Construction of megakaryocyte model and cell bank of megakaryocyte by importing of hTERT recon

Abstract

The invention relates to construction a megakaryocyte model and a cell bank of megakaryocyte by importing of an hTERT recon in the field of medicine. The method is mainly characterized by comprising the following steps: carrying out double enzyme digestion on a plasmid pCIneo-hTERT and a carrier pLXSNneo by incision enzyme EcoR I and Xho I; connecting a PCR amplified and gel electrophoresis separated hTERT and pLXSNneo enzyme-digested product by Ligation Mix; constructing a pLXSNnei-hTERT recon; converting a DH5a competent cell to purify, amplify and pick an ammonium-tolerant benzyl penicillin colony to extract a plasmid, carrying out in-vitro passaging on the logarithmically grown megakaryocyte by lipofection transfection to ensure that the recon is integrated with the DNA of the cell; carrying out enlarge cultivation on the G418 screened cell containing a positive recon to clone, and screening a cell of which the cellular morphology, growth curve, karyotype, nude mouse tumorigenesis experiment, transfection cell telomerase activity, hTERT mRNA expression, immunohistochemical, cell generation cycle and apoptosis rate accord with the immortalized cell characteristics and are same or similar to those of a primary cell as an hTERT-mediated in-vitro study megakaryocyte model which is cryopreserved in liquid nitrogen, so as to lay a foundation for the long-term research of the pathogenesis of related diseases in vitro from the cell level.

Description

technical field [0001] The invention relates to the introduction of hTERT (telomerase reverse transcriptase catalytic subunit) recombinants or hTERT-mediated construction of megakaryocyte models and cell banks, mainly used in the field of blood disease research, for the development of megakaryocytes or various related This research provides cell models and preserves its scientific research resources. Background technique [0002] Megakaryocytes are mature cells in normal bone marrow that produce platelets. The cells are huge, and the edges of mature megakaryocytes rupture and fall off to form platelets. According to the development process, megakaryocytes are divided into (1) Primordial megakaryocytes: large cell body, 15-30 μm in diameter, round or irregular. The nuclei are large, round, and irregular. The chromatin is thick and reticular, closely arranged, and there are 2 to 3 nucleoli. Cell mass less, uneven, irregular edge, stained dark blue, no granular, pale perinuc...

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Problems solved by technology

[0008] However, recent studies have found that SV40Tag transfected cells are accompanied by loss of DNA damage repair mechanism, karyotype instability and tumorigenic transformation of a small number of cells

In addition, long-term in vitro culture found that some cells transfected with SV40Tag only prolong their lifespan, or only pass through the senescence period (MI phase), and most of the cells will eventually enter the crisis phase (M2 phase) and die, indicating that the cells have not obtained immortality

At the same time, SV40 virus is tumorigenic and is related to the occurrence and development of certain tumors. Therefore, the cells transfected with SV40Tag also have certain limitations for some researches.