WO2020258946A1 - Method for inducing reprogramming of spinal cord astrocytes into motor neurons - Google Patents

Method for inducing reprogramming of spinal cord astrocytes into motor neurons Download PDF

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WO2020258946A1
WO2020258946A1 PCT/CN2020/080792 CN2020080792W WO2020258946A1 WO 2020258946 A1 WO2020258946 A1 WO 2020258946A1 CN 2020080792 W CN2020080792 W CN 2020080792W WO 2020258946 A1 WO2020258946 A1 WO 2020258946A1
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王晓冬
陈雪
徐磊
吴坚
陈罡
李奕
潘静莹
陈颖
杨日云
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南通大学
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/08—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the nervous system

Definitions

  • the invention belongs to the field of medical technology, and specifically relates to a method for inducing reprogramming of spinal cord astrocytes into motor neurons.
  • SCI spinal cord injury
  • astrocytes play a decisive role in the nutrition, support and regulation of neurons under normal physiological conditions.
  • astrocytes will rapidly activate into reactive astrocytes And continue to multiply, forming a colloidal scar around the injured site.
  • glial scar can control the spread of inflammation and protect surviving neurons and other cells, its formation also constitutes a physical and chemical barrier, which severely hinders the regeneration of axons, and some studies have also found some reactive glue Plasma cells may produce neurotoxic effects. Due to the death or apoptosis of damaged neurons after spinal cord injury, the number of neurons decreases.
  • iPSCs induced pluripotent stem cells
  • iPSCs transcription factors commonly used for reprogramming are highly expressed in various types of tumors, which will make iPSCs have high tumorigenicity; secondly, about 2 weeks after spinal cord injury is the best period for nerve repair, and The time for iPSCs to induce differentiation is too long to complete the differentiation and repair within the optimal repair period. Furthermore, iPSCs cannot guarantee targeted and efficient differentiation into neurons, especially motor neurons, in the complex spinal cord environment. Finally, the process of neural stem cell transplantation is bound to cause secondary damage to nerve tissue. In recent years, researchers have successfully converted pancreatic exocrine cells into ⁇ -cells through direct reprogramming, or induced fibroblasts to differentiate into cardiomyocytes and neurons.
  • the technical problem to be solved by the present invention is to provide a method for inducing reprogramming of spinal astrocytes into motor neurons to solve the problems raised in the background art.
  • the embodiments of the present invention provide a method for inducing reprogramming of spinal astrocytes into motor neurons, which is characterized by including the following steps: (1) Primary rat astrocytes Purified culture, using primary astrocytes from the spinal cord of newborn rats as target cells for inducing reprogramming, and obtaining high-purity astrocytes through in vitro culture of differential adherence method and microwave shaking culture; (2) According to the materials needed for neuron induction and motor neuron formation and development stage, 7 small molecule drugs were selected, SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin, VPA, and these small molecule drugs can affect astrocytes in vitro Carry out induction reprogramming; (3) Observe cell morphology and immunofluorescence cytochemical technology analysis, real-time fluorescence quantitative PCR analysis and statistical analysis methods to analyze the reprogramming and differentiation of astrocytes into motor neurons.
  • step (1) specifically includes the following process:
  • Ultra-clean workbench is irradiated with ultraviolet rays 30min in advance, take out the ice table pre-cooled at -20°C, sterilized surgical instruments, HBSS buffer and plastic petri dishes, etc., place them in the ultra-clean workbench, and add the petri dishes Place a proper amount of HBSS on the ice table for later use;
  • step (2) specifically includes the following process:
  • step (1) When the astrocytes in step (1) are cultured to the P2-P3 generation, add PBS to rinse for 2-3 times, and then add 2ml 0.25% trypsin digestion cells pre-warmed until the adherent cells can be light Blow gently, add 5ml complete culture medium to stop the digestion, add the cell suspension to a 15ml centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1ml complete culture medium to resuspend the cells, and inoculate the cells with 4 ⁇ 104 cells. In a 24-well plate coated with polylysine, place it in a 5% CO2 incubator and cultivate overnight;
  • the immunofluorescence cytochemical technique analysis in the step (3) specifically includes the following processes:
  • (3-1-2) Discard 4% paraformaldehyde, add 300 ⁇ l PBS to each well to rinse 3 times, 5min each time; (3-1-3) Discard PBS, add 300 ⁇ l rupture fluid to each well, incubate for 30min at room temperature ;
  • (3-1-7) Discard PBS, add 300ml of a mixture of secondary antibody and PBS to each well, incubate at 4°C for ⁇ 2h, protect from light;
  • (3-1-8) Discard the secondary antibody mixture, add 300 ⁇ l PBS to each well and rinse 3 times, 5min each time, and treat it in the dark;
  • the real-time fluorescent quantitative PCR analysis in the step (3) specifically includes the following process:
  • the statistical analysis in the step (3) specifically includes the following process: performing data statistics through the software Graphpad prism5, performing statistical analysis on all quantified data and expressing the average value ⁇ standard deviation; the two-sided t test is used to calculate p The statistical significance of the value, p value ⁇ 0.05 is statistically significant.
  • the complete culture medium is prepared by adding 1% double antibody and 10% FBS to DMEM/F12 basic medium.
  • the drug induction culture medium one is made by adding 2 ⁇ M SB431542, 1 ⁇ M RA, 5 ⁇ M Forskolin, 20ng/ml bFGF, 0.25 ⁇ M LDN-193189 to the basic induction medium;
  • the drug induction culture medium two uses It is made by adding 5 ⁇ M Purmorphamine, 10 ⁇ M RA, 20 ⁇ M Forskolin, 200ng/ml bFGF to the basal induction medium;
  • the drug induction medium 3 is made by adding 1mM VPA, 5 ⁇ M Purmorphamine, 10 ⁇ M RA, 20 ⁇ M Forskolin, 200ng/ml bFGF.
  • the base employed inducing medium was added at 1% of the double antibody DMEM / F12 base medium, 1M B27,1M N 2, 2mM L- glutamic acid, 3.5mM glucose formed.
  • the beneficial effects of the above technical solutions of the present invention are as follows: the small molecule drug combination of the present invention can successfully induce reprogramming of rat astrocytes into motor neurons in vitro, and the transdifferentiation rate exceeds 75%; in addition, the The small molecule drug combination can also successfully complete the transdifferentiation of human astrocytes into neurons; the direct reprogramming of astrocytes into neurons in situ may be compared in the treatment of spinal cord injury repair and functional reconstruction Practical ideas.
  • Figure 1 is a GFAP immunofluorescence cytochemical staining diagram of P0 generation astrocytes in the present invention
  • Figure 2 is respectively the GFAP and Nestin immunofluorescence cytochemical staining diagrams of P2 generation astrocytes of the present invention
  • Figure 3 shows the morphology of astrocytes in the small molecule drug group at 1, 3, 9 and 20 days from left to right;
  • Figure 4 shows the morphology of astrocytes in the control group on day 1, 3, 9 and 20 from left to right;
  • Figure 5 shows the GFAP and Tuj1 staining images of the small molecule drug group when astrocytes are induced in vitro for 6 days;
  • Figure 6 is the GFAP and Tuj1 staining images of the control group when astrocytes are induced in vitro for 6 days;
  • Figure 7 is a partial enlarged view of Figure 5;
  • Figure 8 shows the MAP2 and NeuN staining images of astrocytes induced by small molecule drugs for 10 days in vitro;
  • Figure 9 is a MAP2 staining image of astrocytes induced by small molecule drugs in vitro for 16 days;
  • Figure 11 shows the relative expression of related genes in different stages of astrocytes induced by small molecules
  • Figure 12 shows the staining of MAP2, HB9 and CHAT when small molecule drugs induce astrocytes for 16 days;
  • Figure 13 shows the MAP2, HB9 and CHAT staining images of astrocytes induced by small molecule drugs for 30 days;
  • Figure 14 shows the staining of MAP2 and GAD65 when small molecule drugs induce astrocytes for 18 days;
  • Figure 15 shows the staining of MAP2 and VGLUT1 when small molecule drugs induce astrocytes for 18 days;
  • Figure 16 shows the positive rate of neurons HB9, CHAT, VGLUT1 and GAD65 induced by small molecule drugs on astrocytes for 16 days.
  • a method for inducing reprogramming of spinal cord astrocytes into motor neurons includes the following steps:
  • Purification and culture of primary rat astrocytes using primary astrocytes from the spinal cord of newborn rats as target cells for inducing reprogramming, obtained by in vitro culture of differential adherence method and microwave shaking culture High-purity astrocytes; specifically include the following processes:
  • Ultra-clean workbench is irradiated with ultraviolet rays 30min in advance, take out the ice table pre-cooled at -20°C, sterilized surgical instruments, HBSS buffer and plastic petri dishes, etc., place them in the ultra-clean workbench, and add the culture dishes Place an appropriate amount of HBSS on the ice table for later use.
  • step (1) When the astrocytes in step (1) are cultured to the P2-P3 generation, add PBS to rinse 2-3 times, and then add 2ml of pre-warmed 0.25% trypsin digestion cells until the adherent cells can be light Blow gently, add 5ml of complete culture fluid to terminate the digestion, add the cell suspension to a 15ml centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1ml of complete culture fluid to resuspend the cells, and inoculate with 4 ⁇ 10 4 cells In a 24-well plate containing coated polylysine, place it in a 5% CO 2 incubator and culture overnight;
  • the immunofluorescence cytochemical analysis includes the following processes:
  • (3-1-2) Discard 4% paraformaldehyde, add 300 ⁇ l PBS to each well to rinse 3 times, 5min each time; (3-1-3) Discard PBS, add 300 ⁇ l rupture fluid to each well, incubate for 30min at room temperature ;
  • (3-1-7) Discard PBS, add 300ml of a mixture of secondary antibody and PBS to each well, incubate at 4°C for ⁇ 2h, protect from light;
  • (3-1-8) Discard the secondary antibody mixture, add 300 ⁇ l PBS to each well and rinse 3 times, 5min each time, and treat it in the dark;
  • fluorescence quantitative PCR analysis specifically includes the following processes:
  • the statistical analysis specifically includes the following process: data statistics through the software Graphpad prism5, statistical analysis of all quantified data and expressed in average ⁇ standard deviation; two-sided t test is used to calculate the statistical significance of the p value, p A value of ⁇ 0.05 is statistically significant.
  • the complete culture medium is prepared by adding 1% double antibody and 10% FBS to DMEM/F12 basic medium.
  • the first drug induction culture medium is made by adding 2 ⁇ M SB431542, 1 ⁇ M RA, 5 ⁇ M Forskolin, 20ng/ml bFGF, 0.25 ⁇ M LDN-193189 to the basic induction culture solution;
  • the second drug induction culture solution It is made by adding 5 ⁇ M Purmorphamine, 10 ⁇ M RA, 20 ⁇ M Forskolin, 200ng/ml bFGF to the basal induction medium;
  • the drug induction culture medium 3 is made by adding 1mM VPA, 5 ⁇ M Purmorphamine, 10 ⁇ M RA, 20 ⁇ M Forskolin, 200ng to the basal induction medium /ml Made of bFGF.
  • the base employed inducing medium was added at 1% of the double antibody DMEM / F12 base medium, 1M B27,1M N 2, 2mM L- glutamic acid, 3.5mM glucose formed.
  • the small molecule drug combination of the present invention can successfully induce and reprogram rat astrocytes into motor neurons in vitro, and the transdifferentiation rate exceeds 75%; in addition, the small molecule drug combination can also successfully complete the human origin star Transdifferentiation of glial cells into neurons; direct reprogramming of astrocytes into neurons in situ may be a more feasible idea in the treatment of spinal cord injury repair and functional reconstruction.
  • the present invention can obtain the following experimental results:
  • Rat spinal cord-derived astrocytes were inoculated into cell culture flasks to remove a small amount of fibroblasts by differential adhesion method, and then cultured with complete medium to promote the division and proliferation of glial cells. Since there are still a small number of neurons, oligodendrocytes and microglia in the P0 generation cells, the cell morphology is still diverse when observed under a phase contrast microscope ( Figure 1). After the P0 generation cell fusion degree reaches about 80%, the contaminated cells are removed by microwave shaking culture and subcultured to the P2 generation, then stained with the unique collagen fibrillary acidic protein (GFAP) in astrocytes to identify GFAP positive cells. More than 95% (Figure 1, Figure 2). Under a phase-contrast microscope, most of the P2 generation cells were flat polygonal, with larger cell bodies, large and obvious nuclei, and a small number of irregular protrusions ( Figure 2).
  • GFAP unique collagen fibrillary acidic protein
  • the purified astrocytes express Nestin
  • neural stem cells may be mixed with astrocytes obtained by extracting spinal cord of newborn rats.
  • Neural stem cells are a type of mother cell with high division potential and self-renewal ability. When the body encounters special conditions, it can proliferate and differentiate to form different types of nerve tissue cells, such as neurons, astrocytes, and oligodendrocytes. Cells etc.
  • astrocytes will also self-renew and proliferate after being activated at the injury site and acquire the potential similar to neural stem cells; in addition, studies have found that astrocytes cultured in vitro, when certain conditions are suitable At the same time, the cell morphology can be changed, and then it will appear a state similar to the activation of in vivo injury, and finally express some neural stem cell markers and even dedifferentiate into neural stem-like cells. Therefore, before reprogramming in this experiment, first eliminate the possible dedifferentiation of astrocytes during the cell purification and culture process, and perform GFAP and neural stem cell markers on astrocytes that are to be induced by small molecule drugs.
  • astrocytes The reprogramming of astrocytes does not only depend on transcription factors, but small molecule drugs also have a certain effect on inducing reprogramming.
  • small molecule drugs also have a certain effect on inducing reprogramming.
  • SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin, and VPA for reprogramming.
  • Astrocytes act in drug-induced culture medium one for 2 days, then replace with drug-induced medium for six days, and finally replace with drug-induced culture medium to act again and again. Change the medium every 2-3 days during the entire induction process .
  • the morphology of astrocytes changed, the cell body gradually shrank, and the cell protrusions extended and became thinner.

Abstract

Provided is a method for inducing reprogramming of spinal cord astrocytes into motor neurons. The method comprises the following steps: (1) purifying and culturing primary rat astrocytes; (2) selecting seven small molecule drugs SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin, and VPA according to substances required for neuron induction and motor neuron formation and development stages, the small molecule drugs inducing reprogramming of the astrocytes in vitro; and (3) observing morphological changes of the cells, and analyzing the reprogramming and differentiation of astrocytes into motor neurons by means of immunofluorescence cytochemistry analysis, real-time fluorescence-based quantitative PCR analysis, and statistical analysis methods.

Description

一种诱导脊髓星形胶质细胞重编程为运动神经元的方法A method for inducing reprogramming of spinal cord astrocytes into motor neurons 技术领域Technical field
本发明属于医学技术领域，具体涉及一种诱导脊髓星形胶质细胞重编程为运动神经元的方法。The invention belongs to the field of medical technology, and specifically relates to a method for inducing reprogramming of spinal cord astrocytes into motor neurons.
背景技术Background technique
脊髓损伤(spinal cord injury,SCI)的临床症状从轻微的感觉、运动损伤到肢体的完全瘫痪，主要取决于受伤部位的损伤程度和范围。星形胶质细胞在正常生理条件下对神经元的营养、支持和调节等方面都起决定性作用，然而当脊髓一旦受到损伤，星形胶质细胞就会迅速活化为反应性星形胶质细胞并不断增殖，在损伤部位周围形成胶质疤痕。尽管胶质疤痕能够控制炎症的蔓延并保护幸存的神经元等细胞，但是它的形成同时又构成了一个物理和化学的屏障，严重阻碍了轴突的再生，而且有研究还发现一些反应性胶质细胞可能会产生神经毒性作用。由于脊髓损伤后受损的神经元会发生死亡或凋亡，造成神经元的数量的下降。即使成年脊髓中还残留少量神经干细胞，也无法足量补充丢失的神经元。这样的结果最终使得受损脊髓的神经元数量无法恢复到正常水平，再加上成年神经元轴突再生能力低下，从而使整个受损脊髓神经组织的修复和功能重建成为医学一大难题。The clinical symptoms of spinal cord injury (SCI) range from mild sensory and motor injuries to complete paralysis of the limbs, mainly depending on the extent and scope of the injury. Astrocytes play a decisive role in the nutrition, support and regulation of neurons under normal physiological conditions. However, once the spinal cord is injured, astrocytes will rapidly activate into reactive astrocytes And continue to multiply, forming a colloidal scar around the injured site. Although glial scar can control the spread of inflammation and protect surviving neurons and other cells, its formation also constitutes a physical and chemical barrier, which severely hinders the regeneration of axons, and some studies have also found some reactive glue Plasma cells may produce neurotoxic effects. Due to the death or apoptosis of damaged neurons after spinal cord injury, the number of neurons decreases. Even if a small amount of neural stem cells remain in the adult spinal cord, it cannot replace the lost neurons in sufficient quantities. As a result, the number of neurons in the damaged spinal cord cannot be restored to normal levels, coupled with the low axon regeneration ability of adult neurons, which makes the repair and functional reconstruction of the entire damaged spinal cord nerve tissue a major medical problem.
细胞移植，特别是神经干细胞移植一直被认为是替代受损细胞、保护神经元和促进神经再生修复的一种可行性方法。但是，移植细胞进入损伤脊髓局部组织后，较难定向分化为终末细胞(分化的随机性较大)。而且干细胞缺少稳定的细胞来源、医学伦理、移植后的免疫抑制等问题限制了它的临床应用。2006年,诱导多能干细胞(induced pluripotent stem cells，iPSCs)技术的问世开辟了再生医学的新时代。然而当其应用于脊髓损伤修复时，研究人员仍发现存在诸多应用限制。首先，常用于重编程的转录因子在各种类型的肿瘤中均高表达，这样会使iPSCs具有较高的成瘤性；其次，脊髓损伤后约2周左右是神经修复的最佳时期，而iPSCs整个诱导分化的时间过长，无法在最佳修复时期内完成分化和修复；再者，iPSCs在复杂的脊髓内环境中根本无法保证定向高效的分化为神经元，特别是运动神经元。最后，神经干细胞移植过程又势必对神经组织造成二次损伤。近年来研究人员成功地通过直接重编程将胰腺外分泌细胞转变成β-细胞，或将成纤维细胞诱导分化为心肌细胞和神经元等报道。那么为何不就地取材，直接将损伤部位活化和大量增殖的星形胶质细胞作为诱导靶细胞，重编程成为神经元(induced neurons，iNs)，一方面可以减轻阻碍轴突生长的胶质疤痕，另一方面也可以直接原位补充因损伤丢失的神经元，一举两得的达到解决脊髓损伤后影响修复再生的两大难 题。Cell transplantation, especially neural stem cell transplantation, has always been considered as a feasible method to replace damaged cells, protect neurons and promote nerve regeneration and repair. However, after transplanted cells enter the local tissues of the injured spinal cord, it is difficult to differentiate into terminal cells (the differentiation is more random). Moreover, stem cells lack a stable cell source, medical ethics, and immunosuppression after transplantation have restricted their clinical applications. In 2006, the advent of induced pluripotent stem cells (iPSCs) technology opened up a new era of regenerative medicine. However, when it is applied to spinal cord injury repair, researchers still find many application limitations. First of all, transcription factors commonly used for reprogramming are highly expressed in various types of tumors, which will make iPSCs have high tumorigenicity; secondly, about 2 weeks after spinal cord injury is the best period for nerve repair, and The time for iPSCs to induce differentiation is too long to complete the differentiation and repair within the optimal repair period. Furthermore, iPSCs cannot guarantee targeted and efficient differentiation into neurons, especially motor neurons, in the complex spinal cord environment. Finally, the process of neural stem cell transplantation is bound to cause secondary damage to nerve tissue. In recent years, researchers have successfully converted pancreatic exocrine cells into β-cells through direct reprogramming, or induced fibroblasts to differentiate into cardiomyocytes and neurons. So why not take the materials locally, directly use the activated and proliferating astrocytes at the injury site as the induction target cells and reprogram them into induced neurons (iNs). On the one hand, it can alleviate the glial scars that hinder the growth of axons On the other hand, it can also directly replenish neurons lost due to injury in situ, which can achieve two major problems that affect repair and regeneration after spinal cord injury.
发明内容Summary of the invention
本发明要解决的技术问题是提供一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，以解决背景技术中所提出的问题。The technical problem to be solved by the present invention is to provide a method for inducing reprogramming of spinal astrocytes into motor neurons to solve the problems raised in the background art.
为解决上述技术问题，本发明的实施例提供一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，其特征在于，包括以下步骤：(1)原代大鼠星形胶质细胞纯化培养，以新生大鼠脊髓的原代星形胶质细胞作为诱导重编程的靶细胞，通过差速贴壁法的体外培养和微波震荡培养获得纯度高的星形胶质细胞；(2)根据神经元诱导以及运动神经元形成发育阶段所需的物质选取了7种小分子药物SB431542、LDN-193189、RA、bFGF、Purmorphamine、Forskolin、VPA，这些小分子药物通过体外对星形胶质细胞进行诱导重编程；(3)观察细胞的形态变化及免疫荧光细胞化学技术分析、实时荧光定量PCR分析和统计学分析方法进行分析星形胶质细胞向运动神经元的重编程及分化情况。In order to solve the above technical problems, the embodiments of the present invention provide a method for inducing reprogramming of spinal astrocytes into motor neurons, which is characterized by including the following steps: (1) Primary rat astrocytes Purified culture, using primary astrocytes from the spinal cord of newborn rats as target cells for inducing reprogramming, and obtaining high-purity astrocytes through in vitro culture of differential adherence method and microwave shaking culture; (2) According to the materials needed for neuron induction and motor neuron formation and development stage, 7 small molecule drugs were selected, SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin, VPA, and these small molecule drugs can affect astrocytes in vitro Carry out induction reprogramming; (3) Observe cell morphology and immunofluorescence cytochemical technology analysis, real-time fluorescence quantitative PCR analysis and statistical analysis methods to analyze the reprogramming and differentiation of astrocytes into motor neurons.
进一步的，所述步骤(1)具体包括以下过程：Further, the step (1) specifically includes the following process:
(1-1)超净工作台提前紫外线照射30min，取出-20℃预冷的冰台、已消毒的手术器械、HBSS缓冲液以及塑料培养皿等放置于超净工作台中，将培养皿中加入适量HBSS置于冰台上备用；(1-1) Ultra-clean workbench is irradiated with ultraviolet rays 30min in advance, take out the ice table pre-cooled at -20℃, sterilized surgical instruments, HBSS buffer and plastic petri dishes, etc., place them in the ultra-clean workbench, and add the petri dishes Place a proper amount of HBSS on the ice table for later use;
(1-2)取新生1日龄大鼠置于放有适量***的50ml离心管中，麻醉后用75％的酒精消毒，并用无菌棉球擦干；(1-2) Take a 1-day-old newborn rat and place it in a 50ml centrifuge tube containing an appropriate amount of ether, disinfect with 75% alcohol after anesthesia, and dry it with a sterile cotton ball;
(1-3)使用组织剪断头的同时，无菌棉球压迫止血，用手固定住大鼠身体使其保持伸展姿态；(1-3) While using the tissue cutting head, the sterile cotton ball is pressed to stop the bleeding, and the rat's body is fixed by hand to maintain the stretched posture;
(1-4)用组织剪沿脊柱方向剪开皮肤，并将颈部肌肉剪开；(1-4) Use tissue scissors to cut the skin along the spine, and cut the neck muscles;
(1-5)用直剪从颈部断处沿脊柱方向将背部脊椎与组织剪开，暴露脊髓；(1-5) Cut the back spine and tissues from the broken part of the neck along the direction of the spine with straight scissors to expose the spinal cord;
(1-6)用显微镊将两侧背根神经节截断并将脊髓挑出置于预冷的HBSS中备用；(1-6) Use micro forceps to cut off the dorsal root ganglia on both sides and pick out the spinal cord in pre-cooled HBSS for later use;
(1-7)将脊髓移至另一装有预冷HBSS的培养皿中置于体视显微镜下，用显微镊剥离表面的脊膜以及表面的血污点，将剥离干净的脊髓取出置于预冷的HBSS中，整个过程在冰台上进行；(1-7) Move the spinal cord to another petri dish containing pre-cooled HBSS and place it under a stereo microscope. Use micro tweezers to peel off the surface of the spinal membrane and blood stains on the surface. In the pre-cooled HBSS, the whole process is carried out on the ice table;
(1-8)使用显微剪将脊髓剪碎并将其置于15ml离心管中，加入2ml 0.25％的胰酶，将离心管放置于37℃水浴中，每5min震荡混匀直至组织块分解，然后向离心管中加入5ml完全培养液终止消化，移入离心机后，以1000rpm转速离心5min，弃去上清加入4ml基培 重悬再以1000rpm转速离心5min，重复2次，最后用4ml完全培养液重悬细胞并接种至25cm 2培养瓶中； (1-8) Use microscissors to cut the spinal cord and place it in a 15ml centrifuge tube, add 2ml 0.25% pancreatin, place the centrifuge tube in a 37℃ water bath, shake and mix every 5 minutes until the tissue mass is decomposed Then, add 5ml complete culture solution to the centrifuge tube to stop the digestion. After moving into the centrifuge, centrifuge at 1000rpm for 5min, discard the supernatant, add 4ml base culture, resuspend and centrifuge at 1000rpm for 5min, repeat twice, and finally complete with 4ml Resuspend cells in culture medium and inoculate them into 25cm 2 culture flask;
(1-9)将培养瓶倒置于CO 2培养箱中培养20min，取出细胞悬液于15ml离心管以1000rpm转速离心5min，去上清并加入完全培养液重悬后，重新将细胞悬液加入25cm 2培养瓶中，置于5％CO 2培养箱中培养，此细胞标记为P0代； (1-9) Put the culture flask upside down in a CO 2 incubator and incubate for 20 minutes, take out the cell suspension and centrifuge at 1000 rpm for 5 minutes in a 15ml centrifuge tube, remove the supernatant and add the complete culture solution to resuspend, then add the cell suspension again Cultured in a 25cm 2 culture flask and placed in a 5% CO 2 incubator, this cell is marked as generation P0;
(1-10)每两天换液直至细胞铺满培养瓶，将培养瓶放置于37℃微波震荡培养箱中以160rpm震荡培养18h去除少突胶质细胞和小胶质细胞；(1-10) Change the medium every two days until the cells cover the culture flask. Place the culture flask in a 37°C microwave shaking incubator and shake culture at 160rpm for 18 hours to remove oligodendrocytes and microglia;
(1-11)震荡培养后弃去培养液，并加入PBS漂洗2-3次，然后再加入预温的1.5ml 0.25％胰酶消化细胞直至细胞能轻轻吹下为止，加入5ml完全培养液终止消化后吹起贴壁细胞，细胞悬液移液到15ml离心管中，1000rpm离心5min后弃去上清，加入1ml完全培养液进行传代；(1-11) After shaking culture, discard the culture medium and add PBS to rinse for 2-3 times, then add pre-warmed 1.5ml 0.25% trypsinized cells until the cells can be gently blown down, add 5ml complete culture medium After terminating the digestion, blow up the adherent cells, transfer the cell suspension to a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, and add 1ml complete culture solution for passage;
(1-12)星形胶质细胞培养至P2-P3代用于后续实验。(1-12) Astrocytes were cultured to generation P2-P3 for subsequent experiments.
进一步的，所述步骤(2)具体包括以下过程：Further, the step (2) specifically includes the following process:
(2-1)取出浸泡在75％酒精中的盖玻片置于24孔细胞培养板中，加入无菌超纯水漂洗3次；(2-1) Take out the cover glass soaked in 75% alcohol and place it in a 24-well cell culture plate, and rinse with sterile ultrapure water for 3 times;
(2-2)吸尽超纯水，加入适量100ng/ml的多聚赖氨酸包被过夜，第二天吸取多余多聚赖氨酸，加入无菌超纯水漂洗3次，吸尽多余液体，晾干备用；清洗过程轻柔，确保多聚赖氨酸不脱落；(2-2) Absorb the ultrapure water, add an appropriate amount of 100ng/ml polylysine to coat overnight, absorb the excess polylysine the next day, add sterile ultrapure water and rinse 3 times to exhaust the excess Liquid, dry for later use; the cleaning process is gentle to ensure that the polylysine does not fall off;
(2-3)待步骤(1)中星形胶质细胞培养至P2-P3代时，加入PBS漂洗2-3次，然后加入预温的2ml 0.25％胰酶消化细胞直至贴壁细胞能轻轻吹下为止，加入5ml完全培养液终止消化后将细胞悬液加入15ml离心管中，1000rpm离心5min后弃去上清，加入1ml完全培养液重悬细胞，以4×104细胞量接种入含有包被多聚赖氨酸的24孔板中，放入5％CO2培养箱培养过夜；(2-3) When the astrocytes in step (1) are cultured to the P2-P3 generation, add PBS to rinse for 2-3 times, and then add 2ml 0.25% trypsin digestion cells pre-warmed until the adherent cells can be light Blow gently, add 5ml complete culture medium to stop the digestion, add the cell suspension to a 15ml centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1ml complete culture medium to resuspend the cells, and inoculate the cells with 4×104 cells. In a 24-well plate coated with polylysine, place it in a 5% CO2 incubator and cultivate overnight;
(2-4)第二天弃去24孔培养板中的培养液，用预温的PBS漂洗2-3次，将药物诱导培养液一混合均匀，加入到培养孔中，培养板放入5％CO2培养箱培养；(2-4) The next day, discard the culture medium in the 24-well culture plate, rinse with pre-warmed PBS for 2-3 times, mix the drug-induced culture medium evenly, add it to the culture well, and place the culture plate into 5 %CO2 incubator culture;
(2-5)药物诱导培养液一培养2天后，细胞培养孔中换入药物诱导培养液二并置于5％CO2培养箱培养；(2-5) After the drug-induced culture medium is cultured for 2 days, the cell culture well is replaced with drug-induced culture medium two and placed in a 5% CO2 incubator for culture;
(2-6)药物诱导培养液二培养8-10天后，细胞培养孔中换入药物诱导培养液三并置于5％CO2培养箱培养，每2-3天换液。(2-6) After the second culturing of the drug-induced culture medium for 8-10 days, the cell culture wells were replaced with the drug-induced culture medium three and placed in a 5% CO2 incubator for culture. The medium was changed every 2-3 days.
进一步的，所述步骤(3)中免疫荧光细胞化学技术分析具体包括以下过程：Further, the immunofluorescence cytochemical technique analysis in the step (3) specifically includes the following processes:
(3-1-1)取出24孔细胞培养板，将待染色孔中的培养液吸尽，加入预温的PBS漂洗2-3次，取出盖玻片放置于另一装有300ml预温4％多聚甲醛的24孔板中，固定10min；(3-1-1) Take out the 24-well cell culture plate, suck up the culture solution in the well to be stained, add pre-warmed PBS to rinse 2-3 times, take out the cover slip and place it in another 300ml pre-warmed 4 In a 24-well plate containing% paraformaldehyde, fix for 10 minutes;
(3-1-2)弃去4％多聚甲醛，每孔加入300μl PBS漂洗3次，每次5min；(3-1-3)弃去PBS，每孔加入300μl破膜液，常温孵育30min；(3-1-2) Discard 4% paraformaldehyde, add 300μl PBS to each well to rinse 3 times, 5min each time; (3-1-3) Discard PBS, add 300μl rupture fluid to each well, incubate for 30min at room temperature ；
(3-1-4)弃去破膜液，每孔加入300μl封闭液，常温孵育30min；(3-1-4) Discard the rupture fluid, add 300μl of blocking fluid to each well, and incubate for 30min at room temperature;
(3-1-5)弃去封闭液，每孔加入300μl一抗与封闭液混合液，4℃孵育过夜；(3-1-5) Discard the blocking solution, add 300μl of primary antibody and blocking solution mixture to each well, and incubate overnight at 4°C;
(3-1-6)第二天弃去一抗，每孔加入300μl PBS漂洗3次，每次5min；(3-1-6) Discard the primary antibody the next day, add 300μl PBS to each well and rinse 3 times for 5 minutes each time;
(3-1-7)弃去PBS，每孔加入300ml二抗与PBS的混合液，4℃孵育≥2h，避光进行处理；(3-1-7) Discard PBS, add 300ml of a mixture of secondary antibody and PBS to each well, incubate at 4°C for ≥2h, protect from light;
(3-1-8)弃去二抗混合液，每孔加入300μl PBS漂洗3次，每次5min，避光进行处理；(3-1-8) Discard the secondary antibody mixture, add 300μl PBS to each well and rinse 3 times, 5min each time, and treat it in the dark;
(3-1-9)加入PBS稀释比1:1000的Hoechst常温孵育10min，弃去Hoechst稀释液，加入300ml PBS漂洗3次，每次5min，避光进行处理；(3-1-9) Add Hoechst with a PBS dilution ratio of 1:1000 and incubate at room temperature for 10 minutes, discard the Hoechst diluent, add 300ml PBS and rinse 3 times, 5 minutes each time, and perform treatment in the dark;
(3-1-10)取粘附性载玻片滴上封片液，将盖玻片进行封片，避光处理；(3-1-10) Take the adhesive slide glass and drop it with mounting liquid, and mount the cover glass to avoid light;
(3-1-11)在激光共聚焦显微镜下观察并拍照记录。(3-1-11) Observed and photographed and recorded under a laser confocal microscope.
进一步的，所述步骤(3)中实时荧光定量PCR分析具体包括以下过程：Further, the real-time fluorescent quantitative PCR analysis in the step (3) specifically includes the following process:
(3-2-1)取出含细胞的培养皿，弃去培养基，并用预热至37℃PBS冲洗3次，加入1ml Trizol轻轻吹打将细胞混匀，并收集细胞悬液于1.5ml EP管中，冰上静置5min；(3-2-1) Take out the culture dish containing the cells, discard the culture medium, and rinse with PBS preheated to 37°C for 3 times, add 1ml Trizol and gently pipette to mix the cells, and collect the cell suspension in 1.5ml EP In the tube, let stand on ice for 5 minutes;
(3-2-2)向EP管中加入250μl氯仿震荡混匀，并置于-20℃冰箱中静置1-2h；取出EP管12000rpm，4℃，离心5分钟；(3-2-2) Add 250μl of chloroform to the EP tube, shake and mix, and place it in a refrigerator at -20℃ for 1-2h; take out the EP tube at 12000rpm, 4℃, and centrifuge for 5 minutes;
(3-2-3)去上层清液于另一EP管中，向其中加入等体积的异丙醇，颠倒混匀，12000rpm，4℃，离心10min；(3-2-3) Remove the supernatant and put it in another EP tube, add an equal volume of isopropanol to it, mix upside down, and centrifuge at 12000rpm, 4°C for 10min;
(3-2-4)小心弃去液体加入1ml 75％的乙醇，7500rpm，4℃，离心5min，弃去液体，重复此步骤；(3-2-4) Carefully discard the liquid, add 1ml 75% ethanol, 7500rpm, 4℃, centrifuge for 5min, discard the liquid, repeat this step;
(3-2-5)加入1ml无水乙醇，7500rpm，4℃，离心5min，弃去液体，并将EP管置于超净台中吹干；(3-2-5) Add 1ml of absolute ethanol, 7500rpm, 4℃, centrifuge for 5min, discard the liquid, and place the EP tube in an ultraclean table to blow dry;
(3-2-6)向EP管中加入20μl DEPC水溶解RNA，并将RNA进行逆转录，剩余的保存于-80℃冰箱；(3-2-6) Add 20μl DEPC water to the EP tube to dissolve RNA, reverse transcription of RNA, and store the rest in a refrigerator at -80℃;
(3-2-7)取逆转录试剂盒，以下表的体系配比及PCR反应条件对RNA进行逆转录：(3-2-7) Take the reverse transcription kit, and perform reverse transcription on RNA with the system ratio and PCR reaction conditions in the following table:
Figure PCTCN2020080792-appb-000001
Figure PCTCN2020080792-appb-000001
(3-2-8)逆转录获得的cDNA用于qPCR扩增，剩余的保存于-80℃冰箱中；(3-2-8) cDNA obtained by reverse transcription is used for qPCR amplification, and the rest is stored in a refrigerator at -80℃;
(3-2-9)取FastStart Universal SYBR Green Master中试剂置于冰盒上，以下表的体系配比及反应条件进行PCR扩增：(3-2-9) Put the reagents in FastStart Universal SYBR Green Master on the ice box, and perform PCR amplification with the system ratio and reaction conditions in the following table:
Figure PCTCN2020080792-appb-000002
Figure PCTCN2020080792-appb-000002
Figure PCTCN2020080792-appb-000003
Figure PCTCN2020080792-appb-000003
(3-2-10)PCR扩增结束后对溶解曲线进行分析，确保PCR产物特异性。(3-2-10) Analyze the melting curve after PCR amplification to ensure the specificity of PCR products.
进一步的，所述步骤(3)中统计学分析具体包括以下过程：通过软件Graphpad prism5进行数据统计，对所有量化数据进行统计分析并以平均值±标准差表示；双侧t检验用于计算p值的统计显着性，p值<0.05具有统计学意义。Further, the statistical analysis in the step (3) specifically includes the following process: performing data statistics through the software Graphpad prism5, performing statistical analysis on all quantified data and expressing the average value ± standard deviation; the two-sided t test is used to calculate p The statistical significance of the value, p value <0.05 is statistically significant.
其中，所述完全培养液采用在DMEM/F12基础培养基中添加1％的双抗和10％的FBS的方法制成。Wherein, the complete culture medium is prepared by adding 1% double antibody and 10% FBS to DMEM/F12 basic medium.
1.其中，所述药物诱导培养液一采用在基础诱导培养液中添加2μM SB431542、1μM RA、5μM Forskolin、20ng/ml bFGF、0.25μM LDN-193189、制成；所述药物诱导培养液二采用在基础诱导培养基中添加5μM Purmorphamine、10μM RA、20μM Forskolin、200ng/ml bFGF制成；所述药物诱导培养液三采用在基础诱导培养基中添加1mM VPA、5μM Purmorphamine、10μM RA、20μM Forskolin、200ng/ml bFGF制成。1. Wherein, the drug induction culture medium one is made by adding 2μM SB431542, 1μM RA, 5μM Forskolin, 20ng/ml bFGF, 0.25μM LDN-193189 to the basic induction medium; the drug induction culture medium two uses It is made by adding 5μM Purmorphamine, 10μM RA, 20μM Forskolin, 200ng/ml bFGF to the basal induction medium; the drug induction medium 3 is made by adding 1mM VPA, 5μM Purmorphamine, 10μM RA, 20μM Forskolin, 200ng/ml bFGF.
上述的，所述基础诱导培养液采用在DMEM/F12基础培养基中添加1％的双抗、1M B27、1M N 2、2mM L-谷氨酸、3.5mM葡萄糖制成。 Above, the base employed inducing medium was added at 1% of the double antibody DMEM / F12 base medium, 1M B27,1M N 2, 2mM L- glutamic acid, 3.5mM glucose formed.
本发明的上述技术方案的有益效果如下：本发明的小分子药物组合能在体外成功地将大鼠星形胶质细胞诱导重编程为运动神经元，且转分化率超过75％；另外，该小分子药物组合也能成功完成人源星形胶质细胞向神经元的转分化；对星形胶质细胞的原位直接重编程成神经元，可能在脊髓损伤修复及功能重建的治疗中比较切实可行的思路。The beneficial effects of the above technical solutions of the present invention are as follows: the small molecule drug combination of the present invention can successfully induce reprogramming of rat astrocytes into motor neurons in vitro, and the transdifferentiation rate exceeds 75%; in addition, the The small molecule drug combination can also successfully complete the transdifferentiation of human astrocytes into neurons; the direct reprogramming of astrocytes into neurons in situ may be compared in the treatment of spinal cord injury repair and functional reconstruction Practical ideas.
附图说明Description of the drawings
图1为本发明中P0代星形胶质细胞的GFAP免疫荧光细胞化学染色图；Figure 1 is a GFAP immunofluorescence cytochemical staining diagram of P0 generation astrocytes in the present invention;
图2分别为本发明中P2代星形胶质细胞的GFAP和Nestin免疫荧光细胞化学染色图；Figure 2 is respectively the GFAP and Nestin immunofluorescence cytochemical staining diagrams of P2 generation astrocytes of the present invention;
图3从左至右分别为小分子药物组在1天、3天、9天、20天时星形胶质细胞形态图；Figure 3 shows the morphology of astrocytes in the small molecule drug group at 1, 3, 9 and 20 days from left to right;
图4从左至右分别为对照组在1天、3天、9天、20天时星形胶质细胞形态图；Figure 4 shows the morphology of astrocytes in the control group on day 1, 3, 9 and 20 from left to right;
图5分别为体外诱导星形胶质细胞6天时小分子药物组GFAP和Tuj1染色图；Figure 5 shows the GFAP and Tuj1 staining images of the small molecule drug group when astrocytes are induced in vitro for 6 days;
图6分别为体外诱导星形胶质细胞6天时对照组GFAP和Tuj1染色图；Figure 6 is the GFAP and Tuj1 staining images of the control group when astrocytes are induced in vitro for 6 days;
图7为图5的局部放大图；Figure 7 is a partial enlarged view of Figure 5;
图8分别为小分子药物体外诱导星形胶质细胞10天时MAP2和NeuN染色图；Figure 8 shows the MAP2 and NeuN staining images of astrocytes induced by small molecule drugs for 10 days in vitro;
图9为小分子药物体外诱导星形胶质细胞16天时MAP2染色图；Figure 9 is a MAP2 staining image of astrocytes induced by small molecule drugs in vitro for 16 days;
图10为小分子药物诱导星形胶质细胞转分化为神经元的转分化率(mean±SEM,n＝3independent experiments)；Figure 10 shows the transdifferentiation rate of astrocytes into neurons induced by small molecule drugs (mean±SEM, n=3independent experiments);
图11为小分子诱导星形胶质细胞不同时期相关基因的相对表达情况；Figure 11 shows the relative expression of related genes in different stages of astrocytes induced by small molecules;
图12分别为小分子药物诱导星形胶质细胞16天时MAP2、HB9以及CHAT染色图；Figure 12 shows the staining of MAP2, HB9 and CHAT when small molecule drugs induce astrocytes for 16 days;
图13分别为小分子药物诱导星形胶质细胞30天时MAP2、HB9以及CHAT染色图；Figure 13 shows the MAP2, HB9 and CHAT staining images of astrocytes induced by small molecule drugs for 30 days;
图14分别为小分子药物诱导星形胶质细胞18天时MAP2以及GAD65染色图；Figure 14 shows the staining of MAP2 and GAD65 when small molecule drugs induce astrocytes for 18 days;
图15分别为小分子药物诱导星形胶质细胞18天时MAP2以及VGLUT1染色图；Figure 15 shows the staining of MAP2 and VGLUT1 when small molecule drugs induce astrocytes for 18 days;
图16为小分子药物诱导星形胶质细胞16天时诱导神经元HB9、CHAT、VGLUT1以及GAD65阳性率。Figure 16 shows the positive rate of neurons HB9, CHAT, VGLUT1 and GAD65 induced by small molecule drugs on astrocytes for 16 days.
具体实施方式Detailed ways
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below in conjunction with the drawings and specific embodiments.
一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，包括以下步骤：A method for inducing reprogramming of spinal cord astrocytes into motor neurons includes the following steps:
(1)原代大鼠星形胶质细胞纯化培养，以新生大鼠脊髓的原代星形胶质细胞作为诱导重编程的靶细胞，通过差速贴壁法的体外培养和微波震荡培养获得纯度高的星形胶质细胞；具体包括以下过程：(1) Purification and culture of primary rat astrocytes, using primary astrocytes from the spinal cord of newborn rats as target cells for inducing reprogramming, obtained by in vitro culture of differential adherence method and microwave shaking culture High-purity astrocytes; specifically include the following processes:
(1-1)超净工作台提前紫外线照射30min，取出-20℃预冷的冰台、已消毒的手术器械、HBSS缓冲液以及塑料培养皿等放置于超净工作台中，将培养皿中加入适量HBSS置于冰台上备用。(1-1) Ultra-clean workbench is irradiated with ultraviolet rays 30min in advance, take out the ice table pre-cooled at -20℃, sterilized surgical instruments, HBSS buffer and plastic petri dishes, etc., place them in the ultra-clean workbench, and add the culture dishes Place an appropriate amount of HBSS on the ice table for later use.
(1-2)取新生1日龄大鼠置于放有适量***的50ml离心管中，麻醉后用75％的酒精消毒，并用无菌棉球擦干。(1-2) Take a 1-day-old newborn rat and place it in a 50ml centrifuge tube containing an appropriate amount of ether, disinfect with 75% alcohol after anesthesia, and dry it with a sterile cotton ball.
(1-3)使用组织剪断头的同时，无菌棉球压迫止血，用手固定住大鼠身体使其保持伸展姿态。(1-3) While using the tissue cutting head, the sterile cotton ball is pressed to stop the bleeding, and the rat's body is fixed by hand to maintain the stretched posture.
(1-4)用组织剪沿脊柱方向剪开皮肤，并将颈部肌肉剪开。(1-4) Use tissue scissors to cut the skin along the spine, and cut the neck muscles.
(1-5)用直剪从颈部断处沿脊柱方向将背部脊椎与组织剪开，暴露脊髓。(1-5) Use straight shears to cut the back spine and tissue along the direction of the spine from the neck section to expose the spinal cord.
(1-6)用显微镊将两侧背根神经节截断并将脊髓挑出置于预冷的HBSS中备用。(1-6) Cut off the dorsal root ganglia on both sides with micro forceps and pick out the spinal cord and place it in pre-cooled HBSS for later use.
(1-7)将脊髓移至另一装有预冷HBSS的培养皿中置于体视显微镜下，用显微镊剥离表面的脊膜以及表面的血污点，将剥离干净的脊髓取出置于预冷的HBSS中，整个过程在冰台上进行。(1-7) Move the spinal cord to another petri dish containing pre-cooled HBSS and place it under a stereo microscope. Use micro tweezers to peel off the surface of the spinal membrane and blood stains on the surface. In the pre-cooled HBSS, the whole process is carried out on the ice table.
(1-8)使用显微剪将脊髓剪碎并将其置于15ml离心管中，加入2ml 0.25％的胰酶，将离心管放置于37℃水浴中，每5min震荡混匀直至组织块分解，然后向离心管中加入5ml完全培养液终止消化，移入离心机后，以1000rpm转速离心5min，弃去上清加入4ml基培重悬再以1000rpm转速离心5min，重复2次，最后用4ml完全培养液重悬细胞并接种至25cm 2培养瓶中。 (1-8) Use microscissors to cut the spinal cord and place it in a 15ml centrifuge tube, add 2ml 0.25% pancreatin, place the centrifuge tube in a 37℃ water bath, shake and mix every 5 minutes until the tissue mass is decomposed Then, add 5ml complete culture solution to the centrifuge tube to stop the digestion. After moving into the centrifuge, centrifuge at 1000rpm for 5min, discard the supernatant, add 4ml base culture, resuspend and centrifuge at 1000rpm for 5min, repeat twice, and finally complete with 4ml Resuspend the cells in the culture medium and inoculate them into a 25cm 2 culture flask.
(1-9)将培养瓶倒置于CO 2培养箱中培养20min，取出细胞悬液于15ml离心管以1000rpm转速离心5min，去上清并加入完全培养液重悬后，重新将细胞悬液加入25cm 2培养瓶中，置于5％CO 2培养箱中培养，此细胞标记为P0代。 (1-9) Put the culture flask upside down in a CO 2 incubator and incubate for 20 minutes, take out the cell suspension and centrifuge at 1000 rpm for 5 minutes in a 15ml centrifuge tube, remove the supernatant and add the complete culture solution to resuspend, then add the cell suspension again Cultured in a 25cm 2 culture flask and placed in a 5% CO 2 incubator, the cells are labeled as generation P0.
(1-10)每两天换液直至细胞铺满培养瓶，将培养瓶放置于37℃微波震荡培养箱中以160rpm震荡培养18h去除少突胶质细胞和小胶质细胞。(1-10) Change the medium every two days until the cells cover the culture flask. Place the culture flask in a 37°C microwave shaking incubator and shake culture at 160 rpm for 18 hours to remove oligodendrocytes and microglia.
(1-11)震荡培养后弃去培养液，并加入PBS漂洗2-3次，然后再加入预温的1.5ml 0.25％胰酶消化细胞直至细胞能轻轻吹下为止，加入5ml完全培养液终止消化后吹起贴壁细胞，细胞悬液移液到15ml离心管中，1000rpm离心5min后弃去上清，加入1ml完全培养液进行传代。(1-11) After shaking culture, discard the culture medium and add PBS to rinse for 2-3 times, then add pre-warmed 1.5ml 0.25% trypsinized cells until the cells can be gently blown down, add 5ml complete culture medium After terminating the digestion, blow up the adherent cells, transfer the cell suspension to a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, and add 1ml complete culture solution for passage.
(1-12)星形胶质细胞培养至P2-P3代用于后续实验。(1-12) Astrocytes were cultured to generation P2-P3 for subsequent experiments.
(2)根据神经元诱导以及运动神经元形成发育阶段所需的物质选取了7种小分子药物SB431542、LDN-193189、RA、bFGF、Purmorphamine、Forskolin、VPA，这些小分子药物通过体外对星形胶质细胞进行诱导重编程；具体包括以下过程：(2) Seven small molecule drugs SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin, and VPA were selected according to the materials required for neuron induction and motor neuron formation and development. Glial cells undergo induced reprogramming; specifically including the following processes:
(2-1)取出浸泡在75％酒精中的盖玻片置于24孔细胞培养板中，加入无菌超纯水漂洗3次；(2-1) Take out the cover glass soaked in 75% alcohol and place it in a 24-well cell culture plate, and rinse with sterile ultrapure water for 3 times;
(2-2)吸尽超纯水，加入适量100ng/ml的多聚赖氨酸包被过夜，第二天吸取多余多聚赖氨酸，加入无菌超纯水漂洗3次，吸尽多余液体，晾干备用；清洗过程轻柔，确保多聚赖氨酸不脱落；(2-2) Absorb the ultrapure water, add an appropriate amount of 100ng/ml polylysine to coat overnight, absorb the excess polylysine the next day, add sterile ultrapure water and rinse 3 times to exhaust the excess Liquid, dry for later use; the cleaning process is gentle to ensure that the polylysine does not fall off;
(2-3)待步骤(1)中星形胶质细胞培养至P2-P3代时，加入PBS漂洗2-3次，然后加入预温的2ml 0.25％胰酶消化细胞直至贴壁细胞能轻轻吹下为止，加入5ml完全培养液终止消化后将细胞悬液加入15ml离心管中，1000rpm离心5min后弃去上清，加入1ml完全培养液重悬细胞，以4×10 4细胞量接种入含有包被多聚赖氨酸的24孔板中，放入5％CO 2培养箱培养过夜； (2-3) When the astrocytes in step (1) are cultured to the P2-P3 generation, add PBS to rinse 2-3 times, and then add 2ml of pre-warmed 0.25% trypsin digestion cells until the adherent cells can be light Blow gently, add 5ml of complete culture fluid to terminate the digestion, add the cell suspension to a 15ml centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1ml of complete culture fluid to resuspend the cells, and inoculate with 4×10 4 cells In a 24-well plate containing coated polylysine, place it in a 5% CO 2 incubator and culture overnight;
(2-4)第二天弃去24孔培养板中的培养液，用预温的PBS漂洗2-3次，将药物诱导培养液一混合均匀，加入到培养孔中，培养板放入5％CO 2培养箱培养； (2-4) The next day, discard the culture medium in the 24-well culture plate, rinse with pre-warmed PBS for 2-3 times, mix the drug-induced culture medium evenly, add it to the culture wells, put the culture plate into 5 %CO 2 incubator culture;
(2-5)药物诱导培养液一培养2天后，细胞培养孔中换入药物诱导培养液二并置于5％CO 2培养箱培养； (2-5) After the drug-induced culture medium is cultured for 2 days, the cell culture well is replaced with the drug-induced culture medium and placed in a 5% CO 2 incubator for culture;
(2-6)药物诱导培养液二培养8-10天后，细胞培养孔中换入药物诱导培养液三并置于5％CO 2培养箱培养，每2-3天换液。 (2-6) After the second culturing of the drug-induced culture medium for 8-10 days, the cell culture well is replaced with the drug-induced culture medium three and placed in a 5% CO 2 incubator for culture, and the medium is changed every 2-3 days.
(3)观察细胞的形态变化及免疫荧光细胞化学技术分析、实时荧光定量PCR分析和统计学分析方法进行分析星形胶质细胞向运动神经元的重编程及分化情况。(3) Observe the morphological changes of the cells and analyze the immunofluorescence cytochemical technology analysis, real-time fluorescence quantitative PCR analysis and statistical analysis methods to analyze the reprogramming and differentiation of astrocytes into motor neurons.
其中，免疫荧光细胞化学技术分析具体包括以下过程：Among them, the immunofluorescence cytochemical analysis includes the following processes:
(3-1-1)取出24孔细胞培养板，将待染色孔中的培养液吸尽，加入预温的PBS漂洗2-3次，取出盖玻片放置于另一装有300ml预温4％多聚甲醛的24孔板中，固定10min；(3-1-1) Take out the 24-well cell culture plate, suck up the culture solution in the well to be stained, add pre-warmed PBS to rinse 2-3 times, take out the cover slip and place it in another 300ml pre-warmed 4 In a 24-well plate containing% paraformaldehyde, fix for 10 minutes;
(3-1-2)弃去4％多聚甲醛，每孔加入300μl PBS漂洗3次，每次5min；(3-1-3)弃去PBS，每孔加入300μl破膜液，常温孵育30min；(3-1-2) Discard 4% paraformaldehyde, add 300μl PBS to each well to rinse 3 times, 5min each time; (3-1-3) Discard PBS, add 300μl rupture fluid to each well, incubate for 30min at room temperature ；
(3-1-4)弃去破膜液，每孔加入300μl封闭液，常温孵育30min；(3-1-4) Discard the rupture fluid, add 300μl of blocking fluid to each well, and incubate for 30min at room temperature;
(3-1-5)弃去封闭液，每孔加入300μl一抗与封闭液混合液，4℃孵育过夜；(3-1-5) Discard the blocking solution, add 300μl of primary antibody and blocking solution mixture to each well, and incubate overnight at 4°C;
(3-1-6)第二天弃去一抗，每孔加入300μl PBS漂洗3次，每次5min；(3-1-6) Discard the primary antibody the next day, add 300μl PBS to each well and rinse 3 times for 5 minutes each time;
(3-1-7)弃去PBS，每孔加入300ml二抗与PBS的混合液，4℃孵育≥2h，避光进行处理；(3-1-7) Discard PBS, add 300ml of a mixture of secondary antibody and PBS to each well, incubate at 4°C for ≥2h, protect from light;
(3-1-8)弃去二抗混合液，每孔加入300μl PBS漂洗3次，每次5min，避光进行处理；(3-1-8) Discard the secondary antibody mixture, add 300μl PBS to each well and rinse 3 times, 5min each time, and treat it in the dark;
(3-1-9)加入PBS稀释比1:1000的Hoechst常温孵育10min，弃去Hoechst稀释液，加入300ml PBS漂洗3次，每次5min，避光进行处理；(3-1-9) Add Hoechst with a PBS dilution ratio of 1:1000 and incubate at room temperature for 10 minutes, discard the Hoechst diluent, add 300ml PBS and rinse 3 times, 5 minutes each time, and perform treatment in the dark;
(3-1-10)取粘附性载玻片滴上封片液，将盖玻片进行封片，避光处理；(3-1-10) Take the adhesive slide glass and drop it with mounting liquid, and mount the cover glass to avoid light;
(3-1-11)在激光共聚焦显微镜下观察并拍照记录。(3-1-11) Observed and photographed and recorded under a laser confocal microscope.
其中，荧光定量PCR分析具体包括以下过程：Among them, fluorescence quantitative PCR analysis specifically includes the following processes:
(3-2-1)取出含细胞的培养皿，弃去培养基，并用预热至37℃PBS冲洗3次，加入1ml Trizol轻轻吹打将细胞混匀，并收集细胞悬液于1.5ml EP管中，冰上静置5min；(3-2-1) Take out the culture dish containing the cells, discard the culture medium, and rinse with PBS preheated to 37°C for 3 times, add 1ml Trizol and gently pipette to mix the cells, and collect the cell suspension in 1.5ml EP In the tube, let stand on ice for 5 minutes;
(3-2-2)向EP管中加入250μl氯仿震荡混匀，并置于-20℃冰箱中静置1-2h；取出EP管12000rpm，4℃，离心5分钟；(3-2-2) Add 250μl of chloroform to the EP tube, shake and mix, and place it in a refrigerator at -20℃ for 1-2h; take out the EP tube at 12000rpm, 4℃, and centrifuge for 5 minutes;
(3-2-3)去上层清液于另一EP管中，向其中加入等体积的异丙醇，颠倒混匀，12000rpm，4℃，离心10min；(3-2-3) Remove the supernatant and put it in another EP tube, add an equal volume of isopropanol to it, mix upside down, and centrifuge at 12000rpm, 4°C for 10min;
(3-2-4)小心弃去液体加入1ml 75％的乙醇，7500rpm，4℃，离心5min，弃去液体，重复此步骤；(3-2-4) Carefully discard the liquid, add 1ml 75% ethanol, 7500rpm, 4℃, centrifuge for 5min, discard the liquid, repeat this step;
(3-2-5)加入1ml无水乙醇，7500rpm，4℃，离心5min，弃去液体，并将EP管置于超净台中吹干；(3-2-5) Add 1ml of absolute ethanol, 7500rpm, 4℃, centrifuge for 5min, discard the liquid, and place the EP tube in an ultraclean table to blow dry;
(3-2-6)向EP管中加入20μl DEPC水溶解RNA，并将部分RNA进行逆转录(根据每次 实验所需RNA量不同，RNA浓度不低于200ng/μl即可)；剩余的保存于-80℃冰箱；(3-2-6) Add 20μl DEPC water to the EP tube to dissolve RNA, and perform reverse transcription of part of the RNA (according to the amount of RNA required for each experiment, the RNA concentration should not be less than 200ng/μl); Store in the refrigerator at -80℃;
(3-2-7)取逆转录试剂盒，以下表的体系配比及PCR反应条件对RNA进行逆转录：(3-2-7) Take the reverse transcription kit, and perform reverse transcription on RNA with the system ratio and PCR reaction conditions in the following table:
Figure PCTCN2020080792-appb-000004
Figure PCTCN2020080792-appb-000004
(3-2-8)逆转录获得的部分cDNA用于qPCR扩增(根据每次实验所需cDNA量进行确定)，剩余的保存于-80℃冰箱中；(3-2-8) Part of the cDNA obtained by reverse transcription is used for qPCR amplification (determined according to the amount of cDNA required for each experiment), and the rest is stored in a refrigerator at -80°C;
(3-2-9)取FastStart Universal SYBR Green Master中试剂置于冰盒上，以下表的体系配比及反应条件进行PCR扩增：(3-2-9) Put the reagents in FastStart Universal SYBR Green Master on the ice box, and perform PCR amplification with the system ratio and reaction conditions in the following table:
Figure PCTCN2020080792-appb-000005
Figure PCTCN2020080792-appb-000005
Figure PCTCN2020080792-appb-000006
Figure PCTCN2020080792-appb-000006
(3-2-10)PCR扩增结束后对溶解曲线进行分析，确保PCR产物特异性。(3-2-10) Analyze the melting curve after PCR amplification to ensure the specificity of PCR products.
其中，统计学分析具体包括以下过程：通过软件Graphpad prism5进行数据统计，对所有量化数据进行统计分析并以平均值±标准差表示；双侧t检验用于计算p值的统计显着性，p值<0.05具有统计学意义。Among them, the statistical analysis specifically includes the following process: data statistics through the software Graphpad prism5, statistical analysis of all quantified data and expressed in average ± standard deviation; two-sided t test is used to calculate the statistical significance of the p value, p A value of <0.05 is statistically significant.
上述的，所述完全培养液采用在DMEM/F12基础培养基中添加1％的双抗和10％的FBS的方法制成。As mentioned above, the complete culture medium is prepared by adding 1% double antibody and 10% FBS to DMEM/F12 basic medium.
上述的，所述药物诱导培养液一采用在基础诱导培养液中添加2μM SB431542、1μM RA、5μM Forskolin、20ng/ml bFGF、0.25μM LDN-193189、制成；所述药物诱导培养液二采用在基础诱导培养基中添加5μM Purmorphamine、10μM RA、20μM Forskolin、200ng/ml bFGF制成；所述药物诱导培养液三采用在基础诱导培养基中添加1mM VPA、5μM Purmorphamine、10μM RA、20μM Forskolin、200ng/ml bFGF制成。As mentioned above, the first drug induction culture medium is made by adding 2μM SB431542, 1μM RA, 5μM Forskolin, 20ng/ml bFGF, 0.25μM LDN-193189 to the basic induction culture solution; the second drug induction culture solution It is made by adding 5μM Purmorphamine, 10μM RA, 20μM Forskolin, 200ng/ml bFGF to the basal induction medium; the drug induction culture medium 3 is made by adding 1mM VPA, 5μM Purmorphamine, 10μM RA, 20μM Forskolin, 200ng to the basal induction medium /ml Made of bFGF.
上述的，所述基础诱导培养液采用在DMEM/F12基础培养基中添加1％的双抗、1M B27、1M N 2、2mM L-谷氨酸、3.5mM葡萄糖制成。 Above, the base employed inducing medium was added at 1% of the double antibody DMEM / F12 base medium, 1M B27,1M N 2, 2mM L- glutamic acid, 3.5mM glucose formed.
本发明的小分子药物组合能在体外成功地将大鼠星形胶质细胞诱导重编程为运动神经元，且转分化率超过75％；另外，该小分子药物组合也能成功完成人源星形胶质细胞向神经元的转分化；对星形胶质细胞的原位直接重编程成神经元，可能在脊髓损伤修复及功能重建的治疗中比较切实可行的思路。The small molecule drug combination of the present invention can successfully induce and reprogram rat astrocytes into motor neurons in vitro, and the transdifferentiation rate exceeds 75%; in addition, the small molecule drug combination can also successfully complete the human origin star Transdifferentiation of glial cells into neurons; direct reprogramming of astrocytes into neurons in situ may be a more feasible idea in the treatment of spinal cord injury repair and functional reconstruction.
本发明可以得到以下实验结果：The present invention can obtain the following experimental results:
结果1、脊髓星形胶质细胞的形态和纯化率：Results 1. Morphology and purification rate of spinal cord astrocytes:
大鼠脊髓来源的星形胶质细胞在接种至细胞培养瓶中通过差速贴壁法去除少量的成纤维细胞，随后添加完全培养基培养促进胶质细胞的***增殖。由于P0代细胞中还存在少量神经元、少突胶质细胞和小胶质细胞，在相差显微镜下观察细胞形态仍呈多样性(图1)。待到P0代细胞融合度达到80％左右，通过微波震荡培养去除杂细胞并传代培养至P2代后，用星形胶质细胞内特有的胶原纤维酸性蛋白(GFAP)染色，鉴定GFAP阳性细胞达95％以上(图1、图2)。在相差显微镜下P2代细胞形态多数呈扁平多边形，细胞胞体较大，细胞核大而明显，细胞具有少量不规则凸起(图2)。Rat spinal cord-derived astrocytes were inoculated into cell culture flasks to remove a small amount of fibroblasts by differential adhesion method, and then cultured with complete medium to promote the division and proliferation of glial cells. Since there are still a small number of neurons, oligodendrocytes and microglia in the P0 generation cells, the cell morphology is still diverse when observed under a phase contrast microscope (Figure 1). After the P0 generation cell fusion degree reaches about 80%, the contaminated cells are removed by microwave shaking culture and subcultured to the P2 generation, then stained with the unique collagen fibrillary acidic protein (GFAP) in astrocytes to identify GFAP positive cells. More than 95% (Figure 1, Figure 2). Under a phase-contrast microscope, most of the P2 generation cells were flat polygonal, with larger cell bodies, large and obvious nuclei, and a small number of irregular protrusions (Figure 2).
结果2、纯化的星形胶质细胞表达Nestin情况 Result 2. The purified astrocytes express Nestin
通过提取新生大鼠脊髓而获取的星形胶质细胞中可能混有少量神经干细胞。神经干细胞是一类具有较高***潜能和自我更新能力的母细胞，在机体遭遇特殊情况时可以通过增殖分化形成不同类型的神经组织细胞，如神经元、星形胶质细胞、少突胶质细胞等。大量的研究发现反应性星形胶质细胞在损伤处活化后，也会自我更新增殖并获得类似神经干细胞的潜能；另外，有研究发现在体外培养的星形胶质细胞，当某些条件适宜时可以使细胞形态发生改变，进而出现类似体内损伤时活化的状态，并且最终表达部分神经干细胞标志物甚至去分化成类 神经干细胞。因此在本实验进行重编程之前，首先排除细胞纯化培养过程中，可能造成的星形胶质细胞的去分化现象，对准备进行小分子药物诱导的星形胶质细胞进行GFAP及神经干细胞标志物巢蛋白Nestin的双标免疫细胞化学染色鉴定。结果显示，经过纯化传代培养的星形胶质细胞并未表达Nestin，这就表明体外培养的细胞中没有混入神经干细胞，并且星形胶质细胞自身也没有出现去分化成类神经干细胞现象(图2)。A small amount of neural stem cells may be mixed with astrocytes obtained by extracting spinal cord of newborn rats. Neural stem cells are a type of mother cell with high division potential and self-renewal ability. When the body encounters special conditions, it can proliferate and differentiate to form different types of nerve tissue cells, such as neurons, astrocytes, and oligodendrocytes. Cells etc. A large number of studies have found that reactive astrocytes will also self-renew and proliferate after being activated at the injury site and acquire the potential similar to neural stem cells; in addition, studies have found that astrocytes cultured in vitro, when certain conditions are suitable At the same time, the cell morphology can be changed, and then it will appear a state similar to the activation of in vivo injury, and finally express some neural stem cell markers and even dedifferentiate into neural stem-like cells. Therefore, before reprogramming in this experiment, first eliminate the possible dedifferentiation of astrocytes during the cell purification and culture process, and perform GFAP and neural stem cell markers on astrocytes that are to be induced by small molecule drugs. Double-labeled immunocytochemical staining identification of Nestin. The results showed that the purified and subcultured astrocytes did not express Nestin, which indicated that the cells cultured in vitro were not mixed with neural stem cells, and the astrocytes themselves did not dedifferentiate into neural stem-like cells (Figure 2).
结果3、小分子药物组合诱导星形胶质细胞的形态变化 Result 3. The combination of small molecule drugs induces morphological changes of astrocytes
星形胶质细胞的重编程并不仅依赖于转录因子，小分子药物也有一定的诱导重编程作用。本次试验我们应用SB431542、LDN-193189、RA、bFGF、Purmorphamine、Forskolin、VPA来进行重编程。星形胶质细胞首先在药物诱导培养液一中作用2天，随后替换为药物诱导培养液二作用6天，最后替换为药物诱导培养液三再作用，整个诱导过程每2-3天换液。我们观察到在小分子药物的诱导过程中，星形胶质细胞形态发生改变，胞体逐渐缩小、细胞突起延伸且变细；而对照组细胞形态虽然也有部分改变，但并没有向神经元形态转变，这可能是细胞处于无血清培养条件，引起细胞形态变化；并且，随培养时间的延长两组均有部分细胞裂解死亡(图3、图4)。The reprogramming of astrocytes does not only depend on transcription factors, but small molecule drugs also have a certain effect on inducing reprogramming. In this experiment, we used SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin, and VPA for reprogramming. Astrocytes act in drug-induced culture medium one for 2 days, then replace with drug-induced medium for six days, and finally replace with drug-induced culture medium to act again and again. Change the medium every 2-3 days during the entire induction process . We observed that during the induction of small molecule drugs, the morphology of astrocytes changed, the cell body gradually shrank, and the cell protrusions extended and became thinner. Although the cell morphology of the control group was partially changed, it did not change to neuronal morphology. It may be that the cells were cultured in serum-free conditions, which caused cell morphology changes; and, with the extension of the culture time, some of the cells in both groups were lysed and died (Figure 3, Figure 4).
结果4、小分子药物组合诱导星形胶质细胞重编程为成熟神经元 Result 4. The combination of small molecule drugs induces astrocytes to reprogram into mature neurons
星形胶质细胞形态向神经元形态转变之后，我们通过细胞免疫荧光染色发现小分子药物诱导6天左右星形胶质细胞除了表达GFAP外也表达Tuj1，表明这些星形胶质细胞开始逐渐向神经元转变(图5、图6、图7)。随后在10天左右检测发现诱导细胞大量表达MAP2以及细胞神经核标志物NeuN(图8)；对诱导重编程效率进行统计分析，发现MAP2阳性的神经元转分化率超过80％，其中几乎所有的MAP2阳性神经元都表达NeuN，提示诱导神经元已转化为成熟神经元(图9)。以上结果说明小分子药物可以诱导星形胶质细胞逐步发育为成熟神经元。After the morphology of astrocytes changed to neuronal morphology, we found through cellular immunofluorescence staining that small molecule drugs induced astrocytes to express Tuj1 in addition to GFAP for about 6 days, indicating that these astrocytes began to gradually change Neuron transformation (Figure 5, Figure 6, Figure 7). Then, about 10 days later, it was detected that the induced cells expressed a large amount of MAP2 and the neuronuclear marker NeuN (Figure 8); the statistical analysis of the induction reprogramming efficiency showed that the transdifferentiation rate of MAP2-positive neurons exceeded 80%, and almost all of them MAP2 positive neurons all expressed NeuN, suggesting that induced neurons have transformed into mature neurons (Figure 9). The above results indicate that small molecule drugs can induce astrocytes to gradually develop into mature neurons.
我们利用RT-PCR技术检测不同时期神经元和星形胶质细胞相关基因，在小分子药物诱导过程中的细胞表达情况，结果显示随着小分子药物诱导时间的延长，神经元相关的NGN2和NEUROD1基因以及运动神经元相关的NKX6.1、OLIG2和ISL1等基因表达都有所上调，且ISL1在诱导第十天时达到峰值后表达逐渐降低(图11)。We used RT-PCR technology to detect neuron and astrocyte-related genes at different stages, and the cell expression during the induction of small molecule drugs. The results showed that as the induction time of small molecule drugs prolonged, neuron-related NGN2 and The expression of NEUROD1 gene and motor neuron-related genes such as NKX6.1, OLIG2 and ISL1 were all up-regulated, and the expression of ISL1 gradually decreased after reaching a peak on the tenth day of induction (Figure 11).
结果5、小分子药物组合诱导星形胶质细胞重编程为运动神经元Result 5. The combination of small molecule drugs induces astrocytes to reprogram into motor neurons
在体外神经前体细胞向运动神经元分化过程中，通常应用低浓度的RA和SHH或者Purmorphamine的联合诱导来完成。为此，我们应用高浓度RA和Purmorphamine以及其他 小分子药物对星形胶质细胞进行诱导，能使诱导的细胞表达泛神经元标志物MAP2及运动神经元特异性标记物HB9和CHAT(图12、图13)，且经统计分析显示HB9和CHAT阳性细胞率＞90％(图16)，而谷氨酸能神经元和γ-氨基丁酸能神经元的标志物GAD65和VGLUT1均为阴性(图14、图15)。以上结果提示该小分子药物组合能诱导星形胶质细胞重编程为神经元，并且几乎全部成为运动神经元，而没有向其他种类的神经元分化。In the process of differentiation of neural precursor cells into motor neurons in vitro, the combined induction of low concentrations of RA and SHH or Purmorphamine is usually used. To this end, we used high concentrations of RA and Purmorphamine and other small molecule drugs to induce astrocytes, which can make the induced cells express pan-neuronal markers MAP2 and motor neuron-specific markers HB9 and CHAT (Figure 12 , Figure 13), and statistical analysis showed that the rate of HB9 and CHAT positive cells was >90% (Figure 16), while the markers GAD65 and VGLUT1 of glutamatergic neurons and γ-aminobutyric neurons were negative ( Figure 14, Figure 15). The above results suggest that the small molecule drug combination can induce astrocytes to reprogram into neurons, and almost all become motor neurons, without differentiation into other types of neurons.
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above are the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and modifications can be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims (9)

  1. 一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，其特征在于，包括以下步骤：(1)原代大鼠星形胶质细胞纯化培养，以新生大鼠脊髓的原代星形胶质细胞作为诱导重编程的靶细胞，通过差速贴壁法的体外培养和微波震荡培养获得纯度高的星形胶质细胞；(2)根据神经元诱导以及运动神经元形成发育阶段所需的物质选取了7种小分子药物SB431542、LDN-193189、RA、bFGF、Purmorphamine、Forskolin、VPA，这些小分子药物通过体外对星形胶质细胞进行诱导重编程；(3)观察细胞的形态变化及免疫荧光细胞化学技术分析、实时荧光定量PCR分析和统计学分析方法进行分析星形胶质细胞向运动神经元的重编程及分化情况。A method for inducing reprogramming of spinal cord astrocytes into motor neurons is characterized in that it comprises the following steps: (1) Purification and culture of primary rat astrocytes, using primary astrocytes from the spinal cord of newborn rats As the target cells for inducing reprogramming, astrocytes with high purity are obtained through in vitro culture and microwave shaking culture by differential adherence method; (2) According to the developmental stage of neuron induction and motor neuron formation The required substances were selected from 7 small molecule drugs SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin, and VPA. These small molecule drugs induce reprogramming of astrocytes in vitro; (3) Observe the cell morphology Change and immunofluorescence cytochemical technology analysis, real-time fluorescence quantitative PCR analysis and statistical analysis methods to analyze the reprogramming and differentiation of astrocytes into motor neurons.
  2. 根据权利要求1所述的一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，其特征在于，所述步骤(1)具体包括以下过程：The method for inducing reprogramming of spinal astrocytes into motor neurons according to claim 1, wherein the step (1) specifically includes the following process:
    (1-1)超净工作台提前紫外线照射30min，取出-20℃预冷的冰台、已消毒的手术器械、HBSS缓冲液以及塑料培养皿等放置于超净工作台中，将培养皿中加入适量HBSS置于冰台上备用；(1-1) Ultra-clean workbench is irradiated with ultraviolet rays 30min in advance, take out the ice table pre-cooled at -20℃, sterilized surgical instruments, HBSS buffer and plastic petri dishes, etc., place them in the ultra-clean workbench, and add the petri dishes Place a proper amount of HBSS on the ice table for later use;
    (1-2)取新生1日龄大鼠置于放有适量***的50ml离心管中，麻醉后用75％的酒精消毒，并用无菌棉球擦干；(1-2) Take a 1-day-old newborn rat and place it in a 50ml centrifuge tube containing an appropriate amount of ether, disinfect with 75% alcohol after anesthesia, and dry it with a sterile cotton ball;
    (1-3)使用组织剪断头的同时，无菌棉球压迫止血，用手固定住大鼠身体使其保持伸展姿态；(1-3) While using the tissue cutting head, the sterile cotton ball is pressed to stop the bleeding, and the rat's body is fixed by hand to maintain the stretched posture;
    (1-4)用组织剪沿脊柱方向剪开皮肤，并将颈部肌肉剪开；(1-4) Use tissue scissors to cut the skin along the spine, and cut the neck muscles;
    (1-5)用直剪从颈部断处沿脊柱方向将背部脊椎与组织剪开，暴露脊髓；(1-5) Cut the back spine and tissues from the broken part of the neck along the direction of the spine with straight scissors to expose the spinal cord;
    (1-6)用显微镊将两侧背根神经节截断并将脊髓挑出置于预冷的HBSS中备用；(1-6) Use micro forceps to cut off the dorsal root ganglia on both sides and pick out the spinal cord in pre-cooled HBSS for later use;
    (1-7)将脊髓移至另一装有预冷HBSS的培养皿中置于体视显微镜下，用显微镊剥离表面的脊膜以及表面的血污点，将剥离干净的脊髓取出置于预冷的HBSS中，整个过程在冰台上进行；(1-7) Move the spinal cord to another petri dish containing pre-cooled HBSS and place it under a stereo microscope. Use micro tweezers to peel off the surface of the spinal membrane and blood stains on the surface. In the pre-cooled HBSS, the whole process is carried out on the ice table;
    (1-8)使用显微剪将脊髓剪碎并将其置于15ml离心管中，加入2ml 0.25％的胰酶，将离心管放置于37℃水浴中，每5min震荡混匀直至组织块分解，然后向离心管中加入5ml完全培养液终止消化，移入离心机后，以1000rpm转速离心5min，弃去上清加入4ml基培重悬再以1000rpm转速离心5min，重复2次，最后用4ml完全培养液重悬细胞并接种至25cm 2培养瓶中； (1-8) Use microscissors to cut the spinal cord and place it in a 15ml centrifuge tube, add 2ml 0.25% pancreatin, place the centrifuge tube in a 37℃ water bath, shake and mix every 5 minutes until the tissue mass is decomposed Then, add 5ml complete culture solution to the centrifuge tube to stop the digestion. After moving into the centrifuge, centrifuge at 1000rpm for 5min, discard the supernatant, add 4ml base culture, resuspend and centrifuge at 1000rpm for 5min, repeat twice, and finally complete with 4ml Resuspend cells in culture medium and inoculate them into 25cm 2 culture flask;
    (1-9)将培养瓶倒置于CO 2培养箱中培养20min，取出细胞悬液于15ml离心管以1000rpm转速离心5min，去上清并加入完全培养液重悬后，重新将细胞悬液加入25cm 2培养瓶中，置于5％CO 2培养箱中培养，此细胞标记为P0代； (1-9) Put the culture flask upside down in a CO 2 incubator and incubate for 20 minutes, take out the cell suspension and centrifuge at 1000 rpm for 5 minutes in a 15ml centrifuge tube, remove the supernatant and add the complete culture solution to resuspend, then add the cell suspension again Cultured in a 25cm 2 culture flask and placed in a 5% CO 2 incubator, this cell is marked as generation P0;
    (1-10)每两天换液直至细胞铺满培养瓶，将培养瓶放置于37℃微波震荡培养箱中以160rpm震荡培养18h去除少突胶质细胞和小胶质细胞；(1-10) Change the medium every two days until the cells cover the culture flask. Place the culture flask in a 37°C microwave shaking incubator and shake culture at 160rpm for 18 hours to remove oligodendrocytes and microglia;
    (1-11)震荡培养后弃去培养液，并加入PBS漂洗2-3次，然后再加入预温的2ml 0.25％胰酶消化细胞直至细胞能轻轻吹下为止，加入5ml完全培养液终止消化后吹起贴壁细胞，细胞悬液移液到15ml离心管中，1000rpm离心5min后弃去上清，加入1ml完全培养液进行传代；(1-11) After shaking culture, discard the culture medium, add PBS to rinse for 2-3 times, then add 2ml 0.25% trypsinized cells pre-warmed until the cells can be gently blown down, add 5ml complete culture medium to stop After digestion, blow up the adherent cells, pipette the cell suspension into a 15ml centrifuge tube, centrifuge at 1000rpm for 5min, discard the supernatant, and add 1ml complete culture solution for passage;
    (1-12)星形胶质细胞培养至P2-P3代用于后续实验。(1-12) Astrocytes were cultured to generation P2-P3 for subsequent experiments.
  3. 根据权利要求1所述的一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，其特征在于，所述步骤(2)具体包括以下过程：The method for inducing reprogramming of spinal cord astrocytes into motor neurons according to claim 1, wherein the step (2) specifically includes the following process:
    (2-1)取出浸泡在75％酒精中的盖玻片置于24孔细胞培养板中，加入无菌超纯水漂洗3次；(2-1) Take out the cover glass soaked in 75% alcohol and place it in a 24-well cell culture plate, and rinse with sterile ultrapure water for 3 times;
    (2-2)吸尽超纯水，加入适量100ng/ml的多聚赖氨酸包被过夜，第二天吸取多余多聚赖氨酸，加入无菌超纯水漂洗3次，吸尽多余液体，晾干备用；清洗过程轻柔，确保多聚赖氨酸不脱落；(2-2) Absorb the ultrapure water, add an appropriate amount of 100ng/ml polylysine to coat overnight, absorb the excess polylysine the next day, add sterile ultrapure water and rinse 3 times to exhaust the excess Liquid, dry for later use; the cleaning process is gentle to ensure that the polylysine does not fall off;
    (2-3)待步骤(1)中星形胶质细胞培养至P2-P3代时，加入PBS漂洗2-3次，然后加入预温的2ml 0.25％胰酶消化细胞直至贴壁细胞能轻轻吹下为止，加入5ml完全培养液终止消化后将细胞悬液加入15ml离心管中，1000rpm离心5min后弃去上清，加入1ml完全培养液重悬细胞，以4×10 4细胞量接种入含有包被多聚赖氨酸的24孔板中，放入5％CO 2培养箱培养过夜； (2-3) When the astrocytes in step (1) are cultured to the P2-P3 generation, add PBS to rinse 2-3 times, and then add 2ml of pre-warmed 0.25% trypsin digestion cells until the adherent cells can be light Blow gently, add 5ml of complete culture fluid to terminate the digestion, add the cell suspension to a 15ml centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 1ml of complete culture fluid to resuspend the cells, and inoculate with 4×10 4 cells In a 24-well plate containing coated polylysine, place it in a 5% CO 2 incubator and culture overnight;
    (2-4)第二天弃去24孔培养板中的培养液，用预温的PBS漂洗2-3次，将药物诱导培养液一混合均匀，加入到培养孔中，培养板放入5％CO 2培养箱培养； (2-4) The next day, discard the culture medium in the 24-well culture plate, rinse with pre-warmed PBS for 2-3 times, mix the drug-induced culture medium evenly, add it to the culture wells, put the culture plate into 5 %CO 2 incubator culture;
    (2-5)药物诱导培养液一培养2天后，细胞培养孔中换入药物诱导培养液二并置于5％CO 2培养箱培养； (2-5) After the drug-induced culture medium is cultured for 2 days, the cell culture well is replaced with the drug-induced culture medium and placed in a 5% CO 2 incubator for culture;
    (2-6)药物诱导培养液二培养8-10天后，细胞培养孔中换入药物诱导培养液三并置于5％CO 2培养箱培养，每2-3天换液。 (2-6) After the second culturing of the drug-induced culture medium for 8-10 days, the cell culture well is replaced with the drug-induced culture medium three and placed in a 5% CO 2 incubator for culture, and the medium is changed every 2-3 days.
  4. 根据权利要求1所述的一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，其特征在于，所述步骤(3)中免疫荧光细胞化学技术分析具体包括以下过程：The method for inducing reprogramming of spinal cord astrocytes into motor neurons according to claim 1, wherein the immunofluorescence cytochemical analysis in step (3) specifically includes the following process:
    (3-1-1)取出24孔细胞培养板，将待染色孔中的培养液吸尽，加入预温的PBS漂洗2-3次，取出盖玻片放置于另一装有300ml预温4％多聚甲醛的24孔板中，固定10min；(3-1-1) Take out the 24-well cell culture plate, suck up the culture solution in the well to be stained, add pre-warmed PBS to rinse 2-3 times, take out the cover slip and place it in another 300ml pre-warmed 4 In a 24-well plate containing% paraformaldehyde, fix for 10 minutes;
    (3-1-2)弃去4％多聚甲醛，每孔加入300μl PBS漂洗3次，每次5min；(3-1-3)弃去PBS，每孔加入300μl破膜液，常温孵育30min；(3-1-2) Discard 4% paraformaldehyde, add 300μl PBS to each well to rinse 3 times, 5min each time; (3-1-3) Discard PBS, add 300μl rupture fluid to each well, incubate for 30min at room temperature ；
    (3-1-4)弃去破膜液，每孔加入300μl封闭液，常温孵育30min；(3-1-4) Discard the rupture fluid, add 300μl of blocking fluid to each well, and incubate for 30min at room temperature;
    (3-1-5)弃去封闭液，每孔加入300μl一抗与封闭液混合液，4℃孵育过夜；(3-1-5) Discard the blocking solution, add 300μl of primary antibody and blocking solution mixture to each well, and incubate overnight at 4°C;
    (3-1-6)第二天弃去一抗，每孔加入300μl PBS漂洗3次，每次5min；(3-1-6) Discard the primary antibody the next day, add 300μl PBS to each well and rinse 3 times for 5 minutes each time;
    (3-1-7)弃去PBS，每孔加入300ml二抗与PBS的混合液，4℃孵育≥2h，避光进行处理；(3-1-7) Discard PBS, add 300ml of a mixture of secondary antibody and PBS to each well, incubate at 4°C for ≥2h, protect from light;
    (3-1-8)弃去二抗混合液，每孔加入300μl PBS漂洗3次，每次5min，避光进行处理；(3-1-8) Discard the secondary antibody mixture, add 300μl PBS to each well and rinse 3 times, 5min each time, and treat it in the dark;
    (3-1-9)加入PBS稀释比1:1000的Hoechst常温孵育10min，弃去Hoechst稀释液，加入300ml PBS漂洗3次，每次5min，避光进行处理；(3-1-9) Add Hoechst with a PBS dilution ratio of 1:1000 and incubate at room temperature for 10 minutes, discard the Hoechst diluent, add 300ml PBS and rinse 3 times, 5 minutes each time, and perform treatment in the dark;
    (3-1-10)取粘附性载玻片滴上封片液，将盖玻片进行封片，避光处理；(3-1-10) Take the adhesive slide glass and drop it with mounting liquid, and mount the cover glass to avoid light;
    (3-1-11)在激光共聚焦显微镜下观察并拍照记录。(3-1-11) Observed and photographed and recorded under a laser confocal microscope.
  5. 根据权利要求1所述的一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，其特征在于，所述步骤(3)中实时荧光定量PCR分析具体包括以下过程：The method for inducing reprogramming of spinal astrocytes into motor neurons according to claim 1, wherein the real-time fluorescent quantitative PCR analysis in the step (3) specifically includes the following process:
    (3-2-1)取出含细胞的培养皿，弃去培养基，并用预热至37℃的PBS冲洗3次，加入1ml Trizol轻轻吹打将细胞混匀，并收集细胞悬液于1.5ml EP管中，冰上静置5min；(3-2-1) Take out the culture dish containing the cells, discard the culture medium, and rinse 3 times with PBS preheated to 37°C, add 1ml Trizol and gently pipette to mix the cells, and collect the cell suspension in 1.5ml In the EP tube, let stand on ice for 5 minutes;
    (3-2-2)向EP管中加入250μl氯仿震荡混匀，并置于-20℃冰箱中静置1-2h；取出EP管12000rpm，4℃，离心5分钟；(3-2-2) Add 250μl of chloroform to the EP tube, shake and mix, and place it in a refrigerator at -20℃ for 1-2h; take out the EP tube at 12000rpm, 4℃, and centrifuge for 5 minutes;
    (3-2-3)去上层清液于另一EP管中，向其中加入等体积的异丙醇，颠倒混匀，12000rpm，4℃，离心10min；(3-2-3) Remove the supernatant and put it in another EP tube, add an equal volume of isopropanol to it, mix upside down, and centrifuge at 12000rpm, 4°C for 10min;
    (3-2-4)小心弃去液体加入1ml 75％的乙醇，7500rpm，4℃，离心5min，弃去液体，重复此步骤；(3-2-4) Carefully discard the liquid, add 1ml 75% ethanol, 7500rpm, 4℃, centrifuge for 5min, discard the liquid, repeat this step;
    (3-2-5)加入1ml无水乙醇，7500rpm，4℃，离心5min，弃去液体，并将EP管置于超净台中吹干；(3-2-5) Add 1ml of absolute ethanol, 7500rpm, 4℃, centrifuge for 5min, discard the liquid, and place the EP tube in an ultraclean table to blow dry;
    (3-2-6)向EP管中加入20μl DEPC水溶解RNA，并将RNA进行逆转录，剩余的保存于-80℃冰箱；(3-2-6) Add 20μl DEPC water to the EP tube to dissolve RNA, reverse transcription of RNA, and store the rest in a refrigerator at -80℃;
    (3-2-7)取逆转录试剂盒，以下表的体系配比及PCR反应条件对RNA进行逆转录：(3-2-7) Take the reverse transcription kit, and perform reverse transcription on RNA with the system ratio and PCR reaction conditions in the following table:
    Figure PCTCN2020080792-appb-100001
    Figure PCTCN2020080792-appb-100001
    (3-2-8)逆转录获得的cDNA用于qPCR扩增，剩余的保存于-80℃冰箱中；(3-2-8) cDNA obtained by reverse transcription is used for qPCR amplification, and the rest is stored in a refrigerator at -80℃;
    (3-2-9)取FastStart Universal SYBR Green Master中试剂置于冰盒上，以下表的体系配比及反应条件进行PCR扩增：(3-2-9) Put the reagents in FastStart Universal SYBR Green Master on the ice box, and perform PCR amplification with the system ratio and reaction conditions in the following table:
    Figure PCTCN2020080792-appb-100002
    Figure PCTCN2020080792-appb-100002
    (3-2-10)PCR扩增结束后对溶解曲线进行分析，确保PCR产物特异性。(3-2-10) Analyze the melting curve after PCR amplification to ensure the specificity of PCR products.
  6. 根据权利要求1所述的一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，其特征在于，所述步骤(3)中统计学分析具体包括以下过程：通过软件Graphpad prism5进行数据统计，对所有量化数据进行统计分析并以平均值±标准差表示；双侧t检验用于计算p值的统计显着性，p值<0.05具有统计学意义。The method for inducing reprogramming of spinal cord astrocytes into motor neurons according to claim 1, wherein the statistical analysis in step (3) specifically includes the following process: data is performed through the software Graphpad prism5 Statistics: Perform statistical analysis on all quantified data and express them as mean±standard deviation; two-sided t-test is used to calculate the statistical significance of the p value, and the p value<0.05 is statistically significant.
  7. 根据权利要求2所述的一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，其特征在于，所述完全培养液采用在DMEM/F12基础培养基中添加1％的双抗和10％的FBS的方法制成。The method for inducing reprogramming of spinal astrocytes into motor neurons according to claim 2, characterized in that the complete culture medium is prepared by adding 1% bi-antibody to DMEM/F12 basal medium and 10% FBS method.
  8. 根据权利要求3所述的一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，其特征在于，所述药物诱导培养液一采用在基础诱导培养液中添加2μM SB431542、1μM RA、5μM Forskolin、20ng/ml bFGF、0.25μM LDN-193189、制成；所述药物诱导培养液二采用在基础诱导培养基中添加5μM Purmorphamine、10μM RA、20μM Forskolin、200ng/ml bFGF制成；所述药物诱导培养液三采用在基础诱导培养基中添加1mM VPA、5μM Purmorphamine、10μM RA、20μM Forskolin、200ng/ml bFGF制成。The method for inducing reprogramming of spinal cord astrocytes into motor neurons according to claim 3, characterized in that the drug-induced culture medium is added with 2μM SB431542, 1μM RA, 5μM Forskolin, 20ng/ml bFGF, 0.25μM LDN-193189, made; the drug induction culture medium 2 is made by adding 5μM Purmorphamine, 10μM RA, 20μM Forskolin, 200ng/ml bFGF to the basic induction medium; Drug induction culture medium 3 is made by adding 1mM VPA, 5μM Purmorphamine, 10μM RA, 20μM Forskolin, 200ng/ml bFGF to the basic induction medium.
  9. 根据权利要求8所述的一种诱导脊髓星形胶质细胞重编程为运动神经元的方法，其特征在于，所述基础诱导培养液采用在DMEM/F12基础培养基中添加1％的双抗、1M B27、1M N 2、2mM L-谷氨酸、3.5mM葡萄糖制成。 The method for inducing reprogramming of spinal cord astrocytes into motor neurons according to claim 8, wherein the basic induction culture medium adopts DMEM/F12 basic medium with 1% double antibody , 1M B27, 1M N 2 , 2mM L-glutamic acid, 3.5mM glucose.
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