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Urine-derived stem cell preparation, preparation method thereof and application thereof in preparation of medicines for resisting acute immune rejection after organ transplantation

A stem cell preparation and immune rejection technology, applied in the field of biomedicine, can solve the problems of insufficient identification schemes, different methods of obtaining urine-derived stem cells, and difficulty in standardization, and achieve the effect of reducing immune rejection damage and strong operability

Pending Publication Date: 2019-07-26
THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] However, whether urine-derived stem cells have the ability to participate in the regulation of acute immune rejection after organ transplantation is still unknown, and the methods of obtaining urine-derived stem cells are different, and the identification scheme is not comprehensive enough, and it is difficult to standardize, which limits its application to a certain extent

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  • Urine-derived stem cell preparation, preparation method thereof and application thereof in preparation of medicines for resisting acute immune rejection after organ transplantation
  • Urine-derived stem cell preparation, preparation method thereof and application thereof in preparation of medicines for resisting acute immune rejection after organ transplantation
  • Urine-derived stem cell preparation, preparation method thereof and application thereof in preparation of medicines for resisting acute immune rejection after organ transplantation

Examples

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Effect test

Embodiment 1

[0123] Example 1 Preparation and Detection of Urine-derived Stem Cell Preparation

[0124] 1. The acquisition of the urine-derived stem cell preparation:

[0125] 1.1 The separation method of urine-derived stem cells is as follows:

[0126] 1) Take 150-200mL of clean middle urine from healthy volunteers;

[0127] 2) Divide the urine into 50mL centrifuge tubes and centrifuge at 400g for 10min;

[0128] 3) Use an aspirator to slowly suck off the supernatant, leaving about 3-5mL of urine;

[0129] 4) Add about 20-30mL of phosphate-buffered saline (PBS) containing penicillin-streptomycin mixture, mix gently by pipetting, and centrifuge again at 400g for 10min;

[0130] 5) Use an aspirator to slowly suck off the supernatant until the remaining liquid is less than 1mL;

[0131] 6) Add 1 mL of specific urine-derived stem cell medium, resuspend the remaining pellet, and gently pipette several times;

[0132] 7) The above-mentioned cell suspension was evenly inoculated in a 24-wel...

Embodiment 2

[0204] Example 2 Animal Experiments Using Urine-derived Stem Cell Preparations for Immune Rejection After Kidney Transplantation

[0205] 2.1 Establishment of acute rejection animal model

[0206] A model in which Brown Norway rats (200g) were used as donors and Lewis rats (300g) were used as recipients was used to ensure that the weights of the rats were as uniform as possible. The donor adopts a "ten" incision on the abdomen, and frees the fat tissue around the kidney. Perfuse 10mL of pre-cooled heparin sodium hyperosmotic citrate-purine mixture through the artery at a uniform speed. It can be seen that the donor kidney turns yellowish brown without mottled surface, and the perfusate flowing out of the renal vein is clear. The donor kidney was taken out, trimmed on ice, and preserved in a mixture of ice and water. The recipient adopts abdominal midline incision, free renal blood vessels and abdominal aorta, upper and lower arterial clamps on the exposed abdominal aorta, cu...

Embodiment 3

[0234] Example 3 Animal Experiments Using Urine-derived Stem Cell Preparations for Immune Rejection After Heart Transplantation

[0235] 3.1 Establishment of acute rejection model of heterotopic heart transplantation in mice

[0236] The heart of the balb / c strain mouse was transplanted heterotopically to the neck of the C57BL / 6J strain mouse. The limbs of the donor mice were fixedly shaved, sterilized with 75% alcohol, opened to expose the inferior vena cava, injected 1ml of 20u / ml heparin saline, transected the inferior vena cava and aorta, and transected the diaphragm, from the bottom to the top of the bilateral clavicle midline Until the clavicle was cut; the heart was stopped with 4°C normal saline, and the whole heart was trimmed and stored in 4°C normal saline. Recipient mice were cut an oblique incision about 1 cm in length from the midpoint of the mandible to the midpoint of the right clavicle, the subcutaneous tissue and the external jugular vein were freed, and the...

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Abstract

The invention relates to a urine-derived stem cell preparation and a preparation method thereof, and an application of the preparation in preparation of medicines for resisting immune rejection reaction after organ transplantation. The method comprises the steps: selecting clean middle section urine of healthy volunteers; carrying out separated culture and in-vitro amplification by using a culturemedium, and detecting the in-vitro proliferation capacity; detecting the expression level of surface molecular markers by flow cytometry, and detecting the expression level of kidney-associated proteins by immunoblotting; take the third-sixth generations of urinary stem cells identified, meeting the requirements and with confluence of 80% in logarithmic growth phase; preparing the urine-derived stem cell preparation by further digestion, centrifugation and physiological saline resuspension. The urine-derived stem cell preparation has the advantages of wide source, low acquisition cost and high separation efficiency. The urine-derived stem cell preparation can remarkably prolong the survival time of rats after kidney transplantation, reduce the levels of urea nitrogen and creatinine and reduce the infiltration of T cells in kidney tissues; meanwhile, the beating time of a transplant in the mouse heart transplantation can be obviously prolonged.

Description

[0001] 【Technical field】 [0002] The invention belongs to the technical field of biomedicine, and specifically relates to a urine-derived stem cell preparation and a preparation method thereof, and the application of the preparation as an anti-immune rejection drug after organ transplantation, especially the application of the preparation anti-immune rejection drug after kidney transplantation and heart transplantation. [0003] 【Background technique】 [0004] How to reduce the loss of graft function caused by immune rejection is the key to ensure the clinical effect of organ transplantation. Although with the development and use of new immunosuppressants, the patient's immune system is suppressed, and the rejection level is greatly reduced, but because the immunosuppressive drugs themselves have liver and kidney toxicity, and have large side effects, the patient's compliance is poor; and because the immune system is suppressed, It is easy to cause infection, tumor and other e...

Claims

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Application Information

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IPC IPC(8): A61K35/28A61P37/06C12N5/0775
CPCA61K35/28A61K9/0019A61P37/06C12N5/0668C12N2509/00Y02A50/30
Inventor 王长希刘龙山李希芮毕梓荣韦勇成
Owner THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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