CN102735847A - Application of human interleukin 22 in preparation of marker for liver transplantation rejection reaction - Google Patents
Application of human interleukin 22 in preparation of marker for liver transplantation rejection reaction Download PDFInfo
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- CN102735847A CN102735847A CN2012102186222A CN201210218622A CN102735847A CN 102735847 A CN102735847 A CN 102735847A CN 2012102186222 A CN2012102186222 A CN 2012102186222A CN 201210218622 A CN201210218622 A CN 201210218622A CN 102735847 A CN102735847 A CN 102735847A
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Abstract
The present invention relates to the field of medicine, and especially to an application of human interleukin (IL) 22 in preparation of a marker for a liver transplantation rejection reaction. In liver transplantation, clinical study results show that TH17 related factors of IL-17 and IL-22 are significantly increased in sera of patients after liver transplantation surgeries; compared with IL-17, the IL-17 in liver tissues of patients in a rejection group is significantly increased in the 1st day and the 3rd day after the liver transplantation surgeries, and is gradually reduced in the next 5-7 days, and the IL-22 in liver tissues of patients in a rejection group after the liver transplantation surgeries is significantly increased in the 1st day, and is gradually reduced from the 3rd day, wherein expressions of the IL-17 and the IL-22 of the patients in the rejection group provide statistical significances compared with the tolerance group after the liver transplantation surgeries.
Description
Technical field
The invention belongs to medical domain, the mark of particularly liver transfer operation rejection.
Background technology
The liver transfer operation operation is meant that the liver of implanting a health through operation in patient's body, makes whole a kind of surgical intervention means that latter stage, hepatopath's liver function was well recovered.Liver plantation position is different according to supplying, and can be divided into orthotopic liver transplantation art and heterotopic liver transplantation.Orthotopic liver transplantation (OLT) has become the mature technology of liver surgery, and as far as hepatopath in many whole latter stages, it is unique efficacious therapy means.Although the successful Application of immunodepressant is arranged, and, remain early stage donor damage and transplant one of main hazard factor of back survival rate by the acute transplant rejection reaction that the lymphocyte, monocyte and other inflammatory cells that soak into cause.There are some researches show that reigning IFN γ is not to be indispensable to the graft damage in the rejection, hint also has other factors to participate in causing the inflammatory reaction of rejection.Current research shows that the TH17 cell is participated in the immune response of graft-rejection.IL-22 is one of member of IL-10 family; Mainly by TH17 cell, TH22 cell, NK cell and the generations such as (LTi) of lymphoid tissue inducing cell; IL-22 mainly acts on skin, liver and kidney, the effect of performance inherent immunity, injury protection and promotion regeneration.IL-22 has protection and urgees scorching double action liver, and research proof IL-22 divides the liver cell regeneration after excising to play important facilitation to liver, and up-to-date digital proof IL-22 participates in the protective effect of hepatic ischemia-reperfusion injury.Effects such as the liver cell regeneration of supposition IL-22 participation after liver transplantation and ischemia-reperfusion.But, also do not have IL-22 (human interleukin 2 2) to participate in the report of the immune response of graft-rejection so far.
Summary of the invention
One of the object of the invention is to provide new application of the human interleukin 22, and this is applied as judges that the liver transfer operation rejection provides new thinking.
For realizing above-mentioned purpose, technical scheme of the present invention is:
The application of human interleukin 22 in the mark of preparation liver transfer operation rejection.
Further, said liver transfer operation rejection is that the liver transfer operation of original position property is repelled.
Further, the application of human interleukin 22 albumen in the mark of 1-3 days liver transfer operation rejections of preparation liver transfer operation.
Further, the application of human interleukin 22 mRNA in the mark of preparation liver transfer operation rejection.
Technique scheme confirms its function through following experiment: successfully set up the rat orthotopic liver transplantation animal model; Be divided into repulsion group and tolerance group (each 15); Get postoperative 1,3,5; 7 day rat blood plasma and hepatic tissue sample, the expression of cell, albumen and the mRNA of IL-17 (interleukin-17) and IL-22 (interleukin-22) in application immunohistochemical staining, Western blot and its hepatic tissue of fluorescence quantitative PCR detection.The result shows that repulsion group IL-17 PC is significantly higher than the tolerance group; In hepatic tissue, the lymphocyte positive rate of IL-17 and IL-22, protein expression level and mRNA expression repulsion group are significantly higher than the tolerance group.Thus can the application of confirmer's interleukin-22 2 in the mark of preparation liver transfer operation rejection.In the prior art: IL-17 can act on the defence of autoimmune disease and pathogen and also participate in graft rejection, in kidney, heart and the lung transplantation of people and animal, finds the rising of IL-17 level.In liver transfer operation, there is clinical research to show that TH17 correlation factor IL-17 and IL-23 significantly increase in the patients serum of after liver transplantation.Compare with IL-17, IL-17 postoperative in repulsion group hepatic tissue raise obviously in the 1st day and the 3rd day, and in subsequently 5,7 days, reduced gradually, and IL-22 raise at repulsion group postoperative and obviously and from the 3rd day descends gradually on the 1st day.The IL-17 of repulsion group and IL-22 express all has statistical significance after surgery than the tolerance group.
Description of drawings
Fig. 1 is the HE histotomy, and wherein, TOL is meant the tolerance group, and REJ is meant the repulsion group.
Fig. 2 is the RAI scoring analysis chart of HE histotomy Banff.
Fig. 3 is the liver function index situation of change analysis chart after the Rat Liver Transplantation modelling, and wherein, A is for being index with ALT, and B is for being index with AST.
Fig. 4 is the situation of change of IL-17 and IL-22 in 1,3,5, the 7 day rat hepatic tissues of SABC demonstration transplanting back.
Fig. 5 transplants back 1,3,5,7, IL-17, IL-22 in the day rat hepatic tissue for Western blotting detects.
Fig. 6 transplants back 1,3,5 for Western blotting detects; 7, IL-17, IL-22 mutation analysis figure in the day rat hepatic tissue, wherein A is the mutation analysis figure of IL-17; B is the mutation analysis figure of IL-22; In the analysis chart, horizontal ordinate is the time, and ordinate is the relative concentration of measuring with ultraviolet spectrophotometer (with confidential reference items GAPDH ratio).
Fig. 7 is for transplanting back 1,3,5; The mutation analysis figure of the mRNA of IL-17, IL-22 in the 7 day rat hepatic tissues, wherein, a is the mutation analysis figure of IL-17; B is the mutation analysis figure of IL-22, and horizontal ordinate is the time, and ordinate is the expression (with confidential reference items β-Actin ratio) of mRNA.
Embodiment
In order to make the object of the invention, technical scheme and a bit clearer, will combine accompanying drawing that the preferred embodiments of the present invention are carried out detailed description below.
Present embodiment confirms that through disclosing the expression and the effect of IL-22 in the acute rejection of liver transplantation reaction IL-22 can be used to prepare the mark of liver transfer operation rejection.Through experiment confirm, to compare with tolerance treated animal model, IL-17 in its hepatic tissue of repulsion treated animal and lymphocyte positive rate, protein expression level and the mRNA expression of IL-22 also obviously increase.We find that also STAT3 mRNA expression raises in the repulsion group hepatic tissue simultaneously.These results show that IL-22 participates in the generating process of after liver transplantation acute rejection, and infer that it is through activating STAT3 path performance effect.
Method statistic method data in the present embodiment are represented with mean ± standard deviation, adopt the capable one-way analysis of variance of SPSS13.0 statistical software, and the t that relatively goes between group checks.P＜0.05 is for having statistical significance.
Embodiment one animal modeling
The foundation of 1 animal used as test and rat orthotopic liver transplantation model
Male BN (RT1
n) rat and LEW (RT1
1) rat, SPF level animal, body weight 220-270g purchases the Experimental Animal Center in big level ground hospital of Third Military Medical University.Raise in constant temperature, cleaning ambient, keep 12 hours circadian rhythms, observe animal used as test and use and management principle.Confession, the fasting in preceding 8 hours of acceptor art, postoperative can't help diet.Rat Liver Transplantation is repelled model and is divided into repulsion group and tolerance group, and the repulsion group is LEW (RT1
1) rat is donor BN (RTl
n) rat is acceptor, tolerance group BN (RTl
n) rat is donor LEW (RT1
1) rat is acceptor, 15 pairs every group.Using modified " two oversleeves " method is set up the rat orthotopic liver transplantation animal model, and " two oversleeves " method is concrete with reference to the " CD4 of different model of orthotopic liver rats
+CD25
+The research of regulatory T cells number change [J]. Journal of Immunology, 2011,27 (4): 283-286 ".Put to death the repulsion group on the the the 1st, the 3rd, the 5th, the 7th day and tolerate each 3 of treated animals at after liver transplantation respectively, extract peripheral blood and liver organization and be equipped with inspection.
2 liver organization pathology and immunohistochemical analysis
Cutting hepatic tissue, to place volume fraction be fixing 48 hours of 10% formaldehyde, conventional dehydration, transparent, FFPE, the thick section of 5 μ m, HE dyeing, observation by light microscope.Carry out pathological grading according to liver tissues inflammatory infiltration and downright bad situation.
The result: can see that from HE histotomy shown in Figure 1 the repulsion group than the tolerance group model tangible graft-rejection takes place, repulsion group liver transplantation pathologic finding is all pointed out degeneration of liver cells enlargement under the light microscopic, accompanies a large amount of necrosiss of liver cells; The blood sinus dilatation and congestion; Endotheliocytic swelling is obvious, and it is main inflammatory cell infiltration that whole lobuli hepatis structure disturbance, portal area and central vein occur with the lymphocyte; Cholangiole destroys companion's cell infiltration, meets the pathological manifestations of acute rejection.RAI scoring based on Banff (is repelled the activity index score to portal area, bile duct and venous endothelial inflammation; Total mark is 9 minutes, and RAI 0-2 divides to be decided to be not have and repels, and 2-3 is divided into the impatient property rejection of having a common boundary; 3-4 is divided into slight acute rejection I level; 5-6 is divided into moderate acute rejection II level, and 7-9 is divided into severe acute rejection III level), the result is as shown in Figure 2: the scoring of repulsion group is apparently higher than the scoring of tolerance group.
3 liver functions detect
Place anticoagulant tube in each time point through the about 5mL of inferior caval vein blood drawing, the centrifuging and taking plasma specimen detects the variation of serum liver function alanine aminotransferase (ALT) and aspartic transaminase (AST) with full automatic biochemical apparatus (Olympus).
The result: postoperative is respectively organized Serum ALT, TBIL concentration and variation tendency: 2 groups of liver functions of postoperative all have rising in various degree, can find out repulsion group ALT obviously greater than the tolerance group, and the most obvious the 5th day difference.The TBIL of repulsion group obviously was higher than the tolerance group at the 7th day.(
*P＜0.05), see Fig. 3 for details, the result sees the following form, and table 1 is the situation of change of 0,1,3,5 and 7 day control group and experimental group alanine aminotransferase (ALT), and table 2 is the situation of change of 0,1,3,5 and 7 day control group and experimental group AST.The unit of numeral is U/L in table 1 and the table 2.
Table 1
| 0day | 1day | 3day | 5day | 7day | |
| The ■ control group | 2.33 | 9.33 | 3.00 | 6.67 | 14.67 |
| The experimental group | 2.33 | 92.00 | 74.67 | 447.33 | 267.67 |
Table 2
| 0day | 1day | 3day | 5day | 7day | |
| The ■ control group | 1.33 | 216.67 | 9.33 | 8.33 | 19.00 |
| The experimental group | 6.67 | 172.33 | 328.67 | 1253.00 | 341.67 |
The application of embodiment 2 human interleukin 2s 2 in the mark of preparation liver transfer operation rejection
1 immunohistochemical reaction
With IL-17, IL-22 and INF-gamma antibodies is working fluid (available from Beijing Bo Aosen Bioisystech Co., Ltd), and Immunohistochemical method (available from company of middle China fir Golden Bridge) by specification operation is carried out.
It is obvious than the tolerance group that IL-17 and the IL-22 that can find out the repulsion group in the SABC testing result as shown in Figure 4 is expressed in postoperative 1,3 day.
2Western-blot detects
One is anti-, and: IL-17 purchases in Beijing that Bo Aosen, IL-22 purchase the Cruz in Santa, HRP two anti-, confidential reference items one anti-purchasing in GenScript.Organize total protein with the extraction of tissue homogenate method, all article and 5 * SDS sample-loading buffer are 4: 1 mixed by volume in the taking-up, boil in the boiling water behind the mixing to make albuminous degeneration in 5 minutes.Add after enough electrophoresis liquid by appearance on the equal protein.Changing voltage was to 120V after electrophoresis, 80V ran and concentrate glue.Use confining liquid with correspondence one anti-to be diluted to certain concentration be 1: 500 (volume dilution), confidential reference items one anti-dilution final concentration is 1: 3000 (volume dilution), incubation is 1.5 hours then.Clean 3 times each 5 minutes with TBST.Anti-to be diluted to certain concentration be 1: 5000 (volume dilution) with two to use confining liquid, and incubation is 1.5 hours then.Film is scanned, use the gray-scale value of UVP gel images disposal system Labworks4.6 software analysis purpose band then.
Western blot testing result is as shown in Figure 5, and IL-17 in repulsion group described in the embodiment 1 and the tolerance group hepatic tissue and IL-22 all have rising in various degree before after transplanting, comparing and transplanting.Different is that IL-17 postoperative in repulsion group hepatic tissue raise obviously in the 1st day and the 3rd day, and in subsequently 5,7 days, reduces gradually, and IL-22 raise at repulsion group postoperative and obviously and from the 3rd day descends gradually on the 1st day.The IL-17 of repulsion group and IL-22 express all has statistical significance after surgery than the tolerance group.Show the participation that IL-17 and IL-22 are arranged in the early stage acute rejection after the liver transfer operation.
2Real-time PCR detects
The Trizol method is extracted the total RNA of liver organization.(SmartSpeedTM Plus BIO-RAD) measures RNA quality and concentration, and the result is as shown in Figure 6 with ultraviolet spectrophotometer.
RT-PCR uses TaKaRa company kit, and the operation steps by specification carries out.Real-time quantitative PC R uses
Premix Ex Taq
TMSystem, IL-17A primer: Sense:5 ' ctcaaccgttccact tca3 ', Antisense:5 ' caccagcatcttctccac3 '.IL-22 primer: Sense:5 ' ttccgaggagtcaaagc ca3 ', Antisense:5 ' cgcagggacataaacagca3 '.Get cDNA2 μ L, each 0.5 μ L of upstream and downstream primer, Premix Ex Taq 12.5 μ L, reaction system 25 μ L, mixing.Each sample is done 2 multiple holes.Rotor gene 6300 quantitative PCR appearance are used in PCR reaction, the PCR condition: 95 ℃ 30 seconds; PCR reacts 40 circulations: 95 ℃ 15 seconds, 58 ℃ 15 seconds, 72 ℃ 20 seconds; Solubility curve: 95 ℃ 60 seconds, 60 ℃ 30 seconds, 95 ℃ 30 seconds.As shown in Figure 7, Real-time detects the mRNA level (seeing table 4 for details) of IL-17 (seeing table 3 for details) and IL-22 in the repulsion group postoperative hepatic tissue apparently higher than the tolerance group, and peaks at the 3rd day.The data of table 3 and Fig. 7-a are corresponding, and the data of table 4 and Fig. 7-b are corresponding.
Table 3
Table 4
Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although with reference to preferred embodiment the present invention is specified, those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention; And not breaking away from the aim and the scope of technical scheme of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (4)
1. the application of human interleukin 22 in the mark of preparation liver transfer operation rejection.
2. application according to claim 1 is characterized in that: said liver transfer operation rejection is that the liver transfer operation of original position property is repelled.
3. application according to claim 1 is characterized in that: the application of human interleukin 22 albumen in the mark of 1-3 days liver transfer operation rejections of preparation liver transfer operation.
4. application according to claim 1 is characterized in that: the application of human interleukin 22 mRNA in the mark of preparation liver transfer operation rejection.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008085229A2 (en) * | 2006-11-15 | 2008-07-17 | Arteriocyte Inc. | Cell-based therapies for treating liver disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008085229A2 (en) * | 2006-11-15 | 2008-07-17 | Arteriocyte Inc. | Cell-based therapies for treating liver disease |
Non-Patent Citations (3)
| Title |
|---|
| CHAO YE: "T-helper 17 cell: Adistinctive cell in liver diseases", 《HEPATOLOGY RESEARCH》, vol. 41, 25 November 2010 (2010-11-25), pages 22 - 29 * |
| 杨 波: "Th17细胞及其在相关肝病中的作用", 《临床肝胆病杂志》, vol. 27, no. 2, 28 February 2011 (2011-02-28), pages 206 - 209 * |
| 王小飞: "IL-17 及 IL-22在大鼠肝移植排斥反应中的表达及意义", 《免疫学杂志》, vol. 28, no. 5, 1 May 2012 (2012-05-01) * |
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Application publication date: 20121017 |