Kidney transplant donor-specific urine source cell and its DNA preparation method and application thereof

A specific technology for kidney transplantation, which is applied in the field of biomedicine, can solve the problems of obtaining six-site or eight-site genotype information, the lack of storage time of donor DNA samples, and the inability to accurately determine donor HLA information, etc., to achieve the goal of obtaining The method is completely non-invasive, the acquisition method is safe and non-invasive, and the effect of improving the success rate of sequencing

Pending Publication Date: 2019-10-08
北京博富瑞基因诊断技术有限公司
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] However, HLA high-resolution typing technology has only started to be used in clinical organ transplantation in recent years. In the early years, only low-resolution HLA genotypes were detected; to determine the DSA locus, it was necessary to obtain donor DNA for HLA high-resolution typing testing again. Most of the donor DNA samples cannot meet the testing conditions due to missing or too long storage time
At this time, donor DNA samples need to be obtained again. Some people use transplanted kidney puncture tissue samples to separate donor DNA, resulting in trauma to the transplanted kidney tissue, increasing the risk of bleeding, and wasting precious tissue samples. low success rate
It has also been reported to use urine sediment for HLA single locus screening. However, due to the low content of donor DNA in urine sediment, the method used can only obtain part of the serotype information, because some genotypes have no corresponding serotype typing, so it is difficult to pass This method obtains the donor's clear six-position or eight-position genotype information; at the same time, the results are affected by a large number of recipients' own urethral exfoliated cell DNA, so it is impossible to accurately determine the complete donor HLA information, and at the same time, it is impossible to discover new genotypes [9]

Method used

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  • Kidney transplant donor-specific urine source cell and its DNA preparation method and application thereof
  • Kidney transplant donor-specific urine source cell and its DNA preparation method and application thereof
  • Kidney transplant donor-specific urine source cell and its DNA preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preparation of donor-specific urine-derived cells

[0050] 1.1 The separation and preparation of donor-specific urine-derived cells, the method is as follows:

[0051] 1) Take 150-200mL of clean middle urine from the patient (recipient);

[0052] 2) Divide the urine into 50mL centrifuge tubes and centrifuge at 400g for 10min;

[0053] 3) Use an aspirator to slowly suck off the supernatant, leaving about 3-5mL of urine;

[0054] 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;

[0055] 5) Use an aspirator to slowly suck off the supernatant until the remaining liquid is less than 1mL;

[0056] 6) Add 1 mL of urine-derived cell culture medium, resuspend the remaining precipitate, and gently pipette several times;

[0057] 7) The above-mentioned cell suspension was uniformly inoculated in a 24-well plate coated with 0.1% gelatin solution (Gelatin...

Embodiment 2

[0080] Example 2 Using donor-specific urine-derived cells to detect donor HLA high-resolution typing

[0081] 2.1 Extraction of genomic DNA from donor-specific urine-derived cells (using Tiangen-genomic DNA Kit)

[0082] 1) After the cells proliferate to 1*10 6 Digest each cell with 0.25% EDTA trypsin at 37°C for about 3-5min;

[0083] 2) Add an equal volume of basal medium containing 10% serum to stop the digestion, and gently blow the cells to make them fall off the bottom of the dish;

[0084]3) Transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 200g for 5min;

[0085] 4) Add 1ml of phosphate buffered saline (PBS), resuspend the cells, centrifuge at 10000rpm (~11200g) for 1min, pour off the supernatant, add 200ul of buffer A, shake until completely suspended;

[0086] 5) Add 4 ul RNase A (100 mg / ml), vortex for 15 s, and place at room temperature for 5 min; add 20 ul proteinase K (Proteinase K), and mix well.

[0087] 6) Add 200ul of buffer solutio...

Embodiment 3

[0107] Example 3 Application of donor-specific urine-derived cells to detect high-resolution typing of donor HLA after kidney transplantation in adult kidney transplantation

[0108] 3.1 Using the above methods to detect the results of HLA high-resolution typing after kidney transplantation in adults

[0109] Patient 007, female, 42 years old, donor age 26 years old, nine months after kidney transplantation. Table 3 shows the HLA high-resolution typing results of urine-derived cells and urine sediment after adult kidney transplantation detected in the examples of the present invention. It can be seen that the HLA high-resolution typing results of the recipient itself are consistent with the sequencing results of the recipient's urine sediment. The HLA sequencing results of the recipient's urine-separated cells were consistent with the donor's HLA high-resolution typing results. This method is suitable for adult kidney transplant recipients, and can obtain donor-specific HLA h...

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Abstract

The invention relates to a kidney transplant donor-specific urine source cell and its DNA preparation method, and an application in kidney transplant donor genomics background analysis. The preparation method of the urine source cell of the invention comprises the following steps: (1) taking a middle section of urine of a kidney transplant acceptor, centrifuging the material, and discarding a supernatant; (2) adding a phosphate buffer solution containing a penicillin streptomycin mixture or a primary cell antibiotic to a lower layer obtained by centrifugation, performing resuspending, and thencentrifuging the material again, discarding the supernatant; (3) adding a urine source cell culture medium to a lower layer precipitate obtained by centrifugation, and performing resuspending to obtain a cell suspension; and (4) inoculating the obtained cell suspension into a culture vessel, and performing the cell amplification in vitro to obtain the kidney-transplant donor-specific urine sourcecell. The method obtains the donor-specific urine source cell based on kidney transplant postoperative acceptor urine, obtains donor DNA, performs donor genomics background analysis, is safe, non-invasive, low-cost, and highly efficient for separation, and is easy to obtain a sufficient amount of DNA.

Description

technical field [0001] The invention relates to a method for preparing kidney transplant donor-specific urine-derived cells and DNA thereof and its application in the background analysis of kidney transplant donor genomics (such as HLA high-resolution typing detection, etc.), which belongs to biomedical technology field. Background technique [0002] Kidney transplantation, as the optimal treatment for end-stage renal disease, is widely used clinically. However, the recipient's immune response to the graft after transplantation may cause a variety of adverse prognosis including acute and chronic immune rejection, graft failure, etc., seriously affecting the transplantation effect [1] . A clear donor genomics background is of great significance for the prevention and treatment of various adverse events after transplantation. In the context of genomics, the genotype background of donor human leukocyte antigen (HLA) had the greatest impact on postoperative adverse events. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N15/10C12Q1/6869
CPCC12N5/0686C12N15/101C12Q1/6869C12Q2535/122C12Q2527/107C12Q2521/301
Inventor 王长希刘龙山李希芮韦勇成苏晓均
Owner 北京博富瑞基因诊断技术有限公司
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