人羊膜间充质干细胞和人羊膜上皮细胞移植治疗帕金森病模型大鼠的效应比较

发布时间：2018-03-29 10:32

本文选题：羊膜　切入点：帕金森　出处：《遵义医学院》2012年硕士论文

【摘要】：目的：人羊膜来源的羊膜间充质干细胞(hAD-MSCs)和羊膜上皮细胞(hAECs)都具有多向分化能力,有望成为治疗PD新的细胞供源。本实验探讨hAD-MSCs、 hAECs原位移植治疗PD模型大鼠的疗效,比较不同途径移植hAD-MSCs治疗PD模型大鼠的疗效,并观察移植细胞在病损区的存活与分化。方法：①采用二酶消化法分离培养hAD-MSCs、hAECs细胞,用流式细胞术(FCM)和免疫细胞化学(ICC)染色进行鉴定。②内侧前脑束(MFB)单点注射6-OHDA复制单侧PD大鼠模型。模型动物24只随机分为四组：对照组(n=6)原位注射等量无血清L-DMEM培养基8μl；原位移植hAD-MSCs组(n=6,3×105cells/8μl)；原位移植1AECs组(n=6,3×105cells/8μl);舌下静脉移植hAD-MSCs组(n=6,5×106cells/ml)。细胞移植后每周腹腔注射阿朴吗啡0.5mg/kg诱导大鼠旋转,持续观察10周,监测PD大鼠行为学变化。③免疫荧光技术检测细胞移植后2周和12周两个时间点PD大鼠脑损毁区供体细胞存活及分化,同时采用免疫组化检测各组TH的分布。结果：①分离培养所得hAD-MSCs、hAECs均具有各自典型的生物学特性：第三代hAD-MSCs不表达CD34、HLA-DR、CD19、CD14、CD45、CD80和CD86,表达CD73、CD44、CD105和CD90。第三代hAECs不表达CD34、CD45、CD71、CD80和CD86表达CD44、CD29和CD73；②原位移植hAD-MSCs组第2至8周,与对照组比较,行为学有明显改善,旋转次数分别为13.1±2.7r/min、11.0±1.4r/min、9.3±1.3r/min、7.6±1.0r/min、10.3±1.5r/mim、8.3±3.6r/min和8.9±3.7r/min(均P0.05)；原位移植hAECs组第2至10周,旋转次数分别为10.9±2.6r/min、6±2.2r/min、9.2±2.6r/min、9.2±2.4r/min、8.5±2.6r/min、8.2±1.9r/min、7.2±2.9r/min、8.2±1.9r/min和8.7±3.0r/min,与对照组比较行为学有明显改善(均P0.05)；舌下静脉移植hAD-MSCs组第2至6周,旋转次数分别为分别为7.4±2.0r/min、8.2±4.6r/min、8.5±7.3r/min、9.3±4.4r/min和8.0±5.8r/min,较对照组有明显改善(均P0.05)。原位移植hAECs组行为学改善持续时间较hAD-MSCs原位移植组长2周,原位移植hAD-MSCs组较舌下静脉移植组行为学改善持续时间长2周；③免疫荧光染色结果显示,原位移植hAD-MSCs组和1(?)AECs组移植后2周和12周,病损脑组织中均有植入的细胞存活并表达人神经元细胞微管结合蛋白MAP2,但hAD-MSCs舌下静脉移植组未检测到供体细胞。免疫组化染色显示各移植组病损脑区TH表达均强于较对照组；hAD-MSCs和hAECs原位移植组TH表达较hAD-MSCs舌下静脉移植组强。结论：本研究复证了hAD-MSCs和]hAECs移植对PD大鼠旋转行为具有改善作用,二者可在动物脑损毁区存活较长时间而不被排斥,并分化成DA能神经元样细胞。首次通过行为学研究证明hAECs是较]hAD-MSCs更为理想的治疗PD的候选供体细胞。实验证明供体细胞产生生物学效应与其移植途径有关,就治疗中枢系统疾病而言,原位或局部移植优于静脉注射。
[Abstract]:Objective: human amniotic membrane derived amniotic mesenchymal stem cells (HAD-MSCss) and amniotic epithelial cells (hAECs) have the ability to differentiate into different directions and may be a new cell donor for PD. The purpose of this study was to investigate the effect of hAD-MSCsand hAECs orthotopic transplantation on PD model rats. To compare the effects of hAD-MSCs transplantation on PD model rats, and to observe the survival and differentiation of transplanted cells in diseased area. Methods the cell lines of hAD-MSCsCos were isolated and cultured by two-enzyme digestion. Flow cytometry (FCM) and immunocytochemistry (ICC6) staining were used to identify the unilateral PD rat model induced by single point injection of 6-OHDA into the medial forebrain bundle. Twenty-four rats were randomly divided into four groups: control group (n = 6) and in situ injection of serum-free L-DMEM. Culture medium 8 渭 l; hAD-MSCs group 3 脳 105cells/8 渭 l; 1AECs group 3 脳 105cells/8 渭 l; sublingual vein transplantation hAD-MSCs group 5 脳 10 6 cells / ml. After transplantation, apomorphine 0.5mg/kg was injected intraperitoneally to induce rotation in rats. The behavioral changes of PD rats were continuously observed for 10 weeks. 3 immunofluorescence technique was used to detect the survival and differentiation of donor cells in brain lesion area of PD rats at 2 and 12 weeks after transplantation. Meanwhile, the distribution of th in each group was detected by immunohistochemistry. Results the isolated and cultured hAD-MSCs hAECs had their own typical biological characteristics: the third generation of hAD-MSCs did not express CD34T HLA-DRN CD19, CD14, CD45, CD80 and CD86. The third generation of hAECs did not express CD34, CD45, CD71CD80 and CD86, and the expression of CD442 CD29 and CD732 in orthotopic transplantation hAD-MSCs group was from the 2nd to 8th week, compared with the control group, the third generation of hAECs did not express CD34, CD45, CD71CD80 and CD732 in orthotopic transplantation hAD-MSCs group, compared with the control group, the third generation of hAECs did not express CD34, CD41, CD80 and CD732 in orthotopic transplantation hAD-MSCs group from week 2 to 8, compared with the control group. The number of rotations was significantly improved (13.1 卤2.7rmmin 11.0 卤1.4rpm 9.3 卤1.3rpm 7.6 卤1.0rpm 10.3 卤1.5rmmim 8.3 卤3.6r/min and 8.9 卤3.7rp min, respectively, P 0.05; in orthotopic hAECs group, 10.9 卤2.6rmin / min, 9.2 卤2.6rp / min 9.2 卤2.4rp / min, 8.5 卤2.6rp / min, 8.2 卤2.9rpm / min 7.2 卤2.9rmin / min 8.2 卤2.9rmin / min, 8.7 卤3.0rmins, respectively, compared with the control group (P0.05); in the hAD-MSCs group, the second to sixth week after vein transplantation, the number of rotations was 10.9 卤2.6rmin / min 9.2 卤2.4rp / min 9.2 卤2.6rpm / min 9.2 卤2.6rp / min 8.2 卤1.9rp / min 7.2 卤2.9rmin / min 7.2 卤2.9rmin / min 8.2 卤2.9rmin / min and 8.7 卤3.0rmin / min respectively (P0.05P). The rotation times were 7.4 卤2.0 r / min 8.2 卤4.6 r / min and 8.5 卤7.3 r / min 9.3 卤4.4r/min and 8.0 卤5.8 r / min, respectively, which were significantly better than those in the control group (all P 0.05). The duration of behavioral improvement in orthotopic hAECs group was longer than that in hAD-MSCs orthotopic transplantation group for 2 weeks. The behavior improvement of orthotopic hAD-MSCs group was longer than that of sublingual vein transplantation group. In the AECs group, 2 and 12 weeks after transplantation, In the diseased brain tissue, all the implanted cells survived and expressed human neuronal cell microtubule binding protein MAP2, but no donor cells were detected in the hAD-MSCs sublingual vein transplantation group. Immunohistochemical staining showed that the th expression was strong in the diseased brain regions of each transplantation group. Th expression in hAD-MSCs and hAECs orthotopic transplantation group was stronger than that in hAD-MSCs sublingual vein transplantation group. Conclusion: this study demonstrated that hAD-MSCs and] hAECs transplantation can improve the rotational behavior of PD rats, and they can survive in the brain lesion area for a long time without rejection. HAECs is a better candidate donor cell for PD treatment than hAD-MSCs for the first time. The biological effect of donor cells is related to its transplant pathway. In the treatment of central system diseases, orthotopic or local transplantation is superior to intravenous injection.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.28

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