Ⅲ(94.0±2.2%). Neun (92.2±3.3%). NF (95.0±1.6%), Gale (96.4±1.4%) and GFAP (91.9±2.6%) in the HSCs co-cultured with SCs group andβ-TubulinⅢ(94.5±1.7%). Neun (94.6±2.2%), NF (94.8±2.2%). Galc (96.3±1.8%) and GFAP (94.8±2.1%) in the HSCs co-cultured OECs group were significantly higher than those of the single HSCs cultured group (43.9±8.0%. 49.8±9.3%.50.5±7.3%.58.7±4.2%.39.1±5.2%). There were significant difference among above groups (p<0.05). while there was no significant difference between the HSCs co-cultured SCs group and HSCs co-cultured with SCs group (P>0.05).5.3 Result of immunostaining in the groups involving NSCs:The immunopositive percentage of Neun (46.7±8.8%) and NF (44.5±11.9%) in NSCs co-cultured with SCs group and Neun (31.5±7.5%). NF (35.2±5.5%) in NSCs co-cultured with OECs group were significantly higher than those of the single NSCs group (24.6±6.0%,28.3±7.8%). There were significant higher among above groups (p< 0.05), while between the NSCs co-cultured with OECs group and NSCs co-cultured with SCs, there was also significant difference (p<0.05). The immunopositve percentage of (3-TubulinIII (85.1±7.1%) in the NSCs co-cultured OECs group was significantly higher than that of the single NSCs group (75.6±13.5 %). there was significant difference between above two groups. And in the NSCs co-cultured with SCs group, the immunopositive percentage ofβ-TubulinIII (40.7±7.8 %) was significantly less than that of the single NSCs group, there was significant difference between above two groups (p<0.05). and there was significant difference between the NSCs co-cultured with OECs groups and NSCs co-cultured with SCs group (p<0.05), also. The immunopositive percentage of Galc (22.9±9.9%) and GFAP (15.7±10.7%) in the NSCs co-cultured with SCs group and Gale (21.7±7.8%). GFAP (18.6±7.2%) in the NSCs co-cultured with OECs group were significantly lower than those of the single NSCs group (43.8±16.5%.33.4±15.8%). there were significant difference between above groups. While there were no significant difference between the NSCs co-cultured with OECs group and NSCs co-cultured SCs group (P>0.05).6. Result of RT-PCR:6.1 The expression of neurotrophic factors and their receptors involving BMSCs:The expression of BDNF, CNTF. IGF-1, NGF. PDGF. TGF-β1. TGF-β2. VEGF and the receptor-TrkC in BMSCs co-cultured with SCs group and BMSCs co-cultured with OECs group had no significant difference between the single BMSCs cultured group (p>0.05). while the expression of TrkB (0.71±0.19) in the BMSCs co-cultured with OECs group was significantly higher than that of the single BMSCs cultured group (control group) (0.43±0.21). and there was significant difference between above two groups (p=0.039,<0.05). The expression of TrkA (0.14±0.13) in the BMSCs co-cultured with SCs group was significantly less than that of the single BMSCs group (0.37±0.20), there was significant difference between above two groups (p=0.034.<0.05). 6.2 The expression of neurotrophic factors and their receptors involving NSCs:There were no significant difference in the expression of CNTF, IGF-1, TGF-β1, TGF-β2 and TrkA among the NSCs co-cultured with OECs group. NSCs co-cultured with SCs group and single NSCs cultured group (p>0.05); While the expression of BDNF (0.09±0.07,0.22±0.13). NGF (0.24±0.12,0.61±0.31). PDGF (0.07±0.08. 0.13±0.06), TrkB (0.32±0.13.0.5±0.11) in NSCs co-cultured with OECs group and NSCs co-cultured with SCs group respectively were significant lower than those of the single NSCs cultured group (0.69±0.46.1.46±0.37.0.44±0.16,1.05±0.44), there were significant difference among above groups (p<0.05). As for VEGF. its expression in the NSCs co-cultured with OECs group (0.16±0.10) was also significantly less than that of the single NSCs cultured group (0.26±0.05), and there was significant difference between above two groups (p<0.05); While as for TrkC, its expression in the NSCs co-cultured SCs group was significantly higher than that of the single NSCs cultured group, and there was significant difference between above two groups (p=0.017,<0.05).6.3 The expression of neurotrophic factors and their receptors involving HSCs:There was no significant difference in the expression of TrkA among the HSCs co-cultured with OECs group, HSCs co-cultured with SCs group and single HSCs cultured group (p>0.05). As for the BDNF, CNTF, PDGF, TGF-β1, TGF-β2, VEGF and TrkB. TrkC, their expression in HSCs co-cultured with SCs group (0.93±0.15, 0.61±0.28.0.64±0.50,1.53±0.25.1.01±0.45.0.19±0.15.1.26±0.33,0.81±0.49) were significantly higher than those of the single HSCs cultured group (0.30±0.23. 0.09±0.09.0.16±0.15.0.95±0.11.0.26±0.23.0.06±0.04,0.58±0.60,0.26±0.10), and there were significant difference between above two groups (p<0.05). And the expression of IGF-1 in the HSCs co-cultured with OECs group (1.30±0.15) was significantly higher than that of the single HSCs cultured group (0.98+0.10), there was significant difference between above two groups (p=0.012,<0.05). Finally, the expression of NGF in the HSCs co-cultured with OECs group (0.72±0.13) was significantly lower than that of the single HSCs cutured group (1.23±0.19). there was significant difference between above two groups (p=0.01,<0.05).Conclusions:1. OECs and SCs markedly promote the proliferation and survival of BMSCs,HSCs and NSCs, and the effect of SCs on BMSCs and HSCs was more obvious than that of OECs; while the influence of SCs and OECs on NSCs showed many variation.The effect of SCs on the size of neurosphere of NSCs more obvious than that of OECs. The effect of SCs on the proliferation and vitality of NSCs exhibited weaker than that of OECs.2. OECs and SCs obviously promote the differentiation of BMSCs, HSCs and NSCs. SCs and OECs significantly improve the neuronal differentiation from BMSCs, with few differentiation into astocytes. SCs and OECs significantly promoted the differentiation of HSCs into neurons, astrocytes and oligodendrocytes, and there were few difference of tendency of corresponding in corresponding differentiation. SCs and OECs significantly promoted the neuronal differentiation from NSCs, while markedly inhibited the differentiation of astrocytes and oligodendrocytes from NSCs. The effect of SCs on the neuronal differentiation from NSCs was more obvious than tha of OECs, but as for the effect of SCs and OECs on the differentiation of NSCs into astrocytes and oligodendrocytes, there was no obvious difference between above two groups.3. SCs inhibited the expression of TrkA by BMSCs; OECs promoted the expression of TrkB by BMSCs. OECs and SCs all inhibited the expression of BDNF, NGF, PDGF and TrkB by NSCs, and OECs also inhibited the expression of VEGF by NSCs; while SCs promoted the increasing expression of TrkC by NSCs. SCs improved the expression of BDNF, CNTF, PDGF, TGF-β1, TGF-β2, VEGF, TrkB and TrkC by HSCs. While OECs only promoted the increasing expression of IGF-lby HSCs, and inhibited the expression of NGF by HSCs.