CN113736726B

CN113736726B - Establishment method of urine cell induced pluripotent stem cells of acute kidney injury patient

Info

Publication number: CN113736726B
Application number: CN202111056695.1A
Authority: CN
Inventors: 金永; 张曼玲; 李荣凤; 曹长春
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2022-09-23
Anticipated expiration: 2041-09-09
Also published as: CN113736726A

Abstract

The invention provides a method for establishing urine cell induced pluripotent stem cells of a patient with acute kidney injury, which comprises the following steps: step 1, separating and culturing urine kidney cells A of a patient with acute kidney injury, analyzing the chromosome karyotype of the cells, and identifying the expression of kidney specific molecules by immunofluorescence; step 2, transferring a mouse-derived OCT4, SOX2, KLF4 and c-MYC four transcription factor expression vector TetO-FUW-OSKM and an activation expression vector FUW-M2 rtTA into urine kidney cells A by a nuclear transfection technology to obtain urine kidney cells B; and 3, culturing the urine kidney cells B, adding doxycycline hydrochloride into a culture solution of the urine kidney cells B for induction, establishing a urine cell induced pluripotent stem cell line, and carrying out immunofluorescence identification on pluripotent molecules of the cell line.

Description

Method for establishing urine cell induced pluripotent stem cells of acute kidney injury patient

Technical Field

The invention belongs to the technical field of establishment of induced pluripotent stem cells, and particularly relates to an establishment method of induced pluripotent stem cells of urine cells of patients with acute kidney injury.

Background

In recent years, with the development of regenerative medicine, research and application of stem cells and other related technologies in the medical field are increasingly paid attention, and the establishment of an Induced Pluripotent Stem Cell (iPSCs) system derived from adult cells makes individualized medical treatment of patients possible and provides a new idea for rescuing kidney injury by using iPSCs. In vitro isolation of urine cells is currently the best way to obtain adult cells from patients non-invasively. Healthy adult kidneys filter approximately 100 liters of body fluid per day, with only 2 liters normally excreted with urine, while approximately 2000-7000 shed cells are present in urine. After Acute Kidney Injury (AKI), the kidney undergoes early Tubular Epithelial Cell (TECs) damage and late repair, and the patient clinically manifests itself as early-stage of injury with little or no urine and increased volume of urine during recovery with epithelioid cells in the urinary sediment.

Since Takahashi and Yamanaka pioneered the establishment of iPSCs, iPSCs derived from various human tissue cells were successfully established, including kidney tissue, especially urine-derived kidney cells. At present, urine kidney cells mainly come from newborn infants, premature infants and fetuses with incompletely developed kidneys, and the urine of children and adults with mature kidneys has extremely low or even no kidney cell obtaining efficiency. Although it has been reported that the efficiency of cell isolation from urine of patients with hereditary kidney diseases is improved, it is still low. In the process of repairing the kidney injury, the urine volume of a patient is gradually recovered, and various cell components in the urinary sediment are continuously increased, so that the optimal time for separating the kidney cells of the patient from the urine is suggested, and the research basis is provided for establishing the pluripotent stem cells induced by the urine cells of the patient with acute kidney injury.

Disclosure of Invention

The invention provides a method for establishing urine cell induced pluripotent stem cells of a patient with acute kidney injury, which has the advantages of being capable of separating urine kidney cells of the patient with acute kidney injury and establishing the induced pluripotent stem cells.

The technical scheme of the invention is realized as follows: a method for establishing urine cell-induced pluripotent stem cells of a patient with acute kidney injury comprises the following steps:

step 1, separating and culturing urine kidney cells A of a patient with acute kidney injury, analyzing the chromosome karyotype of the cells, and identifying the expression of kidney specific molecules by immunofluorescence;

step 2, transferring a mouse OCT4, SOX2, KLF4 and c-MYC four transcription factor expression vector TetO-FUW-OSKM and an activation expression vector FUW-M2 rtTA into a urine kidney cell A by a nuclear transfer technology to obtain a urine kidney cell B;

and 3, culturing the urine kidney cells B by adopting a culture system combining leukemia inhibitory factors and basic fibroblast growth factors, adding doxycycline hydrochloride into a culture solution of the urine kidney cells B for induction, establishing a urine cell induced pluripotent stem cell line, and carrying out immunofluorescence identification on pluripotent molecules of the cell line.

As a preferred embodiment, the method for isolating and culturing urine kidney cell a of acute kidney injury patient comprises the following steps:

step 1, collecting 30-50 ml of morning urine of a patient in the recovery period of acute kidney injury by using a sterile centrifugal tube, and storing at a low temperature of 4 ℃;

step 2, centrifuging the morning urine stored in the step 1 at a low temperature of 4 ℃ to obtain a urine cell tube shape, wherein the centrifugation condition is 800g and 15 min;

step 3, inoculating urine cell tubes into a six-hole culture plate, culturing through a cell culture solution, and after culturing for 24 hours, replacing the cell culture solution with the urine cell culture solution for continuous culture;

and 4, replacing the urine cell culture solution every two days, and obtaining the urine kidney cells A after 7-10 days of culture time.

In a preferred embodiment, the cell culture solution consists of 82% high-sugar DMEM, 15% FBS and 3% Pen Strep;

urine cell culture consisted of Knockout-DMEM, 1% ITS, 200ng/ml FGF9, 50ng/ml BMP7, 0.1. mu. mol/L RA, 1. mu.g/ml Heparin, 10ng/m EGF, 10ng/m activin A, 300ng/ml BMP4, 15% FBS and 1% Pen Strep.

As a preferred embodiment, the method for analyzing the karyotype of the cell comprises the steps of:

step 1, urine kidney cell A is added at 1 × 10 ⁵ Inoculating into cell culture plate, and culturing at 37 deg.C under 5% CO ₂ Performing constant-temperature culture under the condition (1);

step 2, after culturing until the cell confluency is 70-90%, adding 0.02 mu g/ml colchicine into the culture solution in the cell culture plate, continuously culturing for 1h, and taking out;

step 3, adding 0.25% of pancreatin for digestion, blowing cells into single cells after digestion of the culture solution is stopped, centrifuging for 3min at 1000rpm, and then removing supernatant;

step 4, adding 2ml of 0.56% KCL hypotonic solution preheated at 37 ℃, gently blowing and beating the mixture to uniformly mix the cells, placing the mixture in a water bath at 37 ℃ for 30min, centrifuging the mixture for 3min at 1000rpm, and then removing supernatant;

and 5, adding 2ml of fixing solution to generate cell sediment, slightly bouncing the cell sediment, adding 2ml of fixing solution, uniformly blowing the cells, standing at room temperature for 40min, centrifuging at 1000rpm for 3min, then discarding supernatant, adding 2ml of fixing solution, standing at room temperature for 20min, centrifuging at 1000rpm for 3min, then discarding supernatant, and taking the rest as a sample to be tested, wherein the fixing solution is a mixture of methanol and glacial acetic acid, and the mixing volume ratio of the methanol to the glacial acetic acid is 3: 1;

and 6, uniformly blowing the cells of the sample to be detected, dripping the cells onto a glass slide at a distance of 70-80 cm, standing overnight at room temperature, and then, according to Giemsa: water 1: 10, and dyeing for 20min, then washing the staining solution with water, standing at room temperature until the water content is evaporated to dryness, and observing the cell karyotype of the sample to be detected under an oil microscope.

As a preferred embodiment, the method for immunofluorescence identification of kidney specific molecule expression comprises the steps of:

step 1, urine kidney cells are divided into 1 × 10 ⁴ Perwell in twenty-four well cell culture plates at 37 ℃ with 5% CO ₂ Culturing at constant temperature under the condition of (1);

step 2, after culturing for 24 hours, removing the culture solution in the cell culture plate, washing the cell culture plate once with PBS (phosphate buffer solution), fixing the cell culture plate for 10 minutes through 4% paraformaldehyde, washing the cell culture plate for three times with PBS again, wherein the washing time for each time is 5 minutes, and washing the cell culture plate for three times with PBS again after 1% Triton X100 passes through the cell culture plate and is transparent for 1 hour at room temperature, and the washing time for each time is 5 minutes;

and 3, after cleaning, sealing the goat serum by using 10% of goat serum at room temperature for 1h, removing the sealing liquid, adding the protein primary antibody, standing the mixture at 4 ℃ overnight, wherein the ratio of the primary antibody to the 10% of goat serum is 1: 200 of a carrier;

step 4, after standing overnight, removing the primary antibody, washing with PBS for three times, adding the secondary antibody after washing for 5min each time in a dark place, removing the secondary antibody after incubating for 1h in a dark place at room temperature, and adding fluoroserine into cells ^TM Observation of the staining results under a fluorescent microscope with withDAPI, secondary antibodies andthe proportion of 10% goat serum is 1: 1000.

as a preferred embodiment, the method for transferring the mouse OCT4, SOX2, KLF4 and c-MYC four transcription factor expression vector TetO-FUW-OSKM and the activation expression vector FUW-M2 rtTA into urine kidney cells A by the nuclear transfection technology comprises the following steps:

step 1, culturing 100ml of Escherichia coli containing TetO-FUW-OSKM plasmid overnight, and culturing 100ml of Escherichia coli containing FUW-M2 rtTA plasmid overnight;

step 2, selecting restriction enzyme Fspl to respectively perform enzyme digestion on the plasmids in the step 20, then respectively taking 20 mu l of enzyme digestion plasmids and the original plasmids to perform agarose gel electrophoresis, and identifying whether the plasmids are cut;

step 3, determining that the plasmid is linearized by enzyme digestion, recovering the plasmid by an ethanol precipitation method, and storing at-20 ℃ for later use;

step 4, urine kidney cell A is placed in a cell culture dish and is heated to 37 ℃ with 5% CO ₂ Performing constant-temperature culture under the condition (1);

step 5, observing after culturing for 24h, digesting the cells into single cells by using 0.05% of pancreatin when the cells reach 80-90% of confluence, and adding fibroblast culture solution (prepared by 84% of high-sugar DMEM, 15% of FBS and 1% of Pen Strep) to stop digestion;

step 6, centrifuging for 5min at 1500rpm, removing supernatant, and cleaning once by using DPBS;

and 7, preparing a nuclear transfer liquid, and transferring the linearized TetO-FUW-OSKM, FUW-M2 rtTA and pCMV-tdTomato into the urine kidney cell A by using a nuclear transfer instrument to obtain a urine kidney cell B.

As a preferred embodiment, the method for establishing a urine cell-induced pluripotent stem cell line comprises the following steps:

step 1, culturing mouse fetal fibroblasts with generation not higher than P4 generation in a culture dish, and treating the cells for 2.5-3 hours by mitomycin-C with concentration of 10 mug/ml when the cells are 95% confluent;

step 2, digesting the cells by using 0.05 percent pancreatin, and centrifuging after terminating digestion of cell culture solution;

step 3, resuspending and counting cells in cell culture medium at 1.0X 10/well ⁵ The cells were added to a four-well plate pretreated with 0.1% gelatin for 30min at 38.5 deg.C with 5% CO ₂ Culturing at constant temperature to obtain a feeding layer;

step 4, verifying whether the TetO-FUW-OSKM and the FUW-M2 rtTA are transferred into urine kidney cells A simultaneously through PCR, and placing the cells in a four-hole culture plate after verification;

step 5, digesting with 0.05 percent pancreatin, centrifuging after the cell culture solution is digested, and discarding the culture solution;

step 6, resuspending and counting the cells by using a stem cell culture solution;

step 7, culturing the cells in a four-well plate with 1.5X 10 wells per well ⁴ The cells were added to the feeder layer at 38.5 ℃ with 5% CO ₂ Performing constant-temperature culture under the condition of (2);

and 8, changing liquid every day for observation, and after the clone grows out, carrying out passage by a mechanical method, and then changing the passage into pancreatin single cell passage.

As a preferred embodiment, the method for verifying whether the TetO-FUW-OSKM and FUW-M2 rtTA are simultaneously transferred into the urine kidney cell A by PCR comprises the steps of screening single clone cell strains with drug resistance by using a neomycin resistance gene in a pCMV-tdTomato plasmid, then respectively extracting genome DNA of each cell strain, and verifying whether the TetO-FUW-OSKM and FUW-M2 rtTA are simultaneously transferred into the cell by PCR;

TetO-FUW-OSKM includes the following components as shown in SEQ ID NO: 1, and the primer shown as SEQ ID NO: 2, or a reverse primer;

FUW-M2 rtTA includes the amino acid sequence shown as SEQ ID NO: 3, and an upstream primer shown as seq id no: 4, or a reverse primer as shown in the figure.

As a preferred embodiment, the stem cell culture fluid is composed of 40% KnockOut ^TM DMEM, 10% KnockOut ^TM SR, 24% DMEM/F12, 24% Neurobasal Medium, 0.5% N-2 Supplement, 1% B-27 Supplement, 0.125. mu.g/ml BSA, 2.5. mu.g/ml insulin, 1. mu. mol/L L-Glutamin,0.05. mu. mol/l of 2-Mercaptoethanol, 0.5% NEAA, 1% PenStrep, 3. mu. mol/l of CHIR99021, 1. mu. mol/l of PD0325901, 2. mu. mol/l of SB431542, 2000UI/ml of LIF, 16ng/ml of bFGF, 50ng/ml of VC and 2. mu.g/ml of DOX.

As a preferred embodiment, the method for immunofluorescence identification of pluripotent molecules of a cell line comprises the steps of:

step 1, removing stem cell culture solution in a four-hole culture plate, washing the stem cell culture solution once with PBS for 5min, fixing the stem cell culture solution for 10min with 4% paraformaldehyde, washing the stem cell culture solution for three times with PBS again, wherein the washing time for each time is 5min, and washing the stem cell culture solution for three times with PBS again after 1% Triton X100 passes through the stem cell culture solution for 1h at room temperature, and the washing time for each time is 5 min;

and 2, after cleaning, sealing the goat serum by using 10% of goat serum at room temperature for 1h, removing the sealing liquid, adding a protein primary antibody, standing the mixture at 4 ℃ overnight, wherein the ratio of the primary antibody to the 10% of goat serum is 1: 200 of a carrier;

step 3, after standing overnight, removing the primary antibody, washing with PBS for three times, adding the secondary antibody after each washing time is 5min in a dark place, removing the secondary antibody after incubation for 1h in a dark place at room temperature, and adding Fluoroshield into cells ^TM And observing a dyeing result under a fluorescence microscope by using withDAPI, wherein the ratio of the secondary antibody to the 10% goat serum is 1: 1000.

after the technical scheme is adopted, the invention has the beneficial effects that:

the invention uses the established urine kidney cell separation method to separate and culture urine cells of patients with acute kidney injury, analyzes the chromosome karyotype of the cells and identifies the specific molecular expression of the kidney by immunofluorescence; the method comprises the steps of transferring rat OCT4, SOX2, KLF4 and c-MYC four transcription Factor expression vectors TetO-FUW-OSKM and an activation expression vector FUW-M2 rtTA into Urine kidney cells by a nuclear transfer technology, culturing a culture system of Leukemia Inhibitory Factor (LIF) and basic Fibroblast Growth Factor (bFGF), inducing by adding doxycycline hydrochloride (DOX) into a culture solution, establishing a human Urine cell induced pluripotent stem cell line (UiPS), and carrying out immunofluorescence identification on multi-molecules of the cell line.

Urine kidney cells of acute kidney injury patients can be stably cultured in vitro, the cells are epithelioid, have normal karyotype and stronger proliferation capacity, and simultaneously express a plurality of kidney specific molecules (Pax8, E-cadherin and AQP1) and kidney development related molecules (Pax2, WT1, Six1 and Six 2); urine kidney cells of a cotransfection TET-ON induction expression vector form UiPS cell clone through induction culture, the UiPS cell clone is in three-dimensional growth and can be subjected to unicellular subculture, alkaline phosphatase staining is positive, and various stem cell pluripotency factors (OCT4, SOX2, TRA-1-60) are expressed at the same time. The invention successfully establishes an induced pluripotent stem cell line from urine cells of patients with acute kidney injury.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic of urine kidney cell culture;

FIG. 2 is a schematic illustration of the karyotyping of human urine kidney cells;

FIG. 3 is a schematic representation of kidney specific marker molecules in human urine kidney cells;

FIG. 4 is a schematic representation of a kidney development-related molecule in human urine kidney cells;

FIG. 5 is a schematic diagram of a TET-ON inducible expression vector;

FIG. 6 is a schematic diagram of urine kidney cells transfected with TetO-FUW-OSKM and FUW-M2 rtTA inducible expression plasmids;

FIG. 7 is a schematic diagram of the establishment of UiPSC and alkaline phosphatase staining (AP staining);

fig. 8 is a schematic diagram of the expression of pluripotent stem cell factors in uipscs.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Referring to fig. 1 to 8, a method for establishing urine cell-induced pluripotent stem cells of a patient with acute kidney injury is as follows:

separating and culturing urine kidney cells of a patient with acute kidney injury;

as shown in figure 1, the acute kidney injury convalescent patient is collected by a special device such as a sterile centrifuge tube, the morning urine volume is about 30-50 ml, the patient is stored at low temperature (4 ℃), and transferred to a laboratory within 2 hours for low-temperature centrifugation (4 ℃, 800g, 15min) to obtain urine cell casts, which were inoculated into a six-well culture plate for culture (82% high-sugar DMEM, 15% FBS, and 3% Pen Strep) and, after 24 hours of culture, and (3) replacing the cell culture solution with urine cell culture solution (Knockout-DMEM, 1% ITS, 200ng/ml FGF9, 50ng/ml BMP7, 0.1 mu mol/L RA, 1 mu g/ml Heparin, 10ng/m EGF, 10ng/m activin A, 300ng/ml BMP4, 15% FBS and 1% Pen Strep), culturing for 7-10 days, replacing the culture solution once every two days, and finally obtaining urine cells.

In the figure, P0 is primary, 2 d-7 d is a graph of 2-7 days of culture, and the scale is 50 μm; p1 is passage 1, and the scale is 50 μm; p4 passage 4, 100 μm scale.

Karyotyping urine cells;

as shown in FIG. 2, urine kidney cells were treated at 1X 10 ⁵ Inoculating into 60mm cell culture plate, and culturing at constant temperature (37 deg.C, 5% CO) ₂ ) Culturing until cell confluence is about 80%, adding 0.02 μ g/ml colchicine into the culture solution, culturing for 1 hr, taking out, digesting with 0.25% pancreatin, and collecting the culture solutionAfter digestion is stopped, the cells are blown into single cells, centrifuged at 1000rpm for 3min, supernatant is discarded, 2ml of 0.56% KCl hypotonic solution preheated at 37 ℃ is added, the cells are lightly blown and mixed, water bath at 37 ℃ is carried out for 30min, centrifuged at 1000rpm for 3min, supernatant is discarded, 2ml of stationary liquid (methanol: glacial acetic acid ═ 3: 1) is added, cell precipitation is flicked up, then 2ml of stationary liquid is added, the cells are blown uniformly, and the mixture is kept stand at room temperature for 40 min. Centrifuging at 1000rpm for 3min, discarding supernatant, adding 2ml fixative, standing at room temperature for 20min, centrifuging at 1000rpm for 3min, discarding excess liquid to obtain about 200 μ l, blowing the cells uniformly, dropping the cells onto a glass slide at a distance of 70-80 cm, and standing at room temperature overnight. According to Giemsa: water 1: preparing a staining solution according to the proportion of 10, staining for 20min, washing the staining solution with water, and standing at room temperature until the water is evaporated to dryness. The cell karyotype was observed under an oil microscope.

In the figure, the urine cell lines (HUC1 and HUC2) were subjected to karyotype analysis after 2 passages (P2) and 1 passage (P1), respectively, and 10 μm was used as a scale.

Immunological identification of urine kidney cells;

as shown in FIG. 3 and FIG. 4, urine kidney cells were processed at 1X 10 ⁴ One cell/well was inoculated into twenty-four well cell culture plates and placed in a constant temperature culture (37 ℃, 5% CO) ₂ ) After 24 hours of culture, the cell culture solution was discarded, washed once with PBS for 5min, fixed with 4% paraformaldehyde for 10min, and then washed three times with PBS for 5min each. Cells were permeabilized with 1% Triton for 1h at room temperature, followed by three additional washes with PBS for 5min each. Blocking with 10% goat serum at room temperature for 1h, removing blocking solution, and adding primary antibody of Pax8, E-cadherin, AQP1, Pax2, WT1, Six1, Six2 protein at 4 deg.C overnight. Primary antibody is dissolved in 10% goat serum, and the proportion is 1: 200.

after overnight, the primary antibody was removed, washed three times with PBS for 5min each, and a secondary antibody was added in the dark, dissolved in 10% goat serum, in a ratio of 1: after incubation for 1h at room temperature in the dark, the secondary antibody was removed 1000. Adding appropriate amount of Fluoroshield into cells ^TM The staining results were observed under a fluorescent microscope with WithDAPI (SIGMA, USA).

The information of the primary antibody and the secondary antibody is shown in the following table:

the scales selected in fig. 3 and 4 are both 50 μm.

Establishing a urine kidney cell line integrating an induction expression vector;

as shown in FIG. 6, the urine kidney cells were transformed with inducible expression system plasmid vectors TetO-FUW-OSKM, FUW-M2 rtTA and red protein fluorescence expression vector pCMV-tdTomato

100ml of E.coli containing TetO-FUW-OSKM plasmid and FUW-M2 rtTA plasmid were cultured overnight, respectively, and the plasmids were extracted using endotoxin-free plasmid macroextraction kit and the plasmid concentration was determined. The plasmid was digested overnight with restriction enzymes Fsp I, and 20. mu.l of each digested plasmid was subjected to agarose gel electrophoresis with the original plasmid to determine whether the plasmid was cleaved. After the plasmid is determined to be linearized by enzyme digestion, the plasmid is recovered by an ethanol precipitation method and stored at-20 ℃ for later use.

Primary urine kidney cells were thawed in a 60mm dish and observed after 24h when the cells reached approximately 80-90% confluency, they were digested into single cells with 0.05% pancreatic enzyme, after termination of digestion by addition of fibroblast culture medium (84% high sugar DMEM, 15% FBS and 1% Pen Strep), centrifuged at 1500rpm for 5min, the supernatant was discarded and washed once with DPBS. According to the kit Amaxa ^TM The Basic nucleofector Kit Primary fibers (Lonza, USA) was used to prepare a nuclear transfer solution, and linearized TetO-FUW-OSKM (4. mu.g), FUW-M2 rtTA (4. mu.g) and pCMV-tdTomato (0.5. mu.g) were co-transferred into urine cells using a nuclear transfer instrument.

In the figure, A: the plasmids TetO-FUW-OSKM and FUW-M2 rtTA are linearized by Fsp I restriction enzyme, and are respectively 1 and 3, and are used as

original plasmid contrast

2 and 4, and are linearized plasmids, wherein M1 is 1Kb DNA ladder, and M2 is DL5000DNA ladder;

b: HUC transfects TetO-FUW-OSKM, FUW-M2 rtTA and pCMV-tdTomato, and after the drug screening is carried out for 8d, the cell clones express the red fluorescence condition, and a ruler is selected to be 100 mu M;

c: the monoclonal cell lines TetO-FUW-OSKM and FUW-M2 rtTA double-conversion PCR molecular identification is carried out, wherein M1 is 1Kb DNA ladder.

Screening a transgenic cell line and identifying single cell clone;

the neomycin resistance gene in the pCMV-tdTomato plasmid is utilized to screen out a monoclonal cell strain with drug resistance. Then extracting genome DNA of each cell strain respectively, and identifying whether the TetO-FUW-OSKM and FUW-M2 rtTA are transferred into cells simultaneously through PCR, wherein the primer sequences are as follows:

TetO－FUW－OSKM：

an upstream primer: 5'-GAGGGAGACCGAGGAGTTCAA-3' (SEQ ID NO.1),

a downstream primer: 5'-GCAGCGTATCCACATAGCGTAA-3' (SEQ ID NO. 2);

FUW－M2rtTA：

an upstream primer: 5'-GGGGAGGGTAAGTGAGGC-3' (SEQ ID NO.3),

a downstream primer: 5'-GCTGACAGGTGGTGGCAAT-3' (SEQ ID NO. 4).

Establishment of urine cell-induced pluripotent stem cells:

induction of pluripotent stem cells: including feeder cell preparation and iPS induction.

Preparing feeder layer cells: thawing mouse fetal fibroblasts with passage no higher than P4, culturing in culture dish, treating cells with mitomycin-C with final concentration of 10 μ g/ml for 2.5-3 hr when the cells reach 95% confluence, digesting with 0.05% pancreatin, terminating digestion and centrifuging in cell culture solution, resuspending and counting cells with cell culture solution, and counting at 1.0 × 10 per well ⁵ The cells were plated in a four-well plate pretreated with 0.1% gelatin for 30min, and cultured at 38.5 ℃ with 5% CO 2.

inducing by iPS: thawing the PCR-identified double positive strain of OSKM and M2rtTA vector in a four-well plate, digesting the cells with 0.05% pancreatin when the cells reach 95% confluence, terminating the digestion and centrifugation of the cell culture fluid, discarding the culture fluid, and culturing with a stem cell culture fluid (40% KnockOut) ^TM DMEM、10％KnockOut ^TM SR、24％DMEM/F12、24％NeurobasalMedium、0.5％N－2Supplement、1％B－27Supplement, 0.125. mu.g/ml BSA, 2.5. mu.g/ml solin, 1. mu. mol/l Glutamin, 0.05. mu. mol/l 2-Mercaptoethanol, 0.5% NEAA, 1% PenStrep, 3. mu. mol/l CHIR99021, 1. mu. mol/lPD0325901, 2. mu. mol/lSB431542, 2000UI/ml LIF, 16ng/ml bFGF, 50ng/ml VC and 2. mu.g/ml DOX) were resuspended and counted, after which the cells were resuspended and counted in four well plates at 1.5X 10 per well ⁴ The cells were added to a feeder layer prepared one day in advance, 38.5 deg.C, 5% CO ₂ And (5) culturing. Three days later, fluid change and observation are carried out to see whether the clone grows out, and then fluid change and observation are carried out every day. When clone grows out, passage is carried out by a mechanical method, and then, passage is changed into pancreatin single cell passage.

Identification of induced pluripotent stem cells: including alkaline phosphatase staining and cellular immunofluorescence staining.

As shown in fig. 7, alkaline phosphatase staining: and (3) carrying out alkaline phosphatase staining on the UiPS clone subjected to subculture, and preliminarily identifying whether the UiPS clone has pluripotency. The procedure was followed using the AlkalinePhosphataseDetectionkit (MILLIPORE, Germany). After staining, the cells were photographed under an inverted microscope.

In the figure, A: the double-rotating cell line of TetO-FUW-OSKM and FUW-M2 rtTA is induced by a stem cell culture system for 8 days to form UiPS cell clone, and the ruler is selected to be 100 mu M;

b: cell clones (400 x) that grew 3 days after the passage of the UiPS single cell;

c: the UiPS cell line was stained with alkaline phosphatase (AP staining) at 100 μm scale.

As shown in fig. 8, cellular immunofluorescence staining: the iPS cell culture solution was discarded, washed once with PBS for 5min, fixed with 4% paraformaldehyde for 10min, and then washed three times with PBS for 5min each time. Cells stained with proteins such as OCT4, SOX2, and Tra-1-60 were permeabilized with 1% Triton at room temperature for 1 hour, followed by three washes with PBS for 5min each. Blocking was performed using 10% goat serum for 1h at room temperature, after which the blocking solution was removed and primary antibody was added overnight at 4 ℃. Primary antibody is dissolved in 10% goat serum, and the proportion is 1: 100. after overnight, the primary antibody was removed, washed three times with PBS for 5min each, and secondary antibody was added in the dark, dissolved in 10% goat serum, in a ratio of 1: 1000, protection from light at room temperatureAfter 1h incubation, the secondary antibody was removed. Adding appropriate amount of Fluoroshield into cells ^TM The staining results were observed under a fluorescent microscope with WithDAPI (SIGMA, USA).

The scale in the figure is 50 μm.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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Claims

1. A method for establishing urine cell-induced pluripotent stem cells of a patient with acute kidney injury is characterized by comprising the following steps:

step 1, separating and culturing urine kidney cells of acute kidney injury patients, namely urine kidney cells A, analyzing chromosome karyotype of the cells, and identifying kidney specific molecule expression by immunofluorescence;

step 2, transferring a mouse-derived OCT4, SOX2, KLF4 and c-MYC four transcription factor expression vector TetO-FUW-OSKM and an activation expression vector FUW-M2 rtTA into urine kidney cells A by a nuclear transfection technology to obtain urine kidney cells B;

step 3, culturing the urine kidney cells B by adopting a culture system combining leukemia inhibitory factor with basic fibroblast growth factor, adding doxycycline hydrochloride into a culture solution of the urine kidney cells B for induction, establishing a urine cell induced pluripotent stem cell line, and performing immunofluorescence identification on pluripotent molecules of the cell line;

the method for separating and culturing urine kidney cells of the acute kidney injury patient in the step 1 comprises the following steps:

step 100, collecting 30-50 ml of morning urine of a patient in the recovery period of acute kidney injury by using a sterile centrifugal tube, and storing at a low temperature of 4 ℃;

step 101, centrifuging the morning urine stored in the step 100 at a low temperature of 4 ℃ to obtain a urine cell tube shape, wherein the centrifugation condition is 800g and 15 min;

102, inoculating urine cell tubes into a six-hole culture plate, culturing by using a cell culture solution, and after culturing for 24 hours, replacing the cell culture solution with the urine cell culture solution for continuous culture;

step 103, replacing the urine cell culture solution every two days, and obtaining urine kidney cells after 7-10 days of culture time, namely urine kidney cells A;

the cell culture solution in the step 102 consists of 82% of high-sugar DMEM, 15% of FBS and 3% of Pen Strep;

the urine cell culture solution consists of Knockout DMEM, 1% ITS, 200ng/ml FGF9, 50ng/ml BMP7, 0.1 mu mol/L RA, 1 mu g/ml Heparin, 10ng/ml EGF, 10ng/ml activinA, 300ng/ml BMP4, 15% FBS and 1% Pen Strep.

2. The method for establishing the urine cell-induced pluripotent stem cell of the acute kidney injury patient according to claim 1, wherein the method for analyzing the karyotype of the cells in step 1 comprises the following steps:

step 110, urine kidney cells A are added at a ratio of 1 × 10 ⁵ Seeded in cell culture plates and incubated at 37 ℃ with 5% CO ₂ Performing constant-temperature culture under the condition (1);

step 111, after culturing until the cell confluency is 70-90%, adding 0.02 mu g/ml colchicine into the culture solution in the cell culture plate, continuously culturing for 1h, and taking out;

step 112, adding 0.25% of pancreatin for digestion, blowing cells into single cells after digestion of a culture solution is stopped, centrifuging for 3min at 1000rpm, and then removing supernatant;

113, adding 2ml of 0.56% KCl hypotonic solution preheated at 37 ℃, slightly blowing and beating the mixture to uniformly mix the cells, placing the mixture in a water bath at 37 ℃ for 30min, centrifuging the mixture for 3min at 1000rpm, and then removing supernatant;

step 114, adding 2ml of a fixing solution to generate cell sediment, slightly bouncing the cell sediment, adding 2ml of the fixing solution, uniformly blowing the cells, standing at room temperature for 40min, centrifuging at 1000rpm for 3min, then discarding supernatant, adding 2ml of the fixing solution, standing at room temperature for 20min, centrifuging at 1000rpm for 3min, then discarding supernatant, and taking the rest as a sample to be tested, wherein the fixing solution is a mixture of methanol and glacial acetic acid, and the mixing volume ratio of the methanol to the glacial acetic acid is 3: 1;

and step 115, uniformly blowing the cells of the sample to be detected, dripping the cells onto a glass slide at a distance of 70-80 cm, standing overnight at room temperature, and then, according to Giemsa: water 1: 10, and dyeing for 20min, then washing the staining solution with water, standing at room temperature until the water content is evaporated to dryness, and observing the cell karyotype of the sample to be detected under an oil microscope.

3. The method for establishing the urine cell-induced pluripotent stem cell of the acute kidney injury patient according to claim 1, wherein the immunofluorescence identification method for the expression of the kidney-specific molecule in the step 1 comprises the following steps:

step 120, urine kidney cells are treated at a ratio of 1 × 10 ⁴ Perwell in twenty-four well cell culture plates at 37 ℃ with 5% CO ₂ Culturing at constant temperature under the condition of (1);

step 121, after culturing for 24 hours, discarding the culture solution in the cell culture plate, washing the cell culture plate once with PBS (phosphate buffer solution) for 5min, fixing the cell culture plate with 4% paraformaldehyde for 10min, washing the cell culture plate again with PBS for three times, wherein the washing time of each time is 5min, and washing the cell culture plate again with PBS for three times after passing through 1% Triton which is transparent for 1 hour at room temperature, and the washing time of each time is 5 min;

and step 122, after the washing is finished, sealing the goat serum by using 10% of goat serum at room temperature for 1h, removing the sealing liquid, adding a protein primary antibody, standing the mixture at 4 ℃ overnight, wherein the ratio of the primary antibody to the 10% of goat serum is 1: 200;

after standing overnight, removing the primary antibody, washing with PBS for three times, adding a secondary antibody in a dark place after washing for 5min each time, removing the secondary antibody after incubation for 1h in a dark place at room temperature, adding Fluoroshield with DAPI into cells, observing a dyeing result under a fluorescence microscope, wherein the ratio of the secondary antibody to 10% goat serum is 1: 1000.

4. the method for establishing the urine cell-induced pluripotent stem cell of the acute kidney injury patient according to claim 1, wherein the method for transferring the murine OCT4, SOX2, KLF4 and c-MYC transcription factor expression vector TetO-FUW-OSKM and the activating expression vector FUW-M2 rtTA into the urine kidney cell A by the nuclear transfection technology in the step 2 comprises the following steps:

20, culturing 100ml of Escherichia coli containing TetO-FUW-OSKM plasmid overnight, and culturing 100ml of Escherichia coli containing FUW-M2 rtTA plasmid overnight;

step 21, selecting restriction endonuclease Fspl to respectively perform enzyme digestion on the plasmids in the step 20, then respectively taking 20 mu l of enzyme-digested plasmids and the original plasmids to perform agarose gel electrophoresis, and identifying whether the plasmids are cut;

step 22, determining that the plasmid is linearized by enzyme digestion, recovering the plasmid by an ethanol precipitation method, and storing at-20 ℃ for later use;

step 23, urine kidney cell A is placed in a cell culture dish and is subjected to 5% CO at 37 DEG C ₂ Performing constant-temperature culture under the condition of (2);

step 24, observing after culturing for 24h, digesting the cells into single cells by using 0.05% of pancreatin when the cells reach 80-90% of confluence, and adding fibroblast culture solution consisting of 84% of high-sugar DMEM, 15% of FBS and 1% of Pen Strep to stop digestion;

step 25, centrifuging for 5min at 1500rpm, removing supernatant, and cleaning once by using DPBS;

and step 26, preparing a nuclear transfer liquid, and transferring the linearized TetO-FUW-OSKM, FUW-M2 rtTA and pCMV-tdTomato into the urine kidney cell A by using a nuclear transfer instrument to obtain a urine kidney cell B.

5. The method for establishing the urine cell-induced pluripotent stem cell line of the acute kidney injury patient according to claim 1, wherein the method for establishing the urine cell-induced pluripotent stem cell line in the step 3 comprises the following steps:

step 300, culturing mouse fetal fibroblasts with generation not higher than P4 generation in a culture dish, and treating the cells for 2.5-3 hours by mitomycin-C with concentration of 10 mug/ml when the cells reach 95% confluence;

step 301, digesting cells with 0.05% pancreatin, terminating digestion of cell culture fluid, and centrifuging;

step 302, resuspend and count cells in cell culture medium at 1.0X 10 per well ⁵ Adding each cell into 0.1% gelatin for 30minIn a four-well plate, 5% CO at 38.5 ℃ ₂ Culturing at constant temperature to obtain a feeding layer;

303, verifying whether the TetO-FUW-OSKM and the FUW-M2 rtTA are transferred into urine kidney cells A at the same time through PCR, and placing the cells into a four-hole culture plate after verification;

304, digesting with 0.05 percent pancreatin, centrifuging after the digestion of the cell culture solution is stopped, and discarding the culture solution;

step 305, resuspending and counting the cells by using a stem cell culture solution;

step 306, 1.5X 10 wells per four-well plate ⁴ The cells were added to the feeder layer at 38.5 ℃ with 5% CO ₂ Performing constant-temperature culture under the condition (1);

and 307, changing liquid every day, observing, and after the clone grows out, carrying out passage by a mechanical method, and then carrying out passage by pancreatin single cells.

6. The method for establishing the urine cell-induced pluripotent stem cells of the acute kidney injury patient as claimed in claim 5, wherein the step 303 of verifying whether the TetO-FUW-OSKM and FUW-M2 rtTA are simultaneously transferred into the urine kidney cells A by PCR comprises the following steps: screening out monoclonal cell strains with drug resistance by using a neomycin resistance gene in a pCMV-tdTomato plasmid, then respectively extracting genome DNA of each cell strain, and identifying whether the TetO-FUW-OSKM and FUW-M2 rtTA are simultaneously transferred into cells by PCR;

identification of TetO-FUW-OSKM includes the amino acid sequence as shown in SEQ ID NO: 1, and the sequence shown in SEQ ID NO: 2, or a reverse primer;

identification FUW-M2 rtTA includes the amino acid sequence as shown in SEQ ID NO: 3, and the upstream primer as set forth in SEQ ID NO: 4, or a reverse primer as shown in the figure.

7. The method for establishing urine cell-induced pluripotent stem cells of a patient with acute kidney injury according to claim 6, wherein the stem cell culture solution in step 305 comprises 40% KnockOut DMEM, 10% KnockOut SR, 24% DMEM/F12, 24% Neurobasal Medium, 0.5% N-2 Supplement, 1% B-27 Supplement, 0.125. mu.g/ml BSA, 2.5. mu.g/ml insulin, 1. mu.mol/L L-Glutamin, 0.05. mu.mol/L2-Mercaptoethanol, 0.5% NEAA, 1% Pen, 3. mu. mol/L CHIR99021, 1. mu. mol/L PD0325901, 2. mu. mol/L SB 432, 2000UI/ml LIF, 16. ang/ml FGF, and 50. mu. ml VC.

8. The method for establishing the urine cell-induced pluripotent stem cell of the acute kidney injury patient according to claim 5, wherein the step 3 of immunofluorescent identification of the pluripotent molecule of the cell line comprises the following steps:

step 310, removing stem cell culture solution in the four-hole culture plate, washing the stem cell culture solution with PBS for 5min once, fixing the stem cell culture solution with 4% paraformaldehyde for 10min, washing the stem cell culture solution with PBS for three times again, wherein the washing time for each time is 5min, and washing the stem cell culture solution with PBS for three times again after 1% Triton is permeated for 1h at room temperature, and the washing time for each time is 5 min;

and 311, after the cleaning is finished, sealing the goat serum by using 10% of goat serum at room temperature for 1h, removing the sealing liquid, adding a protein primary antibody, standing the mixture at 4 ℃ overnight, wherein the ratio of the primary antibody to the 10% of goat serum is 1: 200 of a carrier;

and 312, after standing overnight, removing the primary antibody, washing with PBS for three times, adding a secondary antibody in a dark place after washing for 5min each time, removing the secondary antibody after incubating for 1h in a dark place at room temperature, adding fluoroserine with DAPI into cells, and observing a dyeing result under a fluorescence microscope, wherein the ratio of the secondary antibody to 10% goat serum is 1: 1000.