Construction of Turner syndrome cell model and cell bank of Turner syndrome cell by virtue of importing of hTERT

Abstract

The invention relates to construction of an hTERT-mediated Turner syndrome cell model and a cell bank of Turner syndrome cell by virtue of importing of hTERT, belonging to the medical field. The construction is mainly characterized in that a plasmid pCIneo-hTERT and a vector pLXSNneo are subjected to double enzyme digestion of incision enzymes EcoR I and Xho I, and hTERT and pLXSNneo enzyme-digested products subjected to PCR amplification and gel electrophoresis separation are linked by virtue of Ligation Mix, so as to construct a pLXSNneo-hTERT recombinant; a DH5a competent cell is converted for purification and amplification and an ampicillin-resistant colony is picked to extract the plasmid; the Turner syndrome cell in logarithmic growth undergoes in-vitro passage by virtue of lipofection transfection; the recombinant is integrated with cell DNA, enlarged cultivation is carried out on a cell which is screened out by virtue of G418 and contains a positive recombinant and the cell is cloned; a cell which is consistent with the properties of an immortalized cell in cellular morphology, growth curve, karyotype, nude mice carcinogenicity test, transfection cell telomerase activity, hTERT mRNA expression, immunohistochemistry, cell generation cycle and apoptosis rate and is same or similar to a primary cell is screened out as an hTERT-mediated Turner syndrome cell, and is cryopreserved in liquid nitrogen, so as to lay a foundation for long-term in vitro research on pathogenic mechanism from the view of cellular level.

Description

technical field [0001] The invention relates to hTERT (telomerase reverse transcriptase catalytic subunit) introduction (mediated) Turner syndrome cell model and construction of cell bank, mainly used in the field of human reproductive health research, providing cells for in vitro research of Turner syndrome model and save its research resources. Background technique [0002] Turner syndrome manifests as abnormalities such as short stature, primary amenorrhea, webbed neck, cubitus valgus, dysplasia of secondary features, absence of ovaries, and infertility caused by the lack of one X chromosome in females. Some patients have mild mental retardation, or are accompanied by congenital malformations such as heart, kidney, and bone. The disease was reported by Turner in 1938, so it is called Turner syndrome. It is impossible for patients with this disease to establish reproductive function. For the development and maintenance of secondary sexual characteristics, estrogen repla...

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Problems solved by technology

[0008] However, there have been no literature reports on the construction of immortal cell models and cell banks using hTERT to study the pathogenesis of Turner syndrome from the cellular level in vitro, and it is impossible to carry out research on related projects

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