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Method for preparing immortalized posterior longitudinal ligament cells of patient with posterior longitudinal ligament ossification

A technology of posterior longitudinal ligament and immortalization, applied in the field of genetic engineering, can solve problems such as limited cell number, loss of proliferation ability, and decreased cell viability, and achieve the effect of simplifying the research process

Pending Publication Date: 2021-08-31
SHANGHAI CHANGZHENG HOSPITAL
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

In addition, after 10-15 generations of cell culture, the posterior longitudinal ligament cells basically lose their proliferation ability, and the decline in cell viability limits the number of available cells in the research process.

Method used

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  • Method for preparing immortalized posterior longitudinal ligament cells of patient with posterior longitudinal ligament ossification
  • Method for preparing immortalized posterior longitudinal ligament cells of patient with posterior longitudinal ligament ossification
  • Method for preparing immortalized posterior longitudinal ligament cells of patient with posterior longitudinal ligament ossification

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Embodiment

[0035] Such as Figure 1-5 As shown, the embodiment of the present invention provides a method for preparing immortalized posterior longitudinal ligament cells from patients with ossification of the posterior longitudinal ligament, the method comprising the following steps:

[0036] S1. Construction of longitudinal ligament cells after immortalization;

[0037] 1) Determination of SV40Tantigen gene and HygromycinB resistance gene;

[0038] 2) Clone the overexpressed SV40Tantige lentiviral plasmid and carry HygromycinB resistance;

[0039] Construction of SV40 overexpression lentiviral vector;

[0040] The SV40 and HygromycinB genes were respectively cloned by PCR, HygromycinB was connected to SV40 by T2A, and constructed into a lentiviral expression vector; the primers used in the PCR process were:

[0041] SV40-F(TTTTCCTACAGATCCTTAATTAAgatggataaagttttaaacagagaggaa);

[0042] SV40-R(CTCGACGTCACCGCATGTTAGCAGACTTCCTCTGCCCTCtgtttcaggttcagggggagg);

[0043] Hygromycin B-F (TG...

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Abstract

The invention provides a method for preparing immortalized posterior longitudinal ligament cells of a patient with posterior longitudinal ligament ossification, and relates to the technical field of gene engineering. The method for preparing the immortalized posterior longitudinal ligament cells of the patient with the posterior longitudinal ligament ossification comprises the following steps: S1, constructing the immortalized posterior longitudinal ligament cells; (1) determining an SV40Tant i gen gene and a Hygromyc i nB resistance gene; (2) cloning overexpressing SV40Tant i ge lentivirus plasmids, and carrying Hygromyc i nB resistance; and (3) packaging the lentivirus. According to the invention, the establishment of the immortalized posterior ligament cell line can simplify the research process, provides sufficient research cells with stable characters for research, and is widely applied to the research of human diseases, the immortalized posterior ligament cell line is successfully and efficiently established by constructing overexpression lentiviral plasmids together with the SV40Tant i gen gene and the Hygromyc i nB resistance gene, and the cell line is used for researching pathogenesis of ossification diseases of posterior longitudinal ligaments.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for preparing immortalized posterior longitudinal ligament cells from patients with posterior longitudinal ligament ossification. Background technique [0002] Ossification of the posterior longitudinal ligament is a common heterotopic ossification of the spinal ligament, with a prevalence of 1.9%–4.3%. Epidemiological surveys have found that OPLL mainly occurs in the cervical and thoracic vertebrae. Once the corresponding spinal cord function is damaged, the resulting neurological dysfunction is often extensive and has a serious impact on the quality of life and even survival time of patients. Relevant etiological studies have shown that age, obesity, diet, diabetes, mechanical stimulation caused by exercise or abnormal activities of the spine, and certain endocrine diseases can increase the risk of OPLL, indicating that there are many possible pathogenesis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/65C12N15/37C12N5/10C12N5/071
CPCC12N15/86C12N15/65C07K14/005C12N5/066C12N2740/15043C12N2800/107C12N2710/20022C12N2510/04C12N2509/10C12N2509/00
Inventor 徐辰张子凡刘洋沈晓龙刘宁张子程袁文
Owner SHANGHAI CHANGZHENG HOSPITAL
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