CN111939178A - 一种治疗神经退行性疾病的Ommaya囊及其制备方法 - Google Patents
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Abstract
本发明公开了一种治疗神经退行性疾病的Ommaya囊及其制备方法,该Ommaya囊内含有人羊膜上皮干细胞。本发明提供的一种治疗神经退行性疾病的Ommaya囊可实现脑室内移植的精准定位,可确保干细胞移植成功,此外,该Ommaya囊可以进行1000次穿刺而不损坏,实现了给予神经退行性疾病患者多次移植人羊膜上皮干细胞的目的。采用该Ommaya囊进行治疗神经退行性疾病,创伤小、安全性高,治疗效果好,同时该Ommaya囊也有望成为神经系统疑难疾病干细胞移植治疗的特色技术。
Description
技术领域
本发明涉及医疗设备领域,尤其涉及一种治疗神经退行性疾病的Ommaya囊及其制备方法。
背景技术
神经退行性疾病是神经元结构或功能逐渐丧失甚至死亡而导致功能障碍的一类疾病,包括肌萎缩侧索硬化症、帕金森病、阿尔茨海默病以及脊髓性肌萎缩症等。目前,这类疾病病因尚不明确也无法治愈,严重威胁着人类健康与日常生活。
以帕金森病(Parkinson disease,PD)为例,PD是严重危害中老年人身体健康的常见的慢性神经退行性疾病,病理改变是脑部多巴胺能神经元退行性病变。全世界约有1000万人患有帕金森病。我国PD的发病率为81人/10万,起病年龄平均为55岁。随着社会人口的老龄化,其患病率逐年上升,死亡率亦逐年增加。
PD典型的临床症状包括有静止性震颤、肢体僵硬、运动迟缓、平衡障碍等。多种因素包括遗传、环境毒素、氧化应激、自身免疫、细胞凋亡等密切相互作用,导致中脑黑质多巴胺能神经元进行性变性、坏死,脑内与运动控制、记忆、认知相关的神经递质多巴胺含量减少。而且,神经毒性蛋白聚集物也会引起神经炎症反应,从而进一步导致神经元缺失,形成恶性循环并加剧疾病的进展。研究表明,至少有50%以上的多巴胺能神经元丧失时才会出现临床症状。
目前对帕金森病的治疗方法是补充多巴胺药物的对症治疗,不能阻止疾病的进展。随着PD病情的不断进展,需要逐渐加大药物的剂量才能维持疗效,其药物不良反应问题亦逐渐显现,如出现肌肉抽动、尿频、便秘等;外科治疗例如立体定向射频毁损和DBS手术治疗,仅对部分PD患者有效,但并不适用于所有病人。
目前,其他神经退行性疾病的治疗方法也存在各种不足。近年来,国际医学界一直希望能够找到对因治疗神经退行性疾病的方法,其中干细胞移植治疗备受关注。随着研究的不断深入,科学家逐渐将研究的重点放在了人源神经干细胞、视网膜色素上皮干细胞、羊膜上皮干细胞上,它们被认为是最有可能成为神经退行性疾病移植治疗的理想细胞种子来源。
人羊膜上皮干细胞(human amniotic epithelial stem cells,hAESCs)来源于孕妇产后废弃的胎盘羊膜。研究证实hAESCs具有多巴胺能神经元功能,能够分泌多巴胺以及其它相关神经递质,并且hAESCs能够分泌并释放多种神经营养因子,促进神经组织内源性修复。另一方面,hAESCs具有调节免疫的功能,可以抑制神经组织内炎性反应,从而减少了由神经炎性反应所致的持续神经组织损伤。与其它干细胞不同的是,hAESCs没有端粒酶,不能无限增殖,移植时无致瘤性。而且,该细胞来源广泛、没有伦理限制、具有较好的应用安全性。
在应用hAESCs治疗PD动物模型的研究中,其安全性和有效性已经得到验证。实验证实,hAESCs体内移植显著增高纹状体区及脑脊液中DA及其代谢产物含量,在脑损伤区域的酪氨酸羟化酶阳性神经元数量亦显著增多;移植到侧脑室的hAESCs能够贴壁生长、迁移到受损区域,促进内源性神经干细胞分化。
除了干细胞的种子来源,尚有诸多因素影响干细胞治疗效果,例如细胞移植方式、移植数量、细胞存活率等等。多数研究认为,外源移植的干细胞主要不是以神经整合方式发挥细胞替代修复作用,而是以旁分泌神经递质或营养因子、发挥免疫调节功能而起到神经修复作用。研究证实,直接移植干细胞进入脑内,可以极大地发挥其治疗作用。干细胞的脑内移植离不开立体定向或机器人辅助技术。采用无框架机器人辅助下的脑内神经核团或脑室内干细胞移植技术,能够真正实现精准的干细胞脑内移植,其移植特点是创伤小、定位精准(定位误差不超过1毫米)。
侧脑室移植相对于纹状体移植,未来的应用会更安全、更方便和更广泛,因为作为脑实质的纹状体不适合长期治疗的反复穿刺,这样会造成二次损伤;而单次移植只能在短期内控制疾病进程。所以,为了长期有效治疗,侧脑室移植可能是最佳的移植方式。而移植到侧脑室的干细胞会贴壁迁移到受损区域发挥作用,并促进内源性的神经干细胞分化;通过Ommaya药物囊,可以实现经头皮下多次脑室内注射干细胞,这种方法对患者的损伤最小。文献报道,脐带、骨髓等来源的间充质干细胞联合Ommaya囊用于阿尔茨海默氏病、肌萎缩侧索硬化和中风等疾病的研究[1~3]。但目前未有人羊膜上皮干细胞联合Ommaya囊用于治疗神经退行性疾病的报道。
参考文献:
[1]Lee J,et al.Cerebrospinal fluid from Alzheimer's disease patientsas an optimal formulation for therapeutic application of mesenchymal stemcells in Alzheimer's disease.Scientific Reports,24Jan 2019,9(1):564.
[2]W Baek,et al.Stem cell transplantation into the intraventricularspace via an Ommaya reservoir in a patient with amyotrophic lateral sclerosis[J].Journal of Neurosurgical Sciences,2012,56(3):261-263.
[3]Fauzi A A,et al.Intraventricular Transplantation of AutologousBone Marrow Mesenchymal Stem Cells via Ommaya Reservoir in PersistentVegetative State Patients after Haemorrhagic Stroke:Report of Two Cases&Review of the Literature:[J].Journal of Stem Cells&Regenerative Medicine,2016,12(2):100-104.
发明内容
本发明针对现有技术的不足,提供了一种治疗神经退行性疾病的Ommaya囊及其制备方法。
为实现上述目的,本发明采用以下技术方案:
本发明的第一方面提供了一种治疗神经退行性疾病的Ommaya囊,该Ommaya囊内含有人羊膜上皮干细胞。
进一步地,上述人羊膜上皮干细胞的浓度为3000-7000万个细胞/2毫升。
进一步优选地,人羊膜上皮干细胞的浓度为5000万个细胞/2毫升。
进一步地,上述人羊膜上皮干细胞通过以下方法制备:
步骤一,获取39周以前剖腹产婴儿胎盘,从胎盘内面剥离羊膜;
步骤二,采用0.05%胰酶/EDTA对洗净的羊膜进行消化,然后加入与消化液等体积的消化终止液终止消化;然后离心,弃掉上清液;
步骤三,再采用羊膜完全培养基重悬沉淀,然后加入与所述羊膜完全培养基等体积的消化终止液,离心,弃掉上清液;用羊膜完全培养基重悬沉淀,最后过筛,计数,将所得所述人羊膜上皮干细胞接种至培养皿中或置于冻存液中液氮冻存备用。
本发明的第二方面是提供上述治疗神经退行性疾病的Ommaya囊的制备方法,包括如下步骤:
步骤一,将受试者的头部MRI、神经传导束DTI检测术、CT定位数据依次输入机器人手术计划系统,进行侧脑室前角穿刺点定位和操作规划;
步骤二,对受试者进行全麻;受试者取平卧位,以无创塑型软垫做头颈部充气固定;
步骤三,按定位在前额头皮切2.5厘米直切口,钻孔5毫米1枚,在机器人机械臂引导下进行侧脑室前角穿刺,见脑脊液流出后,穿刺管连接Ommaya囊,并在头皮下埋置Ommaya囊;
步骤四,Ommaya囊安装完成后,依次消毒后缝合头皮;以微量注射器经头皮下抽吸所述Ommaya囊内脑脊液用以判断Ommaya囊是否安装成功;
步骤五,5-7天时,在确定受试者术后安全情况下,先抽取与细胞悬液等量的脑脊液,然后将人羊膜上皮干细胞注射入Ommaya囊,便制备了治疗神经退行性疾病的Ommaya囊。
进一步地,步骤五中人羊膜上皮干细胞注射的次数为4~8次,每次注射间隔的时间为1~3个月。
本发明采用以上技术方案,与现有技术相比,具有如下技术效果:
本发明提供的一种治疗神经退行性疾病的Ommaya囊可实现脑室内移植的精准定位,可确保干细胞移植成功,此外,该Ommaya囊可以进行1000次穿刺而不损坏,实现了给予神经退行性疾病患者多次移植人羊膜上皮干细胞的目的。采用该Ommaya囊进行治疗神经退行性疾病,创伤小、安全性高,治疗效果好,同时该Ommaya囊也有望成为神经系统疑难疾病干细胞移植治疗的特色技术。
附图说明
图1为本发明一实施例中帕金森病受试者的UPDRS表开期评分结果;
图2为本发明一实施例中帕金森病受试者的UPDRS表关期评分结果;
图3为本发明一实施例中帕金森病受试者的SCHWAB&ENGLAND评分结果;
图4为本发明一实施例中帕金森病受试者的PDQ-39生存质量量表评分结果;
图5为本发明一实施例中帕金森病受试者的汉密尔顿抑郁量表评分结果;
图6为本发明一实施例中帕金森病受试者的健康调查简表(SF-36)评分结果。
具体实施方式
本发明提供了一种治疗神经退行性疾病的Ommaya囊,该Ommaya囊内含有人羊膜上皮干细胞。
在本发明一优选的实施例中,上述人羊膜上皮干细胞的浓度为3000-7000万个细胞/2毫升。更优地,人羊膜上皮干细胞的浓度为5000万个细胞/2毫升。
在本发明一优选的实施例中,上述人羊膜上皮干细胞通过以下方法制备:
步骤一,获取39周以前剖腹产婴儿胎盘,从胎盘内面剥离羊膜;
步骤二,采用0.05%胰酶/EDTA对洗净的羊膜进行消化,然后加入与消化液等体积的消化终止液终止消化;然后离心,弃掉上清液;
步骤三,再采用羊膜完全培养基重悬沉淀,然后加入与所述羊膜完全培养基等体积的消化终止液,离心,弃掉上清液;用羊膜完全培养基重悬沉淀,最后过筛,计数,将所得所述人羊膜上皮干细胞接种至培养皿中或置于冻存液中液氮冻存备用。
本发明还提供了上述治疗神经退行性疾病的Ommaya囊的制备方法,包括如下步骤:
步骤一,将受试者的头部MRI、神经传导束DTI检测术、CT定位数据依次输入华志机器人手术计划系统,进行侧脑室前角穿刺点定位和操作规划;
步骤二,对受试者进行全麻;受试者取平卧位,以无创塑型软垫做头颈部充气固定;
步骤三,按定位在前额头皮切2.5厘米直切口,钻孔5毫米1枚,在机器人机械臂引导下进行侧脑室前角穿刺,见脑脊液流出后,穿刺管连接Ommaya囊,并在头皮下埋置Ommaya囊;
步骤四,Ommaya囊安装完成后,依次消毒后缝合头皮;以微量注射器经头皮下抽吸所述Ommaya囊内脑脊液用以判断Ommaya囊是否安装成功;
步骤五,5-7天时,在颅脑CT等检查确定受试者术后安全情况下,先抽取2毫升脑脊液,然后将人羊膜上皮干细胞注射入Ommaya囊,便制备了治疗神经退行性疾病的Ommaya囊。
在本发明一优选的实施例中,步骤五中人羊膜上皮干细胞注射的次数为4~8次,每次注射间隔的时间为1~3个月。
下面通过具体实施例对本发明进行详细和具体的介绍,以使更好的理解本发明,但是下述实施例并不限制本发明范围。
实施例1
本实施例提供临床级别人羊膜上皮干细胞的制备方法,为了避免产道微生物污染,选用了剖腹产胎儿胎盘。由于足月后分娩信号刺激,羊膜会发生凋亡,因此宜用早产胎儿胎盘(38周以前)。经产妇授权同意后,取健康产妇(HIV、梅毒、甲肝、乙肝、丙肝等血清学反应均显示为阴性)剖腹产后的胎盘组织,十字刀切割胎盘,通过机械分离得到整张羊膜。该制备方法具体包括如下步骤:
步骤一,获取39周以前剖腹产婴儿胎盘,从胎盘内面剥离羊膜,将之浸入含有F12/DMEM(含1×青霉素-链霉素和两性霉素)基础培养基的离心管中。4℃冷链运送至实验室细胞间。
步骤二,将羊膜取出,并将每张羊膜置于40ml CMF-HBSS(含1×青霉素-链霉素和两性霉素)中清洗去粘液,并用镊子刮去贴近绒毛膜层的间充质层及粘液,重复3次,每次洗涤均换新容器和新HBSS液。将洗净的羊膜转入新容器,加10ml 0.05%胰酶/EDTA,颠倒30s,弃液。将羊膜转入新容器,加20ml 0.05%胰酶/EDTA,37℃水浴孵育10min弃液。羊膜转入新容器,25ml胰酶/EDTA 37℃水浴孵育40min,保存消化液。
步骤三,初次消化后的羊膜转入新容器,25ml胰酶/EDTA 37℃水浴孵育40min,保存消化液。加入等体积消化终止液(F12/DMEM含5%FBS,1×L-谷氨酸,1×丙酮酸),400g离心10min。
步骤四,弃掉上清液,用羊膜完全培养基(F12/DMEM含5%KSR(KnockOut血清替代物)、1×L-谷氨酰胺、1×丙酮酸、1×ps(青霉素-链霉素))重悬沉淀。加入等体积消化终止液,400g离心10min。弃液,再用完全培养基重悬沉淀。过100μm筛,计数,以105cells/cm2接种至培养皿或置于冻存液(90%FBS,10%DMSO)中液氮冻存备用。
步骤五,对所得人羊膜上皮细胞进行检测:接种1×107个细胞后,待hAESCs贴壁后换培养液,之后三天换一次培养液。待细胞长满平板后将细胞消化下来进行冻存:15cm培养皿加5ml胰酶,10min后镜下观察,细胞变圆且平面晃动平皿时细胞全变为悬浮状态时加等量消化终止液终止消化。用微量移液器按同一方向将培养皿上的细胞吹下,移入15ml离心管,300g离心3min后收集细胞然后进行细胞计数。在冻存管内加入冻存液,标明冻存日期、批次及细胞数量之后将细胞放入冻存管,然后立刻将冻存管放入冻存盒中并将冻存盒放进-80℃冰箱,12h后取出冻存盒,将细胞移入液氮罐中保存。经检测,所得人羊膜上皮细胞纯度高,上皮细胞标记物CD324阳性率>95%;无血细胞污染,血细胞标志物CD34、CD45均<2%。
实施例2
本实施例提供采用本发明提供的治疗帕金森病的Ommaya囊治疗帕金森的方法,该方法采用无框架脑立体定向技术实现了脑内神经核团和脑室内移植的精准定位,定位误差不超过1毫米,靶向穿刺技术是确保干细胞移植成功的关键。安装Ommaya药物囊于前额头皮下,药物囊导管端直达前额侧脑室。Ommaya药物囊可以进行1000次穿刺而不损坏,实现了给予PD患者多次移植hAESCs的目的。该项技术采用全麻手术,减少了因病人异常活动造成的损伤,头部切口一个,仅2.5厘米,颅骨钻孔5毫米,手术出血10毫升以内。该移植技术具有创伤小、安全性高、可重复移植干细胞优点,有望成为神经系统疑难疾病干细胞移植治疗的特色技术。
以下所提到的PD患者都是经过初步筛查后,住院进行全面检查和PD相关量表评估,并签署了临床研究知情同意书、手术知情同意书和干细胞移植知情同意书等等告知书。
具体地,上述方法包括如下步骤:
1.PD患者术前做1毫米薄层头部MRI扫描,拷贝扫描数据;手术当天,在PD患者前额头部贴定位Marker,CT室进行定位扫描同时拷贝数据,将该受试者的头部MRI、神经传导束DTI检测术、CT定位数据依次输入华志机器人手术计划系统,进行侧脑室前角穿刺点定位和手术规划;
2.对PD患者进行全麻,全麻生效后,PD受试者取平卧位,以无创塑型软垫做头颈部充气固定;按定位在前额头皮切2.5厘米直切口,钻孔5毫米1枚,在机器人机械臂引导下进行侧脑室前角穿刺,见脑脊液流出后,穿刺管连接Ommaya,并在头皮下埋置Ommaya囊。Ommaya囊安装完成后,依次消毒后缝合头皮。以微量注射器经头皮下抽吸Ommaya囊内脑脊液,以顺利抽出脑脊液视为手术成功。
3.术后密切观察受试者生命体征,排除颅内出血、水肿、感染等情况。在术后5-7天时,在确定PD患者术后安全情况下,先抽取2毫升脑脊液,然后将5000万个细胞/2毫升的hAESCs经Ommaya囊注射至受试者侧脑室内,随后严密观察3天后出院。
4.在术后5-7天、术后1月±5天、2月±5天、3月±5天,每次移植细胞量为5000万(2毫升);根据PD患者的治疗情况,移植的次数为4~8次,每次注射间隔的时间为1~3个月。细胞移植前,先对Ommaya囊局部消毒、采用经头皮下穿刺Ommaya囊注射法,不需要麻醉,全程2分钟即可完成干细胞移植治疗。
验证实施例
在临床实验中,每位PD受试者通过Ommaya囊接受了4次脑室内hAESCs精准移植,并在不同治疗时间点,接受有效性评价。统计了各种量表的评分情况,结果如图1-6所示。
由图1可知,经过干细胞移植后,PD受试者在开期的UPDRS评分呈现下降趋势,证明其临床症状正在逐渐好转。由图2可知,经过干细胞移植后,PD受试者在关期的UPDRS评分呈现下降趋势,显示其临床症状获得一定程度改善。由图3可知,经过干细胞移植后,PD受试者的SCHWAB&ENGLAND评分呈现下降趋势,显示其日常生活能力受限程度降低。由图4可知,经过干细胞移植后,PD受试者的PDQ-39生存质量的量表评分呈现下降趋势,显示其生存质量在好转。由图5可知,经过干细胞移植后,PD受试者的汉密尔顿抑郁量表评分呈现下降趋势,显示其抑郁状态获得改善。由图6可知,经过干细胞移植后,PD受试者的健康调查简表(SF-36)评分呈现上升趋势,显示其健康状况逐渐好转。
此外,未见干细胞脑室内移植后的不良反应和免疫排斥反应,初步证实了hAESCs脑内移植的安全性;5例PD受试者的临床症状和PD量表评分证实,他们的肢体僵硬、震颤和运动迟缓得到了一定程度改善,抑郁、焦虑症状好转,睡眠质量提高和饮食增加,面部皮肤变得细腻光滑,大便干燥和多尿等情况亦出现好转。
以上对本发明的具体实施例进行了详细描述,但其只是作为范例,本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言,任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此,在不脱离本发明的精神和范围下所作的均等变换和修改,都应涵盖在本发明的范围内。
Claims (5)
1.一种治疗神经退行性疾病的Ommaya囊,其特征在于,所述Ommaya囊内含有人羊膜上皮干细胞。
2.根据权利要求1中所述治疗神经退行性疾病的Ommaya囊,其特征在于,所述人羊膜上皮干细胞的浓度为3000-7000万个细胞/2毫升。
3.根据权利要求1所述治疗神经退行性疾病的Ommaya囊,其特征在于,所述人羊膜上皮干细胞通过以下方法制备:
步骤一,获取39周以前剖腹产婴儿胎盘,从胎盘内面剥离羊膜;
步骤二,采用0.05%胰酶/EDTA对洗净的羊膜进行消化,然后加入与消化液等体积的消化终止液终止消化;然后离心,弃掉上清液;
步骤三,再采用羊膜完全培养基重悬沉淀,然后加入与羊膜完全培养基等体积的消化终止液,离心,弃掉上清液;用羊膜完全培养基重悬沉淀,最后过筛,计数,将所得所述人羊膜上皮干细胞接种至培养皿中或置于冻存液中液氮冻存备用。
4.一种如权利要求1-3任一项所述治疗神经退行性疾病的Ommaya囊的制备方法,其特征在于,包括如下步骤:
步骤一,将神经退行性疾病受试者的头部MRI、神经传导束DTI检测术、CT定位数据依次输入机器人手术计划系统,进行侧脑室前角穿刺点定位和操作规划;
步骤二,对所述受试者进行全麻;所述受试者取平卧位,以无创塑型软垫做头颈部充气固定;
步骤三,按定位在所述受试者前额头皮切2.5厘米直切口,钻孔5毫米1枚,在机器人机械臂引导下进行侧脑室前角穿刺,见脑脊液流出后,穿刺管连接Ommaya囊,并在头皮下埋置所述Ommaya囊;
步骤四,所述Ommaya囊安装完成后,依次消毒后缝合头皮;以微量注射器经头皮下抽吸所述Ommaya囊内脑脊液用以判断Ommaya囊是否安装成功;
步骤五,5-7天时,在确定所述受试者术后安全情况下,先抽取与细胞悬液等量的脑脊液,然后将所述人羊膜上皮干细胞注射入所述Ommaya囊,便制备了所述治疗神经退行性疾病的Ommaya囊。
5.根据权利要求4所述治疗神经退行性疾病的Ommaya囊的制备方法,其特征在于,步骤五所述人羊膜上皮干细胞注射的次数为4~8次,每次注射间隔的时间为1-3个月。
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