CN102618490B - Method for inducing retinal stem cells to differentiate into photosensory cells - Google Patents
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Abstract
本发明公开了一种诱导视网膜干细胞分化为感光细胞的方法，通过抑制视网膜干细胞的IGF IRα受体使视网膜干细胞分化为感光细胞；与现有技术相比，本发明通过抑制视网膜干细胞的IGF IRα受体使视网膜干细胞分化为感光细胞的方法，不导入病毒基因和进行基因操作，无视网膜成熟细胞的混杂污染，且分化效率高，操作简单。The invention discloses a method for inducing retinal stem cells to differentiate into photoreceptor cells. The retinal stem cells are differentiated into photoreceptor cells by inhibiting the IGF IRα receptor of the retinal stem cells; compared with the prior art, the invention inhibits the IGF IRα receptor of the retinal stem cells. The method for in vivo differentiation of retinal stem cells into photoreceptor cells does not introduce virus genes or perform genetic manipulation, and does not have mixed pollution of retinal mature cells, and the differentiation efficiency is high and the operation is simple.
Description
技术领域 technical field
本发明涉及生物技术领域，特别涉及诱导视网膜干细胞分化为感光细胞的方法。The invention relates to the field of biotechnology, in particular to a method for inducing retinal stem cells to differentiate into photoreceptor cells.
背景技术 Background technique
视网膜色素变性(Retinitis pigmentosa，简称RP)是遗传性盲目最常见的病因之一。由于RP患者视力持续性下降的原因是感光细胞的变性和凋亡，而感光细胞不能再生，移植健康的视网膜组织或者感光细胞，已经成为目前最具前景的治疗策略。目前国内外对视网膜干细胞(Retinal stem cells，简称RSCs)定向分化为感光细胞的机制展开了广泛的研究工作，阐述了许多促进RSCs向感光细胞分化的物质及相关的信号途径，如基因沉默和转染等，但始终面临这样一个问题：在促分化条件下，RSCs向感光细胞的分化增加，但因导入了病毒基因和实施了基因操作，存在在体研究安全性的问题和向其它类型的细胞分化问题，而从已有成熟细胞分化的视网膜(胚胎晚期、成体视网膜等)中分离得到的RSCs，还会有其它成熟细胞类型的混杂，这将不利于RSCs诱导分化为感光细胞的在体实验、临床研究和临床应用。Retinitis pigmentosa (RP) is one of the most common causes of hereditary blindness. Since the cause of persistent vision loss in RP patients is the degeneration and apoptosis of photoreceptor cells, which cannot regenerate, transplantation of healthy retinal tissue or photoreceptor cells has become the most promising therapeutic strategy. At present, extensive research work has been carried out on the mechanism of retinal stem cells (RSCs) directional differentiation into photoreceptor cells at home and abroad, and many substances and related signaling pathways that promote the differentiation of RSCs into photoreceptor cells have been described, such as gene silencing and transduction. Infection, etc., but always face such a problem: under the condition of promoting differentiation, the differentiation of RSCs into photoreceptor cells increases, but due to the introduction of viral genes and the implementation of genetic manipulation, there are problems in the safety of in vivo research and the introduction of RSCs to other types of cells However, RSCs isolated from retinas that have differentiated mature cells (late embryonic, adult retina, etc.) will also be mixed with other mature cell types, which will not be conducive to in vivo experiments in which RSCs are induced to differentiate into photoreceptor cells , clinical research and clinical application.
因此，需要一种诱导视网膜干细胞分化为感光细胞的方法，不导入病毒基因和进行基因操作，无视网膜成熟细胞的混杂污染，且分化效率高，操作简单。Therefore, there is a need for a method for inducing retinal stem cells to differentiate into photoreceptor cells, which does not introduce virus genes and perform genetic manipulation, does not have mixed contamination of retinal mature cells, and has high differentiation efficiency and simple operation.
发明内容 Contents of the invention
鉴于此，本发明的目的是提供一种诱导视网膜干细胞分化为感光细胞的方法，不导入病毒基因和进行基因操作，无视网膜成熟细胞的混杂污染，且分化效率高，操作简单。In view of this, the purpose of the present invention is to provide a method for inducing retinal stem cells to differentiate into photoreceptor cells, without introducing viral genes and genetic manipulation, without contamination of retinal mature cells, and with high differentiation efficiency and simple operation.
本发明提供了一种诱导视网膜干细胞分化为感光细胞的方法，通过抑制视网膜干细胞的IGF IRα受体使视网膜干细胞分化为感光细胞。不导入病毒基因和进行基因操作。The invention provides a method for inducing retinal stem cells to differentiate into photoreceptor cells, and the retinal stem cells are differentiated into photoreceptor cells by inhibiting the IGFIRα receptor of the retinal stem cells. Viral genes and genetic manipulations are not introduced.
优选的，所述视网膜干细胞为视杯视网膜干细胞。胚胎视杯(Optic cup，简称OC)中富集视网膜干细胞，并称视杯中的视网膜干细胞为视杯视网膜干细胞(OC-RSCs)，视杯视网膜干细胞是视杯发育成胚眼之前的原始视网膜细胞，因此从视杯中取材、培养会获得较原始的、未分化的、更易富集的视网膜干细胞，无各种成熟细胞的混杂污染。Preferably, the retinal stem cells are optic cup retinal stem cells. The embryonic optic cup (OC) is enriched with retinal stem cells, and the retinal stem cells in the optic cup are called optic cup retinal stem cells (OC-RSCs). The optic cup retinal stem cells are the original retina before the optic cup develops into the embryonic eye. Therefore, the retinal stem cells that are more primitive, undifferentiated, and easier to enrich will be obtained from the optic cup, and there will be no mixed pollution of various mature cells.
优选的，所述抑制视网膜干细胞的IGF IRα受体包括如下步骤：Preferably, said suppression of the IGFIRα receptor of retinal stem cells comprises the steps of:
a.接种视网膜干细胞于细胞培养器皿；a. inoculating retinal stem cells in cell culture vessels;
b.加入含抗IGF IRα受体抗体的分化诱导培养基；b. Add the differentiation induction medium containing anti-IGFIRα receptor antibody;
c.放置培养且定期更换步骤b中的培养基至细胞分化。c. Place the culture and periodically replace the medium in step b until the cells differentiate.
优选的，步骤a中所述视网膜干细胞为大鼠E12.5胚胎视杯视网膜干细胞；所述培养器皿为50mL培养瓶；所述接种采用的接种密度为每瓶2×105个细胞。大鼠E12.5胚胎视杯视网膜干细胞全表达IGF IRα受体，如图1所示视杯视网膜干细胞的IGF IRα受体免疫荧光鉴定图，蓝色荧光为细胞核，绿色荧光为IGF IRα受体，说明全部细胞均表达IGF IRα受体。Preferably, the retinal stem cells in step a are rat E12.5 embryo optic cup retinal stem cells; the culture vessel is a 50 mL culture flask; the seeding density used in the inoculation is 2×10 5 cells per bottle. Rat E12.5 embryonic cup retinal stem cells fully express IGF IRα receptors, as shown in Figure 1, the IGF IRα receptor immunofluorescence identification diagram of optic cup retinal stem cells, blue fluorescence is the nucleus, green fluorescence is IGF IRα receptor, It shows that all cells express IGFIRα receptor.
优选的，步骤b中所述含抗IGF IRα受体抗体的分化诱导培养基为1μg/ml抗IGF IRα受体抗体+B27+20ng/mL碱性成纤维细胞生长因子(bFGF)+2.5％胎牛血清+DMEM/F12培养基。抗IGF IRα受体抗体会特异地与IGF IRα受体结合而使失去生理功能。Preferably, the differentiation induction medium containing anti-IGF IRα receptor antibody described in step b is 1 μg/ml anti-IGF IRα receptor antibody+B27+20ng/mL basic fibroblast growth factor (bFGF)+2.5% fetus Bovine serum + DMEM/F12 medium. Anti-IGF IRα receptor antibody will specifically bind to IGF IRα receptor and make it lose its physiological function.
优选的，步骤c中所述放置培养是在37℃和5％CO2细胞培养箱中进行；所述定期更换为每2-3天更换一次至7天。Preferably, the standing culture in step c is carried out in a cell culture incubator at 37°C and 5% CO 2 ; the regular replacement is every 2-3 days to 7 days.
与现有技术相比，本发明通过抑制视网膜干细胞的IGF IRα受体使视网膜干细胞分化为感光细胞的方法，不导入病毒基因和进行基因操作，无视网膜成熟细胞的混杂污染，且分化效率高，操作简单。Compared with the prior art, the present invention differentiates retinal stem cells into photoreceptor cells by inhibiting the IGFIRα receptor of retinal stem cells, does not introduce viral genes and perform genetic manipulation, has no mixed pollution of retinal mature cells, and has high differentiation efficiency. easy to use.
附图说明 Description of drawings
图1为本发明OC-RSCs细胞的IGF IRα受体免疫荧光鉴定图；Fig. 1 is the IGFIRα receptor immunofluorescence identification figure of OC-RSCs cell of the present invention;
图2为本发明OC-RSCs细胞在抑制IGF IRα受体后视紫红蛋白的表达的Western-blot鉴定图。Figure 2 is a Western-blot identification diagram of the expression of rhodopsin in OC-RSCs cells of the present invention after inhibiting IGF IRα receptors.
具体实施方式 Detailed ways
下面将结合实施例和附图来详细说明本发明，这些实施例和附图仅起说明性作用，并不局限于本发明的应用范围。本发明不限于下述实施方式或实施例，凡不违背本发明精神所做出的修改及变形，均应包括在本发明范围之内。The present invention will be described in detail below in conjunction with the embodiments and drawings, which are only for illustration and not limiting the scope of application of the present invention. The present invention is not limited to the following embodiments or examples, and any modifications and variations that do not violate the spirit of the present invention shall be included within the scope of the present invention.
本实施例诱导视网膜干细胞分化为感光细胞的方法，通过抑制视网膜干细胞的IGF IRα受体使视网膜干细胞分化为感光细胞。The method for inducing retinal stem cells to differentiate into photoreceptor cells in this embodiment is to differentiate retinal stem cells into photoreceptor cells by inhibiting the IGFIRα receptor of retinal stem cells.
本实施例中，所述视网膜干细胞为视杯视网膜干细胞，当然也可以是来自成体视网膜、胚胎晚期视网膜等的视网膜干细胞，均能够实现发明目的，但采用视杯视网膜干细胞效果较佳。In this embodiment, the retinal stem cells are optic cup retinal stem cells, and of course they can also be retinal stem cells from adult retina, late embryonic retina, etc., all of which can achieve the purpose of the invention, but the effect of using optic cup retinal stem cells is better.
本实施例中，所述视网膜干细胞的IGF IRα受体包括如下步骤：In the present embodiment, the IGFIRα receptor of the retinal stem cells comprises the following steps:
a.接种视网膜干细胞于细胞培养器皿；a. inoculating retinal stem cells in cell culture vessels;
本步骤中所述视网膜干细胞为大鼠E12.5胚胎视杯视网膜干细胞；所述培养器皿为50mL培养瓶；所述接种采用的接种密度为每瓶2×105个细胞。大鼠E12.5胚胎的视杯视网膜干细胞全表达IGF IRα受体，如图1所示视杯视网膜干细胞的IGF IRα受体免疫荧光鉴定图，蓝色荧光为细胞核，绿色荧光为IGF IRα受体，说明全部细胞均表达IGF IRα受体。The retinal stem cells in this step are retinal stem cells in the optic cup of rat E12.5 embryos; the culture vessel is a 50 mL culture bottle; the inoculation density used for the inoculation is 2×10 5 cells per bottle. The optic cup retinal stem cells of rat E12.5 embryos fully express the IGF IRα receptor, as shown in Figure 1, the IGF IRα receptor immunofluorescence identification map of the optic cup retinal stem cells, the blue fluorescence is the nucleus, and the green fluorescence is the IGF IRα receptor , indicating that all cells express IGFIRα receptors.
b.加入含抗IGF IRα受体抗体的分化诱导培养基；b. Add the differentiation induction medium containing anti-IGFIRα receptor antibody;
本步骤中所述含抗IGF IRα受体抗体的分化诱导培养基为1μg/ml抗IGF IRα受体抗体+B27+20ng/ml bFGF+2.5％胎牛血清+DMEM/F12培养基。The differentiation induction medium containing anti-IGF IRα receptor antibody described in this step is 1 μg/ml anti-IGF IRα receptor antibody+B27+20ng/ml bFGF+2.5% fetal bovine serum+DMEM/F12 medium.
c.放置培养且定期更换步骤b中的培养基至细胞分化。c. Place the culture and periodically replace the medium in step b until the cells differentiate.
本步骤中所述放置培养是在37℃和5％CO2细胞培养箱中进行；所述定期更换为每2天更换一次至7天。本步骤2-3天更换一次均能够实现发明目的，但2天效果更佳。The standing culture in this step is carried out in a cell culture incubator at 37° C. and 5% CO 2 ; the regular replacement is every 2 days to 7 days. The purpose of the invention can be achieved by changing this step every 2-3 days, but the effect is better in 2 days.
视紫红蛋白(Rhodopsin)是感光细胞的标志蛋白，Rhodopsin表达增加即说明感光细胞分化增加。对分化细胞的Rhodopsin蛋白做Western-blot鉴定，如图2所示，I为对照，即为未抑制IGF IRα受体的OC-RSCs细胞的Rhodopsin表达，II为抑制IGF IRα受体的分化细胞的Rhodopsin表达，II中Rhodopsin表达明显增加，即说明感光细胞分化增加了。Rhodopsin is a marker protein of photoreceptor cells, and the increase of Rhodopsin expression indicates the increase of photoreceptor cell differentiation. The Rhodopsin protein of differentiated cells was identified by Western-blot, as shown in Figure 2, I is the control, that is, the expression of Rhodopsin in OC-RSCs cells that did not inhibit the IGF IRα receptor, II is the expression of differentiated cells that inhibit the IGF IRα receptor The expression of Rhodopsin, the expression of Rhodopsin in II was significantly increased, which means that the differentiation of photoreceptor cells increased.
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be carried out Modifications or equivalent replacements without departing from the spirit and scope of the technical solution of the present invention shall be covered by the claims of the present invention.
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| Zygar CA et al.IGF-1 produced by cone photoreceptors regulates rod progenitor proliferation in the teleost retina.《Bran Res Dev Brain Res》.2005,第154卷(第1期),91-100. |
| 黄小勇.大鼠视杯视网膜干细胞生物学特性和移植的实验研究.《中国优秀博硕士学位论文全文数据库（博士）医药卫生科技辑》.2006,(第1期),全文. * |
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