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CN104073468B - The method of obtaining human OPCs OPCs and media - Google Patents

The method of obtaining human OPCs OPCs and media Download PDF

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CN104073468B
CN104073468B CN 201410336947 CN201410336947A CN104073468B CN 104073468 B CN104073468 B CN 104073468B CN 201410336947 CN201410336947 CN 201410336947 CN 201410336947 A CN201410336947 A CN 201410336947A CN 104073468 B CN104073468 B CN 104073468B
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栾佐
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栾佐
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Abstract

本发明涉及获得人源OPCs的方法和OPCs培养基。 The present invention relates to a method and a medium obtained humanized OPCs of OPCs. 本发明将神经干细胞作为分选来源,通过免疫磁珠的方法分离得到人源OPCs,并找到了适合OPCs体外培养和传代的较经济型的培养基,可以在短时间内获得大量的高纯度的人源OPCs,为临床应用提供了新的选择。 The neural stem cells of the present invention as a source of sorting, isolated OPCs by human immune magnetic beads, and find a more economical medium suitable OPCs and passaged in vitro, a large amount of high purity can be obtained in a short time human OPCs, provides a new choice for clinical application.

Description

获得人源OPCS的方法和OPCs培养基 The method of obtaining human OPCS and medium OPCs

技术领域 FIELD

[0001 ]本发明涉及获得人源OPCs的方法和OPCs培养基。 [0001] The present invention relates to a method and a medium obtained humanized OPCs of OPCs.

背景技术 Background technique

[0002] 脑白质损伤、脊髓损伤、多发性硬化等髓鞘相关疾病由于神经元的髓鞘化障碍或脱髓鞘化,导致了神经元不能正常传递电兴奋,从而导致机体运动等功能失能。 [0002] white matter injury, spinal cord injury, multiple sclerosis and other myelin-associated diseases due myelination of neurons, or demyelination disorders, resulting in neuronal excitability not transmitted properly, resulting in movement of the body functions disabled . 由于目前还没有任何一种药物可以有效地治疗这些髓鞘相关疾病,这给这些疾病的患者及家庭带来了极大的伤害,这使得有效治疗髓鞘相关疾病成为当今急待解决的社会和医学难题。 Because there is no one drug can effectively treat these myelin-related diseases, which gives patients and families of these diseases has brought great harm, which makes it effective in treating myelin-associated diseases become a pressing problem in today's society and medical problem.

[0003] 不论是髓鞘化障碍还是脱髓鞘病变,其根源都是神经元轴突髓鞘程度减少或丢失,因此如果能够恢复神经元轴突的髓鞘化程度,使得神经元电流得以稳定快速地传导,理论上这些疾病就可以被治愈。 [0003] Whether myelination disorders or demyelinating disease, its causes are reduced or lost myelination of axons degree, so if we can restore the degree of myelination of axons and neurons make current stabilized rapidly conducted, in theory, these diseases can be cured. 研究发现,少突胶质前体细胞(oligodendrocyte progenitor ce 11 s,OPCs)是负责中枢神经系统髓鞘化的关键细胞,在中枢神经系统中,OPCs可以分化为少突胶质细胞(oligodendrocyte,0L),0L进一步包绕神经元轴突形成完整的髓鞘结构,从而保证神经元电流的稳定快速传导。 Found, oligodendrocyte precursor cells (oligodendrocyte progenitor ce 11 s, OPCs) are responsible for the central nervous system myelination key cells in the central nervous system, the OPCs can differentiate into oligodendrocytes (oligodendrocyte, 0L ), 0L further formed surrounding the axons intact myelin sheath, thereby ensuring stable fast neuronal conduction currents. 髓鞘相关的疾病一方面是因为疾病破坏了已经形成的髓鞘结构;另一方面更是由于神经系统中OPCs受到了损伤,使得0L失去了源泉。 Myelin-associated disease partly because the disease destroys the myelin sheath has been formed; on the other hand is due to the OPCs nervous system has been damaged, making 0L lost their source. 少突胶质细胞减少,导致脑白质髓鞘化延迟或被破坏,从而使得神经电信号传导异常或者轴突受损,导致神经功能损伤。 Reducing oligodendrocytes, resulting in delay of white matter myelination or destroyed, such that electrical conduction abnormalities or axonal nerve damage, resulting in neurological injury.

[0004] 对脱髓鞘病变患者来说,由于内源性OPCs大量丢失,要促进脑中髓鞘的再生,植入外源成髓鞘细胞是一个有希望的治疗方法。 [0004] For demyelinating lesions, by endogenous OPCs considerable loss of myelin in the brain to promote regeneration, implant exogenous myelinating cells is a promising method of treatment. 由于0PCS是中枢神经系统中成髓鞘细胞的天然来源,OPCs移植可能对治疗脱髓鞘病变起到很好的疗效。 Since 0PCS central nervous system myelin cells into natural sources, OPCs transplant may play a very good effect on the treatment of demyelinating lesions. 实验发现,将啮齿类动物的OPCs移植到新生的啮齿类动物脑内,植入细胞能在宿主脑内广泛迀移、增殖并分化为成熟0L,形成包绕轴突的髓鞘结构并恢复神经功能,表明外源植入OPCs能发挥与内源OPCs同样的功能, OPCs移植可能是治疗髓鞘损伤疾病的一个新的方向,这对脱髓鞘病变治疗具有积极的意义。 It was found that the transplanted rodent OPCs to newborn rodent brain, the implanted cells can be widely Gan host brain shift, proliferate and differentiate into mature 0L, form myelin sheath surrounding nerve axons and recovery function, suggesting that the foreign implanted OPCs endogenous OPCs can play the same function, OPCs transplant may be a new direction for the treatment of diseases of myelin damage, which has positive significance for the treatment of demyelinating lesions.

[0005] 尽管啮齿类动物的OPCs很容易获得,人源OPCs获得却相当困难。 [0005] Although rodent OPCs are readily available source of OPCs people get quite difficult. 依赖于免疫表型, 通过流式细胞术或是免疫磁珠技术从胎儿中枢神经系统组织或是成人脑白质组织中分选可以得到OPCsJindrem等从胎儿或是成人脑组织中分离出了A2B5+PSA-NCAiT表型的细胞, 认为该细胞即为OPCs,将该细胞移植入先天脱髓鞘的小鼠内,可以形成髓鞘。 Depends on the phenotype, can be selected from other OPCsJindrem isolated from fetal or adult brain tissue A2B5 + PSA from fetal tissue or adult central nervous system white matter tissue equatorial by flow cytometry or MACS technology -NCAiT phenotype, namely the OPCs that the cells, the cells are transplanted into mice innate demyelination, may form myelin. 然而由于人源的供体组织较少,分离得到的OPCs数量相当有限,且其存在伦理学问题,因此距离临床应用还非常遥远。 However, due to less human donor tissue, the number of isolated OPCs are limited, and it is present ethical problems, and therefore still very far from clinical application. 另一种可以通过胚胎干细胞或是神经干细胞定向诱导分化获得人源OPCs。 Another can be induced to differentiate by obtaining human embryonic stem cells or OPCs neural stem cells. 美国的Hans等人研究发现,胚胎干细胞体外可以分化为OPCs,且OPCs移植有助于脊髓损伤的神经功能恢复,该方法在美国进入了临床I期的治疗研究。 American Hans et al found that embryonic stem cells can differentiate into OPCs, and the OPCs transplant helps restore neurological function in spinal cord injury, which the United States has entered a Phase I clinical study of the treatment. 但胚胎干细胞的全能性是把双刃剑,既能分化为各种细胞,也有致瘤的风险,因此该方法获得的OPCs在临床应用上还存在一定的致瘤风险。 But totipotent embryonic stem cells are double-edged sword, both differentiate into various cells, but also the risk of tumorigenic, and therefore the OPCs obtained by the method in clinical applications there is still a risk of tumorigenicity. 神经干细胞是一种成体干细胞,由于成体干细胞分化能力受到限制,在一定程度上避免了致瘤的风险,临床应用上更为安全。 Neural stem cell is an adult stem cells, since adult stem cell differentiation is restricted from tumorigenic risk to some extent, safer clinical application. 但这两种细胞的诱导过程都需要较长时间及多种细胞因子的共同作用,这必然导致培养的费用增加,限制细胞的产量。 But both cell induction process will require joint action over a longer time and a variety of cytokines, which will inevitably lead to increased costs of cultivation, to limit production cells.

[0006] 因此,临床的治疗研究需要一种简单获得且经济培养人源OPCs的方法。 [0006] Therefore, the clinical treatment requires a simple and economical method to obtain cultured human source of OPCs. 神经干细胞体外有分化为神经元、星形胶质细胞和少突胶质细胞的能力,且约有16%的细胞表达A 2B5 +PSA-NCAIT表型,同时由于是成体干细胞又在一定程度上避免了致瘤的风险。 Neural stem cells differentiate into neurons, astrocytes and oligodendrocytes cells, and about 16% of the cells express A 2B5 + PSA-NCAIT phenotype, and because they are adult stem cells to a certain extent to avoid the risk of tumorigenic. 神经干细胞还可以在体外大量扩增,可以说神经干细胞是很好的分选OPCs的来源。 Neural stem cells can also be a large number of in vitro amplification can be said neural stem cell is a good source of OPCs sorting.

发明内容 SUMMARY

[0007] 本发明的目的是解决上述问题,提供一种获得人源OPCs的新方法。 [0007] The object of the present invention is to solve the above problems, there is provided a new method for obtaining a human of OPCs. 为此,申请人经过深入研究,将神经干细胞(不论是低代数还是较高代数)作为分选来源,通过免疫磁珠的方法分离得到人源OPCs,并找到了适合OPCs体外培养和传代的较经济型的培养基,可以在短时间内获得大量的高纯度的人源0PCS,为临床应用提供了新的选择。 To this end, the applicant of intensive studies, neural stem cells (either high or low Algebra Algebra) as a source of sorting, humanized OPCs obtained by immunomagnetic bead separation method and found more suitable OPCs and passaged in vitro economical medium, can get a lot of high purity in a short time human 0PCS, provides a new choice for clinical application.

[0008] 本发明提供的获得人源OPCs的方法为,将神经干细胞作为分选来源,通过免疫磁珠的方法分离得到人源OPCs,采用的磁珠为Ant i -PSA-NCAM磁珠。 [0008] The present invention provides a method for obtaining human OPCs, neural stem cells as the source of sorting, isolated OPCs by human immune magnetic beads, magnetic beads used was Ant i -PSA-NCAM beads.

[0009] 优选地,包括以下步骤: [0009] Preferably, the steps comprising:

[0010] ①神经干细胞在胰蛋白酶溶液中被消化成单个细胞,过筛网; [0010] ① neural stem cells into single cells is digested in trypsin solution, through the screen;

[0011] ②离心收集细胞,去上清; [0011] ② The cells were collected by centrifugation, the supernatant was discarded;

[0012] ③细胞重悬在磁珠分选缓冲液中,混匀后在2_8°C中温育; [0012] ③ cells were resuspended in MACS buffer after mixing in 2_8 ° C incubation;

[0013] ④加入Anti-PSA-NCAM磁珠,混匀后在2-8°C中温育; [0013] ④ added Anti-PSA-NCAM beads, at 2-8 ° C incubation mix;

[0014] ⑤加入磁珠分选缓冲液,离心; [0014] ⑤ magnetic bead sorting buffer was added and centrifuged;

[0015] ⑥离心过程中,将分选柱放入磁珠分离器中,加入磁珠分选缓冲液平衡分选柱; [0015] ⑥ during centrifugation, the separation column into the magnetic bead separation vessel, MACS separation column equilibrated with a buffer;

[0016] ⑦离心后,完全弃去上清液,重悬于磁珠分选缓冲液,将细胞悬液加入到分选柱内,收集流出液体,此为未标记的细胞; [0016] ⑦ After centrifugation, the supernatant was discarded completely, resuspended in MACS buffer, the cell suspension was added to the separation column, the liquid effluent is collected, the unlabeled cells;

[0017] ⑧加入磁珠分选缓冲液洗分离柱,离心收集细胞; [0017] ⑧ added MACS separation column wash buffer, cells were collected by centrifugation;

[0018] ⑨加入Ant i-A2B5磁珠,温育,重复⑤-⑧步骤,此次收集细胞应为柱内集合上的细胞,缓冲液洗柱后,将分选柱从磁珠分尚器内取出放置在尚心管内; [0018] ⑨ Ant i-A2B5 beads was added and incubated, repeat step ⑤-⑧, the cells were collected by a cell should be set on the column, the buffer, washing the column, the separation column from the magnetic separation is still remove the core pipe is still placed within;

[0019] ⑩加入磁珠分选缓冲液后,立即推压活塞洗脱掉结合上的细胞,离心,收集到的细胞为型的亚细胞群,即为OPCs。 After [0019] ⑩ magnetic bead sorting buffer added, immediately pushing the piston eluted cells, the combination of centrifugation, collected cells subpopulations type, namely OPCs.

[0020] 更优选地,各步骤的具体操作方法如下: [0020] More preferably, the specific operation method, the steps are as follows:

[0021]①神经干细胞在0.025%胰蛋白酶溶液中被消化成单个细胞,过40μπι尼龙筛网以滤除细胞团块,台酚蓝计算细胞总数;以上"%"是"g/l〇〇ml"的惯常简略表示方式,0.025% 即为0.025g/100ml。 [0021] ① neural stem cells were digested in 0.025% trypsin solution into individual cells, through a nylon mesh to filter 40μπι cell mass, calculating the total number of trypan blue cell; above, "%" is "g / l〇〇ml "the usual shorthand way, namely 0.025% 0.025g / 100ml.

[0022]②取107细胞,300g离心10分钟收集细胞,去上清; [0022] ② take 107 cells, cells were collected by centrifugation for 10 minutes 300g, the supernatant was discarded;

[0023]③107细胞重悬在60μ1的磁珠分选缓冲液中,混匀后在2-8°C中温育10分钟; [0023] ③107 Cells were resuspended in 60μ1 magnetic beads sorting buffer, after mixing incubated at 2-8 ° C 10 min;

[0024] ④加入20μ1的Anti-PSA-NCAM磁珠,混匀后在2-8°C中温育15分钟; [0024] ④ added to 20μ1 of the Anti-PSA-NCAM beads, mixed at 2-8 ° C after incubating 15 minutes;

[0025]⑤加入lml磁珠分选缓冲液,300g离心10分钟; [0025] ⑤ added lml magnetic bead sorting buffer, 300g centrifuged for 10 minutes;

[0026]⑥离心过程中,将MS分选柱放入磁珠分离器中,加入0.5ml的磁珠分选缓冲液平衡分选柱; [0026] ⑥ during centrifugation, the separation column into the MS magnetic bead separation vessel, was added 0.5ml of magnetic bead sorting separation columns equilibrated with a buffer;

[0027]⑦离心后,完全弃去上清液,重悬于0.5ml的磁珠分选缓冲液,将细胞悬液加入到分选柱内,收集流出液体,此为未标记的细胞; [0027] ⑦ After centrifugation, the supernatant was discarded completely, resuspended in 0.5ml beads sorting buffer, the cell suspension was added to the separation column, the liquid effluent is collected, the unlabeled cells;

[0028]⑧加入0.5ml的磁珠分选缓冲液洗分离柱,共三次,离心收集细胞; [0028] ⑧ added 0.5ml of wash buffer magnetic bead sorting separation columns, a total of three times, the cells were collected by centrifugation;

[0029] ⑨加入20μ1的Anti-A2B5磁珠,温育,重复上面⑤-⑧的步骤,此次收集细胞应为柱内集合上的细胞,缓冲液洗柱三次后,将分选柱从磁珠分离器内取出放置在1.5ml的离心管内; [0029] ⑨ added to 20μ1 of Anti-A2B5 beads, incubation, repeat the above steps ⑤-⑧, and the cells were collected by a cell should be set on the column, the column was washed three times with the buffer, the magnetic fraction from the column is selected from taking out the beads separator in 1.5ml centrifuge tube;

[0030] ⑩加入lml磁珠分选缓冲液后,立即推压活塞洗脱掉结合上的细胞,300g离心10分钟,收集到的细胞为型的亚细胞群,即为OPCs。 After [0030] ⑩ added lml magnetic bead sorting buffer, eluted immediately pressing piston on cell binding, 300g centrifugation for 10 minutes, the cells collected subpopulations type, namely OPCs.

[0031] 优选地,利用OPCs培养基对分离得到的人源OPCs进行体外扩增培养,所述培养基由Neurobasal A 1116(1;[11111、1327、匕?6?、肝素和1^飞11^&111;[11组成,如111'(^&8&14 1116(1;[11111、1327和L-glutamin的体积比为97:2:1,bFGF的含量为20-40ng/ml,肝素的含量为5μg/ml。 [0031] Preferably, the culture medium for human use OPCs isolated OPCs expanded in vitro culture, said medium consisting of Neurobasal A 1116 (1;? [11111,1327, dagger 1 ^ 6 ?, heparin and fly 11 ^ & 111; [11 composition, such as 111 '(^ & 8 & 141116 (1; [11111,1327 and L-glutamin volume ratio of 97: 2: 1, bFGF content of 20-40ng / ml, the content of heparin was 5μg / ml.

[0032] 更优选地,体外扩增培养的具体操作方法为:磁珠分选后得到的细胞按照4X104/ cm2密度接种至培养器皿内,在OPCs培养基中培养,置于37°C、5%C02培养箱内,每3-4天,更换2/3培养基为新鲜OPCs培养基,细胞达到80 %-90 %的融合度时,传代培养,传代培养的次数为2-4次。 [0032] More preferably, the specific operation method is cultured in vitro amplification: beads obtained after sorting cells in accordance with 4X104 / cm2 seeded into the culture vessel, the culture medium in OPCs, placed in 37 ° C, 5 % C02 incubator within every 3-4 days, changing the medium with fresh OPCs 2/3 medium, the cells reached 80% -90% confluency, subculture, subculture frequency is 2-4 times.

[0033] 优选地,所述神经干细胞采用神经干细胞培养基进行体外扩增而得,所述神经干细胞培养基由〇1^0/^12、呢士?6?』6?、肝素和1^飞11^311^11组成,01^0/^12、呢和1^111^311^11 的体积比为98:1:1,bFGF的浓度为10-30ng/ml,EGF的浓度为10-30ng/ml。 [0033] Preferably, the neural stem cells, neural stem cells expanded in vitro derived culture, the neural stem cell medium consisting 〇1 0 ^ / ^ 12, it disabilities? 6? '?, heparin and 1 ^ 6 311 ^ 11 ^ 11 fly composition, the volume of 01 ^ 0/12 ^, 1 ^, and then 311 ^ 111 ^ 11 ratio of 98: 1: 1, bFGF at a concentration of 10-30ng / ml, the concentration of EGF is 10- 30ng / ml.

[0034] 更优选地,神经干细胞体外扩增的操作步骤如下: [0034] More preferably, the neural stem cells in vitro amplification procedure is as follows:

[0035]①100g离心5分钟收集神经干细胞; [0035] ①100g centrifuged for 5 minutes to collect the neural stem cells;

[0036]②将上清转移入离心管内,为旧培养基; [0036] ② the supernatant was transferred into a centrifuge tube, the old culture medium;

[0037]③向收集好的神经干细胞内加入lml由0.01M dPBS稀释得到的0.025%胰蛋白酶, 混匀,置37°C培养箱孵育5-7min至细胞球松散; [0037] ③ was added into the neural stem cells collected lml 0.025% trypsin was diluted by a 0.01M dPBS obtained, mixing at 37 ° C incubator until the cells were incubated for 5-7min loose ball;

[0038]④加入1.2mg/ml胰酶抑制剂100μ1,200μ1移液器吸管轻轻吹打成单细胞悬液; [0038] ④ added 1.2mg / ml trypsin inhibitor 100μ1,200μ1 pipette pipette by gently pipetting into single cell suspension;

[0039] ⑤加入15ml dPBS,细胞计数,100g离心5min; [0039] ⑤ added 15ml dPBS, cell count, 100g centrifugation 5min;

[0040] ⑥弃上清,加入2/3新鲜神经干细胞培养基和1/3旧培养基,以2 X 106/mL密度接种于T25ml培养瓶,置细胞培养箱内培养; [0040] ⑥ The supernatant was discarded, a fresh addition of 2/3 and 1/3 neural stem cell culture medium old medium to 2 X 106 / mL culture flask seeded at a density T25ml, cultured cell incubator set;

[0041] ⑦每3-4天更换2/3培养基为新鲜培养基,每10天传代一次。 [0041] ⑦ 2/3 medium was changed every 3-4 days with fresh medium, subcultured once every 10 days.

[0042]本发明提供的获得人源OPCs的方法也包括利用OPCs培养基对其它方式获得的人源OPCs进行体外扩增培养,所述培养基由Neurobasal A medium、B27、bFGF、肝素和L-glutamin组成,Neurobasal A medium、B27和L-glutamin的体积比为97:2:1,bFGF的含量为20-40ng/ml,肝素的含量为5μg/ml。 [0042] A method for obtaining human OPCs of the present invention also provides the use of a human comprising the medium of OPCs OPCs otherwise obtained expanded in vitro culture, said medium consisting of Neurobasal A medium, B27, bFGF, heparin and L- Glutamin composition, Neurobasal a medium, B27, and L-glutamin volume ratio of 97: 2: 1, bFGF content of 20-40ng / ml, heparin content was 5μg / ml.

[0043] 优选地,其它方式获得的人源OPCs体外扩增培养的具体操作方法为:人源OPCs按照4 X 104 /cm2密度接种至培养器皿内,在OPCs培养基中培养,置于37°C、5 % C02培养箱内, 每3-4天,更换2/3培养基为新鲜OPCs培养基,细胞达到80 % -90 %的融合度时,传代培养,传代培养的次数为2-4次。 [0043] Preferably, the specific operation method humanized OPCs otherwise obtained in vitro expansion culture are: human OPCs administered according to 4 X 104 / cm2 to a density within the culture vessel, the culture medium in OPCs, placed in 37 ° C, the within 5% C02 incubator, every 3-4 days, changing the medium with fresh OPCs 2/3 medium, the cells reached 80% -90% confluency, subculturing, the number of subculturing 2-4 times.

[0044] OPCs培养基也是本发明的保护内容,其由Neurobasal A 1^乜11111、8274?6?、肝素和L-glutamin组成,Neurobasal A medium、B27和L-glutamin的体积比为97:2:1,bFGF的含量为20-40ng/ml,肝素的含量为5μg/ml。 ? [0044] OPCs are protected media content of the invention, consisting of Neurobasal A 1 ^ NIE 11111,8274 6 ?, heparin composition and L-glutamin, Neurobasal A medium, B27, and L-glutamin volume ratio of 97: 2 : 1, bFGF content of 20-40ng / ml, heparin content was 5μg / ml.

[0045] 本发明提供的获得人源OPCs的方法易于操作,可以在短时间内获得大量的高纯度的人源〇PCs,为临床应用提供了新的选择。 [0045] The method for obtaining human OPCs of the present invention provides easy to operate, can be obtained in a short time a large amount of high-purity human 〇PCs, provide a new choice for clinical application.

附图说明 BRIEF DESCRIPTION

[0046]图1显示为流式分析分选得到的细胞纯度到达85%以上。 [0046] Figure 1 shows a cell sorting flow analysis purity obtained reaches 85% or more.

[0047]图2显示为分选细胞表达了多种特异OPCs标志:A为分选三天后,细胞为典型的双极或三极形态;BD分别为免疫荧光染色表明分选细胞表达OPCs特异标志04、PDGFaR和SoxlO;E为星形胶质细胞标志GFAP阳性细胞;F为神经元标志Tuj-1阳性细胞。 [0047] FIG. 2 is shown as a plurality of sorted cells express markers specific for OPCs: A is three days after sorting, cells are typically bipolar or tripolar morphology; BD respectively immunofluorescence staining showed that sorting cells expressing specific marker OPCs 04, PDGFaR and SoxlO; E is a marker GFAP-positive astrocytes cells; F. to neuronal marker Tuj-1 positive cells. 比例尺:50μπι (AE),100ym(F)〇 Scale: 50μπι (AE), 100ym (F) billion

[0048]图3显示为分选得到的OPCs可以体外至少培养四代:显微镜观察第二代到第四代OPCs,均为典型的双极或三极形态;免疫荧光染色表明各代数OPCs均高表达少突胶质前体细胞标志04。 [0048] Figure 3 shows sorting of OPCs can be cultured in vitro to obtain at least four generations: microscope OPCs second generation to the fourth generation, are typically bipolar or tripolar morphology; immunofluorescence staining showed that each of the algebraic OPCs are high The expression of oligodendrocyte precursor cell marker 04. 比例尺:50μηι。 Scale: 50μηι.

[0049]图4显示为分选得到的第四代OPCs免疫荧光染色结果:0PCs高表达少突前体细胞标志PDGFaR和SoxlO、极低表达星形胶质细胞标志GFAP和神经元标志Tuj-Ι。 [0049] Figure 4 shows a fourth generation OPCs sorting results of immunofluorescent staining obtained: 0PCs expression of oligodendrocyte precursor cell marker PDGFaR and SoxlO, low expressing markers GFAP astrocytes and neuronal marker Tuj-Ι . 比例尺:50μπι (PDGFaR、Soxl0和GFAP),100ym(Tuj-l)。 Scale: 50μπι (PDGFaR, Soxl0 and GFAP), 100ym (Tuj-l).

[0050]图5显示为第一代到第四代OPCs增殖曲线:经过四代的体外扩增,细胞增殖14倍以上。 [0050] FIG. 5 shows the first generation to the fourth generation OPCs growth curves: four generations in vitro amplification, cell proliferation more than 14 times.

[00511图6显示为OPCs分化为少突细胞。 [00511 FIG. 6 is shown as OPCs differentiate into oligodendrocytes. A:显微镜下观察分化后的细胞形态,细胞呈复杂多分枝结构;B:免疫荧光染色结果表明分化细胞可以表达多分枝的04;C:免疫荧光染色结果表明分化细胞表达Ga 1 c。 A: Cell morphology was observed after differentiation, cells were more complicated branched structure under a microscope; B: Immunofluorescence staining showed that differentiated cells may express multibranched 04; C: Immunofluorescence staining showed that differentiated cells express Ga 1 c. 比例尺:25μπι。 Scale: 25μπι.

具体实施方式 detailed description

[0052]下面为本发明的具体的实施方式。 [0052] The following specific embodiments of the present invention.

[0053] 1从神经干细胞中分选获得OPCs [0053] 1 is selected from neural stem cells obtained equatorial OPCs

[0054] 1.1人神经干细胞体外扩增 [0054] 1.1 In vitro amplification of human neural stem cells

[0055] ① 100g离心5分钟收集神经干细胞; [0055] ① 100g centrifugation for 5 minutes to collect the neural stem cells;

[0056]②将上清转移入离心管内,为旧培养基; [0056] ② the supernatant was transferred into a centrifuge tube, the old culture medium;

[0057]③向收集好的神经干细胞内加入lml 0.025%胰蛋白酶(0.01M dPBS稀释),混匀, 置37°C培养箱孵育5-7min至细胞球松散; [0057] ③ lml 0.025% trypsin was added (diluted with 0.01M dPBS), mixing at 37 ° C incubator for incubating 5-7min loose balls into the cells to neural stem cells collected;

[0058]④加入1.2mg/ml胰酶抑制剂100μ1,200μ1移液器吸管轻轻吹打成单细胞悬液; [0058] ④ added 1.2mg / ml trypsin inhibitor 100μ1,200μ1 pipette pipette by gently pipetting into single cell suspension;

[0059] ⑤加入15ml dPBS,细胞计数,100g离心5min; [0059] ⑤ added 15ml dPBS, cell count, 100g centrifugation 5min;

[0060] ⑥弃上清,加入2/3新鲜神经干细胞培养基和1/3旧培养基,以2 X 106/mL密度接种于T25ml培养瓶,置细胞培养箱内培养。 [0060] ⑥ The supernatant was discarded, a fresh addition of 2/3 and 1/3 neural stem cell culture medium old medium to 2 X 106 / mL culture flask seeded at a density T25ml, cultured cell incubator set.

[0061] ⑦每3-4天更换2/3培养基为新鲜培养基,每10天传代一次。 [0061] ⑦ 2/3 medium was changed every 3-4 days with fresh medium, subcultured once every 10 days.

[0062] 神经干细胞培养基: 组分浓度DMED/F12 98% N2 1% [0062] neural stem cell culture medium: component concentration DMED / F12 98% N2 1%

[0063] bFGF i0-30ng/ml EGF 10-30 ng/ml 肝素5pg/ml L-giutamin 1% [0063] bFGF i0-30ng / ml EGF 10-30 ng / ml heparin 5pg / ml L-giutamin 1%

[0064] 1.2使用磁珠从神经干细胞中分选得到人源的OPCs [0064] 1.2 using magnetic beads selected from the equatorial obtained from neural stem cells of human origin OPCs

[0065] ①神经干细胞在0.025%胰蛋白酶溶液中被消化成单个细胞,过40 μπι尼龙筛网以滤除细胞团块,台酚蓝计算细胞总数; [0065] ① neural stem cells into single cells is digested in 0.025% trypsin solution, 40 μπι through a nylon mesh to filter out the cell aggregates, the total number of cells calculated trypan blue;

[0066] ②取107细胞,300g离心10分钟收集细胞,去上清; [0066] ② take 107 cells, cells were collected by centrifugation for 10 minutes 300g, the supernatant was discarded;

[0067]③107细胞重悬在60μ1的磁珠分选缓冲液中,混匀后在2-8°C中温育10分钟; [0067] ③107 Cells were resuspended in 60μ1 magnetic beads sorting buffer, after mixing incubated at 2-8 ° C 10 min;

[0068] ④加入20μ1的Anti-PSA-NCAM磁珠,混匀后在2-8°C中温育15分钟; [0068] ④ added to 20μ1 of the Anti-PSA-NCAM beads, after mixing incubated at 2-8 ° C 15 min;

[0069]⑤加入lml磁珠分选缓冲液,300g离心10分钟; [0069] ⑤ added lml magnetic bead sorting buffer, 300g centrifuged for 10 minutes;

[0070]⑥离心过程中,将MS分选柱放入磁珠分离器中,加入0.5ml的磁珠分选缓冲液平衡分选柱; [0070] ⑥ during centrifugation, the separation column into the MS magnetic bead separation vessel, was added 0.5ml of magnetic bead sorting separation columns equilibrated with a buffer;

[0071 ]⑦离心后,完全弃去上清液,重悬于0.5ml的磁珠分选缓冲液。 [0071] ⑦ After centrifugation, the supernatant was discarded completely, resuspended in 0.5ml beads sorting buffer. 将细胞悬液加入到分选柱内,收集流出液体,此为未标记的细胞; The cell suspension was added to the separation column, the liquid effluent is collected, the unlabeled cells;

[0072]⑧加入0.5ml的磁珠分选缓冲液洗分离柱,共三次,离心收集细胞; [0072] ⑧ added 0.5ml of wash buffer magnetic bead sorting separation columns, a total of three times, the cells were collected by centrifugation;

[0073]⑨加入Anti-A2B5磁珠,混匀后在2-8°C中温育,重复⑤-⑧步骤,温育后过柱,此次收集细胞应为柱内集合上的细胞,缓冲液洗柱三次后,将MS分选柱从磁珠分离器内取出放置在1.5ml的离心管内; [0073] ⑨ Anti-A2B5 beads were added, the mix was incubated at 2-8 ° C, repeat step ⑤-⑧, passed through the column after the incubation, the cells were collected by a cell should be set on the inner column, buffer after washing the column three times, MS separation column is removed from the separator beads placed within a 1.5ml centrifuge tube;

[0074] ⑩加入lml磁珠分选缓冲液后,立即推压活塞洗脱掉结合上的细胞,300g离心10分钟,收集到的细胞为型的亚细胞群,即为OPCs。 After [0074] ⑩ added lml magnetic bead sorting buffer, eluted immediately pressing piston on cell binding, 300g centrifugation for 10 minutes, the cells collected subpopulations type, namely OPCs.

[0075] 磁珠分选缓冲液:PBS(pH 7.2)+0.5%FBS+2mM EDTA。 [0075] MACS Buffer: PBS (pH 7.2) + 0.5% FBS + 2mM EDTA.

[0076] 通过免疫磁珠的方法,分选出4出5卞3六-从^1_表型亚细胞群,分选纯度高达85%以上。 [0076] by the immune magnetic beads, sorted 5 4 3 Bian six - from 1_ ^ phenotype subpopulations, sorting purity over 85%. 该细胞亚群高表达〇4、H)GFaR和S 〇xl0(0PCs的标志),只有极少数细胞表达GFAP(星形胶质细胞标志)和Tuj-Ι (神经元标志),表明从神经干细胞中可以获得高纯度的较早期的OPCs。 The high expression of cell subpopulations 〇4, H) GFaR 〇xl0 and S (0PCs logo), only a few cells express of GFAP (astrocyte marker) and Tuj-Ι (neuronal marker), indicates that the neural stem cells earlier can be obtained in high purity OPCs. 且从不同代数的神经干细胞中,如低代数P2、P5、P8等,较高代数P25、P30等,都可以使用磁珠分选的方法获得较高纯度的OPCs。 And neural stem cells from different algebra, as low passage P2, P5, P8 and the like, higher algebraic P25, P30, etc., can obtain a higher purity OPCs using magnetic beads sorting method.

[0077] 2 OPCs的体外扩增培养 [0077] 2 OPCs cultured in vitro amplification of

[0078] 临床应用需要大量细胞,因此如果分选得到的细胞可以在体外扩增培养,必将使得过程更简单,费用更低廉。 [0078] Clinical applications require large numbers of cells, and therefore if the sorted cells may be obtained in vitro expansion culture, will make the process easier, cheaper cost. 磁珠分选后得到的细胞按照4X10 4/cm2密度接种至培养器皿内,在OPCs培养基中培养,置于37°C、5 %C02培养箱内。 The resulting beads after cell inoculation sorted according to 4X10 4 / cm2 to a density within the culture vessel, the culture medium in OPCs, placed in 37 ° C, within 5% C02 incubator. 每3-4天,更换2/3培养基为新鲜OPCs 培养基。 Every 3-4 days, changing the medium with fresh OPCs 2/3 medium. 细胞达到80 % -90 %的融合度时,传代培养。 When the cells reached 80% -90% confluency, subculture.

[0079]每7-10天传代一次,传代时,将细胞用吸管轻轻吹起,加入适量的新鲜培养基,以4 X 104/cm2密度接种于细胞培养板中,置细胞培养箱内继续扩增培养。 [0079] passaged once every 7-10 days, at passage, the cells were gently blowing with a pipette, an appropriate amount of fresh medium was added to 4 X 104 / cm2 seeded in cell culture plates, the cell incubator set to continue Expansion cultures. 同时取部分细胞接种于FOL/laminin预处理的48孔板中,4天后进行免疫荧光染色。 At the same time take part in 48-well plates were seeded FOL / laminin pretreated, 4 days after immunofluorescence staining.

[0080]分选得到的高纯度的OPCs可以在维持初始性状不变的情况下在体外至少传代培养四次。 [0080] The separation of high purity OPCs obtained may be cultured in vitro passaged four times in the case of maintaining at least the same initial characters. 这四代的细胞都具有双极或是三极的形态,持续高表达04,第四代细胞高表达SoxlO和PDGFaR,低表达GFAP和Tu j-Ι,并且绝大多数仍处于早期。 Four generations of cells have a bipolar or tripolar morphology, sustained expression 04, the fourth passage cells and expression SoxlO PDGFaR, low expression of GFAP and Tu j-Ι, and the vast majority are in the early.

[0081 ] OPCs分选后计数,取1.5 X105细胞接种到24孔板内,即为P0。10天后,用200μ1移液器轻轻吹起细胞,计数,即为第一代Ρ1。 [0081] OPCs count after sorting, taking 1.5 X105 cells were seeded into 24-well plates, i.e. P0.10 days, gently blowing with a pipette 200μ1 cell count, is the first generation Ρ1. 连续检测4代,绘制出OPCs的增殖曲线。 4 generations continuously detected, plotted growth curves of OPCs. 从增殖曲线可以看出,细胞处于对数生长期,具有较强的增殖能力,经过四代的培养,OPCs数量上可以增殖到14倍以上。 As can be seen from the growth curve, cells in log phase growth, a strong proliferative capacity, four generations of culture, the number of OPCs can be propagated to more than 14 times.

[0082] OPCs 培养基: 组分浓度Neurobasal A medium 97% B27 2% [0082] OPCs media: Component Concentration Neurobasal A medium 97% B27 2%

[0083] bl-Gl· 20-40 rtg/ml 肝素5pg/ml L-glutamin 1% [0083] bl-Gl · 20-40 rtg / ml heparin 5pg / ml L-glutamin 1%

[0084] 通常人源OPCs体外长期培养都需要三种到四种细胞因子的联合应用,而发明者所使用的该OPCs培养基只含有一种细胞因子--bFGF,大大的降低了培养的成本,为大规模临床应用提供了可能。 [0084] OPCs humanized generally require long-term culture in vitro combination of three kinds or four cytokines, OPCs medium which contains only the inventors used a cytokine --bFGF, greatly reduces the cost of culturing It provides the possibility for large-scale clinical application.

[0085] 3 OPCs的体外诱导分化能力 [0085] 3 OPCs in vitro differentiation

[0086]为了确定分选的细胞是否具有分化能力,将其接种到roL-Laminin包被过的孔板中,在OPCs分化培养基中培养。 [0086] In order to determine whether the sorted cells differentiation, inoculated into roL-Laminin coated over the plate, and cultured in the differentiation medium OPCs. 经过14天的培养,部分细胞突触形成了较长的、树枝状的多分枝复杂网状形态,大部分处于非成熟少突胶质细胞阶段,且可以被多分枝的04和Gale所识别。 After 14 days of culture, cells synapse formation portion of a long, complex multi-branched dendritic web form, in the majority of non-oligodendrocyte maturation stage, and can be recognized by many branches 04 and Gale. 表明通过分选得到的OPCs具有分化为少突细胞的能力。 It is shown to have the ability to differentiate into oligodendrocytes by sorting OPCs obtained.

[0087] 下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。 [0087] The following embodiments in conjunction with embodiments of the present invention will be described in detail, those skilled in the art will appreciate that the following examples merely illustrate the invention and should not be construed as limiting the scope of the present invention. 实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。 Examples are the specific conditions are not specified embodiment, in accordance with conventional conditions or conditions recommended by the manufacturer. 以下实施例所用试剂或仪器的生产公司和货号如下表所示,未注明生产厂商者,均为可以通过市购获得的常规产品。 The following examples of the reagents or Num equipment manufacturing company and shown in the following table, the manufacturer does not indicate who are conventional commercially available products through the city.

[0088] [0088]

Figure CN104073468BD00091

[0089] [0089]

Figure CN104073468BD00101

[0090] 实施例1:从第三代人神经干细胞中分选人源OPCs [0090] Example 1: three generations from neural stem cells sorted from human OPCs

[0091] 1第三代神经干细胞消化成单细胞 [0091] a Third Generation digested neural stem cells into a single cell

[0092]① lOOg离心5分钟收集神经干细胞,弃上清; [0092] ① lOOg centrifuged for 5 minutes to collect stem cells, the supernatant was discarded;

[0093] ②加入lml 0.025%胰蛋白酶(0.01M dPBS稀释),混匀,置37°C培养箱孵育5-7min 至细胞球松散; [0093] ② was added (0.01M dPBS dilution) lml 0.025% trypsin, mixing at 37 ° C incubator until the cells were incubated for 5-7min loose ball;

[0094]③加入1.2mg/ml胰酶抑制剂100μΙ,200μ1移液器吸管轻轻吹打成单细胞悬液; [0095] 神经干细胞培养基: 组分浓度DME.D/F12 98% Ν2 1% [0094] ③ added 1.2mg / trypsin inhibitor 100μΙ ml, 200μ1 pipette pipette by gently pipetting into single cell suspension; [0095] neural stem cell culture medium: component concentration DME.D / F12 98% Ν2 1 %

[0096] bFGF 20 ng/ml EGF 20 ng/ml 肝素5pg/ml L-glulamin 1% [0096] bFGF 20 ng / ml EGF 20 ng / ml heparin 5pg / ml L-glulamin 1%

[0097] 2使用磁珠从人神经干细胞中分选OPCs [0097] 2 is selected from OPCs using magnetic beads human neural stem cells from the equatorial

[0098] ①神经干细胞单细胞悬液,过40μπι尼龙筛网以滤除细胞团块,台酚蓝计算细胞总数; [0098] ① single cell suspensions of neural stem cells, through a nylon mesh to filter 40μπι cell clumps, trypan blue calculate total number of cells;

[0099] ②取107细胞,300g离心10分钟收集细胞,去上清; [0099] ② take 107 cells, cells were collected by centrifugation for 10 minutes 300g, the supernatant was discarded;

[0100] ③重悬在60μ1的磁珠分选缓冲液中,混匀后在4°C中温育10分钟; [0100] ③ resuspended in 60μ1 magnetic beads sorting buffer, after mixing were incubated at 4 ° C 10 min;

[0101] ④加入20μ1的Anti-PSA-NCAM磁珠,混匀后在4°C中温育15分钟; [0101] ④ added to 20μ1 of the Anti-PSA-NCAM beads, after mixing at 4 ° C were incubated 15 minutes;

[0102]⑤加入lml磁珠分选缓冲液,300g离心10分钟; [0102] ⑤ added lml magnetic bead sorting buffer, 300g centrifuged for 10 minutes;

[0103]⑥离心过程中,将MS分选柱放入磁珠分离器中,加入0.5ml的磁珠分选缓冲液平衡分选柱; [0103] ⑥ during centrifugation, the separation column into the MS magnetic bead separation vessel, was added 0.5ml of magnetic bead sorting separation columns equilibrated with a buffer;

[0104] ⑦离心后,完全弃去上清液,重悬于0.5ml的磁珠分选缓冲液。 [0104] ⑦ After centrifugation, the supernatant was discarded completely, resuspended in 0.5ml beads sorting buffer. 将细胞悬液加入到分选柱内,收集流出液体,此为未标记的细胞; The cell suspension was added to the separation column, the liquid effluent is collected, the unlabeled cells;

[0105] ⑧加入0.5ml的磁珠分选缓冲液洗分离柱,共三次,离心收集细胞; [0105] ⑧ added 0.5ml of wash buffer magnetic bead sorting separation columns, a total of three times, the cells were collected by centrifugation;

[0106]⑨加入Anti-A2B5磁珠,混匀后在2-8°C中温育,重复⑤-⑧步骤,此次收集细胞应为柱内集合上的细胞,缓冲液洗柱三次后,将MS分选柱从磁珠分离器内取出放置在1.5ml的离心管内; [0106] ⑨ should, after addition of a buffer Anti-A2B5 beads, after mixing incubated at 2-8 ° C, repeat step ⑤-⑧, the cells were collected by washing the column three times with a cell set on the column, the MS separation column is removed from the separator beads placed within a 1.5ml centrifuge tube;

[0107] ⑩加入lml磁珠分选缓冲液后,立即推压活塞洗脱掉结合上的细胞,300g离心10分钟,收集到的细胞为型的亚细胞群,即为OPCs。 After [0107] ⑩ added lml magnetic bead sorting buffer, eluted immediately pressing piston on cell binding, 300g centrifugation for 10 minutes, the cells collected subpopulations type, namely OPCs.

[0108] 磁珠分选缓冲液:PBS(pH 7.2)+0.5%FBS+2mM EDTA。 [0108] MACS Buffer: PBS (pH 7.2) + 0.5% FBS + 2mM EDTA.

[0109] 分选出的细胞检测A2B5+PSA-NCAiT表型亚细胞群纯度,流式结果表明分选纯度高达85%(图1)以上。 [0109] The sorted cells were A2B5 + PSA-NCAiT detected phenotypic subpopulations purity, flow separation results show a purity of 85% (FIG. 1) above.

[0110]同时取部分细胞接种于PDL/laminin预处理的48孔板中,4天后进行免疫荧光染色。 [0110] A portion of the same time in 48-well plates were seeded PDL / laminin pretreated, 4 days after immunofluorescence staining.

[0111] ①将孔板中的细胞用4%多聚甲醛室温固定15min,PBS漂洗3X10min; [0111] ① The plates with the cells in 4% paraformaldehyde fixed at room temperature 15min, PBS rinse 3X10min;

[0112] ②封闭液室温封闭60min,吸去封闭液,PBS漂洗3X lOmin; [0112] ② closed blocking solution at room temperature 60min, absorb the blocking solution, PBS rinsed 3X lOmin;

[0113] ③加入稀释液配制的一抗,4°C湿盒中孵育过夜;PBS漂洗3 X 1 Omin; [0113] ③ Primary antibody dilutions prepared, 4 ° C and incubated overnight in a humid chamber; PBS rinsed 3 X 1 Omin;

[0114] ④加入配制好的二抗,避光,室温孵育45min,PBS漂洗3X10min; [0114] ④ formulated secondary antibody was added, incubated for 45min in the dark, at room temperature, rinsed with PBS 3X10min;

[0115] ⑤荧光显微镜下观察拍照。 Photographed under observation [0115] ⑤ fluorescence microscope.

[0116] 该细胞亚群高表达〇4、PDGFaR和Soxl0(0PCs的标志),只有极少数细胞表达GFAP (星形胶质细胞标志)和Tuj-Ι(神经元标志),表明从神经干细胞中可以获得高纯度的较早期的OPCs (图2)。 [0116] The high expression of cell subpopulations 〇4, PDGFaR and Soxl0 (0PCs logo), only a few cells express of GFAP (astrocyte marker) and Tuj-Ι (neuronal marker), indicates that the neural stem cells earlier the OPCs can be obtained with high purity (FIG. 2).

[0117] 3 OPCs的体外扩增能力及分化能力检测 [0117] 3 OPCs in vitro expansion and differentiation capacity detection

[0118] ① OPCs的体外培养及传代扩增 [0118] ① in vitro amplification and passage of OPCs

[0119] 磁珠分选后得到的细胞按照4X 104/cm2密度接种至培养器皿内,在OPCs培养基中培养,置于37°C、5%⑶2培养箱内。 [0119] Beads obtained after cell sorting according 4X 104 / cm2 seeded into the culture vessel, the culture medium in OPCs, placed in 37 ° C, 5% ⑶2 within the incubator. 每3-4天,更换2/3培养基为新鲜OPCs培养基。 Every 3-4 days, changing the medium with fresh OPCs 2/3 medium. 细胞达到80 % -90 %的融合度时,传代培养。 When the cells reached 80% -90% confluency, subculture.

[0120] 每7-10天传代一次,传代时,将细胞用吸管轻轻吹起,加入适量的新鲜培养基,以4 X 104/cm2密度接种于细胞培养板中,置细胞培养箱内继续扩增培养。 [0120] passaged once every 7-10 days, at passage, the cells were gently blowing with a pipette, an appropriate amount of fresh medium was added to 4 X 104 / cm2 seeded in cell culture plates, the cell incubator set to continue Expansion cultures. 同时取部分细胞接种于FOL/laminin预处理的48孔板中,4天后进行免疫荧光染色。 At the same time take part in 48-well plates were seeded FOL / laminin pretreated, 4 days after immunofluorescence staining.

[0121]分选得到的高纯度的OPCs可以在维持初始性状不变的情况下在体外至少传代培养四次。 [0121] sorting of the OPCs obtained in a high purity can be cultured in vitro passaged four times in the case of maintaining at least the same initial characters. 这四代的细胞都具有双极或是三极的形态,持续高表达〇4(图3)第四代细胞高表达SoxlO和PDGFaR,低表达GFAP和Tuj-Ι,并且绝大多数仍处于早期(图4)。 Four generations of cells have a bipolar or tripolar morphology, sustained expression 〇4 (FIG. 3) of the fourth passage cells and expression SoxlO PDGFaR, low expression of GFAP and Tuj-Ι, and the vast majority are in the early (Figure 4).

[0122] OPCs分选后计数,取1.5 X 105细胞接种到24孔板内,即为P0。10天后,用200μ1移液器轻轻吹起细胞,计数,即为第一代Ρ1。 [0122] OPCs count after sorting, cells were inoculated 1.5 X 105 taken into 24-well plates, i.e. P0.10 days, gently blowing with a pipette 200μ1 cell count, is the first generation Ρ1. 连续检测4代,绘制出OPCs的增殖曲线。 4 generations continuously detected, plotted growth curves of OPCs. 从增殖曲线可以看出,细胞处于对数生长期,具有较强的增殖能力,经过四代的培养,OPCs数量上可以增殖到14倍以上(图5)。 As can be seen from the growth curve, cells in log phase growth, a strong proliferative capacity, four generations of culture, the number of OPCs can be propagated to more than 14 times (Figure 5).

[0123] OPCs 培养基: 组分浓度Neurobasal A medium 97% [0123] OPCs media: Component Concentration Neurobasal A medium 97%

[0124] B27 2% bFGF 20 ng/ml 肝素5μ^η1 [0124] B27 2% bFGF 20 ng / ml heparin 5μ ^ η1

[0125] L-glutamin 1% [0125] L-glutamin 1%

[0126] ②OPCs的诱导分化 [0126] ②OPCs differentiation-inducing

[0127] OPCs传代时,取部分细胞,计数,以1\105/孔接种于?0171&!1^11丨11预处理的24孔板中。 [0127] When OPCs passage, take some cells, count to 1 \ 105 / well were seeded in? 0171 &! ^ 1 24 11 Shu 11 well plates pretreated. 待细胞贴壁后,加入OPCs分化培养基,每2-3天,更换2/3培养基为新鲜0PC分化培养基。 After the adherent cells, OPCs added differentiation medium every 2-3 days, changing the medium with fresh 0PC 2/3 differentiation medium. 14天后收集细胞进行免疫荧光染色观察。 Cells were collected 14 days after immunofluorescence staining. 部分细胞突触形成了较长的、树枝状的多分枝复杂网状形态,大部分处于非成熟少突胶质细胞阶段,且可以被多分枝的04和Gale所识别(图6)。 Cells synapse formation portion of a long, complex multi-branched dendritic web form, in the majority of non-oligodendrocyte maturation stage, and can be recognized by many branches 04 and Gale (FIG. 6). 表明通过分选得到的OPCs具有分化为少突细胞的能力。 It is shown to have the ability to differentiate into oligodendrocytes by sorting OPCs obtained.

[0128] 实施例2:从第三十代人神经干细胞中分选人源OPCs [0128] Example 2: Source selection OPCs from thirty human neural stem cells Generation carve

[0129] 1第三十代神经干细胞消化成单细胞(方法同实施例1) [0129] Thirty-one-generation digested neural stem cells into a single cell (the same method as in Example 1)

[0130] 2使用磁珠从人神经干细胞中分选0PCs(方法同实施例1) [0130] 2 using human neural stem cells from beads sorted from 0PCs (the same method as in Example 1)

[0131] 3 OPCs的体外扩增能力及分化能力检测(方法同实施例1) [0131] 3 OPCs in vitro expansion and differentiation of detection capability (the same method as in Example 1)

[0132]显微镜下观察发现从P30的神经干细胞中分选的OPCs也都具有典型的双极或是三极形态,且高表达0PCS特异标志04、PDGFaR和SoxlO,只有极少数细胞表达星形胶质细胞标志GFAP和神经元标志Tuj-Ι,诱导后部分细胞突触形成了较长的、树枝状的多分枝复杂网状形态,且可以被多分枝的04和Gale所识别。 The [0132] election microscopy revealed that the neural stem cells from the equatorial P30 OPCs also typical bipolar or tripolar morphology, and expression of specific markers 0PCS 04, PDGFaR and SoxlO, only a few cells expressing astrocytes interstitial cell markers GFAP and neuronal marker Tuj-Ι, fraction of cells induced a longer synapse formation, dendritic multi-branched complex mesh shape, and may be identified and multiple branches 04 Gale.

Claims (9)

1. 获得人源OPCs的方法,其特征在于,将神经干细胞作为分选来源,通过免疫磁珠的方法分离得到人源OPCs,采用的磁珠为Anti-PSA-NCAM和Anti-A2B5磁珠。 A method for obtaining human OPCs, characterized in that the neural stem cells as the source of sorting, OPCs obtained by isolating human immune magnetic beads, magnetic beads used for the Anti-PSA-NCAM, and Anti-A2B5 beads.
2. 根据权利要求1所述的方法,其特征在于,包括以下步骤: ① 神经干细胞在胰蛋白酶溶液中被消化成单个细胞,过筛网; ② 离心收集细胞,去上清; ③ 细胞重悬在磁珠分选缓冲液中,混匀后在2-8°C中温育; ④ 加入Ant i-PSA-NCAM磁珠,混匀后在2-8°C中温育; ⑤ 加入磁珠分选缓冲液,离心; ⑥ 离心过程中,将分选柱放入磁珠分离器中,加入磁珠分选缓冲液平衡分选柱; ⑦ 离心后,完全弃去上清液,重悬于磁珠分选缓冲液,将细胞悬液加入到分选柱内,收集流出液体,此为未标记的细胞; ⑧ 加入磁珠分选缓冲液洗分离柱,离心收集细胞; ⑨ 加入Anti-A2B5磁珠,温育,重复⑤-⑧步骤,此次收集细胞应为柱内集合上的细胞,缓冲液洗柱后,将分选柱从磁珠分尚器内取出放置在尚心管内; ⑩ 加入磁珠分选缓冲液后,立即推压活塞洗脱掉结合上的细胞 2. The method according to claim 1, characterized by comprising: ① From neural stem cells were digested in trypsin solution into individual cells, through the screen; ② the cells were collected by centrifugation, the supernatant; ③ the cells were resuspended in MACS buffer after mixing incubated at 2-8 ° C in; ④ added Ant i-PSA-NCAM beads, after mixing incubated at 2-8 ° C in; ⑤ added MACS buffer, centrifuged; ⑥ during centrifugation, the separation column into the magnetic bead separation vessel, MACS separation column equilibrated with a buffer; ⑦ after centrifugation, the supernatant was discarded completely, resuspended beads sorting buffer, the cell suspension was added to the separation column, the liquid effluent is collected, the unlabeled cells; ⑧ added MACS separation column wash buffer, cells were collected by centrifugation; ⑨ beads were added Anti-A2B5 incubation, repeat step ⑤-⑧, the cells were collected by a cell should be set on the column, the buffer, washing the column, the separation column is placed in a still taken from the inner core tube is still magnetic separation; magnetic ⑩ added after the beads sorting buffer, eluted immediately pushing the piston on the cell binding 离心,收集到的细胞为A2B5+PSA-NCAM-表型的亚细胞群,即为OPCs。 Centrifugation, collected cells were A2B5 + subpopulations PSA-NCAM- phenotype, i.e. OPCs.
3. 根据权利要求2所述的方法,其特征在于,各步骤的具体操作方法如下: ① 神经干细胞在0.025 %胰蛋白酶溶液中被消化成单个细胞,过40μπι尼龙筛网以滤除细胞团块,台酚蓝计算细胞总数; ② 取1〇7细胞,300g离心10分钟收集细胞,去上清; ③ 1〇7细胞重悬在60μ1的磁珠分选缓冲液中,混匀后在2-8°C中温育10分钟; ④ 加入20μ1的Anti-PSA-NCAM磁珠,混匀后在2-8°C中温育15分钟; ⑤ 加入lml磁珠分选缓冲液,300g离心10分钟; ⑥ 离心过程中,将分选柱放入磁珠分离器中,加入0.5ml的磁珠分选缓冲液平衡分选柱; ⑦ 离心后,完全弃去上清液,重悬于0.5ml的磁珠分选缓冲液,将细胞悬液加入到分选柱内,收集流出液体,此为未标记的细胞; ⑧ 加入0.5ml的磁珠分选缓冲液洗分离柱,共三次,离心收集细胞; ⑨ 加入20μ1的Anti-A2B5磁珠,温育,重复上面⑤-⑧的步 3. The method according to claim 2, wherein the specific operation method, the steps as follows: ① neural stem cells into single cells is digested in 0.025% trypsin solution, through a nylon mesh to filter out 40μπι cell pellet , calculating the total number of trypan blue cell; ② take 1〇7 cells, 300g cells were collected by centrifugation for 10 minutes, the supernatant; ③ after 1〇7 cells were resuspended in MACS buffer 60μ1, mixing in 2 temperature 8 ° C for 10 minutes; ④ added to 20μ1 of the Anti-PSA-NCAM beads, mixed at 2-8 ° C after incubating 15 minutes; ⑤ added lml magnetic bead sorting buffer, centrifuged for 10 minutes 300g; ⑥ centrifugation, the separation column into the magnetic bead separator, 0.5ml of the magnetic bead sorting separation columns equilibrated with a buffer; ⑦ after centrifugation, the supernatant was discarded completely, resuspended in 0.5ml of beads sorting buffer, the cell suspension was added to the separation column, the liquid effluent is collected, the unlabeled cells; ⑧ beads were added 0.5ml of wash buffer sorting separation columns, a total of three times, the cells were collected by centrifugation; ⑨ Anti-A2B5 was added 20μ1 of beads, incubated, repeat the above step ⑤-⑧ ,此次收集细胞应为柱内集合上的细胞,缓冲液洗柱三次后,将分选柱从磁珠分离器内取出放置在1.5ml的离心管内; ⑩ 加入lml磁珠分选缓冲液后,立即推压活塞洗脱掉结合上的细胞,300g离心10分钟, 收集到的细胞为型的亚细胞群,即为OPCs。 After addition of lml ⑩ MACS buffer;, the cells were collected by a cell should be set on the column, the column was washed three times with buffer, the sorting column is withdrawn from the separator beads placed in the centrifuge tubes 1.5ml , pushing the piston pressure immediately eluted cells on the binding, 300g centrifugation for 10 minutes, the cells collected type subpopulations, namely OPCs.
4. 根据权利要求1所述的方法,其特征在于,利用OPCs培养基对分离得到的人源OPCs进行体外扩增培养,所述培养基由此111'<^383141116(1;[11111、1327、6?6?、肝素和1^-〖111丨3111;[11组成, NeurobasalAmedium、B27和L-glutamin的体积比为97:2:1,bFGF的含量为20_40ng/ml,肝素的含量为Sμg/ml。 4. The method according to claim 1, characterized in that, using the medium of human OPCs isolated OPCs expanded in vitro culture, whereby the culture medium 111 '<^ 383 141 116 (1; [11111,1327 ?, 66 ?, heparin and 1 ^ - 〖111 Shu 3111; [11 composed, NeurobasalAmedium, B27, and L-glutamin volume ratio of 97: 2: 1, bFGF content of 20_40ng / ml, heparin content of Sμg / ml.
5. 根据权利要求4所述的方法,其特征在于,体外扩增培养的具体操作方法为:磁珠分选后得到的细胞按照4 X 104/cm2密度接种至培养器皿内,在OPCs培养基中培养,置于37°C、 5 % C02培养箱内,每3-4天,更换2/3培养基为新鲜OPCs培养基,细胞达到80 % -90 %的融合度时,传代培养,传代培养的次数为2-4次。 The method according to claim 4, wherein the specific operation method is cultured in vitro amplification: sorted beads obtained after the cells were seeded into a culture vessel according to 4 X 104 / cm2 density of the medium in OPCs when cultured, is placed 37 ° C, within 5% C02 incubator, every 3-4 days, changing the medium with fresh OPCs 2/3 medium, the cells reached 80% -90% confluency, subculture passages number of training 2-4 times.
6. 根据权利要求1所述的方法,其特征在于,所述神经干细胞采用神经干细胞培养基进行体外扩增而得,所述神经干细胞培养基由01^/^12川2、6?6?46?、肝素和1^11^11^11组成,DMED/F12、N2和L-glutamin的体积比为98:1:1,bFGF的浓度为10-30ng/ml,EGF的浓度为l〇-30ng/ml〇 6. The method according to claim 1, wherein the neural stem cells, neural stem cells expanded in vitro derived culture, the neural stem cell medium consisted of 01 ^ / ^ 12 River 2,6? 6? heparin and 1 ?, 46 ^ 11 ^ 11 ^ 11 composition, DMED / F12, N2, and L-glutamin a volume ratio of 98: 1: 1, bFGF at a concentration of 10-30ng / ml, the concentration of EGF is l〇- 30ng / ml〇
7. 根据权利要求6所述的方法,其特征在于,神经干细胞体外扩增的操作步骤如下: ① 100g离心5分钟收集神经干细胞; ② 将上清转移入离心管内,为旧培养基; ③ 向收集好的神经干细胞内加入lml由0.0 lMdPBS稀释得到的0.025 %的胰蛋白酶, 混匀,置37°C培养箱孵育5-7min至细胞球松散; ④ 加入1.2mg/ml胰酶抑制剂100μΙ,200μ1移液器吸管轻轻吹打成单细胞悬液; ⑤ 加入15mldPBS,细胞计数,100g离心5min; ⑥ 弃上清,加入2/3新鲜神经干细胞培养基和1/3旧培养基,以2 X 106/mL密度接种于T25ml培养瓶,置细胞培养箱内培养; ⑦ 每3-4天更换2/3培养基为新鲜培养基,每10天传代一次。 7. The method according to claim 6, characterized in that the neural stem cells in vitro amplification procedure is as follows: ① 100g centrifugation for 5 minutes to collect the neural stem cells; ② the supernatant was transferred into a centrifuge tube, the old medium; ③ the the neural stem cells were collected with 0.025% trypsin was added lml of 0.0 lMdPBS obtained by diluting, mixing at 37 ° C incubator for incubating cells to 5-7min loose ball; ④ was added 1.2mg / trypsin inhibitor 100μΙ ml, 200μ1 pipette pipette by gently pipetting into single cell suspension; ⑤ added 15mldPBS, cells were counted, centrifuged at 100g 5min; ⑥ supernatant was discarded, a fresh addition of 2/3 and 1/3 neural stem cell culture medium the old medium, 2 X 106 / mL culture flask seeded at a density T25ml, cultured cell incubator set; ⑦ 2/3 medium is changed every 3-4 days with fresh medium, subcultured once every 10 days.
8. 获得人源OPCs的方法,其特征在于,利用OPCs培养基对人源OPCs进行体外扩增培养, 所述培养基由NeurobasalAmedi um、B27、bFGF、肝素和L-glutamin组成,Neurobasal Amedium、B27和L-glutamin的体积比为97:2:1,bFGF的含量为20-40ng/ml,肝素的含量为5μ g/ml〇 8. The method of obtaining human OPCs, characterized in that, using the medium of human OPCs OPCs expanded in vitro culture, said medium consisting NeurobasalAmedi um, B27, bFGF, heparin and L-glutamin composition, Neurobasal Amedium, B27 L-glutamin and the volume ratio of 97: 2: 1, the content of bFGF is 20-40ng / ml, heparin content of 5μ g / ml〇
9. 根据权利要求8所述的方法,其特征在于,体外扩增培养的具体操作方法为:人源OPCs按照4 X 104/cm2密度接种至培养器皿内,在OPCs培养基中培养,置于37°C、5 %C02培养箱内,每3-4天,更换2/3培养基为新鲜OPCs培养基,细胞达到80 % -90 %的融合度时,传代培养,传代培养的次数为2-4次。 9. The method according to claim 8, wherein the specific operation method is cultured in vitro amplification: OPCs human vaccination in accordance with 4 X 104 / cm2 to a density within the culture vessel, the culture medium in OPCs, placed when 37 ° C, within 5% C02 incubator, every 3-4 days, changing the medium with fresh OPCs 2/3 medium, the cells reached 80% -90% confluency, subculturing, the number of times of subculture 2 -4 times. 10 .OPCs 培养基,其特征在于,由Neurobasal Amedium、B27、bFGF、肝素和L-glutamin 组成,NeurobasalAmedium、B27和L-glutamin的体积比为97:2:1,bFGF的含量20_40ng/ml,肝素的含量为5μg/ml。 10 .OPCs medium, characterized by the Neurobasal Amedium, B27, bFGF, heparin and L-glutamin composition, NeurobasalAmedium, B27, and L-glutamin volume ratio of 97: 2: 1, bFGF content 20_40ng / ml, heparin an amount of 5μg / ml.
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