Reversible immortalized II-type alveolar epithelial cell as well as construction and application thereof

A technology of alveolar cells and alveolar epithelium, applied in the field of cell engineering, can solve the problems of affecting proliferation activity, limited cell volume acquisition, slow proliferation of alveolar cells in vitro, etc., and achieve strong proliferation activity and growth ability, high-efficiency function and mechanism of action

Pending Publication Date: 2022-07-15
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Primary alveolar cells proliferate slowly in vitro, and the amount of cells obtained is limited
At the same time, long-term cultured cells in vitro are prone to self-differentiation, and repeated cryopreservation and recovery will also affect their proliferation activity

Method used

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  • Reversible immortalized II-type alveolar epithelial cell as well as construction and application thereof
  • Reversible immortalized II-type alveolar epithelial cell as well as construction and application thereof
  • Reversible immortalized II-type alveolar epithelial cell as well as construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] In this experiment, primary mouse alveolar cells were extracted and cultured, their morphology was observed, and their proliferation activity and the expression of alveolar-related markers were detected. Then, SSR#41 immortalization tool plasmid and FLP / FRT system were used to construct reversible immortalized mice Alveolar cells, their functional properties were characterized and compared to primary alveolar cells.

[0054] 1 material

[0055] 1.1 Main reagents

[0056] DMEM high glucose medium and fetal bovine serum were purchased from Gibco Company in the United States, Penicillin-Streptomycin, PEI, molecular water (ddH2O), NucleoZOL were purchased from Invitrogen Company in the United States, hygromycin B, DMSO, cell culture flasks were purchased from Thermo in the United States Fisher Scientific, SSR41 plasmid, pCL-Ampho plasmid were purchased from the Laboratory of Molecular Oncology, University of Chicago Medical Center, PC-8 (phenol extraction reagent, PBS, abs...

Embodiment 2

[0109] Sorting and identification of mouse type Ⅱ alveolar epithelial cells imPAC2

[0110] Type II alveolar epithelial cells play an important role in lung development, especially alveolar formation, and are the most potential pulmonary stem cells. During the repair of lung injury, type II alveolar epithelial cells can self-renew and differentiate into type I alveolar epithelial cells to repair the damage and maintain the integrity of the alveolar structure and normal function. Immortalized alveolar cells have been identified in Example 1 of the present invention. In this example, type II alveolar epithelial cells were sorted by magnetic bead method, and cell purity, proliferation activity, surface marker expression, and 3D organoids were detected by flow cytometry. Its biological functions were identified by means such as culture and compared with those before sorting.

[0111] 1 material

[0112] 1.1 Main reagents

[0113] Molecular water (ddH2O), NucleoZOL, and Gelatin ...

Embodiment 3

[0150] In vitro and in vivo experiments to study the differentiation potential of mouse type Ⅱ alveolar epithelial cells imPAC2

[0151] 1 material

[0152] 1.1 Main reagents

[0153] FBS (fetal bovine serum) and DMEM medium were purchased from Gibco Company in the United States, molecular water (ddH2O), Gelatin (gelatin), NucleoZOL, penicillin / streptomycin were purchased from Invitrogen Company in the United States, 7.5M ammonium acetate, PC-8, no Water ethanol, Trypsin (trypsin), methanol, hematoxylin, eosin, mounting resin, Glycogen were purchased from Thermo Fisher Scientific, USA, 5xPCR Buffer, 10xPCR Buffer, dNTP were purchased from NEB, USA, and SYBR Taq was purchased from Bio-Bad, USA , MMLV reverse transcriptase was purchased from New England Biolabs, USA, D-Lueiferin was purchased from GBT Gold Biotechnology, USA, PPCN was purchased from Northwestern University Biomaterials Laboratory, and antibodies were purchased from Santa Cruz Company of USA.

[0154] 1.2 Prepa...

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Abstract

The invention belongs to the technical field of cell engineering, and particularly discloses a reversible immortalized II-type alveolar epithelial cell as well as construction and application thereof. According to the invention, a reversible immortalized alveolar cell strain imPACs is constructed through an SSR # 41 immortalized tool plasmid and a retrovirus system, reversible immortalized mouse type II alveolar epithelial cells are further sorted, and a foundation is laid for knowing the function and action mechanism of the type II alveolar cells. And a better cell model is provided for research on diagnosis and treatment of lung-related diseases. Furthermore, an imPAC2 cell strain is intervened through in-vivo and in-vitro adenovirus Ad-TGF-beta1, an imPAC2 cell line (imPAC2-Si-beta-Catenin) for stable low expression of beta-Catenin is constructed, and an imPAC2 cell line (imPAC2-KramP53) for stable overexpression of Kras and R273H site mutation of p53 of pSEBI-Kras and pSEBI-p53R273H is also constructed, so that a good cell tool is provided for related research of pulmonary fibrosis, tumors and the like.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a reversible immortalized type II alveolar epithelial cell and its construction and application. Background technique [0002] Alveolar is the key part of pulmonary gas exchange function, mainly composed of type I alveolar epithelial cells and type II alveolar epithelial cells, of which progenitor cells are mainly derived from type II alveolar epithelial cells. Abnormal function and respiratory distress syndrome, idiopathic pulmonary fibrosis are closely related. In response to pneumonia or injury, in addition to immune cells such as T cells, mast cells, eosinophils, dendritic cells, macrophages, etc., which play an anti-inflammatory role, alveolar epithelial cells are also considered to be able to secrete various Cytokines directly act on the inflammatory or damaged parts, thus exerting a huge self-protection mechanism in the body. With the progress of regenerative me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/65C12N15/37C12N5/10C12N15/861C12N15/12
CPCC12N15/86C12N15/65C07K14/005C12N5/0688C07K14/495C07K14/4746C07K14/82C07K14/47C12N2740/10043C12N2800/107C12N2710/20022C12N2510/04C12N2710/10343
Inventor 张龄幻罗文萍何通川
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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