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Medicinal preparation for resisting intracellular mycoplasma infection and application thereof

A technology for mycoplasma infection and pharmaceutical preparations, applied in the field of medicine, can solve the problems of cell recontamination, pollution, cell damage, etc., and achieve the effect of no loss of antibacterial effect, low toxicity and drug resistance, and small cell damage

Pending Publication Date: 2022-01-28
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the original drug of sparfloxacin is insoluble in water, only soluble in dimethyl sulfoxide, and dimethyl sulfoxide has a certain damage effect on cells, especially for cells infected with mycoplasma (the cell state is poor at this time). Strong death-inducing effect, cannot be directly used in cell culture occasions
In addition, it is not enough to remove mycoplasma in the public cell culture environment, and the cells may face recontamination or mycoplasma contamination of new cells

Method used

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  • Medicinal preparation for resisting intracellular mycoplasma infection and application thereof
  • Medicinal preparation for resisting intracellular mycoplasma infection and application thereof
  • Medicinal preparation for resisting intracellular mycoplasma infection and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Adopt alkaline solution dissolution method to prepare sparfloxacin anti-mycoplasma intracellulare preparation, the steps are as follows: 4g sodium hydroxide (NaOH) is dissolved in 800ml ultrapure water, after fully dissolving, be settled to 1L, obtain concentration and be 0.1mol / l NaOH solution can be stored at room temperature, protected from light, and sealed for 6 months. NaOH is corrosive, and gloves are required for operation. Dissolve sparfloxacin technical powder fully in 0.1mol / l NaOH solution to obtain sparfloxacin anti-mycoplasma intracellulare preparation with a final concentration of 10 mg / ml, which is filtered through 0.45 μm and 0.22 μm filters successively and stored. Pharmaceutical preparations can be stored at -20° or -80° for long-term storage, and short-term (within 1 month) at 4° and protected from light to avoid repeated freezing and thawing.

Embodiment 2

[0030] Verify that the sparfloxacin anti-intracellular mycoplasma preparation prepared in Example 1 has a clearing effect on intracellular mycoplasma, the steps are as follows: remove the original cell culture medium, replace it with a fresh medium after 3 times with PBS, and replace the above-mentioned sparzafloxacin Star anti-mycoplasma intracellular preparation was added to the culture medium at a ratio of 1:1000 (1 μl sparfloxacin anti-mycoplasma intracellular preparation was dissolved in 1 ml medium); the medium was changed every 1-2 days and continued to be used for 7 days.

[0031] In order to detect the ability of sparfloxacin anti-mycoplasma intracellular preparations to remove mycoplasma in cells, according to the document "Detection of Mycoplasma in cell cultures" (Nat Protoc.2010,5(5):929-34.doi:10.1038 / nprot. 2010.43) method application PCR is detected, and the method is as follows:

[0032] (1) Collect the culture medium of mycoplasma-contaminated cells and the c...

Embodiment 3

[0044] Verify that the sparfloxacin anti-mycoplasma intracellular preparation prepared in Example 1 has the effect on cytotoxicity, the steps are as follows: in the cell culture medium according to 1:1000 (1 μ l sparfloxacin anti-mycoplasma intracellular preparation is dissolved in 1 ml culture medium) Add the above-mentioned sparfloxacin anti-mycoplasma intracellular preparation, then pass the cells according to routine operations, and use CCK-8 to observe the cell viability and growth within 14 days. The results are as follows: figure 2 As shown, the growth status of A549 cells within 14 days was not affected.

[0045] Test the impact of different concentrations of sparfloxacin anti-mycoplasma intracellular preparations on short-term cell survival, the steps are as follows: in the cell culture medium according to 1:1000 (1 μl sparfloxacin anti-mycoplasma intracellular preparations are dissolved in 1ml culture medium), 1:500, 1:200, 1:100, 1:50 and 1:25 were added to the abo...

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Abstract

The invention discloses a medicinal preparation for resisting intracellular mycoplasma infection and an application thereof, and belongs to the technical field of medicines. The pharmaceutical preparation is a sparfloxacin solution with the final concentration of 10 mg / ml, is obtained by dissolving sparfloxacin in a 0.1 mol / l NaOH solution and sequentially filtering through a 0.45 [mu]m filter and a 0.22 mu m filter, is used for preventing cells from infecting mycoplasma and removing the mycoplasma infecting the cells during cell culture, and is continuously used for 7 days every 1-2 days according to the effective use dosage of 0.01-0.1 mg sparfloxacin / ml cell culture medium. The sparfloxacin active compound is dissolved in the NaOH solution to prepare the solution preparation by adopting an alkali solution dissolution method, the solution preparation can replace mycillin to be used for preventing cells from infecting mycoplasmas and removing the mycoplasmas infecting the cells during cell culture, the antibacterial effect is good, toxicity and drug resistance are low, damage to the cells is small while the mycoplasmas are effectively killed, and the mycoplasma can be prevented from polluting the cells again.

Description

technical field [0001] The invention belongs to the technical field of medicine, and specifically relates to a pharmaceutical preparation for resisting intracellular mycoplasma infection and its application, which is used for preventing cells from being infected with mycoplasma and removing mycoplasma from infected cells during cell culture. Background technique [0002] Mycoplasma is the most common microorganism that contaminates / infects cells in cell culture. Studies have shown that up to 35% of cells in laboratories around the world may have mycoplasma contamination, even if the most recognized American type culture collection (American type culture) collection, ATCC) cells, there are also reports of mycoplasma contamination in some cell lines. The harm of mycoplasma-contaminated cells is that it can lead to changes in cell physiology, gene expression, and drug response, resulting in instability and irreproducibility of experimental results. At the same time, mycoplasma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0693C12N5/0688C12N2500/30
Inventor 贺斌峰张静吴奕星
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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