WO2022222138A1 - 一种潜在的可作为阿尔茨海默症药物作用靶点蛋白及其应用 - Google Patents

一种潜在的可作为阿尔茨海默症药物作用靶点蛋白及其应用 Download PDF

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WO2022222138A1
WO2022222138A1 PCT/CN2021/089293 CN2021089293W WO2022222138A1 WO 2022222138 A1 WO2022222138 A1 WO 2022222138A1 CN 2021089293 W CN2021089293 W CN 2021089293W WO 2022222138 A1 WO2022222138 A1 WO 2022222138A1
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ptbp1
gene
protein
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王广基
李昔诺
朱哲英
阿基业
徐进宜
孙渊
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中国药科大学
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Definitions

  • the invention belongs to the field of biomedicine, and relates to a potential target protein that can be used as a drug for Alzheimer's disease and its application.
  • Tau protein is normally found in our brain cells, it maintains the structure of neurons and its stability, and it helps in the transfer of nutrients in cells.
  • Tau misfolded, it becomes viscous and less soluble, aggregates within neurons and forms neurofibrillary tangles, disrupts neuronal function and ultimately leads to neuronal death, and only a small amount of misfolded Tau occurs protein, which can convert its adjacent brain cells into dysfunctional cells [6] .
  • PTBP1 protein Polypyrimidine Tract Binding Protein 1
  • PTBP1 is a subtype of the PTBP family. It plays a crucial role in splicing. PTBP1 is involved in mediating several cellular processes in certain types of cells, including the growth of neuronal cells and the activation of immune cells.
  • PTBP1 plays a role in a variety of diseases, especially in certain cancers (including colorectal cancer, renal cell carcinoma, breast cancer and glioma). Some studies have reported that the PTBP1 gene can improve the state of neurons in Parkinson's disease [8] . Parkinson's patients have different clinical symptoms from AD patients. Parkinson's patients do not lose memory function and experience tremors. Since AD is a complex neurodegenerative disease, the mechanism of AD is extremely complex, which is not only related to the state of neurons, but also to inflammation, A ⁇ protein, and Tau protein.
  • PTBP1 can play a role in inflammation; the expression of A ⁇ protein and Tau protein has not been reported, and whether PTBP1 can also regulate neuronal state in AD patient brain is unknown. At present, in the field of AD research, it is still unclear whether PTBP1 can be used as the target of AD drugs.
  • ARNSTEN A F T, DATTA D, TREDICI K D, et al.Hypothesis:Tau pathology is an initiating factor in sporadic Alzheimer's disease[J].Alzheimer's&Dementia,2021.17(1):115-124
  • the purpose of the present invention is to provide the application of the PTBP1 gene as a therapeutic target in the preparation of a drug for treating Alzheimer's disease in view of the above-mentioned deficiencies of the prior art.
  • Another object of the present invention is to provide an RNP complex for PTBP1 gene editing.
  • Yet another object of the present invention is to provide the use of RNP complexes.
  • PTBP1 gene as a therapeutic target in the preparation of drugs for treating Alzheimer's disease
  • the substance that inhibits the expression of PTBP1 gene is selected from the CRISPR/Cas9 gene editing system for PTBP1 gene knockout, specific antisense oligonucleotides for PTBP1, siRNA/shRNA, zinc Refers to nucleases, transcription activator-like effector nucleases, small molecule inhibitors, adenoviruses, lentiviruses or small molecule compounds.
  • the CRISPR/Cas9 gene editing system for PTBP1 gene knockout includes crRNA, TracrRNA and Cas9 protein targeting the PTBP1 gene; the crRNA targeting the PTBP1 gene is selected from crRNA1, crRNA2 Or any one or more of crRNA3; described crRNA1 sequence is as shown in SEQ ID NO.1, described crRNA2 sequence is as shown in SEQ ID NO.2, described crRNA3 sequence is as shown in SEQ ID NO.3 shown.
  • the crRNA targeting the PTBP1 gene is selected from a combination of crRNA1, crRNA2 and crRNA3.
  • a kind of RNP complex comprises the crRNA, TracrRNA and Cas9 protein of targeting PTBP1 gene;
  • the crRNA of described targeting PTBP1 gene is selected from any one or more in crRNA1, crRNA2 or crRNA3;
  • Described crRNA1 sequence is such as Shown in SEQ ID NO.1, the crRNA2 sequence is shown in SEQ ID NO.2, and the crRNA3 sequence is shown in SEQ ID NO.3.
  • the crRNA targeting the PTBP1 gene is selected from a combination of crRNA1, crRNA2 and crRNA3.
  • PTBP1 gene as a therapeutic target in screening drugs for treating Alzheimer's disease, the accession number of PTBP1 in Genbank is NC_000019.10.
  • ADAM10 was successfully knocked out in SH-SY5Y cells by CRISPR /Cas 9 gene editing technology to build a cell model that fully responds to AD.
  • the expression of /Tau was significantly increased.
  • Inflammasome NLRP3 protein was significantly up-regulated, and the expression of pro-inflammatory factor TNF- ⁇ was significantly increased.
  • the neural differentiation function of cells is weakened, the length of neurons shrinks, the growth rate of cells is significantly slowed down, and the connections of neurons are significantly reduced, which can simulate neuronal necrosis in the brain of AD patients.
  • the present invention found for the first time that PTBP1 protein was significantly increased in ADAM10 KO cells, and the interaction between PTBP1 and Tau was significantly enhanced.
  • the PTBP1 gene is also knocked out in the AD cell model ( ADAM10 KO cell) constructed above through the CRISPR/Cas 9 gene editing technology. After the PTBP1 protein is knocked down, not only A ⁇ 42 and Tau protein can be significantly reduced, but also The inflammasome NLRP3 protein was significantly down-regulated, the cell-neuron differentiation ability was enhanced, the neuron length was significantly longer, and the information exchange between cells was significantly increased.
  • the above data indicate that PTBP1 protein may be a novel target in AD, and drugs synthesized against this target to inhibit the expression of this target gene can treat AD disease more comprehensively.
  • Figure A is a schematic diagram of the positions of CrRNA1, CrRNA2, and CrRNA3 in the ADAM10 gene sequence;
  • Figure B is the sequencing map of gene mutation points.
  • ADAM10 knockout cell lines had point mutations.
  • the sequencing results showed that a cell expansion line knocking out the ADAM10 gene had been initially screened. These cell lines were cultured and expanded, and cryopreserved for subsequent experimental research.
  • ADAM10 in the knockout group was verified at the protein level. As shown in Figure A, compared with the control KO in the blank group, the ADAM10 KO group had a very significant decrease in ADAM10 protein expression, which was consistent with the results of Sanger sequencing. The data further indicated that ADAM10 gene knockout was successful in SH-SY5Y cells using CRISPR/Cas 9 gene editing technology.
  • Accumulation of A ⁇ and intracellular hyperphosphorylated Tau protein are the main neuropathological criteria for the diagnosis of AD, and the ratio of A ⁇ 42/A ⁇ 40 in the plasma of AD patients can be used as a pre-screening index for AD clinically.
  • NLRP3 protein inhibitors may serve as targets for the future treatment of AD.
  • ADAM10 KO group significantly increased NLRP3 and activated the expression of the inflammasome.
  • the growth rate, differentiation and changes of cell density of neuronal cells were observed after knocking out ADAM10 gene in SH-SY5Y cells. Compared with undifferentiated normal SH-SY5Y cells, after neuronal differentiation treatment, normal SH-SY5Y cells have significantly longer neuronal lengths and tighter intercellular connections. After knocking out the ADAM10 gene and then undergoing the same cell differentiation treatment, the length of neurons was significantly shortened, the cells tended to shrink, the connections between cells were significantly reduced, and the ability of nerve cells to transmit information was significantly weakened.
  • Immunofluorescence showed that the expressions of PTBP1 and Tau proteins were significantly increased in ADAM10 KO group compared with blank group.
  • PTBP1 protein in ADAM10 KO group was significantly up-regulated compared with blank control KO. Numerous studies have shown that tau protein plays an important role in neurodegenerative diseases and memory impairment. Compared with the blank control KO, the expression of Tau protein in ADAM10 KO group was significantly increased. The CO-IP results showed that PTBP1 interacted with Tau protein, and compared with the blank group, the interaction between PTBP1 and Tau was obvious. The above experimental results showed that after ADAM10 gene knockout, the significantly shortened neuron length and the weakened cell neural differentiation ability may be due to the interaction between PTBP1 and Tau protein to affect neuronal function.
  • CrRNA1, CrRNA2, and CrRNA3 are the CrRNA sequences that knock out the PTBP1 gene, respectively.
  • CrRNA1 GGCACCCCCUUUUCAGCAAA (SEQ ID ON.4); Cutsite: 804,037; Exon: Exon 4; On Target Score: 0.426.
  • crRNA2 AAUGACAGCAAGAAGUUCAA (SEQ ID ON.5); Cutsite: 804,061; Exon: Exon 4; On Target Score: 0.531.
  • crRNA3 AAAGGUGACAGCCGAAGUGC (SEQ ID ON.6) Cutsite: 804,079; Exon: Exon 4; On Target Score: 0.513.
  • the ADAM10 KO group significantly increased NLRP3 and activated the expression of the inflammasome.
  • Tau protein and PTBP1 protein were significantly increased.
  • follow-up indicators were observed by knocking down the PTBP1 protein in knockout model cells using CRISPR/Cas 9 gene editing technology.
  • the expression of PTBP1 protein was significantly decreased after knockdown of PTBP1 protein.
  • the expression of Tau protein was significantly decreased, and the NLRP3 inflammasome protein was significantly decreased.
  • the level of A ⁇ 42 was measured by ELISA, and the data showed that the level of A ⁇ 42 was significantly reduced after the knockdown of PTBP1 protein.
  • normal SH-SY5Y cells Compared with undifferentiated normal SH-SY5Y cells, after neuronal differentiation treatment, normal SH-SY5Y cells have significantly longer neuronal lengths and tighter intercellular connections. After knocking out the ADAM10 gene and then undergoing the same cell differentiation treatment, the length of neurons was significantly shortened, the cells tended to shrink, the connections between cells were significantly reduced, and the ability of nerve cells to transmit information was significantly weakened. After knockdown of PTBP1 protein expression in model AD cells, neuronal state was recalled.
  • RNA mixture was heated at 95°C for 5 min and cooled at room temperature for 10 min to obtain an annealed RNA mixture. Take 2.9 ⁇ l of the annealed RNA mixture and 1 ⁇ l of Cas 9 protein, mix thoroughly at a ratio of 1.2:1-2:1, and place at room temperature for 10 min. After adding 0.6 ⁇ l of electroporation enhancement solution, the reaction was performed at room temperature for 5 min to obtain the RNP complex.
  • Electroporation of the RNP complex into SH-SY5Y cells to be knocked out preheat the medium containing 15% FBS, take the cells with a density of 80%, and discard the medium. Wash the cells gently with an appropriate amount of PBS solution. After discarding the solution, add an appropriate amount of trypsin to digest the cells at 37°C. After 80% of the cells are detached for a few minutes, the digestion is terminated with a medium containing 15% FBS, 1100rpm. Centrifuge for 10 min and discard the supernatant. Resuspend the cells with 10 ml of PBS solution, mix the cells well, and count 10 ⁇ l of the cell solution.
  • the electroporated cells were gradually diluted to 1 cell/10 ⁇ l, added to a 96-well plate, and supplemented with 200 ⁇ l of medium containing 15% FBS. After stabilizing for 12 hours and waiting for the cells to adhere, the wells containing one cell in the 96-well plate were recorded and screened under a microscope. After screening, continue to wait for these single cells to grow and expand. After the single cells are expanded to a cell density of 80% in a 96-well plate, the cells are transferred to a 12-well plate. After the single cells are expanded to cells in a 12-well plate After the cell density reached 80%, the cells were transferred to a 6-well plate. After the cell density reached 80%, a part of the cells were taken for Sanger sequencing to screen the single cell line that was successfully knocked out. The remaining cells were cultured and replaced every 48 hours. cell culture medium once.
  • the primers of the target gene ADAM10 are designed as follows:
  • the desired DNA bands were cut, and the purification steps were strictly in accordance with the instructions for SK8131 gel recovery.
  • the sequencing reaction system is shown in the following table:
  • the plate was inverted at 600 rmp/min and dried, dried on a clean table for 5 min, added with 10 ⁇ l Hidi Formamide, and shaken on a vortex for 1 min.
  • the 96-well plate was placed in ABI 3730XL sequencer for electrophoresis analysis.
  • the operation of the sequencing electrophoresis instrument is strictly carried out in accordance with the ABI Prism 3730 manual.
  • ADAM10 knockout cell lines had point mutations.
  • the sequencing results (Fig. 1) showed that a cell expansion line knocking out the ADAM10 gene had been initially screened. These cell lines were cultured and expanded, and cryopreserved for subsequent experimental research.
  • ADAM10 in the knockout group was verified at the protein level by Western. As shown in Figure 2A, compared with the blank control KO, the ADAM10 KO group had a very significant decrease in ADAM10 protein expression, which was consistent with the Sanger sequencing results. The data further indicated that ADAM10 gene knockout was successful in SH-SY5Y cells using CRISPR/Cas 9 gene editing technology.
  • the protein level of the inflammasome NLRP3 in the knockout group was verified by Western. As shown in Figure 3, compared with the Control KO group, the ADAM10 KO group significantly increased NLRP3 and activated the expression of the inflammasome.
  • a ⁇ and intracellular hyperphosphorylated Tau protein are the main neuropathological criteria for the diagnosis of AD, and the ratio of A ⁇ 42/A ⁇ 40 in the plasma of AD patients can be used as a pre-screening index for AD clinically. Therefore, we verify A ⁇ ; Tau; S-199Tau; S-214Tau:
  • SH-SY5Y cells were sequentially cultured in different types of media as shown in Table 6 below.
  • the cells were cultured in Growth Media medium to reach a cell density of 70%.
  • the medium formula was then changed to Differentiation media 1 for 7-10 days, during which time the culture medium was changed every 48 hours.
  • the cells were passaged at a ratio of 1:1 in fresh medium Differentiation media 2 for 4-7 days, during which time the medium was changed every 48 hours.
  • the medium was changed to Differentiation media 3 for 7-10 days, during which time the medium was changed every 48 hours.
  • cells differentiate into neurons for subsequent detection and analysis.
  • Differentiated SH-SY5Y neurons were fixed with 4% PFA for 20 min at room temperature. After discarding the solution, the cells were gently rinsed 3 times with 0.1% PBS-T solution for 2 min each time. After fixation, cells were incubated in 5% NGS-T blocking solution for 2 hours at room temperature. Add the primary antibody anti-tubulin III primary antibody (#ab179513, Abcam plc.) at 4°C at a dilution of 1:1000, and incubate overnight. After removing the primary antibody, the cells were gently washed 3 times with 0.1% PBS-T solution.
  • the secondary antibody Alexa Fluor 488 (ab150113, Abcam) was added at room temperature at a dilution of 1:2000, and incubated at room temperature for 1 h. After removing the secondary antibody, cells were fixed on glass slides, and the samples were imaged and detected by confocal microscopy within 24 h.
  • All neuronal differentiation pictures were acquired using a Zeiss 880 confocal microscope.
  • Laser power is set to 4%; gain is set to 650; Digital offset is set to 350; Acquiring speed is set to 1.03s; Z-stack images is set to 5 ⁇ m; an average of 2 shots per film.
  • Images acquired by confocal microscopy were analyzed using the Image J plugin Neuron J. Greater than 3 regions in each image were selected for analysis, and axonal lengths were subsequently measured, and Neuron J was used to measure distances between coordinates.
  • normal SH-SY5Y cells As shown in Figure 4, compared with normal SH-SY5Y cells without neural cell differentiation, after neural cell differentiation treatment, normal SH-SY5Y cells have significantly longer neuronal lengths and tighter intercellular connections . After knocking out the ADAM10 gene and then undergoing the same cell differentiation treatment, the length of neurons was significantly shortened, the cells tended to shrink, the connections between cells were significantly reduced, and the ability of nerve cells to transmit information was significantly weakened.
  • 1Construct RNP complex Take 0.33 ⁇ l of crRNA1 (100 ⁇ M), 0.33 ⁇ l of crRNA2 (100 ⁇ M), 0.33 ⁇ l of crRNA3 (100 ⁇ M) and 1 ⁇ l of Alt-R.CRISPR-Cas9 tracrRNA (100 ⁇ M), mix well and use without The nuclease buffer was diluted to a concentration of 20-80 ⁇ M to obtain the RNA mixture. The RNA mixture was heated at 95°C for 5 min and cooled at room temperature for 10 min to obtain an annealed RNA mixture. Take 2.9 ⁇ l of the annealed RNA mixture and 1 ⁇ l of Cas 9 protein, mix thoroughly at a ratio of 1.2:1-2:1, and place at room temperature for 10 min. After adding 0.6 ⁇ l of electroporation enhancement solution, the reaction was performed at room temperature for 5 min to obtain the RNP complex.
  • Electroporation of the RNP complex into SH-SY5Y cells to be knocked out preheat the medium containing 15% FBS, take the cells with a density of 80%, and discard the medium. Wash the cells gently with an appropriate amount of PBS solution. After discarding the solution, add an appropriate amount of trypsin to digest the cells at 37°C. After 80% of the cells are detached for a few minutes, the digestion is terminated with a medium containing 15% FBS, 1100rpm. Centrifuge for 10 min and discard the supernatant. Resuspend the cells with 10 ml of PBS solution, mix the cells well, and count 10 ⁇ l of the cell solution.
  • Differentiated SH-SY5Y neurons were fixed with 4% PFA for 20 min at room temperature. After discarding the solution, the cells were gently rinsed 3 times with 0.1% PBS-T solution for 2 min each time. After fixation, cells were incubated in 5% NGS-T blocking solution for 2 hours at room temperature. Add primary antibody at 4°C and incubate overnight. After removing the primary antibody, the cells were gently washed 3 times with 0.1% PBS-T solution. The secondary antibody was added at room temperature, and the dilution concentration was strictly in accordance with the instructions, and then incubated at room temperature for 1 h. After removal of the secondary antibody, cells were fixed on glass slides, and the samples were examined by fluorescence microscopy within 24 h.
  • the Co-IP results showed that PTBP1 interacted with Tau protein, and compared with the blank group, the interaction between PTBP1 and Tau was obvious.
  • the above experimental results showed that after ADAM10 gene knockout, the significantly shortened neuron length and the weakened cell neural differentiation ability may be due to the interaction between PTBP1 and Tau protein to affect neuronal function.
  • Stacking gel (5%): ultrapure water 2.7 mL; 30% acrylamide 0.4 mL; 1M Tris-HCl solution (pH 6.8) 0.5 mL; 10% SDS 40 ⁇ l; 10% APS 40 ⁇ l; TEMED 4 ⁇ l.
  • Ammonium persulfate and TEMED are coagulants, and they should be mixed immediately after adding the solution and then poured. After filling the separation gel, gently add ultrapure water, move the pipette when adding ultrapure water, and ensure that the water is on the same level. Let stand until you see a clear line between the separating gel and the water. After the separation gel is solidified, tilt the water to pour out, drain the water, pour in the stacking gel, insert the comb teeth, wait for the stacking gel to solidify and then load the sample.
  • the first stage 75V, 40-60min until the target protein appears a line at the boundary between the separating gel and the stacking gel.
  • the second stage 115V, 60-90min until the target protein migrates to about 1/3 of the bottom of the separation gel.
  • SH-SY5Y cells were sequentially cultured in different types of media as shown in Table 6.
  • the cells were cultured in Growth Media medium to reach a cell density of 70%.
  • the medium formulation was then changed to Differentiation media 1 for 7-10 days, during which time the culture medium was changed every 48 hours.
  • the cells were passaged at a ratio of 1:1 in fresh medium Differentiation media 2 for 4-7 days, during which time the medium was changed every 48 hours.
  • the medium was changed to Differentiation media 3 for 7-10 days, during which time the medium was changed every 48 hours. At this stage, cells differentiate into neurons for subsequent detection and analysis.
  • Differentiated SH-SY5Y neurons were fixed with 4% PFA for 20 min at room temperature. After discarding the solution, the cells were gently rinsed 3 times with 0.1% PBS-T solution for 2 min each time. After fixation, cells were incubated in 5% NGS-T blocking solution for 2 hours at room temperature. Add the primary antibody anti-tubulin III primary antibody (#ab179513, Abcam plc.) at 4°C at a dilution of 1:1000, and incubate overnight. After removing the primary antibody, the cells were gently washed 3 times with 0.1% PBS-T solution.
  • the secondary antibody Alexa Fluor 488 (ab150113, Abcam) was added at room temperature at a dilution of 1:2000, and incubated at room temperature for 1 h. After removing the secondary antibody, cells were fixed on glass slides, and the samples were imaged and detected by confocal microscopy within 24 h.
  • All neuronal differentiation pictures were acquired using a Zeiss 880 confocal microscope.
  • Laser power is set to 4%; gain is set to 650; Digital offset is set to 350; Acquiring speed is set to 1.03s; Z-stack images is set to 5 ⁇ m; an average of 2 shots per film.
  • Images acquired by confocal microscopy were analyzed using the Image J plugin Neuron J. Greater than 3 regions in each image were selected for analysis, and axonal lengths were subsequently measured, and Neuron J was used to measure distances between coordinates.

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Abstract

提供了抑制PTBP1基因表达的物质在制备治疗阿尔兹海默症的药物中的应用。还提供了PTBP1基因作为治疗靶点在筛选治疗阿尔兹海默症的药物中的应用。

Description

一种潜在的可作为阿尔茨海默症药物作用靶点蛋白及其应用 技术领域
本发明属于生物医药领域，涉及一种潜在的可作为阿尔茨海默症药物作用靶点蛋白及其应用。
背景技术
一些科学家认为Aβ的累积引发并驱动了Tau蛋白的积累，导致神经元坏死。目前在临床试验中的大多数药物具有多靶点而不能主要靶向Tau蛋白，并且有大量研究表明其对Tau的回调作用不显著 [1-5]。Tau蛋白通常存在于我们的脑细胞中，它可以维持神经元的结构及其稳定性，并且有助于营养物质在细胞中的转移。当Tau发生错误折叠时会变得粘稠且溶解性降低，在神经元内发生聚集并形成神经原纤维缠结，破坏神经元功能并最终导致神经元死亡，而且仅需少量发生错误折叠的Tau蛋白，即可将其相邻脑细胞转变为功能异常的细胞 [6]。
尽管现针对Aβ与Tau两个靶点的药物在阿尔茨海默症(AD)小鼠模型中有显著性疗效，但在临床上许多针对降低Aβ的抗AD候选化合物未能实质性改变AD患者临床症状或疾病的进程。有研究表明，即使去除AD患者大脑中的淀粉样蛋白斑块，依然存在AD的症状 [3,4,7]。筛选促神经元再生的蛋白靶点有可能是今后AD药物的主要靶标。PTBP1蛋白(Polypyrimidine Tract Binding Protein 1)是PTBP家族中的一个亚型。其在剪接中起着至关重要的作用。PTBP1参与介导某些类型的细胞中的几种细胞过程，包括神经元细胞的生长以及免疫细胞的激活。其功能受各种分子调控，比如microRNA(miRNA)、长非编码RNA(lncRNA)和RNA结合蛋白。PTBP1在多种疾病中起作用，尤其是在某些癌症中(包括结肠直肠癌，肾细胞癌，乳腺癌和神经胶质瘤)。有研究报道PTBP1基因在帕金森疾病中可改善神经元的状态 [8]，帕金森患者与AD患者在临床上的病症不同，帕金森患者记忆功能未丢失，会发生震颤等。由于AD是复杂的神经退行性疾病，AD的机制极其复杂，其不仅与神经元的状态有关，与炎症、Aβ蛋白、Tau蛋白均有相关性。PTBP1是否能对炎症；Aβ蛋白和Tau蛋白的表达有作用目前尚无报道， 且PTBP1是否同样可以调节AD患者脑内的神经元状态是未知的。目前在AD的研究领域，PTBP1是否可以作为AD药物的作用靶点研究尚不清楚。
前期工作在SH-SY5Y上通过敲除ADAM10基因后发现神经元长度变短，细胞生长速度变慢，这是否与PTB蛋白相关？基于此，我们在AD细胞模型中验证PTBP1的表达。
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发明内容
本发明的目的是针对现有技术的上述不足，提供PTBP1基因作为治疗靶点在制备治疗阿尔兹海默症的药物中的应用。
本发明的另一目的是提供一种用于PTBP1基因编辑的RNP复合物。
本发明的又一目的是提供RNP复合物的应用。
本发明的目的可通过以下技术方案实现：
PTBP1基因作为治疗靶点在制备治疗阿尔兹海默症的药物中的应用，所述的PTBP1在Genbank中的登录号为NC_000019.10( https://www.ncbi.nlm.nih.gov/nuccore/NC_000019.10？report＝genbank&fr om＝797452&to＝812316)。
抑制PTBP1基因表达的物质在制备治疗阿尔兹海默症的药物中的应用。
作为本发明的一种优选，所述的抑制PTBP1基因表达的物质选自用于PTBP1基因敲除的CRISPR/Cas9基因编辑系统、针对PTBP1的特异性反义寡核苷酸、siRNA/shRNA、锌指核酸酶、转录激活因子样效应核酸酶、小分子抑制剂、腺病毒、慢病毒或小分子化合物。
作为本发明的进一步优选，所述的用于PTBP1基因敲除的CRISPR/Cas9基因编辑系统包括靶向PTBP1基因的crRNA、TracrRNA和Cas9蛋白；所述的靶向PTBP1基因的crRNA选自crRNA1、crRNA2或crRNA3中的任意一种或多种；所述的crRNA1序列如SEQ ID NO.1所示，所述的crRNA2序列如SEQ ID NO.2所示，所述的crRNA3序列如SEQ ID NO.3所示。
作为本发明的进一步优选，所述的靶向PTBP1基因的crRNA选自crRNA1、crRNA2和crRNA3的组合。
一种RNP复合物，包括靶向PTBP1基因的crRNA、TracrRNA和Cas9蛋白；所述的靶向PTBP1基因的crRNA选自crRNA1、crRNA2或crRNA3中的任意一种或多种；所述的crRNA1序列如SEQ ID NO.1所示，所述的crRNA2序列如SEQ ID NO.2所示，所述的crRNA3序列如SEQ ID NO.3所示。
作为本发明的一种优选，所述的靶向PTBP1基因的crRNA选自crRNA1、crRNA2和crRNA3的组合。
本发明所述的RNP复合物在制备回调Tau蛋白和Aβ 42蛋白的表达和/或增强神经元分化能力、增长神经元长度的药物中的应用。
PTBP1基因作为治疗靶点在筛选治疗阿尔兹海默症的药物中的应用，所述的PTBP1在Genbank中的登录号为NC_000019.10。
有益效果：
本发明前期利用CRISPR/Cas 9基因编辑技术在SH-SY5Y细胞中成功敲除ADAM10构建了更为全面反应AD的细胞模型，研究结果发现敲除ADAM10后，Aβ 42，Aβ 42/Aβ 40与pTau/Tau的表达显著升高。炎症小体NLRP3蛋白显著上调，促炎因子TNF-α的表达显著升高。同时细胞的神经分化功能减弱，神经元长度皱缩，细胞生长速度显著性减慢，神经元的连接显著性减少，可模拟AD患者脑内神经元坏死病症。
在上述模型中，本发明首次发现PTBP1蛋白在ADAM10 KO细胞中显著性升高，且PTBP1与Tau的相互作用显著性增强。本发明还通过CRISPR/Cas 9基因编辑技术在上述构建的AD细胞模型(ADAM10 KO细胞)中敲除了PTBP1基因，PTBP1蛋白被敲低后，不仅Aβ 42与Tau蛋白均可以显著性降低，同时，炎症小体NLRP3蛋白显著性下调，细胞神经元分化能力增强，神经元长度显著性变长，同时细胞间的信息交流明显增多。以上数据表明，PTBP1蛋白在AD中可能是一个新型的靶点，针对此靶点合成的抑制该靶基因表达的药物能够更为全面的治疗AD疾病。
附图说明
图1 Sanger测序验证基因敲除
图A为CrRNA1,CrRNA2,CrRNA3在ADAM10基因基因序列中的位置示意图；图B为基因突变点测序图谱。与空白组Control KO相比，ADAM10敲除细胞株发生点突变。测序结果显示，已初步筛选到敲除ADAM10基因的细胞扩增株。对这些细胞株进行培养扩增，冻存做后续实验研究。
图2敲除ADAM10基因后靶标蛋白的变化
对敲除组的ADAM10在蛋白水平进行验证，如图A所示，与空白组control KO相比，ADAM10 KO组ADAM10蛋白的表达极显著性降低，与Sanger测序结果相吻 合。数据进一步表明运用CRISPR/Cas 9基因编辑技术在SH-SY5Y细胞中敲除ADAM10基因成功。可进行后续实验研究。
Aβ和细胞内高磷酸化Tau蛋白的累积是如今诊断AD的主要神经病理学标准，同时在临床上AD患者血浆中Aβ42/Aβ40比率可作为AD的预筛查指标。
因此我们对Aβ；Tau；S-199Tau；S-214Tau进行验证，如图B-E所示，与空白组control KO相比，ADAM10 KO组Aβ蛋白的表达显著性升高，Tau蛋白，S-199Tau蛋白，S-214Tau蛋白的表达均显著性上调。如图F-I所示，取细胞上清对Aβ42，Aβ40，p-Tau和Tau进行检测。数据表明，与空白组control KO相比，ADAM10 KO组Aβ40蛋白水平无显著性变化，而Aβ42蛋白水平的表达显著性上调，同时Aβ42/Aβ40，pTau/Tau的比值显著性升高。
以上数据表明Aβ与高磷酸化Tau蛋白在ADAM10基因敲除组均显著性高表达。目前数据初步表明，敲除ADAM10基因的细胞可作为AD细胞模型。
图3敲除ADAM10基因后炎症炎症小体NLRP3的变化
NLRP3炎性小体的失调与多种神经退行性疾病的进展有关。有研究表明NLRP3蛋白抑制剂可能会作为未来治疗AD的靶标。与Control KO组比，ADAM10 KO组NLRP3显著性增加，激活了炎症小体的表达。
图4敲除ADAM10基因后神经元状态的变化
观察在SH-SY5Y细胞中敲除ADAM10基因后神经元细胞的生长速度，分化以及细胞密度的变化。与未进行分化处理的正常SH-SY5Y细胞相比，在经过神经细胞分化处理后，正常SH-SY5Y细胞神经元长度显著性变长，细胞间的连接更紧密。而敲除ADAM10基因后再经过相同的细胞分化处理，神经元长度显著性变短，细胞有皱缩趋势，细胞间的连接显著性变少，神经细胞间传递信息能力显著性减弱。
图5免疫荧光观察PTBP1蛋白与Tau蛋白的表达
免疫荧光图显示，与空白组相比，ADAM10 KO组PTBP1与Tau蛋白的表达均显著性升高。
图6 AD细胞模型中PTBP1蛋白与Tau蛋白的相互作用
与空白组Control KO相比，ADAM10 KO组PTBP1蛋白的表达显著上调。大量研究表明，Tau蛋白在神经变性疾病以及记忆功能损伤中发挥着重要的作用。与空白组Control KO相比，ADAM10 KO组Tau蛋白的表达显著升高。CO-IP结果显示， PTBP1与Tau蛋白有相互作用，且与空白组相比，PTBP1与Tau的相互作用明显。以上实验结果表明，ADAM10基因敲除后，神经元长度显著性变短以及细胞神经分化能力减弱有可能是由于PTBP1与Tau蛋白发生相互作用从而影响神经元功能。
图7 PTBP1的结构区域
其中CrRNA1、CrRNA2、CrRNA3分别为敲除PTBP1基因的CrRNA序列。
图8 CrRNA1示意图
其中CrRNA1:GGCACCCCCUUUUCAGCAAA(SEQ ID ON.4)；Cutsite:804,037；Exon:Exon 4；On Target Score：0.426.
图9 crRNA2示意图
其中crRNA2:AAUGACAGCAAGAAGUUCAA(SEQ ID ON.5)；Cutsite:804,061；Exon:Exon 4；On Target Score：0.531.
图10 crRNA3示意图
其中crRNA3:AAAGGUGACAGCCGAAGUGC(SEQ ID ON.6)Cutsite:804,079；Exon:Exon 4；On Target Score：0.513.
图11敲低PTBP1蛋白后靶标蛋白的变化
与Control KO组比，ADAM10 KO组NLRP3显著性增加，激活了炎症小体的表达。同时，Tau蛋白和PTBP1蛋白显著增加。使用CRISPR/Cas 9基因编辑技术在敲除模型细胞中通过敲低PTBP1蛋白来观察后续指标。与ADAM10 KO组比，敲低PTBP1蛋白后，PTBP1蛋白的表达显著降低。同时Tau蛋白的表达显著降低，NLRP3炎症小体蛋白显著降低。
图12敲低PTBP1蛋白后靶标蛋白的变化
ELISA测定Aβ 42的水平，数据表明，PTBP1蛋白敲低后Aβ42水平显著性回调。
图13敲低PTBP1蛋白后神经元的变化
与未进行分化处理的正常SH-SY5Y细胞相比，在经过神经细胞分化处理后，正常SH-SY5Y细胞神经元长度显著性变长，细胞间的连接更紧密。而敲除ADAM10基因后再经过相同的细胞分化处理，神经元长度显著性变短，细胞有皱缩趋势，细胞间的连接显著性变少，神经细胞间传递信息能力显著性减弱。在模型AD细胞中敲低PTBP1蛋白的表达后，神经元状态有所回调。
具体实施方式
本发明通过下面的实施例进行详细的解释，但并不意味着本发明仅限于此。
实施例1AD细胞模型构建
(1)构建CRISPR/CAS9基因编辑方法
以下过程均在无菌操作台中全程无菌的条件下操作：
①构建针对ADAM10基因的RNP复合物：分别取0.33μl的crRNA ADAM101(GGCACCCCCUUUUCAGCAAA(SEQ ID ON.1)，100μM)，0.33μl的crRNA ADAM102(AAUGACAGCAAGAAGUUCAA(SEQ ID ON.2)，100μM)，0.33μl的crRNA ADAM103(AAAGGUGACAGCCGAAGUGC(SEQ ID ON.3)，100μM)与1μl Alt-R.CRISPR-Cas9tracrRNA(100μM)充分混合均匀后使用无核酸酶的缓冲液稀释到浓度为20-80μM，得到RNA混合物。将RNA混合物在95℃下加热5min，在室温中冷却10min，得到退火RNA混合物。取退火RNA混合物2.9μl与Cas 9蛋白1μl按照1.2：1-2:1的比例充分混合后放置室温10min。加入0.6μl电转加强液后在室温中反应5min，得到RNP复合物。
②将RNP复合物电转导入待敲除基因的SH-SY5Y细胞中：预热含15％FBS培养基，取密度为80％的细胞，弃去培养基。使用适量的PBS溶液清缓的清洗细胞，弃去溶液后，加入适量的胰蛋白酶于37℃下消化细胞，等数分钟80％细胞脱壁后，使用含15％FBS的培养基终止消化，1100rpm离心10min弃去上清。用10ml PBS溶液重悬细胞，充分混匀细胞后在取10μl细胞溶液计数。取250,000个细胞在1100rpm离心10min，弃去PBS上清，使用15.5μl的P3溶液重悬细胞。加入4.5μl的RNP复合物，得到20μl体系的细胞溶液。将20μl体系的细胞溶液加入16孔的电转孔板中，将孔板放入Lonza-4D电转仪中，选择SH-SY5Y程序进行电转。
(2)构建单细胞克隆技术方法
将电转好的细胞逐级稀释到1个细胞/10μl，加入96孔板中，补足200μl含15％FBS的培养基。稳定12小时等待细胞贴壁后，在显微镜下观察并筛选记录出96孔板中含一个细胞的孔。筛选后，继续等待这些单细胞生长扩增，待单细胞在96孔板中扩增到细胞密度为80％后将细胞转移至12孔板中，待单细胞在12孔板中扩增到细胞密度为80％后将细胞转移至6孔板中，待细胞密度为80％后， 取一部分细胞进行Sanger测序，筛选敲除成功的单细胞系，剩下的细胞继续培养，每隔48小时换细胞培养液一次。
(3)Sanger测序
①细胞DNA样本的提取
取细胞弃去培养基，加入适量的PBS溶液润洗细胞后弃去溶液，加入适量的Trpsin消化细胞数分钟后加入含15％FBS培养基终止消化，1100rpm离心10minPBS溶液充分重悬后取少量细胞计数，取500，000个细胞严格按照。基因组DNA小量抽提试剂盒(离心柱式；D00D0063)的说明书操作提取细胞DNA。
②聚合酶链式反应(PCR)
如表1所示，目的基因ADAM10的引物设计如下：
表1目的基因ADAM10的引物
Figure PCTCN2021089293-appb-000001
③反应体系如下
表2 PCR反应体系
Figure PCTCN2021089293-appb-000002
④PCR反应条件
表3 PCR反应程序
Figure PCTCN2021089293-appb-000003
⑤PCR产物纯化
根据PCR产物电泳结果切割所需的DNA目的条带，纯化步骤严格按照说明书SK8131胶回收操作。
⑥Sanger测序阶段
测序反应体系如下表所示：
表4 Sanger测序反应体系
Figure PCTCN2021089293-appb-000004
反应条件:
表5 Sanger测序反应条件
Figure PCTCN2021089293-appb-000005
将PCR反应板从PCR仪上取下后每孔加4μl EDTA Mix(0.5mol/L)EDTA，3mol/L醋酸钠，灭菌去离子水比例混合和60μl 95％乙醇，在冰水浴中反应30min。
将溶液于4000g离心20min，轻轻将板倒置，600rmp/min甩干后每孔加150μl 70％乙醇，4000g离心5min。将板倒置600rmp/min甩干，于超净台上晾5min，加10μl Hidi Formamide，漩涡器上振荡1min。
将反应板放在PCR仪上96℃，3min迅速将反应板取下放入冰浴冷却，等待上样。
将96孔板置于ABI 3730XL测序仪电泳分析。测序电泳仪操作按照ABI Prism 3730使用手册严格执行操作。
与空白组Control KO相比，ADAM10敲除细胞株发生点突变。测序结果(图1)显示，已初步筛选到敲除ADAM10基因的细胞扩增株。对这些细胞株进行培养扩增，冻存做后续实验研究。
实施例2
2.1 AD细胞模型中ADAM10靶标蛋白和炎症炎症小体NLRP3的测定
通过Western对敲除组的ADAM10在蛋白水平进行验证。如图2A所示，与空白组control KO相比，ADAM10 KO组ADAM10蛋白的表达极显著性降低，与Sanger测序结果相吻合。数据进一步表明运用CRISPR/Cas 9基因编辑技术在SH-SY5Y细胞中敲除ADAM10基因成功。可进行后续实验研究。
通过Western对敲除组的炎症炎症小体NLRP3在蛋白水平进行验证。如图3所示，与Control KO组比，ADAM10 KO组NLRP3显著性增加，激活了炎症小体的表达。
2.2 AD细胞模型中Aβ与Tau蛋白的测定
Aβ和细胞内高磷酸化Tau蛋白的累积是如今诊断AD的主要神经病理学标准，同时在临床上AD患者血浆中Aβ42/Aβ40比率可作为AD的预筛查指标。因此我们对Aβ；Tau；S-199Tau；S-214Tau进行验证：
取细胞弃去培养基，加入适量的PBS溶液润洗细胞后弃去溶液，加入适量的Trpsin消化细胞数分钟后加入含15％FBS培养基终止消化，1100rpm离心10min。PBS溶液充分重悬后取少量细胞计数，取2，000，000个细胞严格按照ELISA试剂盒说明书操作步骤严格进行，每个样本的总蛋白由BCA试剂盒测定，矫正总蛋白后，测定Aβ40；Aβ42；p-Tau及Tau的含量后进行数据统计。如图2B-E所 示，与空白组control KO相比，ADAM10 KO组Aβ蛋白的表达显著性升高，Tau蛋白，S-199Tau蛋白，S-214Tau蛋白的表达均显著性上调。如图2F-I所示，取细胞上清对Aβ42，Aβ40，p-Tau和Tau进行检测。数据表明，与空白组control KO相比，ADAM10 KO组Aβ40蛋白水平无显著性变化，而Aβ42蛋白水平的表达显著性上调，同时Aβ42/Aβ40，pTau/Tau的比值显著性升高。
以上数据表明Aβ与高磷酸化Tau蛋白在ADAM10基因敲除组均显著性高表达。目前数据初步表明，敲除ADAM10基因的细胞可作为AD细胞模型。
2.3 AD细胞模型中神经元状态变化
(1)细胞神经元分化培养
将SH-SY5Y细胞依次培养在如下表6所示的不同类型的培养基中，首先将细胞在Growth Media培养基中培养使其细胞密度达到70％。随后将培养基配方更换为Differentiation media 1培养7-10天，在此期间，每48小时更换培养液一次。将细胞按照1：1的比例传代于新鲜的培养基Differentiation media 2中培养4-7天，在此期间，每48小时更换培养液一次。将培养基换成Differentiation media 3培养7-10天，在此期间，每48小时更换培养液一次。此阶段细胞分化为神经元用于后续的检测与分析。
表6 SH-SY5Y细胞神经分化的培养基组成成分
Figure PCTCN2021089293-appb-000006
(2)共聚焦显微镜观察神经元的生长
将分化的SH-SY5Y神经细胞使用4％PFA在室温下固定20min。弃去溶液后使用0.1％PBS-T溶液轻缓的润洗细胞3次，每次2min。固定后，将细胞放置在5％NGS-T封闭溶液中于室温孵育2小时。在4℃下加入一抗anti-tubulin III primary antibody(#ab179513,Abcam plc.)，一抗稀释浓度为1：1000，孵育过夜。去除一抗后使用0.1％PBS-T溶液轻缓的润洗细胞3次。在室温下加入二抗Alexa Fluor 488(ab150113,Abcam)，二抗稀释浓度为1：2000，于室温下孵育1h。去除二抗后，将细胞固定在载玻片中，在24h内用过共聚焦显微镜对样品进行成像检测。
使用Zeiss880共聚焦显微镜采集所有神经元分化图片。Laser power设置为4％；gain设置为650；Digital offset设置为350；Acquiring speed设置1.03s；Z-stack images设置为5μm；平均每张片子拍2次。
共聚焦显微镜采集的图像使用Image J插件Neuron J进行分析。选定每张图片中大于3个区域进行分析，随后测量轴突长度，并用Neuron J测量坐标之间的距离。
如图4所示，与未经过神经细胞分化的正常的SH-SY5Y细胞相比，在经过神经细胞分化处理后，正常的SH-SY5Y细胞神经元长度显著性变长，细胞间的连接更紧密。而敲除ADAM10基因后再经过相同的细胞分化处理，神经元长度显著性变短，细胞有皱缩趋势，细胞间的连接显著性变少，神经细胞间传递信息能力显著性减弱。
实施例3构建PTBP1的基因敲除细胞系
以下过程均在无菌操作台中全程无菌的条件下操作：
①构建RNP复合物：分别取0.33μl的crRNA1(100μM)，0.33μl的crRNA2(100μM)，0.33μl的crRNA3(100μM)与1μl Alt-R.CRISPR-Cas9 tracrRNA(100μM)充分混合均匀后使用无核酸酶的缓冲液稀释到浓度为20-80μM，得到RNA混合物。将RNA混合物在95℃下加热5min，在室温中冷却10min，得到退火RNA混合物。取退火RNA混合物2.9μl与Cas 9蛋白1μl按照1.2：1-2:1的比例充分混合后放置室温10min。加入0.6μl电转加强液后在室温中反应5min，得到RNP复合物。
②将RNP复合物电转导入待敲除基因的SH-SY5Y细胞中：预热含15％FBS培养基，取密度为80％的细胞，弃去培养基。使用适量的PBS溶液清缓的清洗细胞，弃去溶液后，加入适量的胰蛋白酶于37℃下消化细胞，等数分钟80％细胞脱壁后，使用含15％FBS的培养基终止消化，1100rpm离心10min弃去上清。用10ml PBS溶液重悬细胞，充分混匀细胞后在取10μl细胞溶液计数。取250,000个细胞在1100rpm离心10min，弃去PBS上清，使用15.5μl的P3溶液重悬细胞。加入4.5μl的RNP复合物，得到20μl体系的细胞溶液。将20μl体系的细胞溶液加入16孔的电转孔板中，将孔板放入Lonza-4D电转仪中，选择SH-SY5Y程序进行电转。
如图11所示，与空白组Control KO相比，ADAM10 KO组PTBP1蛋白显著升高，PTBP1基因治疗组PTBP1蛋白的表达显著回调。运用CRISPR/Cas9基因编辑技术敲低PTBP1蛋白成功，可进行后续实验测定。
实施例4免疫荧光观察PTBP1蛋白和Tau蛋白的表达
将分化的SH-SY5Y神经细胞使用4％PFA在室温下固定20min。弃去溶液后使用0.1％PBS-T溶液轻缓的润洗细胞3次，每次2min。固定后，将细胞放置在5％NGS-T封闭溶液中于室温孵育2小时。在4℃下加入一抗按照说明书比例稀释后孵育过夜。去除一抗后使用0.1％PBS-T溶液轻缓的润洗细胞3次。在室温下加入二抗，稀释浓度严格按照说明书操作后于室温下孵育1h。去除二抗后，将细胞固定在载玻片中，在24h内用荧光显微镜对样品进行检测。
如图5所示，与空白组相比，ADAM10 KO组PTBP1与Tau蛋白的表达均显著性升高。
实施例5免疫共沉淀(Co-IP)验证PTBP1蛋白与Tau蛋白的相互作用
提取组织中的蛋白样本后，取100ul的蛋白上清样本，以4：1的比例体积加入上样缓冲液，沸水煮沸5-10min后作为input组的上样样本。取300-400ul进行免疫共沉淀实验。在每个样本中按照抗体说明书加入一定比例的一抗，放置4℃缓慢摇动过夜。每个样本中按照1：100的比例加入Protein A+G磁珠，4℃上下颠倒缓慢混匀4-6h，3200rpm，5min离心后取出样本使用PBS洗涤5次， 按照适当比例加入上样缓冲液沸水煮5-10min。按照Western blotting的方法步骤进行下一步实验，测定相关蛋白的表达。
如图6所示，Co-IP结果显示，PTBP1与Tau蛋白有相互作用，且与空白组相比，PTBP1与Tau的相互作用明显。以上实验结果表明，ADAM10基因敲除后，神经元长度显著性变短以及细胞神经分化能力减弱有可能是由于PTBP1与Tau蛋白发生相互作用从而影响神经元功能。
实施例6
①细胞总蛋白的提取:
弃去培养液后加入2ml预冷PBS进行润洗细胞，弃去PBS洗液。此操作重复2次。将培养瓶置于冰上，细胞计数后取1000,000个细胞，加入100μl含PMSF的裂解液，充分混合后于冰上裂解30min，隔段时间要摇动细胞瓶使细胞充分裂解。裂解后的细胞快速使用刮棒将细胞刮下，使用移液枪吸取细胞碎片和裂解液转移至1.5ml离心管中。于4℃下13000g离心20min。取上清，留取5μl对蛋白含量进行测定。取适量的上清以4：1体积加入上样缓冲液，于沸水煮沸10min。样品放于-20℃保存。
②制备分离胶与浓缩胶
分离胶(15％)：超纯水2.3mL；30％丙烯酰胺5.0mL；1.5M Tris-HCl(pH 8.8)2.5mL；10％SDS 0.1mL；10％APS(过硫酸铵)0.1mL；TEMED 4μl。
分离胶(8％)：超纯水4.6mL；30％丙烯酰胺2.7mL；1.5M Tris-HCl(pH 8.8)2.5mL；10％SDS 0.1mL；10％APS(过硫酸铵)0.1mL；TEMED 8μl。
分离胶(6％)：超纯水5.3mL；30％丙烯酰胺2.0mL；1.5M Tris-HCl(pH 8.8)2.5mL；10％SDS 0.1mL；10％APS(过硫酸铵)0.1mL；TEMED 8μl。
浓缩胶(5％)：超纯水2.7mL；30％丙烯酰胺0.4mL；1M Tris-HCl溶液(pH 6.8)0.5mL；10％SDS 40μl；10％APS 40μl；TEMED 4μl。
过硫酸铵与TEMED为促凝剂，加入溶液后要立即混匀后灌胶。灌好分离胶后，轻缓的加入超纯水，加超纯水时移动移液枪，保证水在同一水平线上。静置直到看到分离胶与水之间出现较明显的界限。等待分离胶凝固后，倾斜倒掉水，沥干水后，再灌入浓缩胶，插上梳齿，等待浓缩胶凝固后上样。
③蛋白样品上样
沿着垂直方向缓慢的拔出梳齿。使用50μl微量上样器取适量的样本，上样总蛋白一般为20μg，将上样器缓慢插入梳齿最底部，以适当的速度打入蛋白样品。
④电泳
第一阶段：75V，40～60min直至看到目的蛋白在分离胶与浓缩胶分界处出现一条线。
第二阶段：115V，60～90min直至目的蛋白迁移于距分离胶底部约1/3处。
⑤转膜
提前剪裁好与胶条大小匹配的PVDF膜，放入甲醇中浸泡活化15-20s后转移到超纯水中放置2min，将PVDF膜放入转膜缓冲液中浸泡10min。取出转膜滤纸放入转膜缓冲液中浸泡10min。等待电泳结束后切割所需分子量范围内的胶条。转膜装置从上至下的顺序依次为阴极碳板，转膜滤纸，胶，PVDF膜，滤纸。按照顺利放好，精确对齐，每一步均要保证无气泡存在。接通电源，1.5mm的胶恒流0.3A转膜时间2h，1mm的胶恒流0.18A转膜时间1.5h。将转膜槽放置在冰浴中防止转膜过热。
⑥封闭
转膜阶段结束后，断开电源取出PVDF膜。于5％的脱脂奶粉中4℃封闭过夜，或37℃封闭1h。
⑦孵育抗体
a.使用5％脱脂牛奶或者5％BSA溶液按照抗体说明书以适当的比例稀释一抗，孵育4℃过夜后于37℃条件下孵育1h。
b.弃去一抗，使用TBST溶液洗膜30min，每5min换一次液。
c.使用5％脱脂牛奶或者5％BSA溶液按照抗体说明书以适当的比例稀释二抗，于37℃条件下孵育1h。
d.弃去二抗，使用TBST溶液洗膜30min，每5min换一次液。
⑧凝胶图像分析
依据膜的面积将每条膜均匀的加入100～200ul的显影液，将条带放入凝胶成像仪中避光显色。对条带进行扫描，拍照。凝胶图象处理系统处理并分析条带的分子量与净光密度值。曝光时间依据条带的曝光难易程度做适当的调整，最起始的曝光时间为5～20s，总曝光时间根据每个条带做适当调整。
如图11所示，与Control KO组比，ADAM10 KO组Tau蛋白，NLRP3炎症小体蛋白显著上调，敲低PTBP1蛋白后，Tau蛋白与NLRP3炎症小体蛋白显著回调，结果表明，PTBP1蛋白的下调能回调炎症因子的水平，并降低Tau蛋白的表达。
实施例7敲低PTBP1蛋白后Aβ 42的变化
取细胞弃去培养基，加入适量的PBS溶液润洗细胞后弃去溶液，加入适量的Trpsin消化细胞数分钟后加入含15％FBS培养基终止消化，1100rpm离心10min。PBS溶液充分重悬后取少量细胞计数，取2，000，000个细胞严格按照ELISA试剂盒说明书操作步骤严格进行，每个样本的总蛋白由BCA试剂盒测定，矫正总蛋白后，测定Aβ 42的含量后进行数据统计。
如图12所示，与Control KO组比，模型组Aβ 42显著上调。PTBP1敲低后，Aβ 42水平显著回调。结果表明，PTBP1蛋白的下调能回调Aβ 42蛋白的表达。
实施例8
(1)细胞神经元分化培养
将SH-SY5Y细胞依次培养在如表6所示的不同类型的培养基中，首先将细胞在Growth Media培养基中培养使其细胞密度达到70％。随后将培养基配方更换为Differentiation media 1培养7-10天，在此期间，每48小时更换培养液一次。将细胞按照1：1的比例传代于新鲜的培养基Differentiation media 2中培养4-7天，在此期间，每48小时更换培养液一次。将培养基换成 Differentiation media 3培养7-10天，在此期间，每48小时更换培养液一次。此阶段细胞分化为神经元用于后续的检测与分析。
(2)共聚焦显微镜观察神经元的生长
将分化的SH-SY5Y神经细胞使用4％PFA在室温下固定20min。弃去溶液后使用0.1％PBS-T溶液轻缓的润洗细胞3次，每次2min。固定后，将细胞放置在5％NGS-T封闭溶液中于室温孵育2小时。在4℃下加入一抗anti-tubulin III primary antibody(#ab179513,Abcam plc.)，一抗稀释浓度为1：1000，孵育过夜。去除一抗后使用0.1％PBS-T溶液轻缓的润洗细胞3次。在室温下加入二抗Alexa Fluor 488(ab150113,Abcam)，二抗稀释浓度为1：2000，于室温下孵育1h。去除二抗后，将细胞固定在载玻片中，在24h内用过共聚焦显微镜对样品进行成像检测。
使用Zeiss880共聚焦显微镜采集所有神经元分化图片。Laser power设置为4％；gain设置为650；Digital offset设置为350；Acquiring speed设置1.03s；Z-stack images设置为5μm；平均每张片子拍2次。
共聚焦显微镜采集的图像使用Image J插件Neuron J进行分析。选定每张图片中大于3个区域进行分析，随后测量轴突长度，并用Neuron J测量坐标之间的距离。
如图13所示，与模型组相比，PTBP1蛋白敲低后缩短的神经元状态显著回调。

Claims (9)

  1. PTBP1基因作为治疗靶点在制备治疗阿尔兹海默症的药物中的应用，所述的PTBP1在Genbank中的登录号为NC_000019.10。
  2. 抑制PTBP1基因表达的物质在制备治疗阿尔兹海默症的药物中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述的抑制PTBP1基因表达的物质选自用于PTBP1基因敲除的CRISPR/Cas9基因编辑系统、针对PTBP1的特异性反义寡核苷酸、siRNA/shRNA、锌指核酸酶、转录激活因子样效应核酸酶、小分子抑制剂、腺病毒、慢病毒或小分子化合物。
  4. 根据权利要求3所述的应用，其特征在于所述的用于PTBP1基因敲除的CRISPR/Cas9基因编辑系统包括靶向PTBP1基因的crRNA、TracrRNA和Cas9蛋白；所述的靶向PTBP1基因的crRNA选自crRNA1、crRNA2或crRNA3中的任意一种或多种；所述的crRNA1序列如SEQ ID NO.1所示，所述的crRNA2序列如SEQ ID NO.2所示，所述的crRNA3序列如SEQ ID NO.3所示。
  5. 根据权利要求4所述的应用，其特征在于，所述的靶向PTBP1基因的crRNA选自crRNA1、crRNA2和crRNA3的组合。
  6. 一种RNP复合物，其特征在于，包括靶向PTBP1基因的crRNA、TracrRNA和Cas9蛋白；所述的靶向PTBP1基因的crRNA选自crRNA1、crRNA2或crRNA3中的任意一种或多种；所述的crRNA1序列如SEQ ID NO.1所示，所述的crRNA2序列如SEQ ID NO.2所示，所述的crRNA3序列如SEQ ID NO.3所示。
  7. 根据权利要求6所述的RNP复合物，其特征在于，所述的靶向PTBP1基因的crRNA选自crRNA1、crRNA2和crRNA3的组合。
  8. 权利要求6或7所述的RNP复合物在制备回调Tau蛋白和Aβ 42蛋白的表达和/或增强神经元分化能力、增长神经元长度的药物中的应用。
  9. PTBP1基因作为治疗靶点在筛选治疗阿尔兹海默症的药物中的应用，所述的PTBP1在Genbank中的登录号为NC_000019.10。
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