BMSC与OEC联合移植对SCT大鼠神经元存活、胶质瘢痕以及神经再生的影响
发布时间：2018-08-09 11:45
【摘要】：[目的]建立大鼠骨髓间充质干细胞(BMSCs)、嗅鞘细胞(OECs)的体外分离、纯化和培养方法,及大鼠全横断脊髓损伤模型,探讨BMSCs、OECs单独移植及BMSCs+OECs联合移植对全横断损伤大鼠脊髓Ⅷ、Ⅸ板层神经元存活的影响、及胶质瘢痕中GFAP和APC的表达和神经纤维再生的变化,为细胞移植治疗脊髓损伤提供理论和实验室依据。 [方法]体外分离培养大鼠BMSCs和OECs并进行细胞染色鉴定；清洁级健康SD雄性大鼠25只,每组5只将动物随机分为假手术组(SG组)、手术对照组(CG组)、BMSC单独移植组、OEC单独移植组和BMSCs+OECs组。体外条件下将骨髓源性间充质干细胞(Dil标记)和OECs (Hoechst33342标记)立体定向移植入慢性期(全横断术后14天)全横断损伤大鼠脊髓的瘢痕处、头侧和尾侧。在脊髓损伤后第6m末灌注固定后取瘢痕处及头尾侧脊髓制作石蜡切片,行苏木素染色及免疫组化染色,分别计数各组脊髓Ⅷ、Ⅸ板层神经元数量及面积；计数各组瘢痕中GFAP和APC阳性细胞数量并分析其免疫染色OD值,同时观察宿主PKCγ、5-HT及CGRP阳性纤维再生情况。SPSS11.5软件包对数据进行统计学处理。 [结果] 1,细胞培养结果：培养的细胞分别经CD44和p75免疫染色,结果呈阳性反应,鉴定为BMSCs和OECs; 2,细胞移植对宿主神经元存活的影响：手术对照组Ⅷ、Ⅸ板层神经元数量和面积均少于假手术组(p0.05)；①比较各实验组VIII板层神经元数量结果显示：BMSC组与手术对照组无显著差异(p0.05), OEC组多于手术对照组(p0.05), BMSC+OEC联合移植组多于BMSC组或OEC组(p0.05)；②比较各组Ⅷ板层神经元面积结果显示：BMSC组与手术对照组无明显变化(p0.05), OEC组大于手术对照组(p0.05),BMSC+OEC联合组大于BMSC组、OEC组(p0.05)；③比较各组Ⅸ板层神经元数量结果显示：BMSC组或OEC组与手术对照组无明显差异(p0.05),BMSC+OEC联合组多与BMSC组(p0.05),BMSC+OEC联合组与OEC组无明显差异(p0.05)；④比较各组Ⅸ板层神经元面积结果显示：BMSC组与手术对照组无明显差异(p0.05), OEC组大于手术对照组(p0.05),BMSC+OEC联合组大于BMSC组或OEC组(p0.05)。 3,细胞移植对宿主瘢痕组织中星形胶质细胞和少突胶质细胞的影响： 3.1,细胞移植对宿主瘢痕中星形胶质细胞形成影响：脊髓全横断损伤后,宿主瘢痕组织中GFAP阳性细胞数目或免疫染色OD值均明显多于假手术组(p0.05)。①比较各细胞移植组GFAP阳性细胞数目结果显示：BMSC组或OEC组均少于手术对照组(p0.05),在两组的瘢痕头段皮质脊髓束(corticol spinal tract, CST)部位与手术组比较无差异(p0.05),两组的瘢痕头段灰质部位和尾段后角部位均少于手术组(p0.05)；BMSC+OEC联合组少于BMSC组或OEC组(p0.05),其中联合组头段CST部位多于BMSC组(p0.05),与OEC组比较无明显差异(p0.05),联合组头段灰质部位少于BMSC组或OEC组(p0.05),联合组尾段后角部位少于BMSC或OEC组(p0.05)。②比较各细胞移植组GFAP免疫染色OD值结果显示：BMSC组或OEC组均弱于手术对照组(p0.05),其中BMSC组瘢痕头段CST部位和瘢痕尾段后角部位均弱于手术组(p0.05),瘢痕头段灰质部位与CG组无明显差异(p0.05), OEC组瘢痕头段CST部位与CG组无明显差异(p0.05),瘢痕头段灰质部位和瘢痕尾段后角部位均弱于手术组(p0.05),BMSC+OEC联合组弱于BMSC或OEC组(p0.05),其中瘢痕头段灰质部位和瘢痕尾段后角部位均弱于BMSC或OEC组(p0.05),瘢痕头段CST部位与BMSC组或OEC组比较均无显著差异(p0.05)。 3.2,细胞移植对宿主瘢痕中少突胶质细胞的影响：脊髓全横断损伤后,宿主瘢痕组织中APC阳性细胞数目或免疫染色OD值均明显少于假手术组(p0.05)。①比较各细胞移植组APC阳性细胞数目结果显示：BMSC组或OEC组均多于手术组(p0.05),其中BMSC组瘢痕头段CST部位多于手术组(p0.05),OEC组瘢痕头段CST部位与手术组无明显差异(p0.05)BMSC或OEC组的瘢痕头段灰质和瘢痕尾段后角部位均多于手术组(p0.05), BMSC+OEC联合组多于BMSC组(p0.05),其中瘢痕头段CST部位与BMSC组无明显差异(p0.05),瘢痕头段灰质部位和瘢痕尾段后角部位均多于BMSC组(p0.05)；BMSC+OEC联合组与OEC组无显著差异(p0.05),但在瘢痕头段CST和灰质部位、瘢痕尾段后角部位均多于OEC组(p0.05)。②比较各细胞移植组APC免疫染色OD值结果显示：BMSC组或OEC组均多于手术组(p0.05),其中两组的瘢痕头段CST部位与手术组比较均无明显差异(p0.05),两组的瘢痕头段灰质和瘢痕尾段后角部位均多于手术组(p0.05), BMSC+OEC联合组强于BMSC组(p0.05),其中瘢痕头段CST和灰质部位强于BMSC组(p0.05),瘢痕尾段后角部位无明显差异(p0.05), BMSC+OEC联合组强于OEC组,其中瘢痕头段CST和灰质部位、瘢痕尾段后角部位均强于OEC组(p0.05)。 4：细胞移植对各组脊髓神经再生的影响：①PKCγ阳性纤维的再生：全横断损伤后各组PKCγ阳性纤维均未发现有轴突再生,瘢痕组织中未发现有PKCγ阳性染色。②5-HT阳性纤维再生：各组脊髓5-HT阳性纤维均进入瘢痕组织,其中手术组进入长度532.60+145.74μm(距离瘢痕头段距离)；BMSC组进入长度1698.00±880.81μm; OEC组进入长度(?)659.80±271.54μm, BMSC+OEC联合移植组进入长度1492.00+337.52μm,比较发现BMSC组和BMSC+OEC联合移植组的5-HT阳性纤维再生长度大于手术组(p0.05),联合移植组移植效果优于OEC组(p0.05),与BMSC组相比无明显差异(p0.05)。③CGRP阳性上行纤维的再生：手术组未进入瘢痕,距瘢痕尾段962.40±441.14μm; BMSC组也未进入瘢痕,距离尾段313.60±109.79μm; OEC组长入瘢痕,进入长度为198.00±85.26μm; BMSC与OEC联合移植组的CGRP上行纤维长入瘢痕,进入长度为531.80+162.62μm,各细胞移植组的CGRP上行纤维的再生长度均大于CG组(p0.05), BMSC与OEC联合移植组CGRP阳性纤维再生长度大于BMSC或OEC单独移植组(p0.05)。 [结论]：1、全横断脊髓损伤导致宿主脊髓Ⅷ、Ⅸ板层神经元死亡,数量减少,残存神经元胞体萎缩,面积减小。 2、BMSC单独移植组未能促进宿主脊髓Ⅷ、Ⅸ板层神经元的存活,也未能抑制宿主神经元的变性萎缩；OEC单独移植可提高宿主脊髓Ⅷ板层神经元的存活,不能促进IX板层神经元存活,OEC单独移植可抑制宿主脊髓Ⅷ、Ⅸ板层神经元的萎缩；BMSC+OEC联合移植组比BMSC组更能促进宿主VIII、IX板层神经元的存活,联合移植组比OEC组更能促进VIII板层神经元存活,在促进IX板层神经元存活方面与OEC无明显差异,在抑制宿主神经元萎缩方面联合移植组比BMSC或OEC单独移植效果更佳。 3：全横断脊髓损伤后脊髓组织中胶质瘢痕形成,GFAP阳性反应增强,脊髓神经纤维轴突出现脱髓鞘病变,APC阳性反应减弱。BMSC或OEC细胞移植均可改善瘢痕微环境,抑制胶质瘢痕的形成并可促进少突胶质细胞的存活,BMSC与OEC联合移植作用效果更佳。 4：脊髓全横断损伤后,PKCγ阳性纤维和CGRP阳性上行纤维不能再生进入瘢痕组织,5-HT阳性纤维可再生进入瘢痕组织一定长度；BMSC与OEC单独或联合移植不能促进PKCγ阳性纤维的再生；BMSC移植或BMSC+OEC联合移植均可促进5-HT阳性纤维再生,OEC移植不能促进5-HT阳性纤维再生；BMSC和OEC单独或联合细胞移植均可促进CGRP阳性上行纤维的神经再生,其中OEC移植或BMSC+OEC联合移植可促进CGRP上行阳性纤维进入瘢痕组织,联合细胞移植的促再生作用更佳。
[Abstract]:[Objective] to establish the isolation, purification and culture of rat bone marrow mesenchymal stem cells (BMSCs), olfactory ensheathing cells (OECs) in vitro, and the model of rat total transection spinal cord injury. The effects of BMSCs, OECs alone and BMSCs+OECs on the survival of the spinal cord VIII, IX lamellar neurons in the rat spinal cord and the GFAP and APC in the glial scar were investigated. Expression and regeneration of nerve fibers provide theoretical and laboratory evidence for cell transplantation in the treatment of spinal cord injury.
[Methods] BMSCs and OECs were isolated and cultured in vitro. 25 healthy SD male rats in clean grade were randomly divided into sham operation group (group SG), operation control group (CG group), BMSC alone transplantation group, OEC alone transplantation group and BMSCs+OECs group. In vitro, bone marrow derived mesenchymal stem cells (Dil markers) were used in vitro. And OECs (Hoechst33342) stereotactic transplantation into the chronic phase (14 days after total transection) the scars of the spinal cord, the cephaloside and the caudal side of the spinal cord of the rats with all transverse transection. After the spinal cord injury, the paraffin section was made from the cicatricial and the tail and side spinal cord after the spinal cord injury, and the hematoxylin staining and immunohistochemical staining were performed, and the Group VIII and IX were counted respectively. The number and area of the lamellar neurons, count the number of GFAP and APC positive cells in the cicatricial and analyze their immune OD values, and observe the.SPSS11.5 software package of PKC gamma, 5-HT and CGRP positive fibers in the host. The data are statistically processed.
[results]
1, cell culture results: the cultured cells were immunized by CD44 and p75 respectively, and the results were positive. They were identified as BMSCs and OECs.
2, the effect of cell transplantation on the survival of the host neurons: the operation control group VIII, the number and area of the layer IX lamellar neurons were less than that of the sham operation group (P0.05). (1) the number of VIII lamellar neurons in the experimental group showed that there was no significant difference between the BMSC group and the operation control group (P0.05), and the OEC group was more than the operation control group (P0.05), and the BMSC+OEC combined transplantation Group more than group BMSC or group OEC (P0.05); (2) compared group VIII lamellar neuron area results showed that there was no significant change in group BMSC and operation control group (P0.05), OEC group was larger than operation control group (P0.05), BMSC+OEC combined group was larger than BMSC group, OEC group (P0.05); thirdly, the number of neurons in each group IX lamellar neurons showed: BMSC group or group and operation There was no significant difference in the control group (P0.05). There was no significant difference between the BMSC+OEC group and the BMSC group (P0.05), and there was no significant difference between the BMSC+OEC combined group and the OEC group (P0.05). (4) there was no significant difference between the group IX lamellar neuron area (P0.05) and the operation control group (P0.05), and the OEC group was larger than the operation control group (P0.05). P0.05).
3, the effect of cell transplantation on astrocytes and oligodendrocytes in host scar tissue:
3.1, the effect of cell transplantation on the formation of astrocytes in the host scar: after the whole spinal cord injury, the number of GFAP positive cells in the scar tissue of the host or the immunized OD value were significantly more than that of the sham operation group (P0.05). (1) the number of GFAP positive cells in each cell transplantation group showed that the BMSC group or the OEC group were all less than those in the operation control group (p0.0 5) there was no difference in the position of corticol spinal tract, CST in the two groups of cicatricial segments (P0.05). The gray matter part of the two groups and the posterior corner of the tail segment were less than that of the operation group (P0.05), and the joint group of BMSC+OEC was less than the BMSC group or the OEC group (P0.05), and the head segment of the joint group was more than that of the BMSC group. There was no significant difference in the group (P0.05). The head segment of the joint group was less than that of group BMSC or OEC (P0.05), and the posterior corner of the tail segment of the combined group was less than that of the BMSC or OEC group (P0.05). The posterior corner of the tail segment was weaker than that of the operation group (P0.05). There was no significant difference between the gray head part of the cicatricial segment and the CG group (P0.05). There was no significant difference between the CST part of the cicatricial segment and the CG group (P0.05). The cicatricial gray matter and the posterior corner of the tail segment were weaker than the operation group (P0.05), and the BMSC+OEC joint group was weaker than the BMSC or OEC group (P0.05), and the cicatricial segment was in the head segment. Gray matter and posterior horn of scar tail were weaker than those of group BMSC or OEC (P0.05). There was no significant difference in CST location between scar tissue and BMSC group or OEC group (P0.05).
3.2, the effect of cell transplantation on oligodendrocytes in the host scar: after the whole spinal cord transection, the number of APC positive cells in the host scar tissue or the immunized OD value were significantly less than that of the sham operation group (P0.05). (1) the number of APC positive cells in each cell transplantation group showed that the BMSC group or the OEC group were more than the operation group (P0.05), of which BM The head segment of cicatricial segment of CST in group SC was more than that of the operation group (P0.05). There was no significant difference between the CST part of the head segment of the scar of the group OEC and the operation group (P0.05) the cicatricial gray matter and the posterior corner of the tail segment of the group of BMSC or OEC were more than that of the operation group (P0.05), and the joint group of BMSC+OEC was more than the BMSC group (P0.05). The site of the gray matter and the posterior corner of the tail segment of the scar were more than that of the BMSC group (P0.05), and there was no significant difference between the BMSC+OEC group and the OEC group (P0.05), but the posterior corner of the tail segment of the scar was more than that of the OEC group (P0.05). In the operation group (P0.05), there was no significant difference in the head segment CST of the two groups from the operation group (P0.05). The two groups of cicatricial cicatricial gray matter and the posterior corner of the tail segment were more than the operation group (P0.05), and the BMSC+OEC combined group was stronger than the BMSC group (P0.05), and the head CST and gray matter in the cicatricial segment were stronger than the BMSC group (P0.05), and the posterior corner of the scar tail segment was not. Obvious difference (P0.05), BMSC+OEC combined group was stronger than group OEC, and the scar section CST and gray matter location, scar tail part posterior horn were stronger than OEC group (P0.05).
4: the effect of cell transplantation on the regeneration of spinal nerve: (1) regeneration of PKC gamma positive fibers: no axon regeneration was found in all groups of PKC gamma positive fibers after all transection injury. No PKC gamma positive staining was found in scar tissue. (2) 5-HT positive fibers were regenerated: all 5-HT positive fibers in the spinal cord all entered the scar tissue, among which the operation group entered into the scar tissue. The length of 532.60+145.74 mu m (distance from the head distance of scar), the length of BMSC group was 1698 + 880.81 m, and the entry length of OEC group was 659.80 + 271.54 u m, and the length of BMSC+OEC combined transplantation group was 1492.00+337.52 u m. The length of 5-HT positive fiber in BMSC and BMSC+OEC combined transplantation group was greater than that of the operation group. The effect of group transplantation was better than that of group OEC (P0.05), and there was no significant difference (P0.05) compared with group BMSC (P0.05). (3) the regeneration of CGRP positive upper fiber: the operation group did not enter the scar and was 962.40 + 441.14 mu m from the tail section of the scar; the BMSC group did not enter the scar and the tail section was 313.60 + 109.79 mu m; the OEC group entered the scar, and the length was 198 + 85.26 mu m; BMSC and OEC were moved together. The length of the upper CGRP fiber in the plant group entered the scar and entered the length of 531.80+162.62 mu m. The length of the regenerative length of the CGRP uplink fiber in each cell transplantation group was greater than that of the CG group (P0.05). The regeneration length of the CGRP positive fiber in the BMSC and OEC combined transplantation group was greater than that of the BMSC or OEC alone group (P0.05).
[conclusion]:1, totally transected spinal cord injury causes the death of the VIII and the lamellar neurons of the spinal cord, and the number of neurons in the spinal cord is reduced.
2, BMSC alone failed to promote the survival of the host spinal cord VIII, the survival of the lamellar neurons and the degeneration and atrophy of the host neurons. OEC alone could improve the survival of the neurons in the VIII lamina of the host spinal cord, and could not promote the survival of the IX lamellar neurons. OEC alone could inhibit the atrophy of the VIII in the host spinal cord, and the atrophy of the layer IX lamellar neurons; BMSC The +OEC combined transplantation group can promote the survival of the host VIII and IX lamellar neurons more than the BMSC group. The combined transplantation group can promote the survival of the VIII lamellar neurons more than the OEC group, and there is no significant difference in promoting the survival of the IX lamellar neurons with OEC. The combined transplantation group is better than BMSC or OEC alone in the inhibition of the atrophy of the host neurons.
3: the formation of glial scar in spinal cord tissue after all transverse spinal cord injury, GFAP positive reaction was enhanced, demyelinating lesion of spinal nerve fiber axons appeared, and APC positive reaction weakened.BMSC or OEC cell transplantation to improve scar microenvironment, inhibit the formation of glial scar and promote the survival of oligodendrocytes. BMSC and OEC combined transplantation. The effect is better.
4: after all spinal cord transection injury, PKC gamma positive fiber and CGRP positive upper fiber can not be regenerated into scar tissue, and 5-HT positive fibers are renewable into scar tissue for a certain length; BMSC and OEC alone or combined transplantation can not promote the regeneration of PKC gamma positive fibers; BMSC transplantation or BMSC+ OEC combined transplantation can promote the regeneration of 5-HT positive fibers. OEC transplantation can not promote the regeneration of 5-HT positive fibers; both BMSC and OEC alone or combined cell transplantation can promote the nerve regeneration of CGRP positive upper fibers, in which OEC transplantation or BMSC+OEC combined transplantation can promote the positive fibers of CGRP to enter the scar tissue, and the combined cell transplantation has a better role of promoting the regenerative effect.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332;R651.2
[Abstract]:[Objective] to establish the isolation, purification and culture of rat bone marrow mesenchymal stem cells (BMSCs), olfactory ensheathing cells (OECs) in vitro, and the model of rat total transection spinal cord injury. The effects of BMSCs, OECs alone and BMSCs+OECs on the survival of the spinal cord VIII, IX lamellar neurons in the rat spinal cord and the GFAP and APC in the glial scar were investigated. Expression and regeneration of nerve fibers provide theoretical and laboratory evidence for cell transplantation in the treatment of spinal cord injury.
[Methods] BMSCs and OECs were isolated and cultured in vitro. 25 healthy SD male rats in clean grade were randomly divided into sham operation group (group SG), operation control group (CG group), BMSC alone transplantation group, OEC alone transplantation group and BMSCs+OECs group. In vitro, bone marrow derived mesenchymal stem cells (Dil markers) were used in vitro. And OECs (Hoechst33342) stereotactic transplantation into the chronic phase (14 days after total transection) the scars of the spinal cord, the cephaloside and the caudal side of the spinal cord of the rats with all transverse transection. After the spinal cord injury, the paraffin section was made from the cicatricial and the tail and side spinal cord after the spinal cord injury, and the hematoxylin staining and immunohistochemical staining were performed, and the Group VIII and IX were counted respectively. The number and area of the lamellar neurons, count the number of GFAP and APC positive cells in the cicatricial and analyze their immune OD values, and observe the.SPSS11.5 software package of PKC gamma, 5-HT and CGRP positive fibers in the host. The data are statistically processed.
[results]
1, cell culture results: the cultured cells were immunized by CD44 and p75 respectively, and the results were positive. They were identified as BMSCs and OECs.
2, the effect of cell transplantation on the survival of the host neurons: the operation control group VIII, the number and area of the layer IX lamellar neurons were less than that of the sham operation group (P0.05). (1) the number of VIII lamellar neurons in the experimental group showed that there was no significant difference between the BMSC group and the operation control group (P0.05), and the OEC group was more than the operation control group (P0.05), and the BMSC+OEC combined transplantation Group more than group BMSC or group OEC (P0.05); (2) compared group VIII lamellar neuron area results showed that there was no significant change in group BMSC and operation control group (P0.05), OEC group was larger than operation control group (P0.05), BMSC+OEC combined group was larger than BMSC group, OEC group (P0.05); thirdly, the number of neurons in each group IX lamellar neurons showed: BMSC group or group and operation There was no significant difference in the control group (P0.05). There was no significant difference between the BMSC+OEC group and the BMSC group (P0.05), and there was no significant difference between the BMSC+OEC combined group and the OEC group (P0.05). (4) there was no significant difference between the group IX lamellar neuron area (P0.05) and the operation control group (P0.05), and the OEC group was larger than the operation control group (P0.05). P0.05).
3, the effect of cell transplantation on astrocytes and oligodendrocytes in host scar tissue:
3.1, the effect of cell transplantation on the formation of astrocytes in the host scar: after the whole spinal cord injury, the number of GFAP positive cells in the scar tissue of the host or the immunized OD value were significantly more than that of the sham operation group (P0.05). (1) the number of GFAP positive cells in each cell transplantation group showed that the BMSC group or the OEC group were all less than those in the operation control group (p0.0 5) there was no difference in the position of corticol spinal tract, CST in the two groups of cicatricial segments (P0.05). The gray matter part of the two groups and the posterior corner of the tail segment were less than that of the operation group (P0.05), and the joint group of BMSC+OEC was less than the BMSC group or the OEC group (P0.05), and the head segment of the joint group was more than that of the BMSC group. There was no significant difference in the group (P0.05). The head segment of the joint group was less than that of group BMSC or OEC (P0.05), and the posterior corner of the tail segment of the combined group was less than that of the BMSC or OEC group (P0.05). The posterior corner of the tail segment was weaker than that of the operation group (P0.05). There was no significant difference between the gray head part of the cicatricial segment and the CG group (P0.05). There was no significant difference between the CST part of the cicatricial segment and the CG group (P0.05). The cicatricial gray matter and the posterior corner of the tail segment were weaker than the operation group (P0.05), and the BMSC+OEC joint group was weaker than the BMSC or OEC group (P0.05), and the cicatricial segment was in the head segment. Gray matter and posterior horn of scar tail were weaker than those of group BMSC or OEC (P0.05). There was no significant difference in CST location between scar tissue and BMSC group or OEC group (P0.05).
3.2, the effect of cell transplantation on oligodendrocytes in the host scar: after the whole spinal cord transection, the number of APC positive cells in the host scar tissue or the immunized OD value were significantly less than that of the sham operation group (P0.05). (1) the number of APC positive cells in each cell transplantation group showed that the BMSC group or the OEC group were more than the operation group (P0.05), of which BM The head segment of cicatricial segment of CST in group SC was more than that of the operation group (P0.05). There was no significant difference between the CST part of the head segment of the scar of the group OEC and the operation group (P0.05) the cicatricial gray matter and the posterior corner of the tail segment of the group of BMSC or OEC were more than that of the operation group (P0.05), and the joint group of BMSC+OEC was more than the BMSC group (P0.05). The site of the gray matter and the posterior corner of the tail segment of the scar were more than that of the BMSC group (P0.05), and there was no significant difference between the BMSC+OEC group and the OEC group (P0.05), but the posterior corner of the tail segment of the scar was more than that of the OEC group (P0.05). In the operation group (P0.05), there was no significant difference in the head segment CST of the two groups from the operation group (P0.05). The two groups of cicatricial cicatricial gray matter and the posterior corner of the tail segment were more than the operation group (P0.05), and the BMSC+OEC combined group was stronger than the BMSC group (P0.05), and the head CST and gray matter in the cicatricial segment were stronger than the BMSC group (P0.05), and the posterior corner of the scar tail segment was not. Obvious difference (P0.05), BMSC+OEC combined group was stronger than group OEC, and the scar section CST and gray matter location, scar tail part posterior horn were stronger than OEC group (P0.05).
4: the effect of cell transplantation on the regeneration of spinal nerve: (1) regeneration of PKC gamma positive fibers: no axon regeneration was found in all groups of PKC gamma positive fibers after all transection injury. No PKC gamma positive staining was found in scar tissue. (2) 5-HT positive fibers were regenerated: all 5-HT positive fibers in the spinal cord all entered the scar tissue, among which the operation group entered into the scar tissue. The length of 532.60+145.74 mu m (distance from the head distance of scar), the length of BMSC group was 1698 + 880.81 m, and the entry length of OEC group was 659.80 + 271.54 u m, and the length of BMSC+OEC combined transplantation group was 1492.00+337.52 u m. The length of 5-HT positive fiber in BMSC and BMSC+OEC combined transplantation group was greater than that of the operation group. The effect of group transplantation was better than that of group OEC (P0.05), and there was no significant difference (P0.05) compared with group BMSC (P0.05). (3) the regeneration of CGRP positive upper fiber: the operation group did not enter the scar and was 962.40 + 441.14 mu m from the tail section of the scar; the BMSC group did not enter the scar and the tail section was 313.60 + 109.79 mu m; the OEC group entered the scar, and the length was 198 + 85.26 mu m; BMSC and OEC were moved together. The length of the upper CGRP fiber in the plant group entered the scar and entered the length of 531.80+162.62 mu m. The length of the regenerative length of the CGRP uplink fiber in each cell transplantation group was greater than that of the CG group (P0.05). The regeneration length of the CGRP positive fiber in the BMSC and OEC combined transplantation group was greater than that of the BMSC or OEC alone group (P0.05).
[conclusion]:1, totally transected spinal cord injury causes the death of the VIII and the lamellar neurons of the spinal cord, and the number of neurons in the spinal cord is reduced.
2, BMSC alone failed to promote the survival of the host spinal cord VIII, the survival of the lamellar neurons and the degeneration and atrophy of the host neurons. OEC alone could improve the survival of the neurons in the VIII lamina of the host spinal cord, and could not promote the survival of the IX lamellar neurons. OEC alone could inhibit the atrophy of the VIII in the host spinal cord, and the atrophy of the layer IX lamellar neurons; BMSC The +OEC combined transplantation group can promote the survival of the host VIII and IX lamellar neurons more than the BMSC group. The combined transplantation group can promote the survival of the VIII lamellar neurons more than the OEC group, and there is no significant difference in promoting the survival of the IX lamellar neurons with OEC. The combined transplantation group is better than BMSC or OEC alone in the inhibition of the atrophy of the host neurons.
3: the formation of glial scar in spinal cord tissue after all transverse spinal cord injury, GFAP positive reaction was enhanced, demyelinating lesion of spinal nerve fiber axons appeared, and APC positive reaction weakened.BMSC or OEC cell transplantation to improve scar microenvironment, inhibit the formation of glial scar and promote the survival of oligodendrocytes. BMSC and OEC combined transplantation. The effect is better.
4: after all spinal cord transection injury, PKC gamma positive fiber and CGRP positive upper fiber can not be regenerated into scar tissue, and 5-HT positive fibers are renewable into scar tissue for a certain length; BMSC and OEC alone or combined transplantation can not promote the regeneration of PKC gamma positive fibers; BMSC transplantation or BMSC+ OEC combined transplantation can promote the regeneration of 5-HT positive fibers. OEC transplantation can not promote the regeneration of 5-HT positive fibers; both BMSC and OEC alone or combined cell transplantation can promote the nerve regeneration of CGRP positive upper fibers, in which OEC transplantation or BMSC+OEC combined transplantation can promote the positive fibers of CGRP to enter the scar tissue, and the combined cell transplantation has a better role of promoting the regenerative effect.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332;R651.2
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