【摘要】：[目的]建立大鼠骨髓間充質(zhì)干細(xì)胞(BMSCs)、嗅鞘細(xì)胞(OECs)的體外分離、純化和培養(yǎng)方法,及大鼠全橫斷脊髓損傷模型,探討B(tài)MSCs、OECs單獨(dú)移植及BMSCs+OECs聯(lián)合移植對(duì)全橫斷損傷大鼠脊髓Ⅷ、Ⅸ板層神經(jīng)元存活的影響、及膠質(zhì)瘢痕中GFAP和APC的表達(dá)和神經(jīng)纖維再生的變化,為細(xì)胞移植治療脊髓損傷提供理論和實(shí)驗(yàn)室依據(jù)。 [方法]體外分離培養(yǎng)大鼠BMSCs和OECs并進(jìn)行細(xì)胞染色鑒定；清潔級(jí)健康SD雄性大鼠25只,每組5只將動(dòng)物隨機(jī)分為假手術(shù)組(SG組)、手術(shù)對(duì)照組(CG組)、BMSC單獨(dú)移植組、OEC單獨(dú)移植組和BMSCs+OECs組。體外條件下將骨髓源性間充質(zhì)干細(xì)胞(Dil標(biāo)記)和OECs (Hoechst33342標(biāo)記)立體定向移植入慢性期(全橫斷術(shù)后14天)全橫斷損傷大鼠脊髓的瘢痕處、頭側(cè)和尾側(cè)。在脊髓損傷后第6m末灌注固定后取瘢痕處及頭尾側(cè)脊髓制作石蠟切片,行蘇木素染色及免疫組化染色,分別計(jì)數(shù)各組脊髓Ⅷ、Ⅸ板層神經(jīng)元數(shù)量及面積；計(jì)數(shù)各組瘢痕中GFAP和APC陽(yáng)性細(xì)胞數(shù)量并分析其免疫染色OD值,同時(shí)觀察宿主PKCγ、5-HT及CGRP陽(yáng)性纖維再生情況。SPSS11.5軟件包對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)處理。 [結(jié)果] 1,細(xì)胞培養(yǎng)結(jié)果：培養(yǎng)的細(xì)胞分別經(jīng)CD44和p75免疫染色,結(jié)果呈陽(yáng)性反應(yīng),鑒定為BMSCs和OECs; 2,細(xì)胞移植對(duì)宿主神經(jīng)元存活的影響：手術(shù)對(duì)照組Ⅷ、Ⅸ板層神經(jīng)元數(shù)量和面積均少于假手術(shù)組(p0.05)；①比較各實(shí)驗(yàn)組VIII板層神經(jīng)元數(shù)量結(jié)果顯示：BMSC組與手術(shù)對(duì)照組無(wú)顯著差異(p0.05), OEC組多于手術(shù)對(duì)照組(p0.05), BMSC+OEC聯(lián)合移植組多于BMSC組或OEC組(p0.05)；②比較各組Ⅷ板層神經(jīng)元面積結(jié)果顯示：BMSC組與手術(shù)對(duì)照組無(wú)明顯變化(p0.05), OEC組大于手術(shù)對(duì)照組(p0.05),BMSC+OEC聯(lián)合組大于BMSC組、OEC組(p0.05)；③比較各組Ⅸ板層神經(jīng)元數(shù)量結(jié)果顯示：BMSC組或OEC組與手術(shù)對(duì)照組無(wú)明顯差異(p0.05),BMSC+OEC聯(lián)合組多與BMSC組(p0.05),BMSC+OEC聯(lián)合組與OEC組無(wú)明顯差異(p0.05)；④比較各組Ⅸ板層神經(jīng)元面積結(jié)果顯示：BMSC組與手術(shù)對(duì)照組無(wú)明顯差異(p0.05), OEC組大于手術(shù)對(duì)照組(p0.05),BMSC+OEC聯(lián)合組大于BMSC組或OEC組(p0.05)。 3,細(xì)胞移植對(duì)宿主瘢痕組織中星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞的影響： 3.1,細(xì)胞移植對(duì)宿主瘢痕中星形膠質(zhì)細(xì)胞形成影響：脊髓全橫斷損傷后,宿主瘢痕組織中GFAP陽(yáng)性細(xì)胞數(shù)目或免疫染色OD值均明顯多于假手術(shù)組(p0.05)。①比較各細(xì)胞移植組GFAP陽(yáng)性細(xì)胞數(shù)目結(jié)果顯示：BMSC組或OEC組均少于手術(shù)對(duì)照組(p0.05),在兩組的瘢痕頭段皮質(zhì)脊髓束(corticol spinal tract, CST)部位與手術(shù)組比較無(wú)差異(p0.05),兩組的瘢痕頭段灰質(zhì)部位和尾段后角部位均少于手術(shù)組(p0.05)；BMSC+OEC聯(lián)合組少于BMSC組或OEC組(p0.05),其中聯(lián)合組頭段CST部位多于BMSC組(p0.05),與OEC組比較無(wú)明顯差異(p0.05),聯(lián)合組頭段灰質(zhì)部位少于BMSC組或OEC組(p0.05),聯(lián)合組尾段后角部位少于BMSC或OEC組(p0.05)。②比較各細(xì)胞移植組GFAP免疫染色OD值結(jié)果顯示：BMSC組或OEC組均弱于手術(shù)對(duì)照組(p0.05),其中BMSC組瘢痕頭段CST部位和瘢痕尾段后角部位均弱于手術(shù)組(p0.05),瘢痕頭段灰質(zhì)部位與CG組無(wú)明顯差異(p0.05), OEC組瘢痕頭段CST部位與CG組無(wú)明顯差異(p0.05),瘢痕頭段灰質(zhì)部位和瘢痕尾段后角部位均弱于手術(shù)組(p0.05),BMSC+OEC聯(lián)合組弱于BMSC或OEC組(p0.05),其中瘢痕頭段灰質(zhì)部位和瘢痕尾段后角部位均弱于BMSC或OEC組(p0.05),瘢痕頭段CST部位與BMSC組或OEC組比較均無(wú)顯著差異(p0.05)。 3.2,細(xì)胞移植對(duì)宿主瘢痕中少突膠質(zhì)細(xì)胞的影響：脊髓全橫斷損傷后,宿主瘢痕組織中APC陽(yáng)性細(xì)胞數(shù)目或免疫染色OD值均明顯少于假手術(shù)組(p0.05)。①比較各細(xì)胞移植組APC陽(yáng)性細(xì)胞數(shù)目結(jié)果顯示：BMSC組或OEC組均多于手術(shù)組(p0.05),其中BMSC組瘢痕頭段CST部位多于手術(shù)組(p0.05),OEC組瘢痕頭段CST部位與手術(shù)組無(wú)明顯差異(p0.05)BMSC或OEC組的瘢痕頭段灰質(zhì)和瘢痕尾段后角部位均多于手術(shù)組(p0.05), BMSC+OEC聯(lián)合組多于BMSC組(p0.05),其中瘢痕頭段CST部位與BMSC組無(wú)明顯差異(p0.05),瘢痕頭段灰質(zhì)部位和瘢痕尾段后角部位均多于BMSC組(p0.05)；BMSC+OEC聯(lián)合組與OEC組無(wú)顯著差異(p0.05),但在瘢痕頭段CST和灰質(zhì)部位、瘢痕尾段后角部位均多于OEC組(p0.05)。②比較各細(xì)胞移植組APC免疫染色OD值結(jié)果顯示：BMSC組或OEC組均多于手術(shù)組(p0.05),其中兩組的瘢痕頭段CST部位與手術(shù)組比較均無(wú)明顯差異(p0.05),兩組的瘢痕頭段灰質(zhì)和瘢痕尾段后角部位均多于手術(shù)組(p0.05), BMSC+OEC聯(lián)合組強(qiáng)于BMSC組(p0.05),其中瘢痕頭段CST和灰質(zhì)部位強(qiáng)于BMSC組(p0.05),瘢痕尾段后角部位無(wú)明顯差異(p0.05), BMSC+OEC聯(lián)合組強(qiáng)于OEC組,其中瘢痕頭段CST和灰質(zhì)部位、瘢痕尾段后角部位均強(qiáng)于OEC組(p0.05)。 4：細(xì)胞移植對(duì)各組脊髓神經(jīng)再生的影響：①PKCγ陽(yáng)性纖維的再生：全橫斷損傷后各組PKCγ陽(yáng)性纖維均未發(fā)現(xiàn)有軸突再生,瘢痕組織中未發(fā)現(xiàn)有PKCγ陽(yáng)性染色。②5-HT陽(yáng)性纖維再生：各組脊髓5-HT陽(yáng)性纖維均進(jìn)入瘢痕組織,其中手術(shù)組進(jìn)入長(zhǎng)度532.60+145.74μm(距離瘢痕頭段距離)；BMSC組進(jìn)入長(zhǎng)度1698.00±880.81μm; OEC組進(jìn)入長(zhǎng)度(?)659.80±271.54μm, BMSC+OEC聯(lián)合移植組進(jìn)入長(zhǎng)度1492.00+337.52μm,比較發(fā)現(xiàn)BMSC組和BMSC+OEC聯(lián)合移植組的5-HT陽(yáng)性纖維再生長(zhǎng)度大于手術(shù)組(p0.05),聯(lián)合移植組移植效果優(yōu)于OEC組(p0.05),與BMSC組相比無(wú)明顯差異(p0.05)。③CGRP陽(yáng)性上行纖維的再生：手術(shù)組未進(jìn)入瘢痕,距瘢痕尾段962.40±441.14μm; BMSC組也未進(jìn)入瘢痕,距離尾段313.60±109.79μm; OEC組長(zhǎng)入瘢痕,進(jìn)入長(zhǎng)度為198.00±85.26μm; BMSC與OEC聯(lián)合移植組的CGRP上行纖維長(zhǎng)入瘢痕,進(jìn)入長(zhǎng)度為531.80+162.62μm,各細(xì)胞移植組的CGRP上行纖維的再生長(zhǎng)度均大于CG組(p0.05), BMSC與OEC聯(lián)合移植組CGRP陽(yáng)性纖維再生長(zhǎng)度大于BMSC或OEC單獨(dú)移植組(p0.05)。 [結(jié)論]：1、全橫斷脊髓損傷導(dǎo)致宿主脊髓Ⅷ、Ⅸ板層神經(jīng)元死亡,數(shù)量減少,殘存神經(jīng)元胞體萎縮,面積減小。 2、BMSC單獨(dú)移植組未能促進(jìn)宿主脊髓Ⅷ、Ⅸ板層神經(jīng)元的存活,也未能抑制宿主神經(jīng)元的變性萎縮；OEC單獨(dú)移植可提高宿主脊髓Ⅷ板層神經(jīng)元的存活,不能促進(jìn)IX板層神經(jīng)元存活,OEC單獨(dú)移植可抑制宿主脊髓Ⅷ、Ⅸ板層神經(jīng)元的萎縮；BMSC+OEC聯(lián)合移植組比BMSC組更能促進(jìn)宿主VIII、IX板層神經(jīng)元的存活,聯(lián)合移植組比OEC組更能促進(jìn)VIII板層神經(jīng)元存活,在促進(jìn)IX板層神經(jīng)元存活方面與OEC無(wú)明顯差異,在抑制宿主神經(jīng)元萎縮方面聯(lián)合移植組比BMSC或OEC單獨(dú)移植效果更佳。 3：全橫斷脊髓損傷后脊髓組織中膠質(zhì)瘢痕形成,GFAP陽(yáng)性反應(yīng)增強(qiáng),脊髓神經(jīng)纖維軸突出現(xiàn)脫髓鞘病變,APC陽(yáng)性反應(yīng)減弱。BMSC或OEC細(xì)胞移植均可改善瘢痕微環(huán)境,抑制膠質(zhì)瘢痕的形成并可促進(jìn)少突膠質(zhì)細(xì)胞的存活,BMSC與OEC聯(lián)合移植作用效果更佳。 4：脊髓全橫斷損傷后,PKCγ陽(yáng)性纖維和CGRP陽(yáng)性上行纖維不能再生進(jìn)入瘢痕組織,5-HT陽(yáng)性纖維可再生進(jìn)入瘢痕組織一定長(zhǎng)度；BMSC與OEC單獨(dú)或聯(lián)合移植不能促進(jìn)PKCγ陽(yáng)性纖維的再生；BMSC移植或BMSC+OEC聯(lián)合移植均可促進(jìn)5-HT陽(yáng)性纖維再生,OEC移植不能促進(jìn)5-HT陽(yáng)性纖維再生；BMSC和OEC單獨(dú)或聯(lián)合細(xì)胞移植均可促進(jìn)CGRP陽(yáng)性上行纖維的神經(jīng)再生,其中OEC移植或BMSC+OEC聯(lián)合移植可促進(jìn)CGRP上行陽(yáng)性纖維進(jìn)入瘢痕組織,聯(lián)合細(xì)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.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R-332;R651.2

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王靜;吳曉雄;宋雁賓;;異基因聯(lián)合臍帶間充質(zhì)干細(xì)胞移植術(shù)后患者的護(hù)理[J];軍醫(yī)進(jìn)修學(xué)院學(xué)報(bào);2011年08期

2 黃小梅;黃丹;劉月英;胡麗芳;;1例胰、腎聯(lián)合移植術(shù)患者圍術(shù)期的護(hù)理[J];實(shí)用臨床醫(yī)學(xué);2011年04期

3 沈t，

本文編號(hào)：2173972