CN104535774B - New detection index for drug sensitivity under state of inflammatory bowel disease and application of new detection index in design of drug therapy scheme - Google Patents

Abstract

The invention provides a new detection index for drug sensitivity under the state of an inflammatory bowel disease and application of the new detection index in the design of a drug therapy scheme, relates to multiple fields of pharmaceutical pharmacokinetics, pharmacology, cytobiology and molecular biology, creatively reveals natural drug-resistant phenomena of the inflammatory bowel disease and expounds related mechanisms. According to the new detection index, the phosphorylation of STAT3 is promoted by virtue of cell factors TNF-a, IL17 and LPS, which are excessively secreted in mice with the inflammatory bowel disease, in a way of STAT3/Nf-kb, then the phosphorylation and the nuclear translocation of P65 are induced, the expression of peripheral blood monouclear cells P-gp is promoted, and the drug concentration of an immune inhibitor in the cells is reduced, the treatment effect of the immune inhibitor is inhibited, and the natural drug resistance is generated; by administering a P-gp specific inhibitor, the drug resistance phenomenon can be reverted. The new detection index prompts that P-gp expression quantity of peripheral blood lymphocyte can serve as an evaluation index of the natural drug resistance of patients with IBD (inflammatory bowel disease), and by virtue of the combination of the P-gp inhibitor, the decrease of the pharmaceutical effect caused by the natural drug resistance can be reverted, so that a new thought is provided for the treatment scheme of IBD.

Description

The new Testing index of drug susceptibility and its control in medicine under a kind of inflammatory bowel disease state Treat the application in conceptual design

Technical field

The present invention relates to the multiple fields such as pharmacokinetics, pharmacology, cell biology, molecular biology, innovation Disclose to property the natural drug resistance phenomenon of inflammatory bowel disease, it was found that a kind of new inspection based on PMBC P-gp expressions Surveying index is used for the detection of drug susceptibility under inflammatory bowel disease state, and illustrates related mechanism, while proposing combination P-gp suppressions The therapeutic scheme of preparation is possibly used for reversing inflammatory bowel disease natural drug resistance.

Background technology

Inflammatory bowel disease (IBD) is a kind of chronic gut inflammation disease, mainly including Crohn disease (CD) and exedens knot Two kinds of enteritis (UC).It is not only prevailing in the U.S. and European countries, also raises year by year in the incidence of disease of China.The cause of disease of IBD and Pathogenesis is not yet clear and definite, may be related to many factors such as eating habit, environmental factor, inherent cause, microorganism infection.This Outward, because patient IBD is often accompanied by the phenomenon of immunologic derangement, it is also regarded as a kind of autoimmune disease.Clinical treatment is often Mitigate intestinal inflammation using immunodepressant or anti-inflammatory agent, but repeatedly morbidity and the generation of drug resistance phenomenon be usually Limit its final clinical efficacy.

The drug resistance produced during treatment of autoimmune diseases can be divided into natural drug resistance and acquired resistance two Kind, it is related to PMBC P- glycoprotein (P-gp) expression rising.Natural drug resistance refers to patient in first administration Occur as soon as drug resistance phenomenon.In autoimmune disease pathogenic process, excessive inflammatory factor is transported to extracellular by P-gp Abnormal inflammatory reaction is produced, and feeds back stimulation P-gp expression and further raised.Because most immunodepressant are the substrates of P-gp, Therefore elevated P-gp can will discharge immunocyte outside immunodepressant, cause treatment of autoimmune diseases to there may be day The phenomenon of right resistance.Acquired resistance refers to that patient engenders the phenomenon declined to drug susceptibility after multiple dosing.Greatly Quantity research shows often occur serious drug resistance reaction after autoimmune disease patient's Long-Time Service immunodepressant, that is, exempt from Epidemic disease inhibitor is expressed by inducing peripheral blood monocyte P-gp, intracellular drug concentration is reduced, so as to produce acquired resistance.For Control disease, mitigation pathological symptom, patient IBD need to be taken including the panimmunity inhibitor including glucocorticoid, but Clinical research confirmation long-term prescription can cause PMBC P-gp rises to ultimately cause curative effect of medication decline.Although with regard to The acquired resistance produced in IBD therapeutic processes has been found to, but patient IBD rarely has research with the presence or absence of natural drug resistance.It is right The ignorance of natural drug resistance may cause clinical treatment to be delayed and mistake increases dosage, accelerate the generation of acquired resistance.Cause This, it is very necessary and important to carry out Scientific evaluation to before the first medication of the patient IBD or repeatedly drug susceptibility after medication, assesses As a result contribute to optimizing the therapeutic scheme of IBD, improve the final curative effect of short-term and long term immunosuppressant medication.

The content of the invention

It is contemplated that confirming the generation of IBD natural drug resistance phenomenons, and the related mechanism that this phenomenon occurs is illustrated, i.e., Cytokine TNF-α, the IL17 and LPS of excessive secretion is promoted by STAT3/Nf- κ b approach in inflammatory bowel disease mouse body STAT3 phosphorylations, and then induce P65 phosphorylations and nuclear translocation to promote PMBC P-gp expression, reduce intracellular immunity The drug concentration of inhibitor, suppresses its therapeutic effect, so as to cause the generation of natural drug resistance.And give P-gp specific inhibitors This resistance phenomenon can be reversed.Quantitative PCR and LC-MS/MS testing results show peripheral blood mononuclear in inflammatory bowel disease Mice Body Cell mdr1 expression is raised, intracellular levels of drugs is low compared with normal mouse；ELISA testing results confirm inflammatory bowel disease mice plasma Middle TNF-α, IL- β, IL-6, IL-17, LPS secretion level are raised, and In vitro cell experiment shows that above-mentioned inflammatory factor and LPS are equal Inducible mdr1 expression is raised；Western Blot experiments show TNF-α, and LPS and IL17 is by inducing P65 phosphorylations and core Indexing promotes PMBC P-gp expression.IBD periphery blood stranguries in patient body are simulated by inflammatory stimulus or transfection experiment The physiological status of bar cell, gives the phenomenon that reversible intracellular drug concentration declines after specific P-gp inhibitor.

Description of the drawings

There is natural drug resistance phenomenon in Fig. 1, TNBS modeling mouse, and PMBC P-gp expressions are raised, and outer row makees With enhancing, the reduction of intracellular cyclosporine CYSA accumulation levels.

Fig. 2：Inflammatory factor (TNF-α, IL- β, IL-6, IL-17) and LPS levels significantly rise in TNBS modeling mice plasmas It is high

Fig. 3：Inflammatory factor (TNF-α, IL- β, IL-6, IL-17) and the inducible humanized's macrophage THP-1 of LPS and people Arrange transporter P-gp expression outside source property lymphocyte CCRF-CEM to raise

Fig. 4：IL-17 can activate STAT3/Nf- κ b approach, and LPS, TNF-α can activate P65 phosphorylations and nuclear translocation

Fig. 5：P-gp inhibitor reversible IBD natural drug resistance phenomenons.

Specific embodiment

Medicine and reagent

Cell culture：Humanized's macrophage THP-1 and humanized's lymphocyte CCRF-CEM is purchased from U.S.'s culture presevation Center (American Type Culture Collection, ATCC, Rockville, MD), in 37 DEG C, 5%CO₂Under the conditions of Cellar culture, culture medium is the culture mediums of RPMI 1640, containing 10% calf serum, and penicillin 62.5mg/L, streptomysin 100 Mg/L, cultivates in tissue cultures retorts bottle, and every centrifugation in 2～3 days liquid is changed.RPMI1640 culture mediums are purchased from GIBCO (Gland Island, New York), calf serum is purchased from Hyclone (Logan, Utah, USA), and cell culture consumptive material is purchased from Costar (Cambridge, MA, USA).

Animal feeding：Cleaning grade BALB/c mouse, body weight 18-22g, week old 8-10 week, by Nanjing General Hospital, Nanjing Military Area Command, PLA Experimental Animal Center is provided.Of the right age BALB/c mouse is raised under the conditions of feeding standard, temperature：22-24 DEG C, relative humidity： 60%, illumination, dark：12h/12h, free diet, drinking-water are tested after adapting to one week.

PCR：Mouse mdr1a and internal reference actin, people MDR1 and internal reference ACTIN primers are purchased from Invitrogen (Gibco- Invitrogen, USA)；Trizol total RNA extraction reagents, RT-PCR kit, Real-time PCRMaster Mix (SYBR Green) it is purchased from Takara (Dalian is precious biological, Japan).

LC-MS/MS：Methyl alcohol (chromatographically pure), acetonitrile (chromatographically pure) are purchased from Sigma-Aldrich (St.Louis, MO).Formic acid And other analysis pure chemistry reagents are purchased from Nanjing chemical reagent factory (Jiangsu, China).Ultra-pure water is by Milli-Q system It is prepared by (Millipore, Milford, MA, USA).

Westem：RIPA lysates, 5 × sample-loading buffer, 1.5 M Tris-HCl pH 8.8,1 M Tris-HCl pH 6.8th, 10%SDS, an anti-two removal resistant liquid, BCA determination of protein concentration kits are purchased from green skies biotechnology research institute (river Soviet Union, China).Phenylmethylsulfonyl fluoride (phenylmethylsulfonyl fluoride, PMSF) gives birth to emerging biotechnology purchased from Nanjing Co., Ltd (Jiangsu, China).Glycine (electrophoresis level), SDS (electrophoresis level), Tris base (electrophoresis level), Ammonium Persulfate 98.5 (ammonium persulfate, APS) is purchased from Amerso (USA).30%Acrylamide/Bis Solution (29：1)、 TEMED (N, N, N ', N '-tetramethyl ethylene diamine, TEMED) is purchased from Bio-Rad.Enhanced chemical luminescence reagent box (Enhanced Chemiluminescence, ECL) is purchased from Thermo Fisher Scientific(USA).Stat3, P65, P-P65, Lamin B monoclonal antibodies, the anti-rabbit two of horseradish peroxidase-labeled Anti- to be purchased from Cell Signaling Technology (Boston, USA), GAPDH monoclonal antibodies are purchased from Bioworld Technology (MN, USA)..

There is natural drug resistance phenomenon in the TNBS modeling mouse of instantiation one, and PMBC P-gp expressions are raised, Outer row's effect strengthens, and intracellular cyclosporine CYSA accumulation levels are reduced.

TNBS mouse modelings：BALB/c mouse is raised one week in Animal House before modeling.After fasting 24 is little, phenobarbital anesthesia Mouse, 3.5F conduits insert the deep about 5.5cm of enteron aisle from anus.Modeling group is given with the dosage of 100mg/kg and is dissolved in 30% ethanol TNBS, control group gives 30% ethanol with equal administered volume.Mouse is inverted 3 minutes afterwards, the 3rd day or the after administration 7 days eye sockets take blood in the sterile test tube containing heparin and put to death mouse.

PBLC is extracted：Principle of the PBLC based on gradient centrifugation passes through Lympholyte Mammal kits (Cedarlane Laboratories Ltd, Hunby, Ontario, Canada)) extract.To extracting obtained by Venous blood in add equal-volume RPMI-1640 culture mediums to carry out 1: 1 dilution, mix after, gently add to equal-volume point along tube wall (density on chaotropic：1.086±0.001 g/cm³), and form obvious interface with separating liquid.800 × g of Jing are centrifuged 20 minutes Afterwards, careful to draw the vaporific buffy coat of middle white, RPMI-1640 culture mediums are washed twice, after 800g is centrifuged 10 minutes, Appropriate RPMI-1640 culture mediums are resuspended, treat that follow-up test is used.

PBLC P-gp gene expressions：1 is added in the PBLC obtained by extraction MLTrizol, is blown and beaten to clarification and after acellular agglomerate with liquid-transfering gun, in transferring them to without enzyme 1.5mL EP pipes, according to RNA is extracted in the operation of Takara total RNA extraction reagents box, and it is quantitative to carry out RNA.According to RT-PCR kit specification, by mRNA Reverse transcription is cDNA.According to Thermal Cycler DiceTM Real Time System (TakaRa Code：TP800) make Required to carry out PCR experiment operation with specification.Quantitative PCR reaction condition is：

Stage1：Denaturation：95 DEG C, 30sec

Stage2：PCR reacts：95 DEG C, 10 sec；50 DEG C, 30 sec；72 DEG C, 30sec；40cycles

Stage3：Melt curve analysis are analyzed：95 DEG C, 15sec；70 DEG C, 15sec.

The gene order of mouse mdr1a and internal reference actin is as follows：

The AGGGCATTTACTTCAAACTTGTC of Mouse-mdr1a-F 5 ' 3 ',

The CCTGTCTTGGTCATGTGGTC 3 ' of 1 a-R of Mouse-mdr 5 ',

The TCTGGCACCACACCTTCTA of Mouse-actin-F 5 ' 3 ',

Mouse-actin-R 5’AGGCATACAGGGACAGCAC 3’。

PBLC P-gp functions are investigated：It is thin that model group/control group extraction gained periphery hemolymph is given respectively 5 μM of RHO123 of born of the same parents or 10 μ g/ml CYSA, put after being incubated 2 hours in 37 DEG C of shaking tables, medicine are suctioned out rapidly, with 4 DEG C of blank Hank ' s solution swings to be washed 3 times, adds 1mL ultra-pure waters, puts -80 DEG C of refrigerator multigelation tertiary crushing cells.Wherein take 0.1mL thin With the legal albumen of Coomassie brilliant blue, remaining cell suspension puts -20 DEG C for determining to born of the same parents' suspension.

Inflammatory factor (TNF-α, IL- β, IL-6, IL-17) and LPS levels in the TNBS modeling mice plasmas of instantiation two Significantly raise

TNBS mouse modelings：As shown in instantiation one

Blood plasma inflammatory factor and LPS secretion levels are detected：The measure of inflammatory factor is according to commercialization ELISA reagents in blood plasma Box specification is carried out (Excell, Shanghai, China), and the measure of plasma levels of LPS is according to commercialization endotoxin detection kit Specification is carried out (Xiamen BioEndo Technology ， Co.Ltd).

Instantiation three：Inflammatory factor (TNF-α, IL- β, IL-6, IL-17) and the inducible humanized's macrophages of LPS Arrange transporter P-gp gene expressions outside THP-1 and humanized lymphocyte CCRF-CEM to raise

Humanized's macrophage THP-1 and humanized lymphocyte CCRF-CEM are inoculated in 6 orifice plates, are given respectively containing not With concentration inflammatory factor and the serum-free RPMI-16402mL of LPS, withdrawal after being incubated 72 hours in 37 DEG C of incubators, 4 DEG C of Jing Hank ' s wash 3 times and add 1mL Trizol reagents afterwards, blow and beat to clear and after acellular agglomerate, carry out reverse transcription and reality When quantitative PCR experiment, detection MDR1 expression change.Concrete operations scheme is shown in instantiation one.

The gene order of people MDR1 and internal reference ACTIN is as follows：

The GCTGGGAAGATCGCTACTGA of Human-MDR1-F 5 ' 3 ',

The GGTACCTGCAAACTCTGAGCA of Human-MDR1-R 5 ' 3 ',

The GCGTGACATTAAGGAGAAG of Human-ACTIN-F 5 ' 3 ',

Human-ACTIN-R 5’GAAGGAAGGCTGGAAGAG 3’。

Instantiation four：IL-17 can activate STAT3/Nf- κ b approach, and LPS, TNF-α can activate P65 phosphorylations and consideration convey Position

Humanized macrophage THP-1 is incubated in T-25 type blake bottles, gives 50mg/ml TNF-αs or 2 μ g/mlLPS. Stop administration respectively at 0,10,20,40,60,120min, after PBS washes cell once, 1500rpm centrifugation 5min collect cell.Press Plasmosin and core egg are respectively obtained according to nucleoprotein and plasmosin extracts kit specification (triumphant base is biological, Nanjing, China) In vain.Western Blot methods detect nucleus and endochylema p65 and phosphorylation P65 expression.Specific experiment scheme is as follows：

BCA methods determine protein concentration.Protein sample adds 4x sample-loading buffers and boils 10min according to volume ratio 3: 1.System Standby 10% SDS-PAGE, is separated by electrophoresis to sample.After electrophoretic separation, wet electrotransfer is carried out, albumen is gone to into pvdf membrane. Pvdf membrane closed 1 as a child for 37 DEG C in the TBST solution containing 5% skimmed milk power, and 4 DEG C of incubations one are anti-overnight, and corresponding two is anti-37 DEG C Incubation 1 hour, ECL developments, the imaging of Bio-Rad gel imaging instruments.

Humanized lymphocyte CCRF-CEM is incubated in T-25 type blake bottles, gives 50mg/ml TNF-αs, or 100mg/ Ml IL17, or 100mg/ml IL17 10 μM of stat-3 phosphorylation inhibitor Stattic of combination.Respectively at 0,20,40,60, 120,480min stop administration, and after PBS washes cell once, 1500rpm centrifugation 5min collect cell.According to nucleoprotein and endochylema egg White extracts kit specification respectively obtains plasmosin and nucleoprotein.Western Blot methods detect nucleus and endochylema P65, stat3 and phosphorylation stat3 expression.Specific experiment scheme is ibid.

Instantiation five：Suppress monocyte P-gp reversible IBD natural drug resistance phenomenons

To simulate the physiological status of IBD PBLCs in patient body, stimulated by inflammatory factor or transfection makes THP-1 cells or the high expression P-gp of CCRF-CEM cells.Humanized macrophage THP-1 is given after 2 μ g/ml LPS 24h, single To P-gp specific substrates RHO123 or combination P-gp specific inhibitor LY335975, after checking inflammatory stimulus, arrange outside THP-1 Transporter expression is raised, and the outer row functions of P-gp strengthen.Humanized's lymphocyte CCRF-CEM forms the stable height of P-gp by transfection The CCRF-CEM-MDR1 cell lines of expression.By PCR experiment, flow cytometer detection, RHO123 intake experiments from gene, albumen, function Three aspect confirmation CCRF-CEM-MDR1 cell lines can simulate patient's IBD PMBC.Individually give LPS inductions THP-1 and height turn the CCRF-CEM-MDR1 cell line CYSA (10 μ g/ml) of P-gp or while are combined P-gp specific inhibitors After LY3359752h, LC-MS/MS determines the accumulation level of intracellular cyclosporine CYSA, and example one is shown in concrete operations.

Claims (2)

1. detect the reagent of PMBC P-gp in the case where inflammatory bowel disease state is prepared in drug susceptibility diagnostic reagent Using, it is characterised in that the expression of detection P-gp, expression is raised and represents the phenomenon that there is natural drug resistance；

Described P-gp is PMBC P- glycoprotein.

2.P-gp inhibitor is preparing the application in reversing inflammatory bowel disease natural drug resistance phenomenon medicine, it is characterised in that described P-gp inhibitor reverses inflammatory bowel disease natural drug resistance phenomenon by suppressing the activity of P-gp and expressing.