本文選題：羊膜　切入點(diǎn)：帕金森　出處：《遵義醫(yī)學(xué)院》2012年碩士論文

【摘要】：目的：人羊膜來源的羊膜間充質(zhì)干細(xì)胞(hAD-MSCs)和羊膜上皮細(xì)胞(hAECs)都具有多向分化能力,有望成為治療PD新的細(xì)胞供源。本實(shí)驗(yàn)探討hAD-MSCs、 hAECs原位移植治療PD模型大鼠的療效,比較不同途徑移植hAD-MSCs治療PD模型大鼠的療效,并觀察移植細(xì)胞在病損區(qū)的存活與分化。 方法：①采用二酶消化法分離培養(yǎng)hAD-MSCs、hAECs細(xì)胞,用流式細(xì)胞術(shù)(FCM)和免疫細(xì)胞化學(xué)(ICC)染色進(jìn)行鑒定。②內(nèi)側(cè)前腦束(MFB)單點(diǎn)注射6-OHDA復(fù)制單側(cè)PD大鼠模型。模型動(dòng)物24只隨機(jī)分為四組：對照組(n=6)原位注射等量無血清L-DMEM培養(yǎng)基8μl；原位移植hAD-MSCs組(n=6,3×105cells/8μl)；原位移植1AECs組(n=6,3×105cells/8μl);舌下靜脈移植hAD-MSCs組(n=6,5×106cells/ml)。細(xì)胞移植后每周腹腔注射阿樸嗎啡0.5mg/kg誘導(dǎo)大鼠旋轉(zhuǎn),持續(xù)觀察10周,監(jiān)測PD大鼠行為學(xué)變化。③免疫熒光技術(shù)檢測細(xì)胞移植后2周和12周兩個(gè)時(shí)間點(diǎn)PD大鼠腦損毀區(qū)供體細(xì)胞存活及分化,同時(shí)采用免疫組化檢測各組TH的分布。 結(jié)果：①分離培養(yǎng)所得hAD-MSCs、hAECs均具有各自典型的生物學(xué)特性：第三代hAD-MSCs不表達(dá)CD34、HLA-DR、CD19、CD14、CD45、CD80和CD86,表達(dá)CD73、CD44、CD105和CD90。第三代hAECs不表達(dá)CD34、CD45、CD71、CD80和CD86表達(dá)CD44、CD29和CD73；②原位移植hAD-MSCs組第2至8周,與對照組比較,行為學(xué)有明顯改善,旋轉(zhuǎn)次數(shù)分別為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組第2至10周,旋轉(zhuǎn)次數(shù)分別為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,與對照組比較行為學(xué)有明顯改善(均P0.05)；舌下靜脈移植hAD-MSCs組第2至6周,旋轉(zhuǎn)次數(shù)分別為分別為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,較對照組有明顯改善(均P0.05)。原位移植hAECs組行為學(xué)改善持續(xù)時(shí)間較hAD-MSCs原位移植組長2周,原位移植hAD-MSCs組較舌下靜脈移植組行為學(xué)改善持續(xù)時(shí)間長2周；③免疫熒光染色結(jié)果顯示,原位移植hAD-MSCs組和1(?)AECs組移植后2周和12周,病損腦組織中均有植入的細(xì)胞存活并表達(dá)人神經(jīng)元細(xì)胞微管結(jié)合蛋白MAP2,但hAD-MSCs舌下靜脈移植組未檢測到供體細(xì)胞。免疫組化染色顯示各移植組病損腦區(qū)TH表達(dá)均強(qiáng)于較對照組；hAD-MSCs和hAECs原位移植組TH表達(dá)較hAD-MSCs舌下靜脈移植組強(qiáng)。 結(jié)論：本研究復(fù)證了hAD-MSCs和]hAECs移植對PD大鼠旋轉(zhuǎn)行為具有改善作用,二者可在動(dòng)物腦損毀區(qū)存活較長時(shí)間而不被排斥,并分化成DA能神經(jīng)元樣細(xì)胞。首次通過行為學(xué)研究證明hAECs是較]hAD-MSCs更為理想的治療PD的候選供體細(xì)胞。實(shí)驗(yàn)證明供體細(xì)胞產(chǎn)生生物學(xué)效應(yīng)與其移植途徑有關(guān),就治療中樞系統(tǒng)疾病而言,原位或局部移植優(yōu)于靜脈注射。
[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 鹵2.7rmmin 11.0 鹵1.4rpm 9.3 鹵1.3rpm 7.6 鹵1.0rpm 10.3 鹵1.5rmmim 8.3 鹵3.6r/min and 8.9 鹵3.7rp min, respectively, P 0.05; in orthotopic hAECs group, 10.9 鹵2.6rmin / min, 9.2 鹵2.6rp / min 9.2 鹵2.4rp / min, 8.5 鹵2.6rp / min, 8.2 鹵2.9rpm / min 7.2 鹵2.9rmin / min 8.2 鹵2.9rmin / min, 8.7 鹵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 鹵2.6rmin / min 9.2 鹵2.4rp / min 9.2 鹵2.6rpm / min 9.2 鹵2.6rp / min 8.2 鹵1.9rp / min 7.2 鹵2.9rmin / min 7.2 鹵2.9rmin / min 8.2 鹵2.9rmin / min and 8.7 鹵3.0rmin / min respectively (P0.05P). The rotation times were 7.4 鹵2.0 r / min 8.2 鹵4.6 r / min and 8.5 鹵7.3 r / min 9.3 鹵4.4r/min and 8.0 鹵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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329.28

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