CN105688189A - Application of human sDR5-Fc (soluble DR5-Fc) antibody fusion protein used as renal injury treatment medicament - Google Patents

Application of human sDR5-Fc (soluble DR5-Fc) antibody fusion protein used as renal injury treatment medicament Download PDF

Info

Publication number
CN105688189A
CN105688189A CN201610041751.7A CN201610041751A CN105688189A CN 105688189 A CN105688189 A CN 105688189A CN 201610041751 A CN201610041751 A CN 201610041751A CN 105688189 A CN105688189 A CN 105688189A
Authority
CN
China
Prior art keywords
sdr5
renal
fusion protein
antibody fusion
application
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610041751.7A
Other languages
Chinese (zh)
Inventor
马远方
王志增
张军
王耀辉
李小全
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henan University
Original Assignee
Henan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henan University filed Critical Henan University
Priority to CN201610041751.7A priority Critical patent/CN105688189A/en
Publication of CN105688189A publication Critical patent/CN105688189A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention belongs to the field of medicine, and in particular relates to medicinal application of an sDR5-Fc (soluble DR5 and Fc) antibody fusion protein formed by fusing human sDR5 and an Fc segment of a human immunoglobulin, in particular to application of the human sDR5-Fc antibody fusion protein used as a renal injury treatment medicament. According to the invention, experiments prove that in a rat renal ischemia model and an ischemia-reperfusion model, the human sDR5-Fc antibody fusion protein can obviously reduce renal tissue injury, and obviously reduce an apoptosis rate of renal tubular epithelial cells. As a novel candidate medicament for treating acute kidney injury, the human sDR5-Fc antibody fusion protein has great developing potential.

Description

People's sDR5-Fc antibody fusion protein is as the application of Renal injury grade medicine
Technical field
The invention belongs to field of medicaments, be specifically related to people sDR5(solubledeathreceptor5, sDR5) pharmaceutical usage of albumen, especially people sDR5-Fc antibody fusion protein is as the application of renal ischaemia reperfusion injury first aid and medicine。
Background technology
Ischemical reperfusion injury refers to that the blood supply of organ or tissue is interrupted or reduces, cause local organization or organ ischemia's anoxia, the function of histoorgan not only can not be made after Reperfu-sion to be recovered, make the phenomenon that the function caused by its hypoxic-ischemic and dysbolismus and mechanism collapse increase the weight of further on the contrary。Ischemical reperfusion injury is common in organ transplantation, organ partial enucleation, wound and shock etc. clinically, and kidney is due to the characteristic of its blood flow HT, and ischemical reperfusion injury incidence rate is higher。
The ischemia of moment can result in kidney and reversible damage occur, and long-term ischemia-reperfusion can cause renal proximal tubules far-end that irreversible damage occurs。Ischemia-reperfusion injury of kidney is acute injury of kidney (acutekidneyinjury common clinically, AKI), it it is the one of the main reasons causing acute renal failure and transplanted kidney function to be lost, AKI sickness rate in general population is about 0.5-1%, in inpatient, its sickness rate is 5%, and its sickness rate is 30-50% in some Post operation and Intensive Care Unit patient；Except higher sickness rate, AKI patient also has significantly high mortality rate, it is about 28-90%, it is about the identical state of an illness and does not occur 5 times of AKI mortality, and the patient of survival also has chronic renal insufficiency in various degree more, particularly in renal transplantation, ischemia-reperfusion injury of kidney is the key factor that kidney getting up early survival is transplanted in impact, may result in transplanting kidney short-term and long-term functional lesion, including transplanting kidney Primary gastrointrestinal lymphoma, acute rejection in renal transplantation, chronic allograft mistake merit etc.。To this, there is no special effective treatment means at present, serious case needs lifelong renal replacement therapies。Therefore, how to avoid kidney generation ischemical reperfusion injury, there is important clinical meaning。
The response of the damage factor such as hypoxic-ischemic, inflammatory mediator is substantially a kind of stress by cell, and cell should be one of impaired even dead major reason of cell function。In ischemia-reperfusion injury of kidney, there are three kinds of final results in renal cells: cell reversible lesion, necrocytosis and apoptosis。Along with acute renal failure diagnoses and treats the raising of level, renal ischemic is relatively in the past obvious with the interval time of Reperfu-sion to be shortened, renal cells necrosis alleviates to a great extent, therefore in most cases at present, apoptosis in renal tubular epithelial cells accounts for mastery reaction in ischemia-reperfusion injury of kidney。
Death receptor 5 (Deathreceptor, DR5) TRAIL (TNF-relatedapoptosis-inducingligand, TRAIL) death receptor4 activated is combined, can pass through to recruit associated protein and form DISC(death-inducingsignalingcomplex), and then tandem type activation Caspase-8,3 etc., cause apoptosis。The DR5 of total length is containing 411 amino acid whose I type transmembrane glycoproteins。Solubility death receptor 5 (solubleDR5, sDR5) for want of trans-membrane region can not be expressed on cell membrane and be secreted into extracellular。SDR5 expresses relatively low in normal human peripheral blood, because it has the extracellular fragment complete structure combined with TRAIL part, can with the death receptor competitive binding TRAIL molecule on cell membrane, thus block TRAIL induction apoptosis。
There are some researches show: sDR5 can alleviate hepatitis B virus (HBV) and infect the hepatocyte injury caused (referring to Yu-GangLiu by blocking the apoptosis of TRAIL induction, Su-XiaLiu, Xiao-HongLiangetal.BlockadeofTRAILpathwayamelioratesHBV-inducedhepatocyteapoptosisinanacutehepatitismodel.Bioche micalandBiophysicalResearchCommunications352 (2007) 329 334) and improve the cental system damage caused because of ischemia (referring to MinCui, LimeiWang, XiaohongLiangetal.BlockingTRAIL-DR5signalingwithsolubleD R5reducesdelayedneuronaldamageaftertransientglobalcerebr alischemia.NeurobiologyofDisease39 (2010) 138 147)。At present, sDR5, for whether the Renal tissues damage caused because of ischemia equally exists therapeutical effect, there is no report both at home and abroad。
After renal ischaemia and myocardial revascularization, the generation of reperfusion injury and the excessive Apoptosis of renal cells are closely related, for the signal transduction pathway that apoptosis occurs, utilize sDR5-Fc and membranous type DR5 competition binding TRAIL, make TRAIL can not start the generation of apoptosis program, so can reduce Renal tissues damage, improve patient's long-term prognosis。
Summary of the invention
Present invention aim at research people's sDR5-Fc antibody fusion protein and reduce the effect of apoptosis in renal tubular epithelial cells by blocking/close TRAIL-death receptor4, and further provide for the application as Renal injury grade medicine of people's sDR5-Fc fusion protein。
For achieving the above object, the present invention adopts the following technical scheme that
People's sDR5-Fc antibody fusion protein as the application of Renal injury grade medicine, as, it is possible to be the application as acute injury of kidney or Renal Ischemia Reperfusion Injury medicine, it is also possible to be the application as acute renal failure or renal transplant rejection medicine。
People's sDR5-Fc antibody fusion protein blocks the application in the apoptotic agent of TRAIL induction in preparation。Described people's sDR5-Fc antibody fusion protein, by blocking the apoptosis of TRAIL/DR5 path induction, reduces Renal tissues damage, to reach the effect of protection kidney cell。
The application in preparation treatment Renal Ischemia Reperfusion Injury medicine of the protein sequence of people's sDR5-Fc antibody fusion protein, sequence modification and associated biomolecule engineering product。
The Synergistic treatment application in Renal Ischemia Reperfusion Injury of people's sDR5-Fc antibody fusion protein and other medicines。
The present invention utilizes people's sDR5-Fc antibody fusion protein that mammalian cell Chinese hamster ovary celI is originated; C57BL/6 Mouse Kidney ischemia-reperfusion injury model tests the protective effect to Renal Ischemia Reperfusion Injury of people's sDR5-Fc Antibody Fusion; obtain people's sDR5-Fc Antibody Fusion respectively to reduce renal ischemic reperfusion injury degree, reduce apoptosis in renal tubular epithelial cells rate, improve the result of renal function。Experimental result shows: in 24 hours models of Bilateral Renal ischemia 22 minutes and reperfusion, during Reperfu-sion 2h/6h, according to the dosage of 15.0mg/kg, by single intraperitoneal injection sDR5-Fc antibody fusion protein, medication group all can obviously reduce renal ischemic reperfusion injury degree compared with matched group。It addition, people's sDR5-Fc antibody fusion protein can obviously reduce ischemia model mesonephric tubule Epithelial Cell Apoptosis rate。
The present invention is confirmed by zoopery: people's sDR5-Fc antibody fusion protein can block the apoptosis of TRAIL/DR5 path induction; reduce renal ischemic reperfusion injury degree; reach the effect of protection kidney; renal ischaemia and reperfusion injury there is therapeutical effect, as novel drug candidate great exploitation potential and potential applicability in clinical practice。
The action effect of essence and people's sDR5-Fc antibody fusion protein of the present invention in order to be more fully understood that the present invention, is described in detail the application present invention treated in the medicine of renal ischemic reperfusion injury in preparation by concrete drawings and Examples below。It needs to be noted, instantiation and accompanying drawing are merely to understand and explanation, it according to illustrating herein, can be made various correction and change by those skilled in the art or other staff within the scope of the invention, and these are revised and change and also include in the scope of the present invention。
Accompanying drawing explanation
Coomassie brilliant blue staining before and after people's sDR5-Fc protein purification of Fig. 1 purification；
TRAIL is induced the blocking effect of Jurkat cell apoptosis by people's sDR5-Fc albumen of Fig. 2 purification；200ngTRAIL individually or is simultaneously introduced 5 × 10 with 500ng people's sDR5-Fc albumen6Jurkat cell, Apoptosis by Flow Cytometry after 24 hours, add and cultivate basis set conduct comparison；
Fig. 3 mice Bilateral Renal ischemia fills 2h administration model for 22 minutes again and respectively organizes renal function testing result；A: blood urea nitrogen testing result；B: serum creatinine testing result
Fig. 4 mice Bilateral Renal ischemia fills 2h administration model for 22 minutes again and respectively organizes renal tissue HE coloration result；
Fig. 5 mice Bilateral Renal ischemia fills 2h for 22 minutes again and is administered model renal tissue SABC DR5 testing result；
Fig. 6 mice Bilateral Renal ischemia fills 2h for 22 minutes again and is administered model renal tissue SABC TRAIL testing result；
Fig. 7 mice Bilateral Renal ischemia fills 2h for 22 minutes again and is administered model renal tissue SABC Caspase-3 testing result；
Fig. 8 mice Bilateral Renal ischemia fills 2h for 22 minutes again and is administered model renal tissue SABC Caspase-8 testing result；
Fig. 9 mice Bilateral Renal ischemia fills 6h administration model for 22 minutes again and respectively organizes renal function testing result；A: blood urea nitrogen testing result；B: serum creatinine testing result；
Figure 10 mice Bilateral Renal ischemia fills 6h administration model for 22 minutes again and respectively organizes renal tissue HE coloration result；
Figure 11 mice Bilateral Renal ischemia fills 6h for 22 minutes again and is administered model renal tissue SABC DR5 testing result；
Figure 12 mice Bilateral Renal ischemia fills 6h for 22 minutes again and is administered model renal tissue SABC TRAIL testing result；
Figure 13 mice Bilateral Renal ischemia fills 6h for 22 minutes again and is administered model renal tissue SABC Caspase-3 testing result；
Figure 14 mice Bilateral Renal ischemia fills 6h for 22 minutes again and is administered model renal tissue SABC Caspase-8 testing result；
Figure 15 mice Bilateral Renal ischemia fills 6h for 22 minutes again and is administered model renal tissue SABC TUNEL testing result。
Detailed description of the invention
The present invention is further illustrated by the following examples, but protection scope of the present invention is not limited thereto。
Embodiment 1: the preparation of people's sDR5-Fc antibody fusion protein
By DR5 extracellular fragment gene order (182 aminoacid, http://www.uniprot.org/uniprot/O14763) it is cloned in the carrier for expression of eukaryon pGS-Fc with human IgG Fc section, design primer is: sense(5 '-3 '): ggaagcttgccaccATGGAACAACGGGGACAG, antisense(5 '-3 '): aagaattcTCTGGTCGTGGTGCAGGG。Extract high-purity plasmid (OMEGAPlasmidMidiKit) and to adjust concentration be 500ng/ μ l。Recovery CHO/K1 cell, adjusts cell state to best, and instantaneous electricity turns (300v, interval time, 0.125s, persistent period 0.1ms, shocked by electricity three times), obtains high expressed stable cell line through MSX pressurization screening。Current fusion protein yield of the present invention can reach 1.9g/L。Carry out external functional verification with the albumen after purification, detect its human leukemia cell line's Jurkat cell apoptosis that whether can block TRAIL induction。
Fig. 1 shows this antibody fusion protein sDR5-Fc purity and size, and this fusion protein size 45kd is described, purity is more than 90%。
Fig. 2 shows people sDR5-Fc apoptotic signal blocking effect in Jurkat cell。Streaming result shows: after being simultaneously introduced TRAIL and sDR5-Fc, and apoptosis situation is with normally not deal with group (representing in figure) with Normal similar, and then apoptosis ratio is more serious only to add TRAIL group, therefore illustrates that sDR5-Fc has good blocking effect。
Embodiment 2: people's sDR5-Fc antibody fusion protein protective effect in renal ischaemia reperfusion injury
The foundation of renal ischemic reperfusion injury mouse model
The male C57BL/6 mice of cleaning grade 6-8 week old, after weighing, lumbar injection 5% chloral hydrate (7.5ml/kg) is anaesthetized, mice is placed on thermostatic platform, mouse back unhairing, expose skin, operation is come into effect after mouse temperature returns to 36 DEG C, below mice rib, spinal column both sides open the osculum of about 1cm respectively, and the left kidney of mice is more on the low side than right kidney, therefore the osculum outline of left kidney is downward, cut off Musclar layer again, gently kidney is extruded wound by Cotton swab, expose two kidneys；The kidney base of a fruit of right kidney is slightly short, first right kidney is clamped with bulldog clamp, clamp left kidney more at once, left and right kidney folder closes time difference less than 2min, the color of two kidneys is gradually become peony by scarlet, it is 22min that folder closes set of time, two kidneys are put into internal or with infiltration normal saline gauze and covers mouse back, bulldog clamp is unclamped after 22min, can be seen that kidney color progressively becomes again cerise (referring to QingqingWeiandZhengDong, Mousemodelofischemicacutekidneyinjury:technicalnotesandt ricks.AmJPhysiolRenalPhysiol.2012;303:1487-1494.)。Suture muscles layer and skin respectively, puts back to mouse cage after mice is clear-headed on thermostatic platform, at Reperfu-sion 2h/6h lumbar injection sDR5-Fc antibody fusion protein, takes blood after 24h, separates serum, and take kidney specimen。
The mice random packet of kidney base of a fruit ligation will be carried out, it is divided into normal group (representing in figure), sham operated rats (representing with Sham in figure), PBS group and people's sDR5-Fc group with Normal, often group is further divided into three groups according to dosage difference, at least 6 mices of every group, all in triplicate, specific experiment method and result are described below in experiment:
22min after kidney base of a fruit ligation, during Reperfu-sion 2h/6h, according to the dosage of 15.0mg/kg, people's sDR5-Fc group is by fusion protein 0.15ml that mouse peritoneal injection concentration is 2.16mg/ml。
(1) Mouse Kidney Function detection
Heart extracting blood, 3000rpm is centrifuged 10min, takes serum, and-20 degree preserve, and delivers to clinical laboratory of the first Affiliated Hospital of He'nan University detection (Japanese Olympus automatic clinical chemistry analyzer) blood urea nitrogen and serum creatinine。
Fig. 3 shows that sham operated rats and normal group mice serum blood urea nitrogen and creatinine value are all relatively low and there was no significant difference；Bilateral renal ischemia 22min fills 2hPBS matched group, serum urea nitrogen and creatinine value again and raises substantially, with sham operated rats and normal group significant difference；Fill 2h again at ischemia 22min and inject sDR5-Fc antibody fusion protein group; serum urea nitrogen and creatinine value are decreased obviously; and it is not notable with normal group and sham operated rats difference; with PBS control group, there is significant difference, illustrate that sDR5-Fc antibody fusion protein fills 2h again at mice bilateral renal ischemia 22min and has significant protective effect。
Fig. 9 shows that mice bilateral renal ischemia 22min fills 6h administration group renal function testing result again, similar with Fig. 3, illustrates that sDR5-Fc antibody fusion protein fills 6h again at mice bilateral renal ischemia 22min and has significant protective effect equally。
(2) mouse kidney morphological observation HE(hematoxylin-eosin staining method) dyeing
After mouse kidney tissue was fixed in 4% paraformaldehyde more than 48 hours, adjusts position and put into embedding frame, and know title with Pencil with 2B hardness labelling。Rinse 30min with clear water, will be equipped with the embedding frame of tissue and put into automatic embedding machine and carry out dehydration, transparent, waxdip and embedding treatment。Program is provided that 70% ethanol, 2.5h → 85% ethanol, 1h → 95% ethanol I, 1h → 95% ethanol II, 1h → 100% ethanol I, 1h → 100% ethanol II, 1h → dimethylbenzene I, 45min → dimethylbenzene II, 45min → paraffin I, 1h → paraffin II, 1h → paraffin III, 45min。Tissue is taken out from last paraffin box, being placed in embedding machine (open embedding machine in advance and make the melted paraffin wax of the inside), inserted by tissue in the mold box of full melted paraffin, tissue complete packet is embedded in wax stone, being placed on to freeze and quickly cool down on platform, paraffin embedded tissues embeds successfully。With paraffin slicing machine, wax stone is cut into the serial section of 4 μm, is placed in 40 DEG C of warm water, works as section developing, during without wrinkle, be attached on anticreep microscope slide, every slide pastes two parts of tissues, to compare。On 37 DEG C of roasting sheet machines, roasting sheet 1 hours, preserves in film releasing box。
1. 62 DEG C of roasting sheet 30min。
2. dewax to water: dimethylbenzene I, II, III, each 10min → from high to low gradient alcohol dehydration (100% dehydrated alcohol I, II → 95% ethanol → 85% ethanol → 75% ethanol), each 5min → ddH2OI, II, each 5min。
3. Lignum Sappan uniformly dyeing core: haematoxylin dyeing 10min, flowing water 3min washes away haematoxylin loose colour, 1% hydrochloride alcohol 2sec, running water blueing 15min → PBSI, II, III, each 5min。
4. eosin stains: eosin stains 1-3min → PBSI, II, III, each 5min。
5. dehydration: 85% ethanol, 2sec → 95% ethanol, 5min → 100% dehydrated alcohol I, 5min → 100% dehydrated alcohol II, 5mim。
6. transparent: dimethylbenzene I, 10min → dimethylbenzene II, 10min → dimethylbenzene III, 10min。
7. mounting: neutral gum sealing。
The renal tissue specimens paraffin embedding slices taken off each group carries out HE dyeing, observes and takes pictures。
As can be seen from Figure 4: glomerule and the peripheral cell structure of normal group and sham operated rats are normal; sDR5-Fc group morphological structure is similar to sham operated rats to normal group; there is no obvious necrocytosis; and PBS group glomerule has degeneration and downright bad situation; therefore bilateral renal ischemia 22min fills 2h, sDR5-Fc again kidney is had significant protective effect。
Figure 10 shows that bilateral renal ischemia 22min fills 6h, sDR5-Fc again and kidney has significant protective effect。
(3) immunohistochemical observation apoptosis
Renal tissue is carried out homogenate, extracts total protein, detected the expression of TRAIL, DR5, Caspase-3 and Caspase-8 by SABC。
1) SABC Streptavidin-avidin-biotin complex method (SABC method) specifically comprises the following steps that
2) dewaxing, aquation。
3) tissue slice is put into roasting sheet 2 hours in 60 DEG C of baking boxs。
4) section taken out from baking box is immediately placed on dimethylbenzene I immersion 10min → dimethylbenzene II, 10min → dimethylbenzene III, 10min。
5) dehydrated alcohol I, 5min → dehydrated alcohol II, 5min → 95% ethanol I, 5min → 95% ethanol II, 5min → 85% ethanol, 5min → 70% ethanol, 5min → 60% ethanol, 5min → ddH2O, 5min。
6) PBSI, 5min → PBSII, 5min → PBSIII, 5min。
7) 10min, deactivating endogenous peroxydase are closed by the 3%H2O2 lucifuge room temperature of new preparation。
8) PBSI, 5min → PBSII, 5min → PBSIII, 5min。
9) antigen retrieval。Section being put in citrate buffer (pH6.0), put in microwave oven and heat to boiling with high fire, suspend 2 minutes, then heat 20sec with middle high fire, suspend 2 minutes, middle high fire heats 20sec again, so repeats, totally 10 minutes。Will be equipped with the citrate group box of section to take out in microwave oven, put room temperature and naturally dry in the air cool。
10) when recovering to room temperature, PBSI, 5min → PBSII, 5min → PBSIII, 5min。
11) get rid of surplus liquid, drip the 5%BSA reagent in 50 μ lSABC test kits, room temperature 30min at every tissue。
12) getting rid of surplus liquid, do primary antibodie diluent with the 5%BSA in test kit, each tissue drips the 50 μ l antibody diluted, 4 DEG C of overnight incubation of moisture releasing box。
13) SABC SABC method detection DR5 protein expression, primary antibodie dilution ratio 1:200。
SABC SABC method detection trail protein is expressed, primary antibodie dilution ratio 1:200。
SABC SABC method detection CleavedCaspase3 protein expression, primary antibodie dilution ratio 1:2000。
SABC SABC method detection CleavedCaspase8 protein expression, primary antibodie dilution ratio 1:200。
14) primary antibodie is got rid of, PBSI, II, III, respectively wash 3 times, 5min/ time。
15) drying, every tissue dropping 50 μ l biotinylation two resists, moisture releasing box incubated at room 1 hour。
16) get rid of biotinylation two to resist, with PBSI, II, III, respectively wash 3 times, 5min/ time。
17) dry, every tissue dropping 50 μ lSABC, moisture releasing box incubated at room 1 hour。
18) get rid of SABC, with PBSI, II, III, respectively wash 3 times, 5min/ time。
19) preparation DAB(or AEC) nitrite ion, dry slide, drip DAB(or AEC) nitrite ion, under mirror, control colour developing situation。
20) tap water color development stopping is used。PBSI, II, III, respectively wash 3 times, 5min/ time。
21) haematoxylin redyes 5min, and tap water rushes 10min。Hydrochloride alcohol breaks up, 3sec, tap water 10min。
22) dehydration。60% ethanol, 75% ethanol, 80% ethanol, 90% ethanol, dehydrated alcohol I, II, respectively soak 5 minutes successively。
23) transparent。Dimethylbenzene I, II, III, respectively soak 10min。
24) neutral gum mounting, drips 10 μ l neutral gums organizationally, takes coverslip and slowly depress from side, it is to avoid generate bubble, room temperature preservation。
Fig. 5-8 shows that mice bilateral renal ischemia 22min fills 2h again and respectively organizes ImmunohistochemistryResults Results.
Wherein Fig. 5,6 show respectively DR5 and TRAIL PBS group renal tissue express more, and express less in normal group, sham operated rats and sDR5-Fc group, illustrate that PBS group renal tissue cell surface has the expression of DR5 and then causes a series of reaction to ultimately result in apoptosis, sDR5-Fc group is then by competition binding TRAIL, block DR5 and TRAIL to die inducement signal to transmission tune, thus inhibiting apoptosis。Fig. 7,8 expressions of results showing Caspase3 and Caspase8 respectively。Being shown in the expression in the kidney cell of PBS group with Caspase3 and Caspase8, the tune ultimately resulting in cell is died。
Fig. 5-8 illustrates that mice bilateral renal ischemia 22min fills 2h, sDR5-Fc again and kidney has significant protective effect。
Figure 11-14 shows that mice bilateral renal ischemia 22min fills 6h again and respectively organizes ImmunohistochemistryResults Results; 2h administration group is filled again similar with ischemia 22min; Figure 15 shows that PBS group has the DNA break situation that apoptosis causes simultaneously, and therefore Fig. 5-8 and Figure 11-15 illustrates that kidney is had significant protective effect by sDR5-Fc。
Mice ischemical reperfusion injury is had protective effect by conclusion: sDR5-Fc。

Claims (5)

1. people sDR5-Fc antibody fusion protein is as the application of Renal injury grade medicine。
2. apply as claimed in claim 1, it is characterised in that described people's sDR5-Fc antibody fusion protein is as the application of acute injury of kidney or Renal Ischemia Reperfusion Injury medicine。
3. apply as claimed in claim 1, it is characterised in that described people's sDR5-Fc antibody fusion protein is as the application of acute renal failure or renal transplant rejection medicine。
4. the application as described in as arbitrary in claims 1 to 3, it is characterised in that described people's sDR5-Fc antibody fusion protein, by blocking the apoptosis of TRAIL/DR5 path induction, reduces Renal tissues damage, to reach the effect of protection kidney cell。
5. the protein sequence of people sDR5-Fc antibody fusion protein, sequence modification and the associated biomolecule engineering product application in preparation treatment renal ischemic reperfusion injury medicine。
CN201610041751.7A 2016-01-21 2016-01-21 Application of human sDR5-Fc (soluble DR5-Fc) antibody fusion protein used as renal injury treatment medicament Pending CN105688189A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610041751.7A CN105688189A (en) 2016-01-21 2016-01-21 Application of human sDR5-Fc (soluble DR5-Fc) antibody fusion protein used as renal injury treatment medicament

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610041751.7A CN105688189A (en) 2016-01-21 2016-01-21 Application of human sDR5-Fc (soluble DR5-Fc) antibody fusion protein used as renal injury treatment medicament

Publications (1)

Publication Number Publication Date
CN105688189A true CN105688189A (en) 2016-06-22

Family

ID=56228351

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610041751.7A Pending CN105688189A (en) 2016-01-21 2016-01-21 Application of human sDR5-Fc (soluble DR5-Fc) antibody fusion protein used as renal injury treatment medicament

Country Status (1)

Country Link
CN (1) CN105688189A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112386686A (en) * 2019-08-14 2021-02-23 深圳市中科艾深医药有限公司 Application of human sDR5-Fc recombinant fusion protein in preparation of acute kidney injury prevention and treatment drugs

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102949708A (en) * 2012-11-08 2013-03-06 河南大学 Application of human sDR5 (soluble death receptor 5) protein or sDR5-Fc (fragment crystallizable) antibody fusion protein as pharmaceutical drugs for myocardial infarction

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102949708A (en) * 2012-11-08 2013-03-06 河南大学 Application of human sDR5 (soluble death receptor 5) protein or sDR5-Fc (fragment crystallizable) antibody fusion protein as pharmaceutical drugs for myocardial infarction

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TAKAOMI ADACHI等: "Blockade of Death Ligand TRAIL Inhibits Renal Ischemia Reperfusion Injury,第161-170页", 《ACTA HISTOCHEM CYTOCHEM》 *
XIANGFENGLENG等: "Blocking TRAIL-DR5 signaling with soluble DR5 alleviates acute kidney injury in a severely burned mouse model,第3460-3468页", 《INT J CLIN EXP PATHOL》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112386686A (en) * 2019-08-14 2021-02-23 深圳市中科艾深医药有限公司 Application of human sDR5-Fc recombinant fusion protein in preparation of acute kidney injury prevention and treatment drugs

Similar Documents

Publication Publication Date Title
Kuo et al. Tight junction proteins occludin and ZO‐1 as regulators of epithelial proliferation and survival
Yamaguchi et al. Vascular normalization by ROCK inhibitor: therapeutic potential of ripasudil (K-115) eye drop in retinal angiogenesis and hypoxia
Dang et al. Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model
Tomura et al. Effect of decompressive craniectomy on aquaporin-4 expression after lateral fluid percussion injury in rats
Du et al. Hydrogen-rich saline attenuates acute kidney injury after liver transplantation via activating p53-mediated autophagy
Li et al. Retro-orbital injection of FITC-dextran is an effective and economical method for observing mouse retinal vessels
Moccia et al. Nicotinic acid adenine dinucleotide phosphate activates two‐pore channel TPC1 to mediate lysosomal Ca2+ release in endothelial colony‐forming cells
CN114940702B (en) Application of JWA polypeptide in preparation of anti-neovascular eye disease medicine
CN101397343A (en) VEGF receptor fusion protein and its use in preparation of medicament for treating eye disease
Lachapelle et al. Contractile proteins in podocytes: immunocytochemical localization of actin and alpha-actinin in normal and nephrotic rat kidneys
CN104327169B (en) AP25 and its application in treatment neovascular eye diseases medicine is prepared
CN105688189A (en) Application of human sDR5-Fc (soluble DR5-Fc) antibody fusion protein used as renal injury treatment medicament
Irani et al. An Anti–VEGF-B Antibody Fragment Induces Regression of Pre-Existing Blood Vessels in the Rat Cornea
Marzolla et al. Induction of atherosclerotic plaques through activation of mineralocorticoid receptors in apolipoprotein E-deficient mice
Waller et al. Elastin-like polypeptide: VEGF-B fusion protein for treatment of preeclampsia
Liu et al. Permeability of injured blood brain barrier for exogenous bFGF and protection mechanism of bFGF in rat brain ischemia
CN105983093A (en) Applications of angiogenic factor fusion protein in preparing medicines for treating eye diseases relevant to angiogenesis
CN109735447A (en) It is a kind of for separating the devices and methods therefor of islet tissue
KR102200030B1 (en) Composition for preventing heart damage in case of heart arrest containing 8-Oxo-2'-deoxyguanosine as an active ingredient
Fu et al. Porcine partial liver transplantation without veno-venous bypass: An effective model for small-for-size liver graft injury
CN109771642B (en) c-MET agonistic antibodies and uses thereof
El Farissi et al. Ultrastructural characteristics of myocardial reperfusion injury and effect of selective intracoronary hypothermia: an observational study in isolated beating porcine hearts
Xu et al. Islet transplantation promotes podocyte regeneration in a model of diabetic nephropathy
Das et al. Intrasplenic transplantation of hepatocytes after partial hepatectomy in NOD. SCID mice
CN108379555A (en) Applications of the FGF21 in the drug for preparing hypertension and/or angiosis damage that treatment is caused by angiotensinⅡ

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20160622