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壳聚糖水凝胶递送的ABE8e可纠正牙髓干细胞中的PAX9突变。

Chitosan Hydrogel-Delivered ABE8e Corrects PAX9 Mutant in Dental Pulp Stem Cells.

作者信息

Liu Bowen, Zhang Chenjiao, Zhao Han, Gao Jian, Hu Jingchao

机构信息

Outpatient Department of Oral and Maxillofacial Surgery, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Tian Tan Xi Li No. 4, Beijing 100050, China.

Department of General, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Tian Tan Xi Li No. 4, Beijing 100050, China.

出版信息

Gels. 2023 May 25;9(6):436. doi: 10.3390/gels9060436.


DOI:10.3390/gels9060436
PMID:37367107
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10297230/
Abstract

Hypodontia (dental agenesis) is a genetic disorder, and it has been identified that the mutation C175T in PAX9 could lead to hypodontia. Cas9 nickase (nCas9)-mediated homology-directed repair (HDR) and base editing were used for the correction of this mutated point. This study aimed to investigate the effect of HDR and the base editor ABE8e in editing PAX9 mutant. It was found that the chitosan hydrogel was efficient in delivering naked DNA into dental pulp stem cells (DPSCs). To explore the influence of the C175T mutation in PAX9 on the proliferation of DPSCs, hydrogel was employed to deliver PAX9 mutant vector into DPSCs, finding that the PAX9-containing C175T mutation failed to promote the proliferation of DPSCs. Firstly, DPSCs stably carrying PAX9 mutant were constructed. Either an HDR or ABE8e system was delivered into the above-mentioned stable DPSCs, and then the correction efficiency using Sanger sequencing and Western blotting was determined. Meanwhile, the ABE8e presented significantly higher efficiency in correcting C175T compared with HDR. Furthermore, the corrected PAX9 presented enhanced viability and differentiation capacity for osteogenic and neurogenic lineages; the corrected PAX9 even possessed extremely enhanced transcriptional activation ability. In summary, this study has powerful implications for studies into base editors, chitosan hydrogel, and DPSCs in treating hypodontia.

摘要

牙齿发育不全(牙胚缺失)是一种遗传性疾病,已确定PAX9基因中的C175T突变可导致牙齿发育不全。使用Cas9切口酶(nCas9)介导的同源定向修复(HDR)和碱基编辑来纠正这一突变位点。本研究旨在探讨HDR和碱基编辑器ABE8e对编辑PAX9突变体的效果。发现壳聚糖水凝胶能有效地将裸露的DNA递送至牙髓干细胞(DPSC)中。为了探究PAX9基因中C175T突变对DPSC增殖的影响,采用水凝胶将PAX9突变载体递送至DPSC中,发现含有C175T突变的PAX9未能促进DPSC的增殖。首先,构建稳定携带PAX9突变体的DPSC。将HDR或ABE8e系统导入上述稳定的DPSC中,然后使用桑格测序和蛋白质印迹法测定校正效率。同时,与HDR相比,ABE8e在纠正C175T方面表现出显著更高的效率。此外,校正后的PAX9在成骨和神经源性谱系方面表现出增强的活力和分化能力;校正后的PAX9甚至具有极强的转录激活能力。总之,本研究对碱基编辑器、壳聚糖水凝胶和DPSC在治疗牙齿发育不全方面的研究具有重要意义。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/2291c59ac4d9/gels-09-00436-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/16db038cf590/gels-09-00436-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/f14df23172fa/gels-09-00436-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/453cf7bb102b/gels-09-00436-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/d8155cc05204/gels-09-00436-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/d9e8735d25ba/gels-09-00436-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/2291c59ac4d9/gels-09-00436-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/16db038cf590/gels-09-00436-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/f14df23172fa/gels-09-00436-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/453cf7bb102b/gels-09-00436-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/d8155cc05204/gels-09-00436-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/d9e8735d25ba/gels-09-00436-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f52e/10297230/2291c59ac4d9/gels-09-00436-g006.jpg

相似文献

[1]
Chitosan Hydrogel-Delivered ABE8e Corrects PAX9 Mutant in Dental Pulp Stem Cells.

Gels. 2023-5-25

[2]
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Biochem Biophys Res Commun. 2022-11-5

[3]
The Role of Interleukin 6 in Osteogenic and Neurogenic Differentiation Potentials of Dental Pulp Stem Cells.

J Endod. 2019-9-18

[4]
Basic fibroblast growth factor promotes human dental pulp stem cells cultured in 3D porous chitosan scaffolds to neural differentiation.

Int J Neurosci. 2021-7

[5]
Evaluation of Chitosan Hydrogel for Sustained Delivery of VEGF for Odontogenic Differentiation of Dental Pulp Stem Cells.

Stem Cells Int. 2019-12-19

[6]
In vitro osteogenic and odontogenic differentiation of human dental pulp stem cells seeded on carboxymethyl cellulose-hydroxyapatite hybrid hydrogel.

Front Physiol. 2015-10-27

[7]
Chitosan scaffolds induce human dental pulp stem cells to neural differentiation: potential roles for spinal cord injury therapy.

Cell Tissue Res. 2016-10

[8]
In vitro proliferation and osteogenic differentiation of human dental pulp stem cells in injectable thermo-sensitive chitosan/β-glycerophosphate/hydroxyapatite hydrogel.

J Biomater Appl. 2016-9

[9]
Novel gain-of-function mutation of associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia.

Biochem Biophys Rep. 2019-5-17

[10]
Elevated osteogenic potential of stem cells from inflammatory dental pulp tissues by Wnt4 overexpression for treating bone defect in rats.

Ann Palliat Med. 2020-9

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[2]
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[3]
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[4]
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[5]
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[6]
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[7]
Smart stimuli-responsive injectable gels and hydrogels for drug delivery and tissue engineering applications: A review.

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[8]
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Mater Today Bio. 2023-2-16

[9]
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[10]
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