CN114984049A - Application of exosome derived from human umbilical cord mesenchymal stem cells in preparation of medicine for treating acute kidney injury - Google Patents

Abstract

The invention provides application of exosome derived from human umbilical cord mesenchymal stem cells in preparation of a medicine for treating acute kidney injury. According to the invention, biochemical, immunohistochemical and western blot researches show that in a mini pig ischemia reperfusion model, exosomes derived from hUC-MSC (human umbilical cord mesenchymal stem cell) are injected intravenously, so that the renal function can be obviously improved, renal tubular injury and inflammatory reaction can be alleviated, tubular cell proliferation can be promoted, and renal tubular angiogenesis can be stimulated. Furthermore, the expression levels of renal BMP-7 and kloth, two nephroprotective factors, were significantly reduced at 120 min post-ischemia and 72 h post-reperfusion, whereas injection of the hhc-MSC-derived exosomes partially restored their expression. According to the invention, the I/R-induced AKI mini-pig model is successfully constructed, and the therapeutic effect that the I/R-induced AKI is obviously relieved by the exosome derived from the intravenous injection hUC-MSC is observed.

Description

Application of exosome derived from human umbilical cord mesenchymal stem cells in preparation of medicine for treating acute kidney injury

Technical Field

The invention belongs to the field of biological medicine, and relates to exosome derived from human umbilical cord mesenchymal stem cells, in particular to application of exosome derived from human umbilical cord mesenchymal stem cells in preparation of a medicine for treating acute kidney injury.

Background

Acute Kidney Injury (AKI) is a common complication of critically ill patients, and is characterized in that the Glomerular Filtration Rate (GFR), a main index of renal function, is rapidly reduced, the incidence rate is 0.21%, and the mortality rate is high in nearly 5% of hospitalized patients. The causative factors of AKI are complex and include circulating hypovolemia, Ischemia reperfusion (I/R) injury, sepsis, trauma, nephrotoxic drugs, etc. Researchers have constructed animal models and developed preclinical studies aimed at improving both short-term and long-term prognosis in patients with AKI, but with limited progress. Treatment of AKI is still largely based on removal or reversal of pathogenesis, with supportive treatment such as active regulation of blood volume and improvement of electrolyte disturbances while awaiting recovery of renal function, and renal replacement therapy if recovery is too long or impossible. In addition to etiological treatments, there is currently no specific treatment for AKI, and a significant proportion of patients with AKI progress to Chronic Kidney Disease (CKD).

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides the application of the exosome derived from the human umbilical cord mesenchymal stem cells in preparing the medicine for treating the acute kidney injury, and the application of the exosome derived from the human umbilical cord mesenchymal stem cells in preparing the medicine for treating the acute kidney injury aims to solve the technical problem that the effect of the medicine in the prior art for treating the acute kidney injury is poor.

The invention provides application of exosome derived from human umbilical cord mesenchymal stem cells in preparation of a medicament for treating acute kidney injury.

The invention uses the exosome derived from human umbilical cord mesenchymal stem cells (hUC-MSC) to treat an I/R induced AKI miniature pig model by intravenous injection, and then evaluates the protective effect of exosome on kidney through renal function, renal pathology and renal injury markers.

According to the application, biochemical, immunohistochemical and western blot researches, the exosome from the hUC-MSC is injected into a small pig ischemia reperfusion model through veins, so that the renal function can be obviously improved, the renal tubular injury and inflammatory reaction can be alleviated, the expression of PCNA in the renal tubular epithelial cells of the small pig can be promoted, the proliferation and the tissue regeneration of the renal tubular epithelial cells can be promoted, and the generation of renal small blood vessels can be stimulated.

The results of the present study show that infusion of MSC-derived exosomes can partially restore Klotho downregulation by I/R. The results of the invention also show that the expression levels of two kidney protection factors, namely kidney BMP-7 and kloth, are remarkably reduced 72 hours after 120 minutes of ischemia and reperfusion, and the expression of the exosome derived from the hUC-MSC can be partially recovered by injecting the exosome.

AKI is characterized by tubular epithelial cell death and tubular dysfunction with impaired glomerular filtration barrier and vascular endothelial cell injury. In the invention, the I/R-induced AKI mini-pig kidney tissues have the observed that the expression levels of endothelial markers VEGFA, VEGFR2 and CD31 are remarkably reduced, and the application of the exosome can promote the recovery of the endothelial markers, which indicates that the exosome derived from hUC-MSC has the potential of repairing a microvasculature and stimulating the regeneration of blood vessels.

Compared with the prior art, the invention has the advantages of positive and obvious technical effect. The invention is a first large animal research for exploring the treatment effect of the hUC-MSC-derived exosome on AKI, and through successfully constructing an I/R-induced AKI miniature pig model, the I/R-induced AKI treatment effect is obviously relieved by intravenous injection of the hUC-MSC-derived exosome, and no adverse reaction related to treatment is observed.

Drawings

Figure 1 shows the effect of exosomes on piglet kidney function; (A, C) baseline blood creatinine and blood urea nitrogen concentrations in the various groups of miniature pigs; (B, D) serum creatinine and blood urea nitrogen concentrations 72 hours after renal reperfusion in each group of piglets. SCr: (ii) blood creatinine; BUN: blood urea nitrogen; d0: before operation; d3: 72 hours after surgery; and Sham: a sham operation group; I/R: ischemia reperfusion; exo: an exosome. Data are expressed as mean ± sem; *: p is less than 0.05; **: p is less than 0.01.

FIG. 2 shows renal pathology HE staining of miniature pigs; (A) HE staining of piglet kidney tissue, arrows pointing to damaged tubules, Scale bar 100 μm; (B) the results of renal tubular injury scoring calculated from HE staining were averaged for 4 specimens per group, each specimen scored for 3 fields. And Sham: a sham operation group; I/R: ischemia reperfusion; exo: an exosome; veh: and (3) a solvent. Data are expressed as mean ± sem; *: p is less than 0.05; **: p is less than 0.01.

FIG. 3 shows that exosomes reduce the expression of mini-pig kidney injury markers Kim-1 and NGAL; (A) renal tissue NGAL immunohistochemical staining, Scale bar 100 μm; (B) counting positive tubules according to NGAL staining results, wherein each group comprises 4 samples, each sample is counted according to 3 visual fields, and an average value is obtained;

(C) detecting the expression levels of Kim-1 and NGAL in kidney tissues by Western blot, and taking GAPDH as an internal reference; (D-E) calculating a Kim-1 and NGAL relative expression histogram according to the result of the Western blot strip gray value of the kidney tissue. And Sham: a sham operation group; I/R: ischemia reperfusion; exo: an exosome; veh: a solvent; kDa: kilodaltons; . Data are expressed as mean ± sem; *: p is less than 0.05; **: p is less than 0.01.

Figure 4 shows that exosomes reduce the level of apoptosis; (A) TUNEL assay fluorescence photograph of kidney tissue, Scale bar 100 μm; (B) the number of positive cells calculated from TUNEL test fluorograms was averaged for each group of 4 samples, each sample counted according to 3 fields; (C) detecting the expression level of the cleared caspase 3 in the kidney tissue by Western blot, and taking GAPDH as an internal reference; (D) and (3) calculating a relative expression quantity histogram according to the result of the Western blot band gray value of the kidney tissue. And Sham: a sham operation group; I/R: ischemia reperfusion; exo: an exosome; veh: a solvent; kDa: kilodalton; . Data are expressed as mean ± sem; *: p is less than 0.05; **: p is less than 0.01.

Figure 5 shows that exosomes reduce the level of programmed necrosis; (A) renal tissue phosphorylated MLKL immunofluorescent staining, Scale bar 200 μm; (ii) a (B) Relative fluorescence intensities calculated from photographs of phosphorylated MLKL fluorescent staining were averaged for 4 samples per group, each sample calculated from 3 fields; (C) detecting the expression levels of p-MLKL and MLKL in kidney tissues by Western blot, and taking GAPDH as reference protein; and (D-E) performing semi-quantitative calculation according to the result of the grey value of the Western blot band of the kidney tissue, drawing a histogram of relative expression and performing statistical analysis. And Sham: a sham operation group; I/R: ischemia reperfusion; exo: an exosome; veh: a solvent; kDa: kilodalton; data are expressed as mean ± sem; *: p is less than 0.05; **: p is less than 0.01.

Figure 6 shows the effect of exosomes on inflammation indicators; (A) kidney tissue F4/80 immunohistochemical staining, Scale bar 100 μm; (B) calculating the positive rate according to the pictures of F4/80 immunohistochemical staining, each group of 4 samples, each sample is calculated according to 3 fields, and taking the average value; (C-F) detecting the expression levels of inflammatory factors MCP-1, TNF-alpha, IL-1 beta and IL-10 by fluorescent quantitative PCR; (H) detecting the expression levels of p-NF-kappa B, NF-kappa B, p-STAT3 and STAT3 in kidney tissues by Western blot, and taking GAPDH as an internal reference; (G, I-K) histogram of relative expression calculated from the results of the Western blot band intensity values of the kidney tissues. And Sham: a sham operation group; I/R: ischemia reperfusion; exo: an exosome; veh: a solvent; kDa: kilodaltons; data are expressed as mean ± sem; *: p is less than 0.05; **: p is less than 0.01.

FIG. 7 shows the effect of exosomes on vascular indices; (A) immunofluorescent staining for renal tissue vascular indices VEGFA and CD31, Scale bar 200 μm; (B-C) calculating relative fluorescence intensities from immunofluorescence photographs of VEGFA and CD31, 4 samples per group, each sample calculated from 3 fields, and averaging them; (D) detecting the expression levels of CD31, VEGFA and VEGFR2 in kidney tissues by Western blot, and taking alpha-Tubulin as an internal reference; (E-G) calculating a histogram of relative expression according to the result of the Western blot band gray scale value of the kidney tissue. And Sham: a sham operation group; I/R: ischemia reperfusion; exo: an exosome; veh: a solvent; kDa: kilodalton; data are expressed as mean ± sem; *: p is less than 0.05; **: p is less than 0.01.

FIG. 8 shows the effect of exosomes on nephroprotective factor and regeneration index; (A) immunohistochemical staining of the renal tissue regeneration molecule PCNA (200 ×); (B) calculating the positive rate according to immunohistochemical staining photographs of PCNA, wherein each group comprises 4 samples, each sample is calculated according to 3 visual fields, and the average value of the samples is taken; (C) detecting the expression levels of PCNA, Klotho and BMP-7 in the kidney tissues by Western blot, and taking alpha-Tubulin as an internal reference; (D, E-F) calculating relative expression quantity histogram according to the result of Western blot strip gray value of kidney tissue. And Sham: a sham operation group; I/R: ischemia reperfusion; exo: an exosome. Data are expressed as mean ± sem; *: p is less than 0.05; **: p is less than 0.01.

Detailed Description

Example 1: preparation of I/R-induced AKI miniature pig model

1.1 materials

Guangxi Bama miniature pigs were purchased from Thai and Biotech Inc. (license number SCXK (Su) 20170010), female, weighing 15-20 kg. The animal is raised in a large animal raising room of south hospital of east hospital of Shanghai city, the ambient temperature is 20-25 ℃, and the illumination is performed alternately in light and dark for 12 hours every day. 1-3 pigs are fed in each cage, the cages are cleaned every day, and the pigs are disinfected regularly. The feed is fed with standard feed (Taizhou Taihe Biotech Co., Ltd.) and water is freely drunk twice a day. All were acclimatized for 2 weeks prior to the experiment. The protocol was ethically prepared and approved by the animal ethics committee of eastern hospital of Shanghai, affiliated with university of Hospital, and all procedures on experimental Animals followed the American National Research Council (National Research Council) guidelines for the Care and Use of experimental Animals (1996 edition) (Guide for the Care and Use of Laboratory Animals).

1.2 methods

1.2.1 Experimental groups

The 16 piglets were randomly divided into 4 groups of 4 piglets, each group having ischemia duration of 0, 60, 90, 120 minutes.

1.2.2 surgical procedure

(1) Fasting was performed 24 hours before surgery. Sterilizing the operating room environment by ultraviolet irradiation; sterilizing surgical instruments by high-pressure steam; the consumables such as the surgical drape, the surgical gown, the gauze and the like use the disposable packaging products sterilized by the ethylene oxide. The operating room temperature was set to around 25 ℃.

(2) Sutai 50 intramuscular injection anesthesia: 1 bottle of the sutai powder was dissolved in 10mL of physiological saline and administered at 5 mg/kg body weight, i.e., 0.2mL/kg body weight. A proper amount of Shutai solution is extracted by a 10mL syringe, an infusion needle (No. 7 needle head, black) is connected, and gas in the tube is discharged. The best site for intramuscular injection is the junction of the hairy and hairless areas behind the ear, where the thickness of the fat layer is small. The small pig is fixed by the cage, the small pig is prevented from running in a large range, the needle head is pricked into muscle, the syringe piston is pushed, and intramuscular injection anesthesia is completed.

(3) Establishing a venous access: after sufficient anesthesia, the hair on the back of the ear is shaved by an electric shaver, and the skin on the back of the ear is moistened by alcohol gauze to make the veins on the back of the ear appear. The proximal end of the vein is compressed at the root of the ear to fill it. The venous indwelling needle (24G x 0.75) is inserted into the vein from the distal end thereof at an angle as parallel as possible, the needle is inserted about 1mm after the blood return is seen, the flexible tube needle is completely inserted into the blood vessel while the needle core is pulled out, and the indwelling needle is fixed to the ear with a transparent adhesive tape.

(4) Tracheal intubation: the small pig is fixed on the operating table in the supine position, and the upper jaw is fixed on the wood plate with the hole by a bandage. The assistant uses the bandage to raise the mandible of the miniature pig forcefully, the operator holds the laryngoscope with one hand to expose the oral cavity, the oropharynx and the epiglottis of the miniature pig, holds the disposable trachea cannula with one hand and inserts the trachea cannula into the glottis, and extracts the guide wire. Whether the intubation is successful is judged by lung auscultation or observation of the airflow condition at the mouth of the tracheal intubation. If the yellowish liquid is left in the tube, it may be mistakenly entered into the esophagus and the tube needs to be reinserted. After confirming that the end of the tube is positioned in the trachea, about 10mL of air is injected into the balloon and the endotracheal tube is fixed by taping. The size of the tracheal cannula is selected according to the body weight, and a small pig with the body weight of 20kg can adopt a No. 6.5 tracheal cannula.

(5) Whole-body heparinization and fluid infusion: heparin sodium (1mg/kg) is injected through an ear back indwelling needle, then an infusion set and a physiological saline bag are connected, the flow rate is adjusted, and the intraoperative fluid infusion is about 500 mL.

(6) Device connection and setup: an electrotome electrode plate is arranged below the waist and back of the miniature pig. The miniature pig is fixed on an operating table in a supine position. The monitor leads are respectively connected with the left upper Limb (LA), the left Lower Limb (LL) and the right upper limb (RA) of the miniature pig, the finger pulse oxygen detection probe is clamped at the tail (hair needs to be shaved) or the nipple, and the monitor is started in the whole operation process. Connected to a ventilator, 2% isoflurane was used to maintain anesthesia. The breathing rate of the respirator is 1:2, the oxygen flow is 1L/min, the breathing frequency is 15-20 times/min, and the pre-adjusted tidal volume is about 10-15 mL/kg. The room temperature of the operating room is constant at 25 +/-2 ℃.

(7) Skin preparation, disinfection and sheet laying: skin preparation ranged from the costal margin to the anterior abdomen of the groin, shaved and scrubbed clean, and the incision locations were marked with a marker. Sterilizing with iodophor for 3 times, and sequentially spreading small sheets, middle sheets and hole towels.

(8) Left renal ischemia reperfusion: the middle and lower abdomen were opened along the midline of the abdomen, pushing open the bowel and exposing the bilateral kidneys. The left renal artery was isolated and the blood supply was blocked with a non-destructive vascular occlusion band and a luminal cuff, with the left kidney becoming dark in color and increasing in volume. The control group was taped only to the left renal artery and not ligated. After 0 min, 60 min, 90 min or 120 min following ischemia, the suture guide was loosened and the vasoocclusive tape was carefully removed, taking care not to injure the vessels and ureters and observing the color of the left kidney as reddish.

(9) Right nephrectomy: the right kidney was dissociated and the renal pedicle was ligated, the kidney was excised, and the kidney tissue was collected.

(10) Closing the abdomen and suturing: sequentially sewing the peritoneal layer, the muscle layer and the subcutaneous layer, and closing the two sides without leaving dead space; then the triangular needle is used for sewing the skin by adopting an intracutaneous suture method; the incision was again disinfected with iodophor and covered with gauze to protect the incision. And finally, the piglet is transferred to the vicinity of the heater, and the temperature is properly raised to maintain the body temperature of the piglet and promote the piglet to recover from postoperative anesthesia. Meanwhile, the vital signs of the miniature pigs are closely focused until anesthesia resuscitation.

(11) And (3) postoperative treatment: the minipig was anesthetized with sutai again 72 hours post-surgery, the abdominal cavity was opened along the original incision, the left kidney was exposed, the renal pedicle was ligated and the left kidney was excised, leaving the kidney tissue. The piglets were sacrificed by intravenous potassium chloride and the carcasses were disposed of as prescribed.

Example 2: preparation of exosome derived from human umbilical cord mesenchymal stem cells

2.1 culture of human umbilical cord mesenchymal Stem cells

(1) Preparation of culture solution

Taking out the serum substitute from-20 ℃, thawing in 37 ℃ water bath, quickly taking out from a water bath pot after thawing, subpackaging by using a 50mL centrifuge tube, immediately preparing the MSC culture solution or sealing by using a sealing film, and storing at-20 ℃ to avoid repeated freezing and thawing. The MSC culture solution was prepared in a ratio of 94% α -MEM culture solution + 5% serum replacement + 1% penicillin-streptomycin mixed solution. The above MSC culture broth was filtered through a 0.22 μm syringe filter to remove insoluble particles. The filtered MSC culture solution is used for cell culture or is temporarily stored at the temperature of 2-8 ℃ after being sealed by a sealing film and is used within 2 weeks.

(2) Cell resuscitation

Cells frozen in liquid nitrogen or-80 ℃ were removed and rapidly thawed in a 37 ℃ water bath within 1-3 minutes. The cell suspension in the frozen tube is pipetted into an appropriate amount of α -MEM culture medium (generally, more than ten times the volume of the cell suspension), and the cells are washed by mixing. Centrifuge at 800rpm for 5 minutes and discard the supernatant. Resuspending the cell pellet at the bottom of the centrifuge tube with MSC culture solution, and mixing well. And (3) sucking 10 mu L of cell suspension to a cell counting plate, counting the cell suspension by using a full-automatic cell counter, and recording the number and the survival rate of the cells. According to 3X 10 ³ -6×10 ³ Cells are inoculated into a culture flask or a culture dish at a density of cells/square centimeter, and an appropriate amount of culture solution is added, wherein the volume of the culture solution in the culture dish is 10mL in a 10cm culture dish, and the volume of the culture solution in the culture flask is 30mL in a 600mL culture dish. Mixing cell suspension in culture flask or culture dish, uniformly distributing, standing at 37 deg.C and 5% CO ₂ And culturing in a constant temperature incubator under the saturated humidity condition. After several hours, the cells begin to adhere to the wall, and the culture medium is changed once in 1-3 days.

(3) Cell passage

When the cells are cultured to 80-90% of the bottom of the culture vessel, the cells can be subjected to subculture. The cells were removed from the incubator and the supernatant collected with a pasteur dropper. Adding DPBS into the culture bottle or the culture dish, slightly shaking to wash the cells, removing the DPBS by using a vacuum aspirator, and repeating for 2-3 times. An appropriate amount of cell digest was added to a culture dish full of cells, typically 3mL in a 10cm dish and 10mL in a 600mL flask. The vessel was gently shaken and then returned to the incubator and incubated at 37 ℃ for 10-20 minutes.

And (3) after digestion, uniformly blowing and stirring by using a Pasteur dropper to enable the MSC to be in single cell suspension, sucking the MSC into a centrifugal tube, flushing a culture vessel by using culture solution, transferring the obtained cell suspension into the centrifugal tube, diluting the digestion solution, and stopping digestion. Centrifuge at 800rpm for 5 minutes and discard the supernatant. Resuspending the bottom of the centrifuge tube with MSC culture fluidPrecipitating the cells, and mixing. Dividing the cell suspension into 3 equal parts, adding into new culture vessel, respectively, adding culture solution, mixing, adding 5% CO at 37 deg.C ₂ And subculturing in a constant-temperature incubator under the saturated humidity condition. The suspension can be divided into 2-6 equal parts according to the cell density for passage.

(4) Cell cryopreservation

Collecting supernatant, washing DPBS, digesting cells, and performing centrifugation operation and cell passage. Resuspending the cell sediment at the bottom of the centrifuge tube with 1-5mL of cell freezing solution, mixing well, adding into the cell freezing tube about 1mL per tube, and sealing with cut sealing film. The cells are frozen at a temperature reduction rate of about 5 ℃ per hour in a gradient manner, and finally stored at-80 ℃ or in liquid nitrogen. The cells can be placed in a gradient cryopreservation box, and then the cryopreservation box is directly placed in a refrigerator at the temperature of minus 80 ℃, or a cell cryopreservation tube is placed at the temperature of 4 ℃ for 2 hours, then the cell cryopreservation tube is transferred to the temperature of minus 20 ℃ for cryopreservation for 4 hours, and the cell cryopreservation tube can be moved to liquid nitrogen for long-term storage after overnight at the temperature of minus 80 ℃.

(5) Culture supernatant collection and processing

When the cells grow to 80-90% fusion, collecting the supernatant of 5-7 generation hUC-MSC cells, centrifuging at 4 deg.C for 20 min, retaining the supernatant, discarding the bottom cell precipitate, centrifuging at 4 deg.C for 2000g for 30 min, and discarding the bottom cell debris. The cell culture supernatant after cell debris removal by gradient centrifugation is used for extracting exosomes or is temporarily stored at-80 ℃.

2.2 ultracentrifugation method for the extraction of exosomes

(1) Filtering the supernatant with 0.22 μm needle filter to remove microvesicles with diameter of above 220nm, and replacing the filter after the filtration resistance is increased. The filtrate was transferred to an ultracentrifuge tube and placed on ice.

(2) Installing a pipe plug and a pipe cap, paying attention to air exhaust, sealing and strict balancing (the weight difference between two pipes which are symmetrically arranged in a matched mode is not more than 0.05g), wiping off liquid attached to the outer wall of the ultracentrifuge pipe, placing the ultracentrifuge rotor after confirming that no error exists, sealing the rotor cover, placing the rotor cover into an ultracentrifuge base, and preparing ultracentrifuge.

(3) Setting the temperature at 4 ℃ and 120000 g for 2.5 hours, vacuumizing the cavity of the ultracentrifuge, starting centrifugation after the pressure is lower than 200 mu m, and waiting by side observation until reaching the preset rotating speed and leaving without abnormality.

(4) After centrifugation was complete, the vacuum in the chamber was released (vacuum was released after "ready" display on the screen), the centrifuge tube was carefully and smoothly removed, and the remaining supernatant was gently decanted, and a pellet of about 5mm in diameter was visible to the naked eye.

(5) Resuspend the pellet with 15mL DPBS, rinse the exosomes, reduce impurities such as protein clumps, and filter again with a 0.22 μm filter.

(6) Transferring the filtrate to

An Ultra 100K centrifugal filter is centrifuged at 4 ℃ and 2000g for 15 minutes, and the residual solution in the centrifugal filter is observed until 200 and 500 mu L of the DPBS solution of the exosome are absorbed into an EP tube and stored at-80 ℃ for standby.

Example 3

3.1.1 objects and groupings

The number of the Bama miniature pigs is 16, the weight of the Bama miniature pigs is about 20kg, and the female pigs are randomly divided into 4 groups, wherein 4 pigs in each group are respectively as follows:

(1) sham + PBS group (Sham + Vehicle);

(2) sham + exosome group (Sham + Exo);

(3) I/R + PBS group (I/R + Vehicle);

(4) I/R + exosome group (I/R + Exo).

3.1.2 Experimental articles

ProFlex PCR System (Thermo Fischer, USA)

Fluorescent quantitative PCR system (Applied Biosystems, USA)

nanodrop oneC ultramicro spectrophotometer (Thermo Fischer, USA)

Orbitrap Exploris ^TM 480 Mass Spectrometry platform (Thermo Fischer, USA)

RNAeasy (TM) animal RNA extraction kit (Biyuntian, China)

Dithiothreitol (Biyuntian, China)

PrimeScript reverse transcription kit (TaKaRa, Japan)

TB Green Premix Ex Taq (TaKaRa, Japan)

DEPC water (Bi Yun Tian, China)

RNase/DNase removal spray (Biyuntian, China)

Kim-1 primary antibody # ab47635(Abcam, USA)

NF- κ B p65 Primary antibody # sc8008(Santa Cruz, USA)

Phospho-NF-. kappa. B p65 Primary antibody #3033(Cell Signaling Technology, USA)

STAT 3(124H6) primary antibody #9139(Cell Signaling Technology, USA)

Phospho-STAT 3 primary antibody #9145(Cell Signaling Technology, USA)

F4/80 primary antibody #28463-1-AP (Proteitech, USA)

VEGFA-anti # MA1-16626(Invitrogen, USA)

VEGFR2 Primary antibody #26415-1-AP (Proteitech, USA)

CD31 Primary antibody # PA5-32321(Invitrogen, USA)

PCNA primary antibody # ARG62605(Arigo, Taiwan)

Klotho primary antibody #1854-1(Santa Cruz, USA)

BMP 7 primary antibody #4693(Cell Signaling Technology, USA)

alpha-Tubulin primary antibody #2125(Cell Signaling Technology, USA)

MicroAmp Optical 96-well PCR plate-with skirt (Applied Biosystems, USA)

MicroAmp Optical fluorescent quantitative PCR transparent sealing plate film (Applied Biosystems, USA)

0.2mL PCR eight row tube (Axygen, USA)

PCR eight-row flat cover (Axygen, USA)

PCR tube rack box (ai you, China)

3.1.3 Experimental procedures

(1) Preoperative fasting, sutai anesthesia, venous access establishment, blood sampling, tracheal intubation, heparin injection, ventilator and monitor connection, isoflurane inhalation anesthesia, skin preparation, disinfection, drape, laparotomy, isolation and blockage of the left renal artery, and right renal resection as described in example 1.

(2) The exosomes (1X 10) were injected in groups during the operation ⁹ Particles/kg body weight) or an equal volume of PBS.

(3) And (3) after 120 minutes of ischemia, removing the blockage of the left renal artery, closing the abdomen, suturing, stopping isoflurane, disconnecting the breathing machine and the monitor after the miniature pig recovers the spontaneous respiration and revives, and transferring to a feeding room for continuous observation.

(4) And (3) after 72 hours of operation, intramuscular injecting Shutai anesthesia miniature pigs again, taking blood, opening the abdominal cavity along the original incision, exposing the left kidney, ligating the renal pedicle, excising the left kidney, and leaving the renal tissue. The piglets were sacrificed by intravenous potassium chloride and the carcasses were disposed of as prescribed.

3.1.4 porcine Kidney tissue RNA extraction and reverse transcription

The consumables used do not contain RNase or DNase, and the experiment table and the environment are sprayed to remove enzymes possibly existing in the environment in advance.

(1) Preparation of lysate

Dithiothreitol (DTT) was added to the lysate of the kit to a final concentration of 40 mmol/L. DTT is filled in 5g, the relative molecular mass is 154.25g/mol, 8.1 mL is added into a bottle, and the mixture is fully shaken and dissolved to prepare DTT stock solution with the concentration of 4 mol/L. To 1mL of the lysate was added 10. mu.L of DTT stock solution to a final concentration of 40 mmol/L. DTT stock was stored at-20 ℃ and DTT-containing lysates were stored at 4 ℃.

(2) Sample preparation

Cutting kidney tissue (about 15-20mg) of mung bean-sized piglet, placing in an EP tube, adding 100. mu.L of lysate (containing DTT), cutting the tissue pieces with an ophthalmic scissors, and grinding the tissue with a Tiangen electric high-speed tissue grinder for about 30 seconds. After the grinding was complete, the pestle was rinsed with 500. mu.L of lysis solution and the rinse was also collected in the EP tube. After homogenization, the homogenate is gently blown and beaten 8 to 10 times and left at room temperature for 3 to 5 minutes. The mixture was then centrifuged at about 14000 g for 2 minutes and the supernatant was transferred to a new centrifuge tube. After lysis and centrifugation of the tissue sample, the lower part of the centrifuge tube may have some gelatinous material, which is suitable for transfer to the next step as supernatant.

(3) Extraction of RNA by centrifugal column method

1) An equal volume of binding solution (600. mu.L) was added to the homogenate and the mixture was mixed by gentle inversion 3-5 times. At this time, a precipitate may be generated, which is a normal phenomenon.

2) The mixture (including the precipitate) was transferred to a purification column, centrifuged at 12000 g for 30 seconds, and the liquid in the collection tube was decanted. The maximum volume per column pass was 600. mu.L, and 2 column passes should be made. The centrifugal rotating speed and time can be adjusted according to actual conditions, and the same is applied below with the liquid passing completely.

3) Add 600. mu.L of Wash I, centrifuge at 12000 g for 30 seconds and discard the liquid in the collection tube.

4) Add 600. mu.L of Wash II, centrifuge at 12000 g for 30 seconds and discard the liquid in the collection tube. Repeat 2 times.

4) The residual liquid was removed by centrifugation at the highest speed (about 14000-16000 g) for 2 minutes.

5) Putting the RNA purification column into an RNA elution tube provided by the kit, adding 30-50 mu L of eluent, standing at room temperature for 2-3 minutes, cutting off the cover of the elution tube, centrifuging at the highest speed for 30 seconds to obtain a solution, namely purified RNA, and transferring the solution into a new enzyme-free EP tube. The eluent is required to be applied to the center of the cylindrical surface of the purification column so that it is completely absorbed. When the room temperature is lower, the eluent is preheated for a moment at 37 ℃ to help the yield. In addition, the solution after elution is added back to the original purification column again and then is subjected to centrifugal elution once, so that the yield can be improved by about 10% -30%; or eluting once more with a new eluent after the first elution, RNA is obtained in an amount of about 15% -40% of the first elution.

(4) RNA concentration determination

The concentration of the RNA solution was measured with a ultramicro spectrophotometer and zeroed with an eluent, and the amount of the sample was 1. mu.L. The loading volume required for the reverse transcription reaction, i.e., loading volume (μ L) ═ loading amount (500ng)/RNA concentration (ng/μ L), was calculated.

(5) Reverse transcription PCR

1) The kit stored at-20 ℃ was taken out and thawed on ice. The 10. mu.L reaction system, as shown in the table below, was prepared in the volume calculated above, loaded separately into eight-row calandria tubes, capped, shaken, mixed well and centrifuged slightly.

TABLE 3.1 reverse transcription PCR reaction System

2) Placing the eight rows of tubes into a PCR instrument, and setting the program as follows: A) 15 minutes at 37 ℃ (reverse transcription); B)85 ℃, 5 seconds (inactivation reaction of reverse transcriptase); C) keeping at 4 ℃. After confirming that no error exists, starting the program and starting the reaction.

3) The eight rows of tubes containing the reverse transcription product (cDNA) were removed from the instrument, centrifuged slightly, the lid opened, 40. mu.L of distilled water was added to each tube, and the product was diluted 5-fold and immediately used for fluorescent quantitative PCR or buffered at-20 ℃.

3.1.5 fluorescent quantitative PCR

(1) Primer design

The gene indexes to be detected are determined, the corresponding target gene numbers of the pig species are inquired on an NCBI gene platform, and then primers are designed through http:// www.ncbi.nlm.nih.gov/tools/primer-blast/website. The design principle of the primer comprises: 1) the designed primer is as close to the 3' end of the gene as possible, so that the amplification efficiency is ensured to the maximum extent; 2) the primer spans the intron as much as possible, and is not easily affected by the genomic DNA pollution; 3) The annealing temperatures (Tm values) of the two primers do not differ too much, preferably by <2 ℃; 4) the length of the product is preferably 80-200bp, and the longest length is not more than 300 bp; 5) conserved regions were avoided as much as possible. The sequences of the primers used in this study are shown in the following table:

TABLE 3.2 fluorescent quantitative PCR primer sequences

The primers were synthesized according to the designed sequences and purified by PAGE, trusted Suzhou Jinwei Zhi Co. After the synthesized primer sample is received, the primer powder is centrifuged at 12000 rpm for 5 minutes, distilled water is added according to the volume indicated by the label, and the primer powder is dissolved by vortex oscillation to prepare a primer stock solution with the concentration of 100 mu mol/L. Primer working solution with concentration of 10 μmol/L was prepared according to the ratio of stock solution to distilled water of 1: 10. Both the stock solution and the working solution were stored at-20 ℃.

(2) Preparation of the reaction System

The required samples and indexes are designed according to the experimental requirements, and the reaction quantity and the sample distribution on a 96-well plate are planned. The kit was taken out and stored at-20 ℃ and thawed on ice. A20. mu.L reaction system as shown in the following Table was prepared, added to each well of a 96-well plate, the plate was sealed with a sealing plate, air bubbles were removed, shaken and mixed well, and slightly centrifuged.

TABLE 3.3 fluorescent quantitative PCR reaction System

(3) Performing PCR amplification on the sample

Putting the eight-row pipes of the 96-hole PCR plate into a fluorescent quantitative PCR instrument, setting the sample and the target index name, adopting a two-step PCR amplification standard program, and setting the following steps:

1) first-stage pre-denaturation: at 95 ℃, 30 seconds and the cycle number of 1;

2) and (3) second-stage PCR reaction: cycle number of 40 at 95 deg.C, 5 seconds, 60 deg.C, 34 seconds;

3) third stage dissolution curve: 95 deg.C, 15 seconds, 60 deg.C, 95 deg.C, 15 seconds, and the procedure is ended.

After amplification, the 96-well plate was removed, the amplification curve and the lysis curve were analyzed, and the CT value was read.

(4) Calculation of relative expression amount results

GAPDH was used as an internal reference gene to calculate how much the expression of the target gene in the treatment group was doubled compared with that of the control group, and the result was 2 ^-ΔΔCt The calculation formula is as follows:

2 ^-ΔΔCt ＝2- ^{(Ct value of treatment group target gene-Ct value of treatment group reference gene) - (Ct value of control group target gene-Ct value of control group reference gene)}

Wherein, the CT value refers to the number of amplification cycles which are passed when the fluorescence signal of the amplification product reaches a set threshold value in the fluorescent quantitative PCR amplification process, and is generally 15-35.

3.2.1 improving renal function in an I/R induced AKI miniature pig model by infusion of MSC-derived exosomes

The 16-head Bama miniature pigs weighing about 18-20kg were randomly divided into 4 groups, namely Sham + PBS group (Sham + Vehicle), Sham + exosome group (Sham + Exo) I/R + PBS group (I/R + Vehicle) and I/R + exosome group (I/R + Exo). Blood was collected via the anterior vena cava at 72 hours before and after surgery, and renal function was measured using a kit (Nanjing King) with specific values as shown in Table 3.4. Fig. 1A and 1C show no significant difference in baseline values for blood creatinine and blood urea nitrogen between the four treatment groups. After 72 hours of operation, affected by the laparotomy, the blood creatinine of the control group miniature pigs is slightly increased compared with the baseline, and the blood urea nitrogen is not obviously changed. The blood creatinine and blood urea nitrogen values of the piglet in the I/R + PBS group are obviously increased compared with those in the control group, which indicates that an I/R induced AKI model is successfully constructed. The I/R + exosome group minipigs had significantly reduced blood creatinine compared to the simple I/R model group, suggesting that MSC-derived exosomes have a kidney protective effect on I/R-induced AKI (FIG. 1B). Due to the large intra-group individual differences in blood urea nitrogen concentration, there were no statistical differences between the I/R + PBS group and the I/R + exosome group, but the approximate trend was consistent with the blood creatinine values, also supporting that exosomes may have a renal protective effect (fig. 1D).

TABLE 3.4 Effect of exosomes on piglet Kidney function

3.2.2 Exo-body treatment improving Effect on Mini-pig Kidney pathological manifestations

Kidney tissues of the miniature pigs were taken and examined microscopically after 4% paraformaldehyde fixation, paraffin embedding, sectioning and HE staining. The observation under an optical microscope shows that the kidney of the piglet in the sham operation group has no obvious pathological change. In the I/R + PBS group, obvious renal tubule lumen expansion, renal tubular epithelial cell shedding to the lumen, interstitial cell infiltration, interstitial edema and hyaline cast and granular cast formation can be seen. The pathological changes in the I/R + exosome group are obviously improved. Fig. 2A is a representative pathological photograph of each group. The severity of the tubular injury was scored and the results showed that the I/R + PBS group had significant acute tubular injury compared to the sham group, while the I/R + exosome group had significantly reduced injury compared to the simple I/R model group (fig. 2B). The above results indicate that MSC-derived exosomes can reduce tubular injury in an I/R-induced AKI mini-pig model.

3.2.3 exosomes reduce expression of Mini-pig Kidney injury markers Kim-1 and NGAL

To further determine whether MSC-derived exosomes have a renal protective effect on I/R-induced AKI, this section of the study analyzed the expression of kidney injury markers Kim-1 and NGAL in kidney tissue by Western blot and immunohistochemistry. The results show that the expression levels of Kim-1 and NGAL in the kidney of the piglet in the sham operation group are very low, and the expression levels are high after the I/R induces AKI, while the expression level of NGAL in the exosome treatment group is obviously reduced compared with that in the I/R + PBS group, and the Western blot and immunohistochemical results are consistent (FIGS. 3A and C). The image was semi-quantitatively analyzed, and the results showed that the difference in the expression levels of Kim-1 and NGAL between the I/R + PBS group and the I/R + exosome group was statistically significant, indicating that exosomes could significantly reduce the expression level of renal NGAL in AKI animal models (FIGS. 3B and D-E).

3.2.4 MSC-derived exosomes reduce the level of apoptosis in Mini-pig Kidney tissue

Tubular epithelial apoptosis is an important mechanism in the development of AKI. In this section, we evaluated the possible mechanism of MSC-derived exosomes to exert kidney-protective effects by TUNEL assay and detection of the expression level of clear Caspase 3, an important marker of apoptosis. In the kidney of the piglet in the sham group, the proportion of cells undergoing TUNEL positive apoptosis was low, while in the I/R induced AKI piglet kidney, the positive rate was high, and the positive cells were mainly distributed in the renal tubule wall, indicating that tubular epithelial cell apoptosis is the main factor, and the positive rate of the exosome treatment group was significantly reduced compared with the I/R + PBS group (FIGS. 4A-B). Consistent with the results of the TUNEL trial, clear Caspase 3 was low expressed in the sham group and high expressed in the I/R group, and exosome treatment significantly reduced the level of clear Caspase 3, suggesting that exosomes may alleviate AKI by reducing tubular epithelial apoptosis (fig. 4C-D).

3.2.5 Exo-body treatment group piglet kidney programmed necrotic cells are remarkably reduced

The phosphorylation level of MLKL was positively correlated with the degree of programmed necrosis. In this section of the study, we examined the phosphorylation levels of MLKL by immunofluorescence and Western Blot to map the apoptotic cell status. The results show that the results of the two experimental methods are consistent. The expression level of total MLKL was substantially consistent in each group, but there was a difference in the degree of phosphorylation. In the kidney of the piglet in the sham group, the phosphorylation level of MLKL was low, while in the I/R-induced AKI piglet kidney, the positive rate and expression level of p-MLKL were significantly increased, while the phosphorylation level of MLKL in the exosome-treated group was significantly decreased compared to the I/R + PBS group (fig. 5A-E). The above results indicate that inhibition of tubular epithelial cell apoptosis may also be one of the important mechanisms for exosomes to exert AKI kidney protective effects.

3.2.6 Effect of MSC-derived exosomes on Kidney inflammation indicators

F4/80 is a marker for macrophages, and this study analyzed macrophage infiltration by immunohistochemical examination of F4/80. As a result, no obvious F4/80 positive cells were found in the kidney of the piglet in the sham group, while F4/80 positive staining of the renal interstitium, i.e., macrophage infiltration, was seen in the I/R group, and the amount of macrophage infiltration was significantly reduced by MSC-derived exosome treatment (FIG. 6A). Quantitative analysis of the number of positive cells revealed that the I/R group had significantly increased macrophage infiltration, while the exosome-treated group had significantly decreased macrophage infiltration compared to the I/R + PBS group (FIG. 6B).

The fluorescent quantitative PCR is a method of adding a fluorescent group into a PCR reaction system, monitoring the change of the amount of each cycle of amplified product in the whole PCR amplification reaction in real time by using fluorescent signal accumulation, and finally carrying out quantitative analysis on an initial template through a CT value (namely the cycle number) and a standard curve. The fluorescent quantitative PCR result shows that the exosome can promote the mRNA expression of the anti-inflammatory factor IL-10 and inhibit the expression of the proinflammatory cytokines MCP-1, TNF-alpha and IL-1 beta (FIG. 6C-F).

The NF-kB transcription factor family can regulate the expression and the production of inflammatory factors, and the NF-kB pathway can be activated under the action of various stimulations. It is reported that the TWEAK-induced non-classical pathway is activated during the development of AKI. In addition, many aspects of cell growth, survival and death are regulated by proteins in the Signal Transducer and Activator of Transcription (STAT) family. There is evidence that STAT3 plays an important role in the induction and maintenance of the pro-inflammatory microenvironment. In this study, phosphorylation levels of typical inflammatory factors NF-. kappa.B and STAT3 were low in the sham group, and increased significantly after AKI induction by I/R, while the exosome-treated group was significantly decreased compared to the I/R + PBS group (FIGS. 6G-K).

The above results indicate that MSC-derived exosomes may also exert kidney-protective effects by inhibiting I/R-induced inflammatory responses in AKI.

3.2.7 Effect of MSC-derived exosomes on Kidney angiogenesis indicators

Reduced vascular endothelial density is a common phenomenon in animal models of I/R-induced AKI, whereas Vascular Endothelial Growth Factor A (VEGFA) and its receptors (e.g. VEGFR 2) induce neovascularization. In the research, the expression condition of the related indexes of the vascular endothelium is discussed through immunofluorescence and Western Blot, and the results obtained by the two methods have better consistency. The results show that I/R induced down-regulation of VEGFA, VEGFR2 and CD31 (all three endothelial biomarkers) in kidney tissue of AKI mini pig model compared to sham group, but the expression level of exosome treated group was significantly higher than I/R + PBS group but not as high as that of sham group (fig. 7A-G). These results indicate that MSC-derived exosomes can reduce I/R-induced AKI by reducing I/R-induced reduction in vascular endothelial density.

3.2.8 exosome therapy promotes upregulation of nephroprotective factor and proliferation of tubular epithelial cells

Proliferating Cell Nuclear Antigen (PCNA) is closely related to DNA synthesis in cells and is a good indicator of cell proliferation status. The expression condition of PCNA in the kidney of the piglet is detected by two methods of immunohistochemical staining and Western blot, and a consistent result is obtained. PCNA is expressed less in normal kidney tissue (sham) and increased significantly in the I/R induced AKI kidney; administration of exosomes further upregulated the expression level of PCNA (fig. 8A-D). Immunohistochemical staining showed that PCNA positive cells were found mainly in the renal tubules. This population of cells was increased in the I/R-induced AKI kidney and the number of exosomes was further increased by infusion compared to the sham group. These results indicate that MSC-derived exosomes may have a role in promoting I/R-induced tubular epithelial cell regeneration in AKI.

Often accompanied by a decrease in the level of the protective protein Klotho in AKI and CKD. In this study, I/R-induced expression levels of Klotho in the mini-pig model of AKI were significantly reduced compared to the sham group, while expression levels of Klotho rose after treatment with MSC-derived exosomes (fig. 8C and E). Bone morphogenetic protein 7 (BMP-7) is an important factor in kidney development and is highly expressed in renal tubules, glomerular epithelial cells, podocytes, and renal vascular cells. In this study, the expression level of renal BMP-7 was also significantly down-regulated in the case of I/R, while the BMP-7 expression level in the exosome-treated group was significantly higher than in the I/R + PBS group (FIGS. 8C and F). These results indicate that the use of hUC-MSC-derived exosomes partially restores the I/R-induced downregulation of renal protective factors.

Sequence listing

<110> Shanghai City eastern Hospital (affiliated eastern Hospital of Tongji university)

Application of exosome derived from human umbilical cord mesenchymal stem cells in preparation of medicine for treating acute kidney injury

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<213> Artificial Sequence (Artificial Sequence)

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Claims (1)

1. Use of exosome derived from human umbilical cord mesenchymal stem cells in preparation of a medicament for treating acute kidney injury.

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