WO2020258637A1 - 肾移植供体特异性尿源细胞及其dna的制备方法以及其应用 - Google Patents

肾移植供体特异性尿源细胞及其dna的制备方法以及其应用 Download PDF

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WO2020258637A1
WO2020258637A1 PCT/CN2019/115518 CN2019115518W WO2020258637A1 WO 2020258637 A1 WO2020258637 A1 WO 2020258637A1 CN 2019115518 W CN2019115518 W CN 2019115518W WO 2020258637 A1 WO2020258637 A1 WO 2020258637A1
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donor
urine
kidney transplant
hla
dna
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French (fr)
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王长希
刘龙山
李希芮
韦勇成
苏晓均
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中山大学附属第一医院
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
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  • the invention relates to a method for preparing kidney transplantation donor-specific urine-derived cells and their DNA and its application in kidney transplantation donor genomics background analysis (such as HLA high-resolution typing detection, etc.), and belongs to biomedical technology field.
  • Kidney transplantation as the optimal treatment plan for end-stage renal disease, is widely used clinically.
  • the immune response of the recipient to the graft after transplantation may cause a variety of adverse prognosis including acute and chronic immune rejection, graft failure, etc., which seriously affects the transplant effect [1] .
  • a clear donor genomics background is of great significance for the prevention and treatment of various adverse events after transplantation.
  • the genotype background of the donor human leukocyte antigen (HLA) has the greatest influence on the adverse events after surgery.
  • HLA Human leukocyte antigen
  • the results of HLA high-resolution typing include the genotype information of three types of HLA (HLA-A, HLA-B, HLA-C) and three types of HLA (HLA-DR, HLA-DQ, HLA-DP).
  • HLA-A genotype information of three types of HLA
  • HLA-B HLA-C
  • HLA-DR three types of HLA
  • HLA-DQ HLA-DP
  • donor-specific epitopes can be obtained. Compared with the traditional serological response mode, this screening will provide a more scientific and usable matching system, which greatly improves the accuracy of matching, thereby improving postoperative graft function and patient quality of life [5-8] .
  • Donor specific antibody (DSA) mediated rejection is the primary risk factor for long-term graft failure.
  • DSA body-mediated rejection
  • AMR antibody-mediated rejection
  • PRA panel reactive antibodies
  • HLA high-resolution typing technology has only been used in organ transplantation clinics in recent years. In the early years, only low-resolution HLA genotypes were detected. To determine the DSA locus, it is necessary to obtain donor DNA and perform HLA high-resolution typing again. Donor DNA samples are mostly missing or stored for too long and cannot meet the testing conditions. At this time, it is necessary to obtain a new donor DNA sample. Some people use a transplant kidney puncture tissue sample to isolate the donor DNA, which causes trauma to the transplanted kidney tissue, increases the risk of bleeding, wastes valuable tissue samples, and the amount of kidney tissue cells is small, leading to detection The success rate is not high. There are also reports using urine sediment for HLA single site screening.
  • the method used can only obtain partial serotype information, because some genotypes have no corresponding serotypes, so it is difficult to pass
  • This method obtains the donor's clear six-site or eight-site genotype information; at the same time, the results are affected by the DNA of a large number of urethral exfoliated cells of the recipient itself, and the complete donor HLA information cannot be accurately determined, and new genotypes cannot be found. [9] .
  • the purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide a safe and non-invasive method for preparing kidney transplant donor-specific urine-derived cells and their DNA.
  • a sufficient amount of DNA can be obtained to accurately perform Donor genomics background analysis, such as obtaining donor-specific HLA (human leukocyte antigen) high-resolution typing results.
  • HLA human leukocyte antigen
  • the present invention also provides the application of a sufficient amount of kidney transplant donor-specific DNA obtained by the above method in the background analysis of kidney transplant donor genomics, such as the detection method of HLA high-resolution typing.
  • the present invention provides a method for preparing kidney transplant donor-specific urine-derived cells, which includes the following steps:
  • step (2) Add a phosphate buffer containing penicillin or a mixture of primary cell antibiotics to the lower sediment obtained in step (1) for resuspension, then centrifuge again, and discard the supernatant;
  • step (3) Add urine-derived cell culture medium to the lower sediment obtained by centrifugation in step (2), and resuspend to obtain a cell suspension;
  • step (3) The cell suspension obtained in step (3) is inoculated into a culture container, and the cells are expanded in vitro to obtain kidney transplant donor-specific urine-derived cells.
  • Primary cell antibiotics can be purchased from Xinbosheng Biotechnology Company.
  • the present inventors discovered that the mid-section urine of the kidney transplant recipient is used as a cell source, and the kidney transplant donor-specific urine-derived cells are obtained from the kidney transplantation donor-specific urine cells by separation, purification and in vitro expansion.
  • Urine-derived cells are mainly derived from functional kidney tissue, so the source of urine-derived cells in patients after kidney transplantation is mainly the donor kidney. Because the recipient bladder ureteral epithelial cells shed with urine are mostly terminally differentiated cells, they cannot adhere to and proliferate under in vitro culture conditions, while donor-specific urine-derived cells can quickly adhere to the wall and proliferate rapidly. The signal of the receptor will be greatly reduced when the liquid and the expansion of the culture are carried out. In vitro amplification by the method of the present invention significantly expands the signal of donor-specific cells, and greatly reduces and dilutes the signal of recipient cells.
  • the cells can be expanded to the required number as required.
  • the urine-derived cell culture medium contains the following components: REGM medium, DMEM-high Glucose culture Base, non-essential amino acid cell culture supplement, GlutaMAX supplement and fetal bovine serum. Studies have shown that using the urine-derived cell culture medium to culture donor-specific urine-derived cells in vitro can ensure stable proliferation and quickly obtain the amount of DNA required for sequencing.
  • the non-essential amino acid cell culture supplement is NEAA (non-essential amino acids) cell culture supplement.
  • NEAA (non-essential amino acids) cell culture supplements are commercial reagents that can be purchased from Saiye Biotech.
  • the culture container is coated with a gelatin solution.
  • the method of cell expansion in vitro is: after the cell suspension is inoculated in a culture vessel, first Place it in an incubator for static culture, then replace with fresh urine-derived cell culture medium, continue culturing until the cell confluence reaches 60%-80%, and then use EDTA trypsin digestion and passage to expand the cells to the required number.
  • the present invention provides a method for preparing kidney transplant donor-specific urine-derived cell DNA, which includes the following steps:
  • kidney transplant donor-specific urine-derived cells with a number of not less than 1 ⁇ 10 6 ;
  • kidney transplant donor-specific urine-derived cell DNA When using the method of the present invention to prepare kidney transplant donor-specific urine-derived cell DNA, ensure that the number of kidney transplant donor-specific urine-derived cells in step (a) is not less than 1 ⁇ 10 6 to obtain a sufficient amount of high-purity DNA donor HLA sequencing detection.
  • the method for extracting DNA from kidney transplant donor-specific urine-derived cells includes the following steps:
  • step (b2) Add RNase A and proteinase K to the cell suspension obtained in step (b1) for treatment, then add absolute ethanol, shake and mix well, and flocculent precipitation appears;
  • step (b3) Add the solution and flocculent precipitate obtained in step (b2) to the adsorption column, centrifuge to remove the waste liquid, and clean the adsorption column at least once with a rinse solution, and then add the elution buffer to the adsorption membrane of the adsorption column The middle part is placed and centrifuged to collect the elution buffer solution to obtain the specific urine-derived cell DNA of the kidney transplant donor.
  • the present invention provides the application of kidney transplant donor-specific urine-derived cells or kidney transplant donor-specific urine-derived cell DNA prepared by the above-mentioned method in the background analysis of kidney transplantation donor genomics.
  • the genomics background analysis is HLA typing analysis.
  • the present invention obtains transplant donor-specific urine-derived cell DNA from the mid-section urine of a kidney transplant recipient, which can be used for donor genomics background analysis, such as HLA high-resolution sequencing analysis.
  • This method can avoid clinical invasive renal puncture to obtain the donor genome; guarantee the donor specificity of the scoring result of the test; can provide sufficient amount of DNA to ensure the feasibility of sequencing; it has low acquisition cost and accurate analysis
  • the high rate, the acquisition method is completely non-invasive and other advantages.
  • the present invention provides a method for detecting HLA typing of a kidney transplant donor, which includes the following steps: using the above method to prepare kidney transplant donor-specific urine-derived cell DNA, and detecting HLA typing in the obtained DNA to obtain a kidney transplant Donor HLA typing results.
  • the method for detecting HLA typing in the obtained DNA is: high-throughput sequencing is used to detect HLA high-resolution typing in the obtained DNA to obtain HLA high-resolution typing results of kidney transplantation donors.
  • the method for detecting HLA high-resolution typing in DNA obtained by high-throughput sequencing is: first perform HLA-A/B/C/ The DRB1/DQB1/DPB1 locus gene is amplified and the amplicon sequence is collected, and then modified by restriction enzyme digestion, each sample is added with "barcode" information, and then high-throughput sequencing is performed to analyze the data obtained from the sequencing to obtain a kidney transplant donor HLA high-resolution typing results.
  • the kit used for gene amplification of the HLA-A/B/C/DRB1/DQB1/DPB1 locus is NGSgo-AmpX kit (GenDx company); the kit used for the restriction digestion modification is NGSgo LibrX kit (GenDx company); the kit used for adding "barcode" information is the NGSgo-Indx kit (GenDx company).
  • the method of the present invention is based on donor-specific urine-derived cells and DNA obtained from recipient urine after kidney transplantation to perform donor genomics background analysis (taking HLA high-resolution typing detection as an example), and has an acquisition method It is safe, non-invasive, low cost, high separation efficiency, and easy to obtain a sufficient amount of DNA for sequencing analysis.
  • the method of the present invention provides a sufficient amount of DNA, thereby greatly improving the success rate of sequencing; and in the method of the present invention, because the urinary tract of the recipient collected in the urine
  • the exfoliated cells are terminally differentiated cells that cannot effectively adhere to the wall and proliferate in vitro, so the interference of the recipient's exfoliated cell DNA is greatly diluted. Therefore, the method of the present invention can provide a sufficient amount of donor-specific urine-derived cells for kidney transplant patients who cannot obtain high-resolution information of donor HLA, and obtain a sufficient amount of donor-specific DNA from them, which can be used for subsequent high-throughput sequencing. Finish laying the foundation.
  • the method of the present invention for genomics background analysis of kidney transplantation donors can be applied to a variety of populations, regardless of postoperative time, recipient age, renal function and other factors. Using this method to detect long-term surviving recipients after kidney transplantation, it is found that this type of recipient can still accurately detect the donor’s HLA information through this method; at the same time, using this method to test two recipients transplanted with the same donor kidney The results of HLA high-resolution typing are consistent, which further confirms the donor specificity of this method.
  • the present invention can provide more evidence for postoperative detection of donor-specific antibody (DSA) levels and clinical medication adjustment for patients who lack the high-resolution typing of donor HLA, which is of great application value.
  • DSA donor-specific antibody
  • the HLA high-resolution typing detection method of the present invention can be used in the confirmation of the donor-specific antibody (DSA) site of the population reactive antibody (PRA) positive patient, and the HLA high-resolution typing result is compared with the PRA result Yes, you can confirm the DSA site of the donor-specific antibody.
  • DSA donor-specific antibody
  • PRA population reactive antibody
  • Figure 1 is a flow chart of the method for detecting HLA typing of kidney transplant donors of the present invention
  • Figure 2 is a cell proliferation curve diagram of specific urine-derived cells from a kidney transplant donor in Example 1 of the present invention
  • Fig. 3 is a graph of DNA extraction concentration and purity of specific urine-derived cells from kidney transplantation donors in Example 2 of the present invention.
  • the present invention uses the donor-specific urine-derived cells for the detection method of kidney transplantation donor HLA typing as an example, and the process is shown in FIG. 1.
  • PBS phosphate buffered saline
  • PBS phosphate buffered saline
  • REGM medium stock solution (CC-4127, Lonza), DMEM-high Glucose medium (Hyclone, SH30022.01), non-essential amino acid additives (NEAA, 11140050, Gibco), GlutaMAX additives (Gibco, 35050061) and fetal bovine serum ( FBS, Gibco, 12664025) is configured according to specific proportions.
  • liquid A REGM medium stock solution
  • liquid B 90% DMEM-high Glucose basic medium + 10% fetal bovine serum + 1:100 non-essential amino acid additive + 1:100 GlutaMAX additive
  • Liquid A and Liquid B are mixed uniformly in a ratio of one to one, which is a specific medium for urine-derived cells.
  • Figure 2 is the cell proliferation curve test results of urine-derived cells in the example of the present invention; it can be seen that urine-derived cells maintain a relatively vigorous proliferation ability under in vitro culture conditions.
  • Table 1 is a statistical table of the proliferation ability of donor-specific urine-derived cells of kidney transplant recipients (numbered USC-KT001) in different generations (third generation P3, sixth generation P6) in the examples of the present invention. The results show that urine-derived cells can proliferate stably and rapidly in vitro, regardless of the P3 or P6 generation, and can quickly obtain the required cell volume for sequencing in a short period of time.
  • Table 1 Statistical table of cell proliferation ability of different generations (P3, 6) of donor-specific urine-derived cells of recipient KT-001 in the embodiment of the present invention
  • Example 2 Using donor-specific urine-derived cells to detect donor HLA high-resolution typing
  • Figure 3 shows the results of the spectrophotometric detection of DNA of two donor-specific urine-derived cells (numbered USC-KT033 and USC-KT035, respectively) in the embodiment of the present invention. It can be seen that 1*10 6 urine-derived cells can obtain sufficient and high-purity DNA for HLA sequencing detection.
  • Table 2 is the concentration and purity test table of two cases of urine-derived cell DNA (numbered USC-KT033 and USC-KT035 respectively) in the examples of the present invention.
  • A260/280 represents the ratio of the absorbance of the sample at 260 and 280nm, which represents the purity of the DNA sample, and the ratio of pure DNA is greater than 1.8;
  • A260/230 represents the ratio of the absorbance of the sample at 260 and 280nm, which is the detection
  • the second indicator of nucleic acid purity the value of 260/230 is larger than the value of 260/280, usually 1.8-2.2. The results show that both can obtain sufficient and high purity DNA for the next step of sequencing.
  • NGSgo-AmpX kit (GenDx company) to amplify HLA-A/B/C/DRB1/DQB1/DPB1 locus genes and collect amplicon sequences;
  • Table 3 shows the HLA high-resolution typing results of urine-derived cells and urine sediment after adult kidney transplantation 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 results of the recipient's urine sediment sequencing. The HLA sequencing results of isolated cells from the recipient urine are consistent with the HLA high-resolution typing results of the donor. This method is applicable to adult kidney transplant recipients and can obtain donor-specific HLA high-resolution typing results.
  • Table 4 shows the HLA high-resolution typing results of urine-derived cells after kidney transplantation in children detected in the examples of the present invention. It can be seen that the recipient's urine-derived cells are consistent with the donor's HLA high-resolution typing results, but are inconsistent with the recipient. This method is also applicable to children's donor kidneys.
  • Example 5 Use of donor-specific urine-derived cells to detect the application of high-resolution typing of donor HLA after kidney transplantation in long-term survival recipients after kidney transplantation
  • Table 5 shows the HLA high-resolution typing results of urine-derived cells of long-term surviving recipients after kidney transplantation detected in the examples of the present invention, showing that the HLA high-resolution typing of urine-derived cells is inconsistent with the recipient itself. This method can be seen Donor HLA information can still be measured in long-term surviving recipients after kidney transplantation.
  • DQB1*02 02.
  • DQA1*02 The 01 site is a possible DSA site. Further observation of the changes in the DSA site before and after the patient's treatment, and found that if the DSA site cannot be determined, the LSA report does not have an overall pattern. If the DSA site is confirmed, the change can be clearly seen.
  • Table 7 shows the HLA high-resolution typing results of unknown donors detected by urine-derived cells in the examples of the present invention.
  • the recipient's urine was used to successfully obtain donor-specific urine-derived cells, and the donor-specific HLA high-resolution typing results were successfully detected.
  • Table 8 shows the results of the detection of PRA by the liquid-phase chip single antigen microbead method (LSA) of patient 040. Comparing the high-resolution typing results of the donor HLA obtained in Table 9 with the LSA results of the recipient, infer the corresponding DSA sites, showing that DQB1*02: 02, DQA1*02: 01 sites are possible DSA sites . It can be seen that the recipient has produced antibodies that specifically attack the donor’s kidney. Combined with the pathological changes, it can be determined that the patient’s transplanted kidney dysfunction is due to antibody-mediated immune rejection. Therefore, drugs that specifically inhibit B cells, plasma exchange, and high-dose intravenous immunoglobulin therapy can be implemented.
  • LSA liquid-phase chip single antigen microbead method
  • HLA-II antibody positive (+) 15%
  • MFI calibration value range reference ⁇ 1000 negative; 1000-4000 weak positive; 4000-10000 positive; >10000 strong positive
  • the HLA sites detected by antibody include: Class I: HLA-A, B, C; Class II: HLA-DRB1, DQA1, DQB1, DPA1, DPB1
  • kidney transplantation After kidney transplantation, it is necessary to combine the results of the recipient population reactive antibody (PRA) and the donor HLA typing to determine the donor-specific antibody (DSA) to assist in the diagnosis and treatment of antibody-mediated rejection (AMR) .
  • PRA recipient population reactive antibody
  • DSA donor-specific antibody
  • AMR antibody-mediated rejection
  • the recipient Child1010, female was 7 years old; the donor was 1.8 years old, 3 months after kidney transplantation.
  • Table 9 shows the high-resolution typing of the same receptor HLA detected by urine-derived cells and urine separation sediment in the examples of the present invention. The results showed that the urine sediment sequencing failed, while the urine-derived cells were successfully sequenced, and the donor-specific HLA high-resolution typing results were accurately and specifically obtained. It can be seen that the recipient urine sediment sequencing has a failure rate due to the low specific DNA content; and this method can accurately obtain the donor-specific HLA high-resolution typing compared with the urine sediment sequencing.
  • Table 10 is a comparison of the method of the embodiment of the present invention, the urine separation sediment detection method and the donor kidney biopsy detection method, showing that the method used in the present invention can obtain donor-specific HLA high-resolution typing results conveniently, quickly and non-invasively.
  • the technology used in the present invention to obtain unknown donor kidney genomics background information after kidney transplantation has strong operability, convenience and non-invasiveness, safe process, and reliable results, so the patient acceptance is high ; Low cost and high separation efficiency, easy to obtain sufficient amount of DNA for sequencing analysis, greatly improving the success rate of sequencing, and diluting and reducing the influence of the DNA of the terminal epithelial cells of the recipient urinary system, and the obtained donor-specific HLA results are complete and accurate .
  • Comparing the results with the results of the recipient's population reactive antibody can determine whether the recipient produces a donor-specific antibody (DSA), and then guide the clinical kidney transplant recipient's DSA detection, AMR diagnosis, treatment effect evaluation and treatment plan Adjustment.
  • DSA donor-specific antibody

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Abstract

提供了一种肾移植供体特异性尿源细胞及其DNA的制备方法以及其在肾移植供体基因组学背景分析中的应用。该尿源细胞的制备方法包括以下步骤：(1)取肾移植受体的中段尿，离心，弃上清；(2)向离心所得下层沉淀中加入含有青链霉素混合液或原代细胞抗生素的磷酸盐缓冲液进行重悬，然后再次离心，弃上清；(3)向离心所得下层沉淀中加入尿源细胞培养基，重悬，得到细胞悬液；(4)将所得细胞悬液接种于培养容器中，进行细胞体外扩增，得到肾移植供体特异性尿源细胞。基于肾移植术后受体尿液获取的供体特异性尿源细胞，获得供体DNA，进行供体基因组学背景分析，易于获得足够数量的DNA。

Description

肾移植供体特异性尿源细胞及其DNA的制备方法以及其应用 技术领域
本发明涉及一种肾移植供体特异性尿源细胞及其DNA的制备方法及其在肾移植供体基因组学背景分析(如HLA高分辨率分型检测等)中的应用，属于生物医药技术领域。
背景技术
肾移植作为终末期肾脏疾病的最优治疗方案，被广泛应用于临床。然而，移植后受体对移植物产生的免疫反应可能造成包括急慢性免疫排斥、移植物失功等在内的多种不良预后，严重影响移植效果 [1]。而明确的供体基因组学背景，对预防及治疗各种移植术后不良事件的发生，有重要意义。而在基因组学背景中，以供体人白细胞抗原(HLA)的基因型背景对术后不良事件的影响最大。
人类白细胞抗原(human leukocyte antigen，HLA)是人类的主要组织相容性复合体，控制机体识别“自身”和“异己”的过程，参与异体抗原提呈，启动特异的免疫反应。然而在识别异己的同时，它也成为了异体器官移植的主要障碍，是决定移植排斥水平的重要因素。在器官移植时，供体和受体HLA相容程度越高，排斥水平越低，移植成功率和移植物长期存活率越高；反之，则容易发生排斥反应，移植物功能损伤越严重 [2]。因此，获取供受体双方的HLA分型，鉴定HLA的基因型构成，可明确供受体之间的HLA差异，进而科学的决定器官分配。
早期HLA的检测包含血清学检测，如淋巴细胞毒实验；随着分子技术的进步，逐渐过渡到基于多聚酶链式反应(polymerase chain reaction，PCR)的DNA序列分析分子诊断为主导，包含PCR序列特异引物(PCR-SSP)和PCR序列特异的寡核苷酸引物(PCR-SSO)方法 [3]。随着高通量测序技术的发展，采用高通量测序技术获取HLA基因高分辨率分型结果被认为是最可靠的标准。HLA高分辨率分型结果包含3种Ⅰ类HLA(HLA-A，HLA-B，HLA-C)和三种Ⅱ类HLA(HLA-DR，HLA-DQ，HLA-DP)的基因型信息。这一技术推动了器官移植供 受者选择系统的发展，使两者的匹配达到更为精准的层面 [4]。
通过对供体特异性的HLA高分辨率分型结果分析，可以获取供体特异性的抗原表位(epitope)。这种筛选相对于传统血清学反应模式，将提供一个更科学可用的匹配系统，大大提高了配型的精度，从而提高术后移植物功能和患者生存质量 [5-8]。
同时，明确供体的HLA高分辨分型对于器官移植患者术后管理意义重大。供体特异性抗体(donor specific antibody,DSA)介导的排斥反应(antibody-mediated rejection，AMR)是导致移植物远期失功的首要危险因素。AMR的诊断和治疗效果评价依赖于明确DSA位点。临床上，首先检测受者外周血的群体反应性抗体(panel reactive antibodies，PRA)，然后结合和比对供体HLA高分辨分型结果，来最终判定DSA位点，明确AMR的诊断，调整后续治疗。
然而，HLA高分辨分型技术近年才开始应用于器官移植临床，早年仅检测HLA低分辨基因型；若要判定DSA位点，需要获取供体DNA重新做HLA高分辨分型检测，而早年的供体DNA标本多因缺失或保存时间过长不能满足检测条件。此时需要重新获取供体DNA标本，有人采用移植肾穿刺组织标本来分离供体DNA，导致移植肾组织创伤、增加出血风险、浪费宝贵的组织标本，且由于肾组织细胞量较少，导致检测成功率不高。也有报道利用尿沉渣进行HLA单个位点筛查，然而由于尿沉渣中供体DNA含量低，其所用方法只能获得部分血清型信息，因为存在部分基因型无对应血清型分型，因此难以通过该方法获得供体明确的六位点或八位点基因型信息；同时结果受到大量受体本身尿道脱落细胞DNA的影响，无法准确判定完整的供体HLA信息，同时也无法发现新的基因型 [9]。
因此，一种安全、便捷、无创的供体DNA获取技术及相应基因组学背景分析技术(如HLA高分辨分型检测技术等)，具有重大的临床应用前景。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种安全无创的肾移植供体特异性尿源细胞及其DNA的制备方法，采用该方法能获得足够数量的DNA，从而准确进行供体基因组学背景分析，例如获取供体特异性HLA(人类白细胞抗原)高分辨率分型结果。
此外，本发明还提供了由上述方法获取的足量肾移植供体特异性DNA在肾移植供体基因组学背景分析中的应用，如HLA高分辨率分型的检测方法。
为实现上述目的，本发明采取的技术方案为：
首先，本发明提供了一种肾移植供体特异性尿源细胞的制备方法，其包括以下步骤：
(1)取肾移植受体的中段尿，离心，弃上清；
(2)向步骤(1)离心所得下层沉淀中加入含有青链霉素或原代细胞抗生素混合液的磷酸盐缓冲液进行重悬，然后再次离心，弃上清；
(3)向步骤(2)离心所得下层沉淀中加入尿源细胞培养基，重悬，得到细胞悬液；
(4)将步骤(3)所得细胞悬液接种于培养容器中，进行细胞体外扩增，得到肾移植供体特异性尿源细胞。
上述原代细胞抗生素(Primocin TM)可购买自欣博盛生物科技公司。
本发明人发现，以肾移植受体的中段尿作为细胞来源，从中分离纯化并进行体外扩增得到肾移植供体特异性尿源细胞。尿源细胞主要来源于功能性肾组织，因此肾移植术后患者的尿源细胞的来源主要为供体肾脏。由于随尿液脱落的受体膀胱输尿管上皮细胞多为终末分化细胞，在体外培养条件下无法贴壁并增殖，而供体特异的尿源细胞可快速贴壁并迅速增殖，因此随着换液及扩大培养，受体信号将大大降低。本发明方法进行体外扩增显著扩大了供体特异性细胞的信号，极大的降低并稀释了受体细胞的信号。
本发明可根据需要，将细胞扩增至所需数量。
作为本发明所述肾移植供体特异性尿源细胞的制备方法的优选实施方式，所述步骤(3)中，尿源细胞培养基含有下述组分：REGM培养基、DMEM-high  Glucose培养基、非必需氨基酸细胞培养添加物、GlutaMAX添加剂和胎牛血清。研究表明，采用所述尿源细胞培养基体外培养供体特异性尿源细胞，可保证其稳定增殖，快速获得测序所需的DNA量。
作为本发明所述肾移植供体特异性尿源细胞的制备方法的优选实施方式，所述非必需氨基酸细胞培养添加物为NEAA(non-essential amino acids)细胞培养添加物。NEAA(non-essential amino acids)细胞培养添加物是商品化的试剂，可购买于赛业生物。
作为本发明所述肾移植供体特异性尿源细胞的制备方法的优选实施方式，所述步骤(4)中，培养容器经明胶溶液包被处理。
作为本发明所述肾移植供体特异性尿源细胞的制备方法的优选实施方式，所述步骤(4)中，细胞体外扩增的方法为：将细胞悬液接种于培养容器中后，先置于培养箱内静置培养，然后更换新鲜尿源细胞培养基，继续培养至细胞汇合度达到60％-80％，再用EDTA胰酶消化传代，将细胞扩增至所需的数量。
另外，本发明提供了一种肾移植供体特异性尿源细胞DNA的制备方法，其包括以下步骤：
(a)采用上述方法制备数量不低于1×10 6的肾移植供体特异性尿源细胞；
(b)从肾移植供体特异性尿源细胞中提取DNA，得到肾移植供体特异性尿源细胞DNA。
采用本发明方法制备肾移植供体特异性尿源细胞DNA时，保证步骤(a)中肾移植供体特异性尿源细胞数量不低于1×10 6可以获取足够量、高纯度的DNA供HLA测序检测。
作为本发明所述肾移植供体特异性尿源细胞DNA的制备方法的优选实施方式，所述步骤(b)中，从肾移植供体特异性尿源细胞中提取DNA的方法包括以下步骤：
(b1)采用EDTA胰酶对肾移植供体特异性尿源细胞进行消化，终止消化后加入磷酸盐缓冲液重悬细胞，得到细胞悬液；
(b2)向步骤(b1)所得细胞悬液中加入RNA酶A和蛋白酶K进行处理， 然后加入无水乙醇，振荡混匀，出现絮状沉淀；
(b3)将步骤(b2)所得溶液和絮状沉淀均加至吸附柱中，离心，去除废液，再采用漂洗液清洗吸附柱至少一次，然后将洗脱缓冲液加至吸附柱的吸附膜中间部位，放置后离心，收集洗脱缓冲溶液，得到肾移植供体特异性尿源细胞DNA。
另外，本发明提供了上述方法制备得到的肾移植供体特异性尿源细胞或肾移植供体特异性尿源细胞DNA在肾移植供体基因组学背景分析中的应用。优选地，所述基因组学背景分析为HLA分型分析。
本发明从肾移植受体的中段尿获取移植供体特异性尿源细胞DNA，可用于供体基因组学背景分析，例如用于HLA高分辨测序分析。该方法可避免临床为获取供体基因组而行的有创肾穿刺；保证检测所得分型结果的供体特异性；可提供足够量的DNA，保证测序的可行性；具有获取成本低且分析准确率高，获取方法完全无创等优点。
最后，本发明提供了一种肾移植供体HLA分型的检测方法，其包括以下步骤：采用上述方法制备肾移植供体特异性尿源细胞DNA，检测所得DNA中HLA分型，得到肾移植供体HLA分型结果。
作为本发明所述肾移植供体HLA分型的检测方法的优选实施方式，所述检测所得DNA中HLA分型的方法为：采用高通量测序检测所得DNA中HLA高分辨率分型，得到肾移植供体HLA高分辨率分型结果。
作为本发明所述肾移植供体HLA分型的检测方法的优选实施方式，所述高通量测序检测所得DNA中HLA高分辨率分型的方法为：先进行HLA-A/B/C/DRB1/DQB1/DPB1位点基因扩增并汇集扩增子序列，然后进行酶切修饰，将每个样本添加“条码”信息，再进行高通量测序，分析测序所得数据，得到肾移植供体HLA高分辨率分型结果。
作为本发明所述肾移植供体HLA分型的检测方法的优选实施方式，所述HLA-A/B/C/DRB1/DQB1/DPB1位点基因扩增采用的试剂盒为NGSgo-AmpX试剂盒(GenDx公司)；所述酶切修饰采用的试剂盒为NGSgo LibrX试剂盒(GenDx 公司)；所述添加“条码”信息采用的试剂盒为NGSgo-Indx试剂盒(GenDx公司)。
与现有技术相比，本发明的有益效果为：
(1)本发明基于肾移植术后受体尿液获取的供体特异性尿源细胞及DNA进行供体基因组学背景分析(以HLA高分辨率分型检测为例)的方法，具有获取方式安全无创，成本低廉且分离效率高，易于获得足够数量的DNA进行测序分析的优点。
(2)相比尿沉渣直接进行测序分析的方法，本发明方法由于提供了足够量的DNA，进而大大提高测序成功率；并且在本发明方法中，由于尿液中收集到的受者尿路脱落细胞为终末分化细胞，无法有效贴壁及体外增殖，因此大大稀释了受者脱落细胞DNA的干扰。因此，本发明方法可为无法获得供体HLA高分辨信息的肾移植患者提供足够量的供体特异性尿源细胞，并从中获取足够量的供体特异性DNA，为后续高通量测序的完成奠定基础。
(3)本发明进行肾移植供体基因组学背景分析(如HLA高分辨率分型的检测)的方法可以应用于多种人群，不受术后时间，受体年龄，肾功能等因素影响。利用该方法对肾移植术后长期存活受体进行检测，发现该类受体依然可以通过该方法准确检测出供体HLA信息；同时，利用该方法对移植同一供体肾脏的两个受体进行HLA高分辨率分型检测，两者结果一致，进一步确认了该方法的供体特异性。
(4)本发明可为缺乏供体HLA高分辨率分型的患者术后检测供体特异性抗体(DSA)水平及临床用药调整提供更多证据，十分具有应用价值。
(5)本发明HLA高分辨率分型的检测方法可用于群体反应性抗体(PRA)阳性患者确认供体特异性抗体(DSA)位点中，将HLA高分辨率分型结果与PRA结果比对，即可确认供体特异性抗体DSA位点。
附图说明
图1为本发明肾移植供体HLA分型的检测方法流程图；
图2为本发明实施例1中肾移植供体特异性尿源细胞的细胞增殖曲线图；
图3为本发明实施例2中肾移植供体特异性尿源细胞的DNA提取浓度及纯度曲线图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
下述实施例中，本发明以利用该供体特异性尿源细胞进行肾移植供体HLA分型的检测方法为例，流程如图1所示。
实施例1 供体特异性尿源细胞的制备
1.1 所述供体特异性尿源细胞的分离及制备，方法如下:
1)留取患者(受体)清洁中段尿150-200mL；
2)将尿液分装到50mL离心管，400g离心10min；
3)用吸引器缓慢吸去上清，剩余约3-5mL尿液；
4)加入含有青链霉素混合液的磷酸盐缓冲液(PBS)，约20-30mL，轻轻吹打混匀，再次400g离心10min；
5)用吸引器缓慢吸去上清至剩余液体少于1mL；
6)加入1mL尿源细胞培养基，重悬剩余沉淀，轻柔吹打若干次；
7)将上述细胞悬液均匀接种于已用0.1％明胶溶液(Gelatin)包被处理的24孔板中,补足尿源细胞培养基；
8)将培养皿置于37℃，5％CO 2的培养箱内静置培养3天；
9)避光观察是否有细胞贴壁，补加少量培养基，继续静置于37℃，5％CO 2培养箱；
10)在接种后的5-7天，视细胞贴壁状况，用磷酸盐缓冲液(PBS)轻柔吹洗一遍后更换新鲜培养基；
11)视细胞生长状况更换或添加培养基；若细胞形成较大克隆，可用0.25％EDTA胰酶消化分盘及传代；
12)视细胞生长状况进行细胞计数，保证用于提取DNA的细胞量至少为1*10 6个。
1.2 尿源细胞的特定培养基配置如下：
REGM培养基原液(CC-4127,Lonza)、DMEM-high Glucose培养基(Hyclone,SH30022.01)、非必需氨基酸添加剂(NEAA,11140050,Gibco)、 GlutaMAX添加剂(Gibco,35050061)及胎牛血清(FBS,Gibco,12664025)按照特定比例配置。其中包括A液:REGM培养基原液；B液：90％DMEM-high Glucose基础培养基+10％胎牛血清+1：100比例添加的非必需氨基酸添加剂+1：100比例添加的GlutaMAX添加剂；将A液和B液以一比一的比例混合均匀，即为尿源细胞的特定培养基。
1.3 细胞生长曲线绘制
1)取生长状态良好的尿源细胞，用0.25％EDTA胰酶消化为单细胞悬液；
2)200g离心5min，弃上清，加入适量新鲜培养基重悬；
3)按照一定倍数稀释该单细胞悬液；
4)准备血球计数板，洗净晾干；
5)吸取10ul细胞悬液，加样到血球计数板，镜下计数；
6)计算细胞总数，调整细胞浓度到1*10 5/ml；
7)准备24孔板，每孔加入1万个细胞(0.1ml)和0.9ml的新鲜培养基，轻轻晃动摇匀；
8)培养24h后，每隔24h收样，每次3个孔，0.25％EDTA胰酶消化重悬，进行细胞计数；
9)计数结果整理，以时间为横坐标，细胞数为纵坐标绘制生长曲线。
图2为本发明实施例中尿源细胞的细胞增殖曲线检测结果；可见尿源细胞在体外培养条件下保持着较为旺盛的增殖能力。
表1为本发明实施例中肾移植受体(编号USC-KT001)的供体特异性尿源细胞不同代数(第三代P3、第六代P6)的增殖能力统计表。结果显示尿源细胞无论P3或P6代均可以在体外稳定快速增殖，可以在短期内快速获取所需的细胞量进行测序。
表1 本发明实施例中受体KT-001的供体特异性尿源细胞不同代数(P3、6)细胞增殖能力统计表
Figure PCTCN2019115518-appb-000001
Figure PCTCN2019115518-appb-000002
实施例2 利用供体特异性尿源细胞检测供体HLA高分辨率分型
2.1 供体特异性尿源细胞的基因组DNA的提取(使用天根基因组DNA提取试剂盒(Tiangen-genomic DNA Kit))
1)待细胞增殖至1*10 6个细胞量，用0.25％EDTA胰酶在37℃消化约3-5min；
2)加入等体积含有10％血清的基础培养基终止消化，轻轻吹打细胞，使其从皿底脱落；
3)将细胞悬液转移至15mL离心管，200g离心5min；
4)加入1ml磷酸盐缓冲液(PBS)，重悬细胞，10000rpm(～11200g)离心1min，倒尽上清，加200ul缓冲液A，振荡至彻底悬浮；
5)加入4ul RNA酶A(100mg/ml)，涡旋振荡15s，室温放置5min；加入20ul蛋白酶K(Proteinase K)，混匀。
6)加入200ul缓冲液B，充分颠倒混匀，70℃放置10min，溶液变清亮，简短离心以去除管盖内壁的水珠；
7)加200ul无水乙醇，充分振荡混匀15s，出现絮状沉淀，简短离心以去除管盖内壁的水珠；
8)将7)中所得溶液和絮状沉淀都加入吸附柱中(吸附柱放入收集管中)，12000rpm(～13400g)离心30s，倒掉废液，将吸附柱放入收集管中。
9)向吸附柱中加入600ul漂洗液，12000rpm(～13400g)离心30s，倒掉废液，将吸附柱放入收集管中；
10)重复9)操作步骤；
11)将吸附柱放回收集管中，12000rpm(～13400g)离心2min，倒掉废液；
12)将吸附柱置于室温放置数分钟，以彻底晾干吸附材料中残余的漂洗液；
13)将吸附柱转入干净的离心管中，向吸附膜的中间部位悬空滴加50-200ul洗脱缓冲液，室温放置2-5min，12000rpm(～13400×g)离心2min，将溶液收集到离心管中。
图3为本发明实施例中两例供体特异性尿源细胞(编号分别为USC-KT033和USC-KT035)的DNA的分光光度仪检测结果。可见，1*10 6个尿源细胞可以获取足够量、高纯度的DNA供HLA测序检测。
表2为本发明实施例中两例尿源细胞DNA(编号分别为USC-KT033和USC-KT035)的浓度及纯度检测表。A260/280表示样品在260和280nm处的吸光值的比值，代表的是DNA样品的纯度，纯DNA的比值在大于1.8；A260/230表示样品260和280nm处的吸光值的比值，这是检测核酸纯度的第二指标，260/230值要比260/280的值大，通常在1.8-2.2。结果显示，两者均可获取足够量、高纯度的DNA，以便进行下一步测序。
表2 本发明实施例中两例尿源细胞(USC-KT033、035)的DNA浓度及纯度检测表
Figure PCTCN2019115518-appb-000003
2.2 二代测序法(Illumina平台)检测供体特异性尿源细胞的DNA的HLA高分辨率分型
1)确认所得DNA浓度及纯度符合要求(A260/280比值大于1.8,浓度大于500ng/μl)；
2)使用NGSgo-AmpX试剂盒(GenDx公司)进行HLA-A/B/C/DRB1/DQB1/DPB1位点基因扩增并将扩增子序列汇集；
3)利用NGSgo LibrX试剂盒(GenDx公司)进行酶切修饰；
4)利用NGSgo-Indx试剂盒(GenDx公司)将每个样本添加“条码”信息；
5)将所有样本汇集利用illumina MiniSEQ测序平台上机，进行二代测序；
6)测序所得原始数据利用NGSengine软件(GenDx公司)分析后得到HLA分型结果；
7)将供体特异性的HLA高分辨率分型结果与受体信息进行比对，明确高分辨率分型匹配水平。
实施例3 利用供体特异性尿源细胞检测肾移植后供体HLA高分辨率分型在成人肾移植中的应用
3.1 利用上述方法检测成人肾移植术后HLA高分辨率分型结果
患者007，女，42岁，供体年龄26岁，肾移植术后九个月。表3为本发明实施例中检测的成人肾移植术后尿源细胞及尿沉渣的HLA高分辨率分型结果，可见受体本身HLA高分辨率分型结果与受体尿沉渣测序结果一致，而受体尿液分离细胞的HLA测序结果则与供体的HLA高分辨率分型结果一致。该方法在成人肾移植受体中适用，可以获取供体特异的HLA高分辨率分型结果。
表3 本发明实施例中检测的成人肾移植术后尿源细胞及尿沉渣的HLA高分辨率分型结果
Figure PCTCN2019115518-appb-000004
实施例4 利用供体特异性尿源细胞检测肾移植后供体HLA高分辨率分型在儿 童肾移植中的应用
4.1 利用上述方法检测儿童肾移植受体术后HLA高分辨率分型结果
患者Child1004，男，8岁，供体年龄6岁，肾移植术后1个月。表4为本发明实施例中检测的儿童肾移植术后尿源细胞的HLA高分辨率分型结果，可见受体尿源细胞与供体HLA高分辨率分型结果一致，与受体不一致。该方法在儿童供肾中同样适用。
表4 本发明实施例中检测的儿童肾移植术后尿源细胞的HLA高分辨率分型结果
Figure PCTCN2019115518-appb-000005
实施例5 利用供体特异性尿源细胞检测肾移植后供体HLA高分辨率分型在肾移植术后长期存活受体中的应用
5.1 利用上述方法检测肾移植术后长期存活受体(25年)HLA高分辨率分型结果
患者016，男，58岁，肾移植术后25年。表5为本发明实施例中检测的肾移植术后长期存活受体的尿源细胞的HLA高分辨率分型结果，显示尿源细胞HLA高分辨率分型与受体本身不一致，可见该方法在肾移植术后长期存活受体中依然可以测出供体HLA信息。
表5 本发明实施例中检测的肾移植术后长期存活受体的尿源细胞的HLA高分辨率分型
Figure PCTCN2019115518-appb-000006
Figure PCTCN2019115518-appb-000007
实施例6 利用供体特异性尿源细胞检测两例移植同一供体肾脏的HLA高分辨率分型的应用
6.1 利用上述方法检测两例移植同一供体肾脏的HLA高分辨率分型结果
受体1-Child1004，男，8岁；受体2-Child1005，男，16岁；肾移植术后1个月；两个患者，供体为同一人，男，6岁。表6本发明实施例中检测的两例移植同一供体肾脏的HLA高分辨率分型。结果显示，接受同一个体肾脏的两个受体，其尿源细胞均为供体HLA分型。可见该方法可以准确获取供体特异性HLA高分辨率分型结果。
表6 本发明实施例中检测的两例移植同一供体肾脏的HLA高分辨率分型
Figure PCTCN2019115518-appb-000008
实施例7 供体特异性尿源细胞检测在群体反应性抗体(PRA)阳性患者确认供 体特异性抗体(DSA)位点中的应用
患者040，男，年龄47岁，供体信息不详。肾移植术后2年发生排斥反应，液相芯片检测发现群体反应性抗体(PRA)阳性，但由于缺失供体明确的HLA结果，无法判定供体特异性抗体(DSA)。
收集患者尿液，分离扩增尿源细胞，进行HLA测序，获得HLA高分辨率分型结果，与液相芯片单抗原微珠法(LSA)检测PRA的报告结果比对，明确DQB1*02：02、DQA1*02：01位点为可能的DSA位点。进一步观察患者治疗前后DSA位点的变化，发现如果不能确定DSA位点，则LSA报告没有整体规律，确认了DSA位点，则能明晰看到变化。
表7为本发明实施例中通过尿源细胞检测的未知供体的HLA高分辨率分型结果。利用受体的尿液成功获取供体特异性的尿源细胞，并且成功检测出供体特异的HLA高分辨率分型结果。
表7 本发明实施例中通过尿源细胞检测的未知供体的HLA高分辨率分型结果
Figure PCTCN2019115518-appb-000009
表8为患者040的液相芯片单抗原微珠法(LSA)检测PRA的结果展示。将表9中所得的供体HLA高分辨率分型结果与受体LSA结果相比较，推测相应的DSA位点，显示DQB1*02：02、DQA1*02：01位点为可能的DSA位点。可见受体已经产生特异性攻击供体肾脏的抗体，结合病理改变，可以判断患者是由于抗体介导的免疫排斥反应导致的移植肾功能障碍。因此，可以实施特异性抑制B细胞的药物或者血浆置换、大剂量静脉注射免疫球蛋白进行治疗。
表8 患者040的液相芯片单抗原微珠法(LSA)检测PRA的结果展示HLA-I类抗体：阳性(+)  1％
Figure PCTCN2019115518-appb-000010
HLA-II类抗体：阳性(+)  15％
Figure PCTCN2019115518-appb-000011
MFI校准值范围参考:<1000阴性；1000–4000弱阳性；4000–10000中阳性；>10000强阳性
抗体检测的HLA位点包括：I类：HLA-A、B、C；II类：HLA-DRB1、DQA1、DQB1、DPA1、DPB1
在肾移植术后，需结合受体群体反应性抗体(PRA)结果及供体HLA分型，判断供体特异性抗体(DSA)，从而辅助抗体介导的排斥反应(AMR)的诊断及治疗。而对于部分早期缺失供体HLA分型的肾移植受体，无法获取供体的DNA进行HLA分型的检测，因此无法准确的进行AMR诊断及药物治疗评价。本发明方法可有效的解决该问题，简单无创的获得足够量的供体特异性细胞及DNA，进行HLA高分辨率分型检测。
实施例8 供体特异性尿源细胞检测肾移植后供体HLA高分辨率分型方法与尿 沉渣测序的比较
受体Child1010，女，年龄7岁；供体年龄1.8岁，肾移植术后3个月。表9为本发明实施例中分别通过尿源细胞及尿液分离沉渣检测的同一例受体HLA高分辨率分型。结果显示，尿液沉渣测序失败，而尿源细胞测序成功，且准确特异的获得供体特异的HLA高分辨率分型结果。可见，受体尿沉渣测序由于特异DNA含量较低，存在失败率；而该方法相较于尿液沉渣测序可以准确获取供体特异性HLA高分辨率分型。
表9 本发明实施例中分别通过尿源细胞及尿液分离沉渣检测的同一例受体HLA高分辨率分型比较
Figure PCTCN2019115518-appb-000012
表10为本发明实施例方法与尿液分离沉渣检测方法及供肾活检检测方法的比较，显示本发明所使用方法可以方便快捷无创的获取供体特异性的HLA高分辨率分型结果。
表10 本发明实施例方法与尿液分离沉渣检测方法及供肾活检检测方法的比较
Figure PCTCN2019115518-appb-000013
Figure PCTCN2019115518-appb-000014
本发明所使用的获取肾移植术后未知的供肾基因组学背景信息的技术(例如HLA高分辨率分型检测)，可操作性强，方便无创，过程安全，结果可靠，因此患者接受度高；成本低廉且分离效率高，易于获取足够量的DNA进行测序分析，大大提高测序成功率，并且稀释并降低了受体泌尿系统终末上皮细胞DNA的影响，所得供体特异性HLA结果完整准确。将该结果与受体的群体反应性抗体结果比对，可以判定受体是否产生供体特异性抗体(DSA)，进而指导临床肾移植受体的DSA检测、AMR诊断及治疗效果评估和治疗方案调整。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
参考文献
1.Augustine,J.,Kidney transplant:New opportunities and challenges.Cleve Clin J Med,2018.85(2):p.138-144.
2.Konvalinka,A.and K.Tinckam,Utility of HLA Antibody Testing in Kidney Transplantation.J Am Soc Nephrol,2015.26(7):p.1489-502.
3.Erlich,H.,HLA DNA typing:past,present,and future.Tissue Antigens,2012.80(1):p.1-11.
4.Alizadeh,M.,A.Walencik,C.Frassati,P.Moskovtchenko,X.Lafarge,F.Verite,et al.,Evidence for a higher resolution of HLA genotyping by a new NGS-based approach.Transfus Clin Biol,2017.24(3):p.120-123.
5.Kramer,C.S.M.,M.Israeli,A.Mulder,I.I.N.Doxiadis,G.W.Haasnoot,S.Heidt,et al.,The long and winding road towards epitope matching in clinical transplantation.Transpl Int,2019.32(1):p.16-24.
6.Lim,W.H.,G.Wong,S.Heidt,and F.H.J.Claas,Novel aspects of epitope  matching and practical application in kidney transplantation.Kidney Int,2018.93(2):p.314-324.
7.Sypek,M.,J.Kausman,S.Holt,and P.Hughes,HLA Epitope Matching in Kidney Transplantation:An Overview for the General Nephrologist.Am J Kidney Dis,2018.71(5):p.720-731.
8.Picascia,A.,V.Grimaldi,and C.Napoli,From HLA typing to anti-HLA antibody detection and beyond:The road ahead.Transplant Rev(Orlando),2016.30(4):p.187-94.
9.Kwok,J.,L.C.Choi,J.C.Ho,G.S.Chan,M.M.Mok,M.F.Lam,et al.,A Modified Protocol with Improved Detection Rate for Mis-Matched Donor HLA from Low Quantities of DNA in Urine Samples from Kidney Graft Recipients.PLoS One,2016.11(11):p.e0166427.

Claims (10)

  1. 一种肾移植供体特异性尿源细胞的制备方法，其特征在于，包括以下步骤：
    (1)取肾移植受体的中段尿，离心，弃上清；
    (2)向步骤(1)离心所得下层沉淀中加入含有青链霉素混合液或原代细胞抗生素的磷酸盐缓冲液进行重悬，然后再次离心，弃上清；
    (3)向步骤(2)离心所得下层沉淀中加入尿源细胞培养基，重悬，得到细胞悬液；
    (4)将步骤(3)所得细胞悬液接种于培养容器中，进行细胞体外扩增，得到肾移植供体特异性尿源细胞。
  2. 如权利要求1所述的肾移植供体特异性尿源细胞的制备方法，其特征在于，所述步骤(3)中，尿源细胞培养基含有下述组分：REGM培养基、DMEM-high Glucose培养基、非必需氨基酸细胞培养添加物、GlutaMAX添加剂和胎牛血清。
  3. 如权利要求1所述的肾移植供体特异性尿源细胞的制备方法，其特征在于，所述步骤(4)中，培养容器经明胶溶液包被处理。
  4. 如权利要求1所述的肾移植供体特异性尿源细胞的制备方法，其特征在于，所述步骤(4)中，细胞体外扩增的方法为：将细胞悬液接种于培养容器中后，先置于培养箱内静置培养，然后更换新鲜尿源细胞培养基，继续培养至细胞汇合度达到60％-80％，再用EDTA胰酶消化传代，将细胞扩增至所需的数量。
  5. 一种肾移植供体特异性尿源细胞DNA的制备方法，其特征在于，包括以下步骤：
    (a)采用权利要求1～4任一项所述方法制备数量不低于1×10 6的肾移植供体特异性尿源细胞；
    (b)从肾移植供体特异性尿源细胞中提取DNA，得到肾移植供体特异性尿源细胞DNA。
  6. 如权利要求5所述的肾移植供体特异性尿源细胞DNA的制备方法，其特征在于，所述步骤(b)中，从肾移植供体特异性尿源细胞中提取DNA的方法 包括以下步骤：
    (b1)采用EDTA胰酶对肾移植供体特异性尿源细胞进行消化，终止消化后加入磷酸盐缓冲液重悬细胞，得到细胞悬液；
    (b2)向步骤(b1)所得细胞悬液中加入RNA酶A和蛋白酶K进行处理，然后加入无水乙醇，振荡混匀，出现絮状沉淀；
    (b3)将步骤(b2)所得溶液和絮状沉淀均加至吸附柱中，离心，去除废液，再采用漂洗液清洗吸附柱至少一次，然后将洗脱缓冲液加至吸附柱的吸附膜中间部位，放置后离心，收集洗脱缓冲溶液，得到肾移植供体特异性尿源细胞DNA。
  7. 如权利要求1～4任一项所述方法制备得到的肾移植供体特异性尿源细胞或如权利要求5或6所述方法制备得到的肾移植供体特异性尿源细胞DNA在肾移植供体基因组学背景分析中的应用；优选地，所述基因组学背景分析为HLA分型分析。
  8. 一种肾移植供体HLA分型的检测方法，其特征在于，包括以下步骤：采用权利要求5或6所述方法制备肾移植供体特异性尿源细胞DNA，检测所得DNA中HLA分型，得到肾移植供体HLA分型结果。
  9. 如权利要求8所述的肾移植供体HLA分型的检测方法，其特征在于，所述检测所得DNA中HLA分型的方法为：采用高通量测序检测所得DNA中HLA高分辨率分型，得到肾移植供体HLA高分辨率分型结果。
  10. 如权利要求9所述的肾移植供体HLA分型的检测方法，其特征在于，所述高通量测序检测所得DNA中HLA高分辨率分型的方法为：先进行HLA-A/B/C/DRB1/DQB1/DPB1位点基因扩增并汇集扩增子序列，然后进行酶切修饰，将每个样本添加“条码”信息，再进行高通量测序，分析测序所得数据，得到肾移植供体HLA高分辨率分型结果；优选地，所述HLA-A/B/C/DRB1/DQB1/DPB1位点基因扩增采用的试剂盒为NGSgo-AmpX试剂盒；所述酶切修饰采用的试剂盒为NGSgo LibrX试剂盒；所述添加“条码”信息采用的试剂盒为NGSgo-Indx试剂盒。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101314792A (zh) * 2007-05-31 2008-12-03 同昕生物技术（北京）有限公司 肾移植免疫排斥三联基因尿检法及试剂盒和诊断试剂
CN104838269A (zh) * 2012-11-08 2015-08-12 Umc乌得勒支控股有限公司 用于预测对错配的人白细胞抗原的免疫应答的方法
US9746479B2 (en) * 2010-03-09 2017-08-29 Cornell University Methods and compositions to predict and detect acute rejection
CN107893049A (zh) * 2010-12-31 2018-04-10 维也纳农业大学 产生诱导多能干细胞和分化细胞的方法
CN109576216A (zh) * 2017-09-29 2019-04-05 四川大学华西医院 尿来源间充质干细胞的提取及扩增培养方法
CN110051694A (zh) * 2019-04-22 2019-07-26 中山大学附属第一医院 一种尿源干细胞制剂、其制备及其在制备抗器官移植后急性免疫排斥药物的应用
CN110305835A (zh) * 2019-06-26 2019-10-08 中山大学附属第一医院 肾移植供体特异性尿源细胞及其dna的制备方法以及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104212762B (zh) * 2014-08-25 2017-01-18 中国人民解放军总医院 一种通过体外小分子诱导培养尿源性多潜能干细胞的方法
CN108570446A (zh) * 2017-12-05 2018-09-25 皓昇莱生物制药有限公司 一种尿液来源细胞的培养方法
CN108715830B (zh) * 2018-05-04 2019-08-06 中南大学湘雅医院 尿源干细胞外泌体的提取方法及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101314792A (zh) * 2007-05-31 2008-12-03 同昕生物技术（北京）有限公司 肾移植免疫排斥三联基因尿检法及试剂盒和诊断试剂
US9746479B2 (en) * 2010-03-09 2017-08-29 Cornell University Methods and compositions to predict and detect acute rejection
CN107893049A (zh) * 2010-12-31 2018-04-10 维也纳农业大学 产生诱导多能干细胞和分化细胞的方法
CN104838269A (zh) * 2012-11-08 2015-08-12 Umc乌得勒支控股有限公司 用于预测对错配的人白细胞抗原的免疫应答的方法
CN109576216A (zh) * 2017-09-29 2019-04-05 四川大学华西医院 尿来源间充质干细胞的提取及扩增培养方法
CN110051694A (zh) * 2019-04-22 2019-07-26 中山大学附属第一医院 一种尿源干细胞制剂、其制备及其在制备抗器官移植后急性免疫排斥药物的应用
CN110305835A (zh) * 2019-06-26 2019-10-08 中山大学附属第一医院 肾移植供体特异性尿源细胞及其dna的制备方法以及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TING ZHOU, CHRISTINA BENDA, SARAH DUNZINGER, YINGHUA HUANG, JENNY CY HO, JIAYIN YANG, YU WANG, YA ZHANG, QIANG ZHUANG, YANHUA LI, : "Generation of human induced pluripotent stem cells from urine samples", NATURE PROTOCOLS, vol. 7, no. 12, pages 2080 - 2089, XP055140462, ISSN: 17542189, DOI: 10.1038/nprot.2012.115 *

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