脂肪源性干细胞治疗大鼠海绵体神经损伤性勃起功能障碍的研究

发布时间：2018-05-03 08:41

本文选题：脂肪干细胞 + 神经细胞　；参考：《武汉大学》2015年博士论文

【摘要】：第一部分脂肪干细胞的分离培养、鉴定及多向诱导分化的研究目的：据报道脂肪干细胞(Adipose derived stem cells, ADSCs)具有多向分化能力并在特定的诱导剂下能够向神经元样细胞分化,这可能为神经性勃起功能障碍(Erectile dysfunction,ED)的修复提供新的治疗思路。本文旨在探讨脂肪干细胞体外诱导分化成神经细胞的潜能,为恢复阴茎海绵体神经(Carvernous nerve,CN)受损导致勃起功能障碍的研究建立基础。方法：从SD雄性大鼠腹股沟和附睾头端取出脂肪组织,剪碎后用I型胶原酶37℃水浴消化、离心,加培养基重悬后放入培养箱中培养。当传代培养至第3代时用流式细胞仪检测脂肪干细胞表面CD分子,第3代脂肪干细胞在特定诱导剂下诱导分化成脂肪细胞和神经细胞。用两种方法进行神经诱导：方法一是加入诱前诱导剂表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)、脑源性神经营养因子(BDNF)诱导3天后加入神经诱导剂吲哚美辛、胰岛素、3.异丁基.1.甲基黄嘌呤(IBMX)方法二与方法一的区别是没有加入诱前诱导剂BDNF。然后用油红0染色来鉴定诱导分化的脂肪细胞,用免疫荧光法及Western Blot检测诱导分化的神经细胞中表达的胶原纤维酸性蛋白(Glialfibrillary acidic protein,GFAP)和p-微管蛋白(β-tubulinⅢ,Tuj-1).结果：流式细胞仪检测脂肪干细胞表面CD分子的结果为：CD44(+)细胞率为96.0%、CD90(+)细胞率为98.5%、CD45(一)细胞率为0.6%、CD34(一)细胞率为0.5%。脂肪干细胞诱导成脂肪细胞,油红O染色脂滴染成红色。脂肪干细胞在神经诱导剂下诱导成神经细胞,48h后荧光染色GFAP、β-tubulin Ⅲ蛋白表达阳性,方法一和方法二的诱导率分别为74±3.3.3%、65+2.1%和51±1.2%、41±1.1%。且阳性细胞数差异具有统计学意义p0.05)。结论：我们成功从脂肪组织中分离提取并体外培养得到脂肪干细胞。研究表明脂干细胞在体外能够分化成神经细胞,并在方法一作用下向神经细胞分化诱导率高,时间短；在诱前诱导液中加入BDNF为体外神经诱导提供一个新的诱导方法从而能获得更多的神经细胞。脂肪干细胞可能成为治疗神经性勃起功能障碍的理想干细胞。第二部分脂肪干细胞对大鼠阴茎海绵体神经再生修复作用的研究目的：据报道保留海绵体神经的前列腺癌根治术是处于性活跃时期的局限性前列腺癌患者治疗的选择。在接受手术或者前列腺放疗的前列腺癌患者,术后频繁的发生勃起功能障碍(erectile dysfunction, ED)。在大多数情况下,海绵体神经(cavernous nervers, CNs)可能在保留神经的前列腺切除时由于手术操作原因不经意受损。另外有研究显示脂肪干细胞(adipose derived stem cells,ADSCs)分泌的神经营养因子能够促进CN的再生修复。本研究通过大鼠海绵体内注射ADSCs探讨ADSCs是否对CN的再生有促进调节作用,以及评估双侧CN受损导致ED的恢复情况。方法：将30只SD雄性大鼠随机分成3组(每组10只)：一组仅分离双侧CN作为假手术组,其余两组行双侧CN钳夹损伤。钳夹损伤组中一组在损伤后立即海绵体内注射ADSCs,另外一组(损伤对照组)在损伤后不作治疗。3个月后通过电刺激CN测海绵体内压来评估勃起功能。测压后取双侧钳夹损伤CN行甲苯胺蓝染色；取大鼠阴茎中段组织一部分行Masson染色。一部分行WesternBlot检测阴茎组织nNOS蛋白的表达。结果：术后3个月测压结果显示损伤对照组最大海绵体内压(maximal intracavernous pressure,maxICP)为38.6±12.4,最大海绵体内压与平均动脉压(mean arterial pressure,MAP)比值为0.26±0.03,显著低于假手术组(maxICP为108.2±10.8, ICP/MAP比值为0.68±0.08),差异有统计学意义(P0.05)。同时ADSCs治疗组1naxICP (90.1±12.8)和ICP/MAP(0.44±0.05)显著高于对照组(P0.05)。CN甲苯胺蓝染色显示ADSCs治疗组神经轴突数为(92.5±18),损伤对照组神经轴突数为(584±20),差异有统计学意义(P0.05)。Masson染色结果显不ADSCs治疗组平滑肌占胶原纤维比例(9.2±2.8)显著高于对照组(5.4±1.1,p0.05)。结论：这些结果显示海绵体内注射ADSCs能够促进海绵体神经再生和勃起功能的恢复。我们的结果表明阴茎内注射ADSCs够促进神经性ED动物模型勃起功能的恢复。第三部分脂肪干细胞结合自体静脉移植修复大鼠勃起功能障碍的研究目的：海绵体神经(cavernous nervers, CNs)损伤是前列腺癌根治术后发生勃起功能障碍(erectile dysfunction, ED)的主要原因,这种勃起功能的恢复仍具有挑战性。我们主要是探讨脂肪干细胞(adipose derived stem cells,ADSCs)结合自体静脉移植提高双侧CN较长切断损伤大鼠模型勃起功能的可能性。方法：SD大鼠(36只)随机分成四组(每组各9只)。A：假手术组；B：ADSCs结合自体静脉移植治疗组；C：生理盐水结合白体静脉移植治疗组；D：双侧CN离断损伤组。3个月后行海绵体内压(ICP)和平均动脉压(MAP)测定。测压后取大鼠阴茎中段组织一部分行Masson染色,一部分行免疫组化检测阴茎组织中nNOS阳性纤维。自体移植的静脉在显微镜下进行解剖。结果：术后3个月测压结果显示脂肪干细胞结合自体静脉移植治疗组大鼠勃.起功能的恢复程度(maxICP 78.8±12.7)显著高于生理盐水结合自体静脉移植治疗组(maxICP 48.6±15.7)和双侧CN离断损伤组(maxICP 26.5±5.8) (p＜0.05).免疫组化结果显示脂肪干细胞结合自体静脉移植治疗组大鼠阴茎背侧nNOS阳性纤维显著多于双侧CN离断损伤组(p0.05)。Masson染色结果显示脂肪干细胞结合自体静脉移植治疗对大鼠阴茎海绵体内平滑肌占胶原纤维的比例有保护作用。ADSCs治疗组大鼠阴茎组织中平滑肌占胶原纤维比例(8.5±2.1)显著高于双侧CN离断损伤组(4.6±1.2,p0.05)。脂肪干细胞结合自体静脉移植治疗组的移植静脉在显微镜下找到新生的神经。结论：ADSCs结合自体静脉移植能够促进大鼠较长缺损海绵体神经(0.8cm)再生和修复,并在一定程度上能够恢复大鼠的自主勃起功能。
[Abstract]:The purpose of this study was to investigate the potential of Adipose derived stem cells ( ADSCs ) to differentiate into adipocytes and to induce differentiation into neuron - like cells . Results : The results of flow cytometry showed that CD44 ( + ) cell rate was 96.0 % , CD90 ( + ) cell rate was 98.5 % , CD90 ( + ) cell rate was 98.5 % , CD34 ( I ) cell rate was 0.5 % . Adipose stem cells were induced into adipocytes and oil red O - stained lipid droplets were positive . The induction rates were 74 卤 3.3 % , 65 + 2.1 % and 51 卤 1.2 % , 41 卤 1.1 % , respectively , and the difference of positive cells was statistically significant ( p < 0.05 ) . Conclusion : We successfully isolated from adipose tissue and cultured in vitro to obtain adipose - derived stem cells .
It is suggested that BDNF is an ideal stem cell for the treatment of neurological erectile dysfunction . The second part of adipose - derived stem cells is the ideal stem cell for the treatment of neurological erectile dysfunction . Methods : Thirty SD male rats were randomly divided into 3 groups ( 10 rats in each group ) : 30 SD male rats were randomly divided into 3 groups ( 10 rats in each group ) .
Results : The results showed that the maximal intracavernous pressure ( maxICP ) was 38 . 6 卤 12 . 4 , and the mean arterial pressure ( MAP ) ratio was 0 . 26 卤 0 . 03 , which was significantly lower than that of the control group ( P 0 . 05 ) . Conclusion : These results showed that ADSCs were injected into cavernous body to promote the restoration of erectile dysfunction in rats . The results showed that ADSCs were used to promote the recovery of erectile dysfunction in the model of erectile dysfunction after radical prostatectomy . The third part of adipose - derived stem cells combined with autologous vein graft to repair erectile dysfunction in rats .
B : ADSCs combined with autologous vein graft treatment group ;
C : Normal saline was combined with the white body vein graft treatment group .
The results showed that fat stem cells combined with autologous vein graft in the treatment group were significantly higher than those in normal saline group ( maxICP 48.6 卤 15.7 ) and bilateral CN group ( maxICP 26.5 卤 5.8 ) ( p < 0.05 ) . Conclusion : ADSCs can promote the regeneration and repair of long - defect cavernous nerves ( 0.8cm ) in rats and restore the function of spontaneous erections in rats to some extent .

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R698

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