Adenovirus vector-mediated gene transfection method for stereoscopic-state tooth germ ameloblasts

Abstract

The invention discloses an adenovirus vector-mediated gene transfection method for stereoscopic-state tooth germ ameloblasts. The method comprises the following steps that: a separated primary tooth germ with an upward cusp is placed on a microporous filter membrane of a Transwell built-in cabinet and is cultured in a culture medium containing the tooth germ; infectious viral particles containing an AAV-GFP gene are used to infect the cultured tooth germ; green fluorescence appears on a surface layer of the tooth germ 24 hours after infection; in combination with the immunostaining of amelogenin, recombinant tooth germ ameloblasts co-expressed by GFP and the amelogenin are positioned on the tooth germ. The ameloblasts of the Transwell-system in vitro cultured tooth germ maintain a stereoscopic state and have interaction with surrounding matrixes and related cells, and the gene transfection success of the ameloblasts in the mode is favorable for researching the gene function of the ameloblasts in the tooth germ from a three-dimensional perspective.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to gene transfection of tooth germs and gene function research of ameloblasts, in particular to an adenovirus vector-mediated three-dimensional gene transfection method for ameloblasts of tooth germs. Background technique [0002] Gene transfection is the transfer of exogenous gene fragments into host cells for expression. It is one of the important methods to study gene function. The gene transfection of monolayer cultured cells as host cells has been widely used to study its gene function. Adenoviral vectors are currently the most commonly used expression vectors. Usually, recombinant adenoviral vectors include replication-defective and proliferative types; Difficult-to-transfer tissues or cells, such as retina, respiratory epithelium, placenta, and submucosal gland ,, Adenovirus, Adeno-Associated Virus, Lentivirus, and Measles Virus. Molecular and Cellular Neuroscience 2001 May; 17(5)...

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Problems solved by technology

[0006] Compared with other cells, the number of ameloblasts is small, and they are in close contact with the surrounding tissues and cells and difficult to separate. It is difficult to obtain a sufficient number of ameloblasts for primary culture by conventional primary cell culture methods such as mechanical separation or enzyme digestion. Its in vitro gene transfection efficiency is even lower, so that the current international research on the gene function of ameloblasts uses some cell lines such as LS8 (Zhou YL, Snead ML. gene.Journal of Biological Chemistry 2000 April 21, 275(16):12273-80) and PABSo-E (DenBesten PK, Gao C, Li W, Mathews CHE, Gruenert DC, Development and characterization of an SV40 immortalized porcine ameloblast-like cell line, European Journal of Oral Sciences 1999August; 107(4): 276-81.), etc.

LS8 and PABSo-E cell lines cannot fully reflect the three-dimensional growth state of ameloblasts in vivo, and cannot meet the needs of the study of their gene functions, especially from a three-dimensional perspective to study the gene functions of ameloblasts in tooth germs; ameloblasts in During the differentiation process, there is a series of changes in height and growth polarity. These characteristics reflect the different differentiation stages of the cells. In the cell monolayer culture system, these three-dimensional or polar characteristics are difficult to maintain

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