A preservation solution for mitigating early injury of steatotic transplanted livers
By using reconstituted fibroblast growth factor 21 protein during the cold preservation process of liver transplantation, the ischemia-reperfusion injury problem of steatotic donor livers was solved, significantly improving liver function, reducing transplant liver damage and steatosis, and improving the quality of donor livers.
Patent Information
- Application Number
- CN202410617676.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-17
- Publication Date
- 2026-03-20
- Estimated Expiration
- 2044-05-17
AI Technical Summary
Current technologies cannot effectively prevent and treat ischemia-reperfusion injury in steatotic donor livers during liver transplantation, leading to early allogeneic graft dysfunction and poor prognosis, and static cold preservation methods have limited effectiveness.
During the cold preservation of liver transplantation, reconstituted fibroblast growth factor 21 (FGF21) protein was used. By adding fibroblast growth factor 21 to the preservation solution, ischemia-reperfusion injury was improved, tissue damage, apoptosis and inflammation were inhibited, and intrahepatic lipid accumulation was improved.
It significantly improves ischemia-reperfusion injury in transplanted livers with steatosis, reduces the risk of transplanted liver dysfunction, improves the success rate of donor liver use, reverses hepatic steatosis, reduces cell apoptosis and inflammation, and improves intrahepatic lipid accumulation.
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Figure CN119498274B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biomedical technology, and particularly relates to a preservation solution for reducing early injury of steatotic liver grafts. BACKGROUND
[0002] Liver transplantation (LT) is the most effective radical treatment for patients with end-stage liver disease. Early graft injury is common after LT and can lead to early allograft dysfunction, which is closely related to poor prognosis of the graft and the recipient. Ischemia-reperfusion (I / R) injury is the main cause of early graft injury and is also the main determinant of liver shortage.
[0003] In recent years, in order to overcome the contradiction between the demand and supply of organs, the use of steatotic donor livers has increased year by year in major transplant centers. However, steatotic donor livers are more sensitive to I / R injury and are prone to graft dysfunction, which seriously affects the efficacy of transplantation of such donor livers. In the context of steatosis, the occurrence of I / R injury is more like a "second blow", and the damaged liver is prone to some more aggressive forms of disease, such as non-alcoholic steatohepatitis and hepatocellular carcinoma.
[0004] However, there is currently no approved drug in clinical practice for the prevention and treatment of liver injury caused by I / R.
[0005] Static cold storage (SCS) is the mainstream method for ex vivo preservation of donor livers in clinical practice, which reduces the injury of donor livers during preservation by lowering the metabolic rate and oxygen consumption at low temperature. However, even in a low-temperature environment, cells still undergo anaerobic respiration at a lower rate, leading to ATP depletion and lactic acid accumulation in cells, and ultimately causing cell damage and loss of graft function. At the same time, the lack of shear stress stimulation from dynamic blood flow can reduce the production of endogenous nitric oxide and exacerbate endothelial cell damage during reperfusion. Currently, SCS is only suitable for short-term organ preservation, and the effect is not satisfactory.
[0006] Numerous studies have shown that perfusion solution combined with nitric oxide and various vasodilators can significantly improve liver microcirculation, improve donor liver function, and reduce the risk of post-transplant complications. A study from Massachusetts General Hospital also showed that the "degreasing cocktail" strategy combined with perfusion solution can achieve short-term degreasing of liver cells, significantly improving the function of steatotic liver grafts. Therefore, in-depth analysis of the molecular mechanisms of the lipid metabolism regulatory network of liver cells, identification of relevant key regulatory factors, and development of new intervention strategies based on cold preservation technology, as well as active promotion of multi-center and leading clinical research, will be an important research direction for improving the quality of steatotic donor livers and reducing their discard rate in the future.
[0007] Fibroblast growth factor 21 (FGF21) is a secreted factor mainly produced by hepatocytes, which plays an important role in maintaining the energy metabolism homeostasis of the body and has the effect of regulating tissue repair and regeneration. So far, more and more evidence shows that FGF21 has good therapeutic effect in various disease models, such as atherosclerosis, microvascular injury and stroke, and the potential mechanism of its pharmacological effect has not been elucidated. At present, the therapeutic effect of FGF21 is mainly anti-inflammatory, anti-oxidation, maintaining energy metabolism homeostasis and promoting tissue repair, and the effect and mechanism of FGF21 on liver I / R injury are still unclear, which needs further research. SUMMARY
[0008] The purpose of the present application is to provide a drug capable of effectively improving the ischemia-reperfusion injury of a transplanted liver, especially a fatty degeneration donor liver, to improve the quality of a fatty degeneration donor liver and reduce the discard rate when applied to the cold preservation process of liver transplantation.
[0009] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:
[0010] The present application provides the application of fibroblast growth factor 21 in the preparation of a drug for preventing and / or treating liver injury caused by ischemia-reperfusion.
[0011] The fibroblast growth factor 21 can be a recombinant protein, which is a modified form of a natural protein produced by recombinant DNA or RNA genetic engineering technology in a host cell.
[0012] Further, the liver injury is ischemia-reperfusion injury of a transplanted liver. Ischemia-reperfusion during liver transplantation can cause early injury of the transplanted liver, leading to dysfunction of the transplanted liver.
[0013] The present application research shows that exogenous supplementation of fibroblast growth factor 21 protein can significantly improve liver ischemia-reperfusion injury.
[0014] Further, the liver injury is ischemia-reperfusion injury of a fatty liver. Fatty degeneration can aggravate ischemia-reperfusion injury, and the present application research shows that exogenous supplementation of fibroblast growth factor 21 protein can effectively inhibit tissue injury, cell apoptosis and inflammation caused after I / R, and also improve intrahepatic lipid accumulation and reduce triglyceride levels, reversing fatty degeneration of the liver.
[0015] Further, the transplanted liver is a fatty degeneration transplanted liver.
[0016] Further, the manifestations of the liver injury include: sinusoidal congestion, edema, cell vacuolization, necrotic area of hepatocytes, increased number of apoptotic cells, and immune cell infiltration. The above symptoms can be significantly alleviated by exogenous supplementation of fibroblast growth factor 21 protein.
[0017] The application also provides a use of fibroblast growth factor 21 in the preparation of a liver transplant organ preservation solution. The application improves liver cell injury caused by ischemia-reperfusion by adding fibroblast growth factor during the preservation of a liver graft.
[0018] Further, the preservation solution is a preservation solution for static cold preservation. The preservation solution is composed of a conventional organ preservation solution such as UW solution or histidine-tryptophan-ketoglutarate solution (HTK solution) and fibroblast growth factor 21. For example, in rats, 1 mg of fibroblast growth factor 21 per kg of body weight is added to the preservation solution.
[0019] Further, the preservation solution is a preservation solution for static cold preservation of a steatotic liver graft. Studies have shown that static cold preservation of a steatotic liver graft in an organ preservation solution containing fibroblast growth factor 21 not only significantly improves the innate immune injury of the transplanted liver, but also improves the lipid accumulation in the liver.
[0020] The application also provides a preservation solution for reducing early injury of a steatotic liver graft, which comprises fibroblast growth factor 21 recombinant protein and an isotonic liquid.
[0021] Further, the isotonic liquid is histidine-tryptophan-ketoglutarate solution.
[0022] The application has the following beneficial effects:
[0023] The application discloses that fibroblast growth factor 21 is an effective intervention target for early injury of a liver graft. Exogenous supplementation of fibroblast growth factor 21 can effectively improve liver cell injury caused by ischemia-reperfusion. In particular, for a steatotic liver graft, fibroblast growth factor 21 can not only inhibit tissue injury, cell apoptosis, and inflammation caused by ischemia-reperfusion, but also improve lipid accumulation in the liver, reverse liver steatosis, and improve the success rate of use of a steatotic liver graft. Therefore, fibroblast growth factor 21 is applied to the preparation of a drug for preventing and treating liver injury caused by ischemia-reperfusion or a liver transplant organ preservation solution, and has important application value in the field of liver transplantation. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1For the schematic diagram of the mouse I / R injury model construction and drug treatment in Example 1, the rFGF21 pre-treatment group of mice was intravenously injected with rFGF21 (0.5 mg / kg) 1.5 h before the operation, n = 6 mice / group.
[0025] Figure 2 For the representative HE staining (A) and quantitative assessment using Suzuki score (B) of the liver sections of the mice in each group 6 h after reperfusion of the mouse I / R injury model in Example 1, ***p < 0.001.
[0026] Figure 3 For the representative TUNEL staining (A) and comparison of the number of TUNEL+ cells under each high-power field (B) of the liver sections of the mice in each group 6 h after reperfusion of the mouse I / R injury model in Example 1, ***p < 0.001.
[0027] Figure 4 For the comparison of the ALT (A) and AST levels (B) of the serum of the mice in each group 6 h after reperfusion of the mouse I / R injury model in Example 1, ***p < 0.001.
[0028] Figure 5 For the detection of the expression levels of BCL-2 and Cleaved caspase-3 in the liver of each group in Example 1 by Western blot.
[0029] Figure 6 For the schematic diagram of the mouse fatty liver I / R injury model construction and drug treatment in Example 2, the rFGF21 pre-treatment group of mice was intraperitoneally injected with rFGF21 (0.5 mg / kg, 2 weeks); n = 5 mice / group.
[0030] Figure 7 For the comparison of the ALT (A) and AST levels (B) of the serum of the mice in each group 6 h after reperfusion of the mouse fatty liver I / R injury model in Example 2, **p < 0.01, ***p < 0.001.
[0031] Figure 8 For the representative HE staining, TUNEL staining and MPO-IHC staining (A) of the liver sections of the mice in each group 6 h after reperfusion of the mouse fatty liver I / R injury model in Example 2, and the quantitative assessment of liver I / R injury using Suzuki score, the number of TUNEL+ cells and the number of MPO+ cells under each high-power field (B), respectively, ***p < 0.001, ****p < 0.0001.
[0032] Figure 9Oil red O staining of liver sections (A), comparison of NAFLD activity score (B) and comparison of TG content in serum (C) of each group of mice after reperfusion for 6h in the mouse fatty liver I / R injury model in Example 2, **p<0.01, ***p<0.001.
[0033] Figure 10 Schematic diagram of the construction and intervention treatment of the rat steatosis donor liver orthotopic liver transplantation model in Example 3. During the liver transplantation process, the donor liver was implanted after being cold-preserved ex vivo for 4 hours; n=5 rats / group.
[0034] Figure 11 Comparison of ALT (A) and AST (B) levels in serum of rats in each experimental group 24h after liver transplantation in Example 3, *p<0.05, ***p<0.001.
[0035] Figure 12 Representative HE staining, TUNEL staining, MPO-IHC staining and oil red O staining of liver sections of rats in each group 24h after orthotopic liver transplantation in Example 3.
[0036] Figure 13 Quantitative evaluation of liver I / R injury using Suzuki score, TUNEL+ and MPO+ cell counts per high-power field, and NAFLD activity score in Example 3, **p<0.01, ****p<0.0001. DETAILED DESCRIPTION
[0037] The application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the application and are not used to limit the applicable scope of the application. Modifications or replacements of the methods, steps or conditions of the application, without departing from the spirit and essence of the application, all belong to the scope of the application.
[0038] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.
[0039] The experimental methods involved in the following examples are as follows:
[0040] 1. Hematoxylin-eosin staining (HE): First, the liver tissue section was soaked in prepared xylene I and xylene II for 5 min, then in 100% ethanol for 1 min, 95% ethanol for 1 min, 80% ethanol for 1 min, 75% ethanol for 1 min; hematoxylin staining for 5 min, and slight rinsing with tap water, the tissue was observed to change from red to blue; differentiation in 1% hydrochloric acid alcohol for 3 s and return to blue in tap water for 10 min; eosin staining for 1 min, and then tap water; dehydration in alcohol gradient, 75% ethanol for 30 s; 80% ethanol for 30 s; 95% ethanol for 30 s; 100% ethanol for 30 s; transparency: placed in xylene I and xylene II for 2 min each; mounting: mounted with neutral resin in a fume hood; the damage score standard referred to Suzuki's score.
[0041] 2. TdT-mediated dUTP Nick-End Labeling (TUNEL) apoptosis detection: for adherent cells, after washing once with PBS, the cells were fixed with 4% paraformaldehyde for about 30 min, washed once with PBS, and then 0.3% Triton X-100 was added and incubated at room temperature for 5 min; for paraffin sections, the dewaxing process was as described below for immunohistochemical staining, then 20 μg / mL of DNase-free proteinase K was added and placed in a 37°C incubator for 20 min, and then washed with PBS for 3 times; reaction: draw a circle on the section with an immunohistochemical pen according to the size of the tissue, then add about 50 μL of TUNEL detection solution to the sample, and incubate in a 37°C incubator for 60 min in the dark; after the end, wash with PBS for 3 times; mounting: mounted with an anti-fluorescence quenching mounting medium; observation: place the section under a fluorescence microscope and take a photo (excitation wavelength 450 nm, detection wavelength 565 nm).
[0042] 3. Immunohistochemistry (IHC): After cutting, mounting and baking the slice, immerse the tissue slice in xylene I and II for 10 min each, and then immerse in alcohol gradient (100% ethanol-100% ethanol-95% ethanol-80% ethanol-70% ethanol) for 5 min each. Immerse the slice in PBS solution completely, and wash for 3 times, 5 min each time. Place the tissue slice in 0.01 mol / L citric acid repair solution (pH 6.0), and preheat the water bath to 95°C. Repair for 40 min, and then wait for natural cooling. Immerse the slice in PBS solution completely, and wash for 3 times, 5 min each time. Draw a circle around the slice using a suitable immunohistochemical pen. Add 3% hydrogen peroxide on the tissue slice, and cover the tissue completely. Place in a dark room for 10 min. Immerse the slice in PBS solution completely, and wash for 3 times, 5 min each time. Dilute the primary antibody in PBS solution (add PBS solution only for the blank control), and add on the tissue slice. Incubate at 4°C overnight. Immerse the slice in PBS solution completely, and wash for 3 times, 5 min each time. Add the prepared secondary antibody on the tissue slice, and incubate at 37°C for 1 h. Immerse the slice in PBS solution completely, and wash for 3 times, 5 min each time. Prepare DAB developing solution, and add on the slice. Observe the color change under a microscope. After the color develops completely, stop the developing process by placing in tap water. After 30 s in tap water, dye for 5 min with hematoxylin, and then pass through tap water until the color of the tissue on the slice changes from red to blue. Return to blue for 10 min in tap water after differentiation with 1% hydrochloric acid alcohol for 3 s. Dehydrate in alcohol gradient (70% ethanol-80% ethanol-90% ethanol-100% ethanol-100% ethanol) for 1 min each. Place in xylene I and II for 2 min each. Dry naturally in a well-ventilated environment, and then seal with resin. Store in the dark. The results of immunohistochemical staining are scored by two other researchers.
[0043] 4. Oil red O staining of liver tissue: Fresh liver tissue is embedded with OCT embedding agent. Embed the sample on a support table, and cut the slice with a freezing microtome (generally at -22°C), with a thickness of 5 pm. Store in a -20°C refrigerator. Prepare oil red O staining working solution according to the ratio of oil red O solution and ddH2O, which is 3:2. Mix well, stand for a period of time, and then filter with a 0.22 pm filter to ensure use within 2 h. Add an appropriate amount of staining and washing solution to cover the frozen slice for 20 s. Remove the staining and washing solution, and then cover the slice with oil red O staining working solution. Stain for 15 min. After staining, wash with ddH2O, and then observe and take a photo under a microscope. Finally, evaluate and analyze according to the NAFLD activity score (NAS).
[0044] 5. Liver function biochemical tests: The alanine transaminase (ALT) and aspartate aminotransferase (AST) levels in the liver function biochemical tests were all detected by Mindray Medical's BS-220 fully automated biochemical analyzer.
[0045] 6. Statistical Methods: SPSS 26.0 and GraphPad Prism 9 software were used for statistical analysis. Normally distributed continuous data were expressed as mean ± standard deviation (x ± s), and independent samples t-tests were used for comparisons between groups. Skewedly distributed continuous data were expressed as median (interquartile range) (M(IQR)), and Mann-Whitney test was used for comparisons between groups. Chi-square test was used for comparisons of categorical variables. The Kaplan-Meier method was used to calculate survival rates and plot relevant survival curves, and the Log-rank test was used to assess differences in survival rates. All p-values were two-tailed, and p < 0.05 was considered statistically significant.
[0046] The recombinant FGF21 protein (rFGF21) used in the following examples was recombinant human fibroblast growth factor 21 purchased from Suzhou Nearshore Protein Technology Co., Ltd., produced by a mammalian expression system, encoding the target gene of Ala29-Ser210 (protein NCBI number Q9JJN1), with a 6His tag at the C-terminus; HTK solution was purchased from Puchuan Medical Technology (Shanghai) Co., Ltd.
[0047] Example 1: Exogenous supplementation of rFGF21 protein improves liver I / R-mediated histopathological damage
[0048] Considering that the absence of FGF21 can worsen transplant liver injury, this embodiment constructs a mouse liver I / R injury model to further investigate whether FGF21 supplementation can improve liver I / R injury.
[0049] 1. Experimental Methods
[0050] The method for constructing a mouse liver I / R injury model pretreated with rFGF21 is as follows: Figure 1 As shown, normal C57BL / 6 mice weighing approximately 20g were selected to construct a liver I / R injury model and divided into three experimental groups: sham operation group, I / R + 0.9% saline group, and rFGF21 pretreatment group. The rFGF21 pretreatment group was intravenously injected at a dose of 0.5mg / kg 1.5h before surgery. The corresponding solvent for rFGF21 was saline (0.9% NaCl).
[0051] Establishment of a mouse ischemia-reperfusion (I / R) injury model: C57BL / 6 mice weighing approximately 20g were selected and fasted for 12 hours prior to surgery. Mice were anesthetized by intraperitoneal injection of 0.2mL of 4% chloral hydrate. After skin preparation, the mice were thoroughly disinfected with 75% alcohol. A midline abdominal incision was made to open the abdominal cavity, and an abdominal dilator was placed to expose the liver. The intestines were moved to one side using a cotton swab to expose the first hepatic hilum, and the hepatic pedicles of the left and middle lobes were freed. The portal vein and hepatic artery of the left and middle lobes of the liver were clamped with non-invasive microvascular clips. The blood vessels were clamped to induce ischemia in about 70% of the liver. After a few seconds, the color of the left and middle lobes of the liver became lighter, indicating successful ischemia. The abdominal cavity was temporarily closed and the surgical area was covered with wet gauze. The mouse was placed on an electric blanket to maintain body temperature. After the mouse's liver was continuously ischemic for 90 minutes, the abdominal cavity was opened, the microvascular clamps used for clamping were quickly released, and then the abdominal muscles and skin were sutured layer by layer to close the abdominal cavity. The area was then disinfected to complete the surgery. Six hours after blood flow to the ischemic liver was restored, blood was taken from the eyeball, the mouse was euthanized, and the ischemic liver was completely removed for subsequent experiments.
[0052] Liver tissue sections were prepared and stained with hematoxylin and eosin (HE) and turbinates (TUNEL). Serum ALT and AST levels were measured. Western blot was used to detect the expression levels of apoptosis-related proteins in the liver.
[0053] 2. Experimental Results
[0054] like Figure 2 As shown, compared with the control group mice, rFGF21 pretreatment significantly alleviated liver I / R damage, manifested as reduced sinus congestion, edema, cell vacuolation or necrosis.
[0055] like Figure 3 As shown, TUNEL staining also indicated a decrease in the number of apoptotic cells.
[0056] like Figure 4 As shown, after 6 hours of reperfusion, the rFGF21 pretreatment group showed a 74.7% decrease in ALT and a 67.0% decrease in AST compared to the control group.
[0057] like Figure 5 As shown, Western blot analysis revealed that after I / R injury, the rFGF21 pretreatment group showed upregulated expression of the anti-apoptotic protein BCL-2 and downregulated expression of the pro-apoptotic protein C-Caspase3 in the liver compared to the control group.
[0058] The above results fully demonstrate that exogenous supplementation of rFGF21 protein can significantly improve liver injury / reperfusion (I / R) damage. Example 2: Exogenous supplementation of rFGF21 protein significantly reduced fatty liver I / R damage and improved steatosis.
[0059] The present embodiment evaluates the effect of exogenous rFGF21 in fatty liver I / R injury by constructing a mouse fatty liver I / R injury model.
[0060] 1. Experimental method
[0061] Fatty liver model of mice and rats: select 4-week-old C57BL / 6 mice, which are raised in SPF level animal room; stop feeding and keep water for 3h; on the first day, normal feed: high-fat feed is 5:1; on the second day, normal feed: high-fat feed is 2:1; on the third day, normal feed: high-fat feed is 1:1; on the fourth day, completely change to high-fat feed; after 8 weeks of high-fat feed, randomly select 3 mice, and determine the successful establishment of the fatty liver model by liver HE staining and oil red O staining. Select SD rats weighing about 120g, which are raised in SPF level animal room; stop feeding and keep water for 3h; on the first day, normal feed: high-fat feed is 5:1; on the second day, normal feed: high-fat feed is 2:1; on the third day, normal feed: high-fat feed is 1:1; on the fourth day, completely change to high-fat feed; after 8 weeks of high-fat feed, randomly select 3 rats, and determine the successful establishment of the fatty liver model by liver HE staining and oil red O staining.
[0062] The construction of the mouse fatty liver I / R injury model and the administration method of FGF21 recombinant protein are as shown in Figure 6 After 8 weeks of high-fat feed to establish fatty liver mice, the liver I / R injury model is constructed, which is divided into three experimental groups: sham operation group, I / R+0.9% normal saline group and rFGF21 pretreatment group; the mouse rFGF21 is intraperitoneally injected at a dose of 0.5mg / kg daily, and the injection is continuously performed for 2 weeks before the operation. The corresponding solvent of rFGF21 is normal saline (0.9% NaCl).
[0063] Then, liver tissue sections are prepared for HE staining, TUNEL staining and MPO-IHC staining; liver frozen sections are prepared for oil red O staining; the levels of ALT, AST and TG in serum are determined.
[0064] 2. Experimental results
[0065] As shown in Figure 7 , after I / R injury, the serum ALT and AST levels of the mice in the rFGF21 pretreatment group were significantly lower than those in the control group.
[0066] As shown in Figure 8 , HE staining and TUNEL staining results show that fewer necrotic areas of liver cells and apoptotic cells are observed on the liver sections of the rFGF21 pretreatment group; MPO-IHC staining results show that the degree of neutrophil infiltration in the I / R injury liver of the rFGF21 pretreatment group is significantly lower than that of the control group, suggesting that the innate immune injury in fatty liver is improved.
[0067] As shown in Figure 9 , Oil red O staining found that the liver of the rFGF21 pretreatment group showed more dispersed lipid microvesicles, smaller volume of lipid vacuoles, and a significant reduction in NAFLD activity score compared with the control group (A and B). Meanwhile, combined with the comparative analysis of serum TG, it was also confirmed that the degree of liver steatosis in the rFGF21 pretreatment group was lighter (C). Figure 9 Figure 9 C).
[0068] These results all show that exogenous supplementation of rFGF21 can effectively inhibit tissue damage, apoptosis and inflammation caused after I / R, and also improve lipid accumulation in the liver and reduce the level of triglyceride, reverse liver steatosis.
[0069] Example 3: FGF21 recombinant protein preservation solution effectively reduces steatosis of transplanted liver
[0070] This example constructs a steatosis donor liver transplantation model in rats to evaluate the repair and function enhancement of marginal donor liver by FGF21 recombinant protein preservation solution during liver transplantation.
[0071] 1. Experimental method
[0072] The construction of a steatosis donor liver transplantation model in rats and the intervention treatment are as shown in Figure 10 After 8 weeks of high-fat diet feeding to establish fatty liver rats, an orthotopic liver transplantation model was constructed, and divided into three experimental groups: sham operation group, HTK solution group and HTK solution + rFGF21 group; the HTK solution + rFGF21 group was 100 mL of HTK solution with 1 mg / kg of rFGF21 (calculated according to the body weight of the donor rat). During liver transplantation, the donor liver was implanted after 4 hours of cold preservation ex vivo. The method is as follows:
[0073] Establishment of rat orthotopic liver transplantation model (OLT):
[0074] (1) Preparation of donor and recipient: select SD rats with body weight of about 450 g fed with high-fat diet for 8 weeks as the donor of rat OLT, and select normal chow-fed SD rats with slightly larger body weight than the donor as the recipient, and fast for 12 h before operation and drink for 4 h before operation;
[0075] (2) Anesthesia: the donor and recipient rats were anesthetized by isoflurane using a small animal inhalation anesthesia machine;
[0076] (3) Donor surgery: After the donor rat is fully anesthetized, the rat is fixed and disinfected, and then an abdominal cavity is accessed along the midline of the abdomen to expose the liver by placing an abdominal cavity dilator; the liver is disconnected from the surrounding tissue and organ ligaments, and the left phrenic vein is ligated and cut; the thoracic segment of the abdominal aorta is clamped with a vascular clamp, a needle head of an indwelling needle is punctured above the celiac trunk and fixed with a vascular clamp, 10 mL of 4°C UW solution is slowly perfused, and the inferior vena cava is cut to serve as an outflow tract; after the perfusion is completed, the left and right renal veins are then ligated and disconnected, the subhepatic inferior vena cava, portal vein and suprarenal inferior vena cava are isolated and disconnected, the common bile duct is disconnected and ligated; the donor liver is separated and placed in 100 mL of 4°C HTK solution or rFGF21 repair solution for cold preservation for 4 h, and is further processed;
[0077] (4) Liver repair: The Kamada reported "double sleeve method" is used to trim the isolated liver, effectively fixing the portal vein sleeve and the subhepatic inferior vena cava sleeve, covering the vessel wall on the sleeve in an everted manner and fixing it, and slowly perfusing 10 mL of 4°C pre-cooled HTK solution through the portal vein; the trimmed donor liver is placed in a 4°C refrigerator for standby;
[0078] (5) Recipient lower liver: After the recipient rat is anesthetized, the abdominal cavity is accessed in the middle of the abdomen, the common bile duct and the common hepatic artery are isolated and ligated, the subhepatic inferior vena cava is blocked above the right renal vein with atraumatic vascular clamps, the portal vein is blocked above the pyloric vein, and the anhepatic phase is entered; the proximal portal trunk is punctured and the liver is flushed with normal saline until the liver turns white, then the suprarenal inferior vena cava is blocked with atraumatic vascular clamps, and then the entire liver is isolated; the anhepatic phase time should be controlled within 30 min;
[0079] (6) Liver implantation: 10-0 atraumatic suture is used to continuously anastomose the suprarenal inferior vena cava of the donor and the recipient; the "sleeve method" is used to reconstruct the portal vein and the inferior vena cava; the portal vein is opened, and the anhepatic phase is ended; the common bile duct is reconstructed by the biliary stent method, and the recipient is placed in an incubator for postoperative recovery after checking that there is no active bleeding in the abdominal cavity of the recipient;
[0080] (7) Sample collection: After 24 h of recovery of the blood flow of the transplanted liver, the inferior vena cava is taken, the rat is sacrificed, and the rat transplanted liver is completely taken out for subsequent experiments.
[0081] Among them, the FGF21 recombinant protein preservation solution: 1 mg / kg is added to 100 mL of HTK solution as the FGF21 recombinant protein repair solution for each rat with fatty degeneration of the liver.
[0082] 2. Experimental results
[0083] As shown in Figure 11 , the serum ALT and AST levels of the rats in the FGF21 recombinant protein preservation solution group were significantly lower than those in the HTK solution group in the early stage after liver transplantation.
[0084] As Figure 12 and Figure 13 As shown in FIG. 8, FIG. 9, and FIG. 10, HE staining and TUNEL staining results showed that less necrotic area and apoptotic cells were observed in the FGF21 recombinant protein preservative group. MPO-IHC staining results showed that the degree of neutrophil infiltration in the FGF21 recombinant protein preservative group was significantly lower than that in the control group, suggesting that the improvement of the innate immune damage in the fatty liver. Oil red O staining showed that the FGF21 recombinant protein preservative group exhibited more dispersed lipid microvesicles and smaller volume of lipid vacuoles than the HTK solution group, but there was no statistically significant difference in the NAFLD activity score.
[0085] These results all showed that the FGF21 recombinant protein preservative group could effectively improve the tissue damage, apoptosis and inflammation caused by the liver transplantation process, and also had certain potential for improving the lipid accumulation in the liver.
Claims
1. The application of fibroblast growth factor 21 in the preparation of liver transplant organ preservation solution, characterized in that, The preservation solution is used for static cold preservation of livers with steatosis. Fibroblast growth factor 21 improves hepatocyte damage caused by ischemia-reperfusion and reverses hepatic steatosis.
2. The application as described in claim 1, characterized in that, The manifestations of hepatocellular injury include: sinus congestion, edema, cell vacuolation, increased areas of hepatocellular necrosis and apoptotic cells, and immune cell infiltration.
3. The application as described in claim 1, characterized in that, Add 1 mg of fibroblast growth factor 21 per kilogram of body weight to the preservation solution.
4. The application as described in claim 1, characterized in that, The preservation solution includes recombinant fibroblast growth factor 21 protein and isotonic fluid.
5. The application as described in claim 4, characterized in that, The isotonic liquid is histidine-tryptophan-ketoglutarate solution.