Application of immortalized human lung microvascular endothelial cell complex cell factor in preparing biological agent for repairing ischemic tissue injury

A technology of endothelial cells and cytokines, used in cardiovascular system diseases, skin diseases, drug combinations, etc., to achieve obvious tissue regeneration, promote blood vessel regeneration, and regulate immune response.

Active Publication Date: 2014-09-10
三和(南京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] There is no report on the application of immortalized human

Method used

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  • Application of immortalized human lung microvascular endothelial cell complex cell factor in preparing biological agent for repairing ischemic tissue injury
  • Application of immortalized human lung microvascular endothelial cell complex cell factor in preparing biological agent for repairing ischemic tissue injury
  • Application of immortalized human lung microvascular endothelial cell complex cell factor in preparing biological agent for repairing ischemic tissue injury

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of composite cytokines containing immortalized human pulmonary microvascular endothelial cells

[0038] Immortalized human pulmonary microvascular endothelial cells were isolated from vascular endothelial tissue of normal human lung tissue, and co-transfected with a plasmid containing telomerase catalytic component (hTERT) and SV40LTag to prepare an immortalized human pulmonary microvascular endothelial cell line (HPMEC, PromoCell , Germany). After resuscitation, the cells were inoculated in endothelial cell growth medium (Lonza, Swiss) for culture, and passaged when the cell density reached 80-90%, and cytokines were collected when the cells reached the third passage. figure 1 Morphology when cytokines were collected for third-generation immortalized human pulmonary microvascular endothelial cells, and the cell density reached 100%.

[0039] The preparation method of immortalized human pulmonary microvascular endothelial cell compound cytokine ...

Embodiment 2

[0046] Example 2 Detection of the effects of normoxia and hypoxia on the expression of cytokines in human pulmonary microvascular endothelial cells

[0047] During the culture, the cells were divided into triplicates. One part was cultured according to normal oxygen concentration; one part was cultured with anaerobic culture tank (Mitsubishi, Japan) for 24 hours; one part was cultured with anaerobic culture tank (Mitsubishi, Japan) for 48 hours. The oxygen concentration in the anaerobic culture tank was 1%.

[0048]After collecting cytokines from the above three cells, the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration of vascular endothelial growth factor (VEGF) in the sample, and avidin-biotin-horseradish peroxidase was used to detect the concentration of vascular endothelial growth factor (VEGF). The system performs color development, and finally detects the VEGF content among different samples by a microplate read...

Embodiment 3

[0051] Example 3 Detection of the therapeutic effect of immortalized human pulmonary microvascular endothelial cells combined with cytokines in a full-thickness skin defect model

[0052] Sprague Dawley rats were used to prepare the skin defect model, namely ( Figure 4 ) in a1, the prepared immortalized human pulmonary microvascular endothelial cell cytokines and hyaluronic acid were mixed and spread on the wound surface, and the adhesive was used to prevent the loss of colloidal fluid. The control group only applied hyaluronic acid on the wound. After 14 days of photographic observation, from Figure 4 It can be seen that the experimental group is significantly better than the control group.

[0053] Conclusion: Controlled studies have shown that cytokines in immortalized human pulmonary microvascular endothelial cells can promote the recovery of wound healing.

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Abstract

The invention provides an application of an immortalized human lung microvascular endothelial cell complex cell factor in preparing a biological agent for repairing tissue skin full-thickness defects and preparing a biological agent for treating ischemic hypoxic tissue injury. The complex cell factor is a cell factor secreted by a low-oxygen cultured immortalized human lung microvascular endothelial cell, and does not contain a hereditary material (DNA or RNA). According to the application, the immortalized human lung microvascular endothelial cell complex cell factor promotes the formation of a local blood vessel by being locally applied, achieves obvious effect and plays an obvious tissue regeneration role on injury diseases, namely skin tissue defect and the like; the cell complex cell factor has multiple effective components capable of promoting angiogenesis, can promote the revascularization and can be used for repairing the injured tissues, accelerating the growth and healing of the injured tissues, regulating the immune reaction, and the like.

Description

technical field [0001] The invention relates to a biological preparation containing compound cytokines of immortalized human lung microvascular endothelial cells, a therapeutic preparation for treating full-thickness skin damage, and a biological preparation for repairing tissue hypoxic damage. Background technique [0002] Immortalized human pulmonary microvascular endothelial cells are cells derived from human lung microvascular endothelial cells, which have the characteristics of immortalization and can secrete a large amount of cytokines related to angiogenesis. The cells were simultaneously transformed with a plasmid containing the catalytic component of telomerase and the simian virus 40 large T antigen. This kind of cells can be cultured in vitro for more than 24 months and has very strong viability. In addition, the cells were not tumorigenic in nude mouse transplantation assays. (Generation of Human Pulmonary Microvascular Endothelial Cell Lines) [0003] Human p...

Claims

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Application Information

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IPC IPC(8): A61K35/44A61K47/36A61P17/00A61P9/10A61P17/02
Inventor 徐妍郭世磊周炳荣
Owner 三和(南京)生物科技有限公司
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