CN102641502A - Novel use of aromatase inhibitors - Google Patents

Abstract

The invention relates to a novel use of aromatase inhibitors, in particular to application of the aromatase inhibitors in terms of preparation of medicines for treating male bladder outlet obstruction and lower urethral syndromes caused by the same. The medicines can rapidly and effectively treat bladder outlet obstruction of male mice. The novel use can be clinically applied better, provides a fundamental basis for clinical treatment, and is a novel method for treating the bladder outlet obstruction.

Description

The new purposes of aromatase inhibitor

Technical field

The present invention relates to medical usage, be specifically related to the application of aromatase inhibitor in the medicine of the syndrome of the lower urinary tract for preparing control male's FBOO and cause.

Background technology

Along with the prolongation of human longevity, the following urethra of elderly men (lower urinary tract, LUT) the increasing concern that receives society and medical circle.

Epidemiological study show 26% the people of having an appointment among the 40-49 male in year have the following urethra symptom group of moderate and severe (lower urinary tract syndrome, LUTS); In 70 years old male, have an appointment and 46% LUTS occurs.

The epidemiological survey of China shows; In the general crowd in urban district, Shanghai City, extract more than 40 years old 1583 of male residents; Through methods such as international prostate gland symptoms scoring (IPSS) and quality of life (QoL) scorings, the prevalence of investigating LUTS among this crowd reaches the influence to quality of life.The result is in 40-49 year, 50-59 year, 60-69 year with greater than in 70 years old age group, in, severe LUTS (IPSS >=8) prevalence is respectively 8.7%, 19.4%, 32.3% and 40.2%, appear with age growth prevalence ascendant trend; Along with the increase of the LUTS order of severity, patient's QoL scoring is significantly rising (P＜0.01) also.China male LUTS prevalence has a strong impact on male's physiological health and quality of life also constantly near American-European countries.

The main diseases of male's LUTS because of be FBOO (bladder outlet obstruction, BOO), urethra inflammation, urinary incontinence and bladder cancer.

FBOO (BOO) is modal a kind of disease (Groutz et al.2001) in the following urethra symptom group (LUTS).BOO is meant that from urodynamics definition the urine resistance to outflow that neck of bladder and near-end urethra a variety of causes cause raises, the urinary system difficulty, can not urinate and can not the emptying bladder in storage urinate.

BOO often causes the detrusor of bladder dysfunction, produces a series of severe complications then, is interrupted like urine stream; Urinary system difficulty, can not urinate and can not the emptying bladder in storage urine, serious can cause hydronephrosis; Chronic renal insufficiency, long-term dysuria causes abdominal pressure to increase, and also can cause inguinal hernia; Internal hemorrhoid and proctoptosis, renal failure and sudden death etc.

The treatment means of BOO mainly contains Drug therapy, operative treatment at present, wherein:

1, surgical operation therapy

Acceptable clinically at present interventional therapy mainly is: transurethral prostatic resection (TURP), open prostatectomy (OS), the electrified art (TUVP) of per urethra prostate, laser gasification art (VLAP), transurethral microwave iatrotechnics (TUMT), per urethra puncture ablation (TUNA), a matter laser coagulation (ILC), urethra rack art.

Should select to help most patient's modus operandi according to patient's concrete condition clinically.The surgical operation curative effect of standard is obvious, and symptom gets improvement for a long time and dangerous little.But more and more tend to minimally-invasive treatment at present; Symptom improves to a certain extent; Dangerous (some case) is also less than open surgery; But lack relevant persistency evidence at present, do not confirm as yet whether these new techniques have bigger advantage (Yang Yong 2002) than standard surgical (like TURP) on expense and curative effect.

2, Drug therapy

α 1AR antagonist: the main hypotype in prostate is α 1a AR and α 1d AR, and α 1a AR antagonist can improve among the BPH prostate smooth muscle contraction state, and (Andersson 2000; Michel and Vrydag 2006), alleviates the extruding of prostate, thereby improve the symptom of obstruction of bladder urethra.Though the diastole that α 1a AR blocker can bring the prostate smooth muscle with increase the urine flow, improve the symptom of blocking that BPH causes, the scoring of the LUTS that BPH brings does not change.Have only α 1a AR and α 1d AR blocker to unite use, the scoring of LUTS just is able to change (Haynes et al.2003; Sigala et al.2004; Barendrecht et al.2008).Its effectiveness of α 1AR antagonist and safety are confirmed in open expansion clinical trial.But clinical existing all α 1AR antagonisies are all relevant with generation dizzy, weak and orthostatic hypotension.

The 5 inhibitor: testosterone is converted into dihydrotestosterone under the effect of 5 to stimulate prostatic hyperplasia, prostatic hyperplasia be the main diseases therefore that causes FBOO.In present existing hormone therapy, 5 inhibitor finasteride is unique good effectiveness and acceptable safety medicine of in randomized clinical trial, showing.The modal side effect of finasteride is a sexual function badness, descends and sexual impotence like ejaculation minimizing, libido.

The medicine that has gone on the market at present all is the FBOO that causes to the treatment prostatic lesion, does not see the treatment to female/androgen FBOO that causes out of proportion as yet.

Aromatase is a kind of compound enzyme that belongs to Cytochrome P450, and the oxidable 19-methyl of sloughing C19 androgen (androstenedione and testosterone) makes A ring aromatisation, thereby is transformed into C18 estrogen (estrone and estradiol).Therefore, aromatase is the important enzyme of synthetic estrogen in the body.Aromatase inhibitor passes through the binding ability with aromatase, suppresses the level that aromatase activity can reduce the body inner estrogen.Aromatase inhibitor commonly used at present is the tripyrrole derivant, is non-steroidal class competitive inhibitor, like Anastrozole (Anastrozole), letrozole (Letrozole), finrozole (Finrozole) and vorozole (Vorozole) etc.

Letrozole (Letrozole), chemical name is: 1-[two (4-cyano-phenyl) methyl]-1,2,4-triazole; Perhaps be 4,4 '-(1H-1,2,4-triazol-1-yl methylene)-two benzene nitriles; Its CAS registration number is 112809-51-5, is aromatase inhibitor of new generation, is the synthetic benzyl triazole derivative of manual work; Letrozole makes decrease in estrogen through suppressing aromatase, thereby eliminates the stimulation of estrogen to tumor growth.Be mainly used in postmenopausal women with advanced breast carcinoma, be used for the second line treatment after the estrogen antagonist treatment is failed more.

Anastrozole (Anastrozole), chemical name: (tetramethyl-5-(1H-1,2,4-triazol-1-yl methyl)-1,3-benzene diacetonitrile), molecular formula: C ₁₇H ₁₉N ₅, molecular weight: 293.37, the CAS registration number is 120511-73-1.Be the strong nonsteroidal aromatase inhibitor of imitating.Can reduce blood plasma estrogen level, produce the effect that suppresses the breast tumor growth.Be used for postmenopausal women's advanced breast cancer treatment clinically.

Finrozole (Finrozole) and vorozole (Vorozole) also belong to aromatase inhibitor, can be used for postmenopausal women's advanced breast cancer treatment.

Do not see at present the report of aromatase inhibitor aspect the FBOO associated uses as yet.

Summary of the invention

The new purposes that the purpose of this invention is to provide a kind of aromatase inhibitor.

The invention provides the application of aromatase inhibitor in the medicine of the syndrome of the lower urinary tract for preparing treatment male's FBOO and cause.

In the above-mentioned application: the symptom of said FBOO is: frequent micturition, urgent micturition, dysuria, dripping not to the utmost, urination time prolongs.

Said FBOO causes by female/androgen is out of proportion;

Said aromatase inhibitor is the tripyrrole derivant, is preferably Anastrozole, letrozole, finrozole and vorozole.

Said aromatase inhibitor is the preparation that contains aromatase inhibitor, and said preparation can commercially availablely obtain, or forms preparation with pharmaceutically acceptable carrier or diluent.

Said preparation is tablet, capsule, granule, pill, oral liquid or injection, and said injection is injection or freeze-dried powder.

Said pharmaceutically acceptable carrier or diluent are meant the pharmaceutical carrier that pharmaceutical field is conventional, are selected from filler, binding agent, disintegrating agent, lubricant, suspending agent, wetting agent, solvent, surfactant or the correctives one or more.

Said filler is selected from starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline Cellulose or glucose etc.;

Said binding agent is selected from cellulose derivative, alginate, starch, dextrin, gelatin or polyvinylpyrrolidone etc.;

Said disintegrating agent is selected from microcrystalline Cellulose, carboxymethyl starch sodium, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose or cross-linking sodium carboxymethyl cellulose;

Said lubricant is selected from stearic acid, Polyethylene Glycol, calcium carbonate, sodium bicarbonate, micropowder silica gel, Pulvis Talci or magnesium stearate;

Said suspending agent is selected from micropowder silica gel, Cera Flava, cellulose, solid polyethylene glycol;

Said wetting agent is selected from glycerol, tween 80, ethyoxyl castor oil hydrogenated or lecithin;

Said solvent is selected from ethanol, liquid polyethylene glycol, isopropyl alcohol, tween 80, glycerol, propylene glycol or vegetable oil, and said vegetable oil is selected from soybean oil, Oleum Ricini, Oleum Arachidis hypogaeae semen, mediation wet goods;

Said surfactant is selected from dodecylbenzene sodium sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, the fatty acid Pyrusussuriensis is smooth or Polysorbate (tween) etc.;

Said correctives is selected from aspartame, Sucralose, essence, citric acid or saccharin sodium.

The consumption of said aromatase inhibitor is 1-50mg/Kg/d, is preferably 5-25mg/Kg/d.

The new purposes of aromatase inhibitor provided by the invention has the following advantages:

1, the present invention proposes the new purposes of aromatase inhibitor class medicine; Be specifically related to new application at the syndrome of the lower urinary tract of treating male's FBOO and causing; This medicine can be treated FBOO fast and effectively; For clinical treatment is provided fundamental basis, for the treatment FBOO a kind of new method is provided simultaneously;

2, experiment confirm: with the syndrome of the lower urinary tract that aromatase inhibitor is treated FBOO and caused, sx is obvious, and treatment rate is 100%.

Description of drawings

Fig. 1: the structural representation of aromatase expression vector.

Fig. 2: the PCR qualification result, No. 6 positive ,+positive contrast.

Fig. 3: Southern Blot The selection result, 1 positive contrast is the probe that reclaims, 6 is the positive Mus of southern blot.

Fig. 4: the evaluation of mouse propagation urinary system phenotype: the left side two Mus difference: the 1st, the urine retention mice of positive transgenic mice; The wild-type mice of 2 negative matched groups, the anatomy analysis that contains the positive Mus that changes the aromatase gene that the right one Mus is analyzed for Fig. 3 southern blot are the morphological change of the bladder that causes of urine retention.

Fig. 5: bladder body HE dyeing and Van Gieson coloration result, A is the HE coloration result, B is a Van Gieson coloration result; Wherein, 1 is wild-type mice, 2 positive transgenic mices; A representes collagen fiber, and b representes muscle fiber.

Fig. 6: the sxemiquantitative RT-RCR of aromatase gene, A1, A2 are the aromatase transgenic mice, WT1, WT2 are wild-type mice.

Fig. 7: aromatase inhibitor Western Blot testing result, A1, the positive transgenic mice of A2, WT are wild-type mice.

Fig. 8: transgenic mouse that aromatase inhibitor is handled and positive control Mus: A are the transgenic mouse that letrozole is handled; B is the transgenic mouse that Anastrozole is handled; C is the transgenic mouse that finrozole is handled, and among the figure: the negative matched group of A:WT, AROM+ are the urine retention mice, and AROM+latrozol is the letrozole group; AROM+Anastrozole is the Anastrozole group; AROM+Finrozole is the finrozole group.

Fig. 9: histopathological analysis, wherein the negative matched group of A (being WT) is the sphincter of wild-type mice urethral orifice; B is that urine retention mice (being AROM+) is the sphincter of transgenic mouse urethral orifice; C is letrozole group (being AROM+latrozol); D is for being Anastrozole group (AROM+Anastrozole); E is for being finrozole group (AROM+Finrozole).

The specific embodiment

Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.

Embodiment 1: the preparation of mouse model

List of references: Molecular mechanisms of bladder outlet obstruction in transgenic male mice over expressing aromatase (Cyp19a1) .LinW, Rahman NA, Lin J; Zhang H, Gou K, Yu W; Zhu D; Li N, Huhtaniemi I, Li X.Am J Pathol.2011Mar; 178 (3): 1233-44.PMID:21356374.

Experimental animal: the C57BL/6 mice is available from Academy of Military Medicine, PLA's animal center.C57BL/6 is the external inbred line of introducing, and is that B6F129 and B6JEiF78 are hybridized, the inbred line of then producing through Sibling's inbreeding in 129 generations, and the related gene type is a/a.

Test reagent: pUB6/V5-HisA (Cat.No.V25020), available from Invitrogen, Carlsbad, CA; PRC/CMV (Cat.No.10131027), available from Invitrogen, Carlsbad, CA.

Make up animal model with bladder outlet obstructed with the C57BL/6 mice, concrete grammar is following:

One, the structure of recombinant expression carrier (or being the aromatase expression vector)

1, the acquisition of the promoter of Ubiquitin C gene and exon I thereof and intron I

With restricted enzyme BglII and HindIII enzyme action pUB6/V5-HisA; Reclaim promoter and the exon I and the intron I fragment of Ubiquitin C gene, wherein the promoter of Ubiquitin C gene and exon I thereof and intron I and this fragment at the nucleotide sequence of the restriction enzyme site at two ends on the carrier pUB6/V5-HisA shown in artificial sequence in the sequence table 1; Nucleotide is the recognition sequence (A|GATCT) of BglII to artificial sequence 1 from 5 ' terminal 1-6 position, and nucleotide is the recognition sequence (A|AGCTT) of HindIII to artificial sequence 1 from 5 ' terminal 1218-1223 position; Nucleotide is the promoter of fragment Ubiquitin C gene and the sequence of exon I and intron I thereof to artificial sequence 1 from 5 ' terminal 7-1217 position, and its artificial synthesized sequence is seen shown in the artificial sequence 1 in the sequence table.

2, the acquisition of the cDNA of people's fragrance enzyme coding gene:

Two ends have the cDNA full length sequence of people fragrance enzyme coding gene of restriction enzyme site shown in artificial sequence in the sequence table 2; Wherein sequence 2 is the recognition sequence (A|AGCTT) of HindIII from 5 ' terminal 1-6 position nucleotide; 2155-2160 position nucleotide is the recognition sequence (T|CTAGA) of XbalI; The cDNA full length sequence of 7-2154 position nucleotide behaviour aromatase encoding gene, its artificial synthesized sequence is seen shown in the artificial sequence 2 in the sequence table.

3, the polyA signal sequence of bovine growth hormone gene:

The polyA signal sequence that on carrier pRC/CMV, has bovine growth hormone gene, its nucleotide sequence are seen shown in the artificial sequence 3 in the sequence table;

4, the structure of recombinant expression carrier

With restricted enzyme BglII and HindIII enzyme action carrier pRC/CMV; Reclaim big fragment (promptly removing the carrier segments of CMV promoter); The promoter and the exon I thereof of the Ubiquitin C gene that enzyme action in itself and the step (1) is obtained are connected with intron I fragment, obtain recombinant vector pRC/Ubiquitin C;

With restricted enzyme HindIII/XbaI enzyme action recombinant vector pRC/Ubiquitin C, reclaim big fragment; CDNA fragment with restricted enzyme HindIII/XbaI enzyme action people fragrance enzyme coding gene; Connect; Obtain recombinant expression carrier pRC/Ubiquitin C/cDNA (being the aromatase expression vector); Its structure is as shown in Figure 1, obtains dna fragmentation " the cDNA+bGH polyA of the promoter of Ubiquitin C gene, exon I and intron I+ people fragrance enzyme coding gene ", and its nucleotide sequence is seen shown in the artificial sequence 4 in the sequence table.

Two, the structure of transgenic mice

The aromatase expression vector that makes up is carried out double digestion with Bgl II and Dra I; Reclaim dna fragmentation; Be the cDNA+bGH polyA of promoter, exon I and the intron I+ people fragrance enzyme coding gene of Ubiquitin C gene; Above-mentioned dna fragmentation is processed the suspension of 2ng/ μ L, and micro-procaryotic injection is gone in the C57BL/6 mouse fertilized egg; Embryo transfer goes into to treat in the pregnant parent uterus; Produce transgenic mice, the transgenic mice meter that obtains is done F0 generation.Concrete experimental procedure is following:

(1) superovulation (ultra row) with get ovum

1, superovulation: get the 4-6 ultra ovulation of the female Mus do donor in age in week; Lumbar injection pregnant mare serum (PMSG) and human chorionic gonadotropin (hCG) are induced female Mus ovulation; Be spaced apart 45h the two inject time, the ovulation occur in the injection hCG after 13h, ID is every Mus 8ul.Every female Mus places in the normal public mouse cage of raising separately behind the injection hCG, checks vaginal suppository morning next day.

2, get ovum: put to death the female Mus that vaginal suppository is arranged with the cervical vertebra dislocation method; Under a bit of uterine scissors of complete taenia tubae; Place the PBS balanced salt solution; Under anatomical lens, find out fimbriae of uterine tube portion, germ cell is gone out, remove behind the cingens granular cell immediately with hyaluronidase and ovum changed washing promptly can be used for microinjection 4 times to the fresh medium with the syringe of band syringe needle.

(2) injection foreign DNA:

Microinjection need be used the micrurgy appearance, is used for controlling micro-ovum pin and the microinjection pin held.Get the fertilized egg cell and the foreign DNA suspension that have prepared, under inverted microscope, do microinjection.Earlier hold germ cell with holding the ovum pin during injection, the reuse entry needle is drawn the foreign DNA suspension, promotes entry needle then, makes its male pronucleus that thrusts germ cell, and 1-2 μ L DNA is injected (DNA concentration: 2ng/ μ L).Injection finishes, and slowly extracts entry needle out, and germ cell is put into new culture fluid, makes its recovery.The germ cell of 50-80% is still kept fit after microinjection.

(3) transplanting of injection back germ cell (or embryo):

Injection there is the germ cell of foreign DNA be transplanted in the fallopian tube of the female Mus of post-coitum 0.5d pseudo-fetus the same day at micrurgy.Foreign DNA can directly import in the protokaryon, and the foreign DNA of importing is integrated with the acceptor gene group before the spilting of an egg.After treating the female Mus output of pseudo-fetus young baby, available young Mus tail extracts genomic DNA and detects.

Following implantation method is adopted in this experiment: with pentobarbital sodium anesthesia receptor Mus, from the thick conduit of pseudo-fetus Mus vagina to 10 millimeters of cervical canal insertion external diameters, the fine duct with 5 millimeters of diameters inserts thick conduit inboard then, extend into a body of uterus or a side cornua uteri.Blastaea being preserved liquid together with 2 milliliters transplant into through fine duct, specifically is through conduit blastaea to be blown into intrauterine 1-3 minute with the carbon dioxide that dry ice generates.

Also can adopt following implantation method: with pentobarbital sodium anesthesia receptor Mus; Last rib bones level of center line is cut 1cm left and right sides otch along receptor Mus back; Push otch gently aside, the visible white adipose pad that is positioned at the ovary top. clamp the white adipose pad with pincet and it is moved to external, clamp with hopkins' vascular clamp; And lean on its gravity to play fixation, in case fallopian tube withdrawal body cavity.Inhaling the preparation of 15-20 germ cell with ovum shifting tube transplants. and mice is forwarded to carefully search out fallopian tube under the anatomical lens.It is thus clear that fallopian tube is held by the cyst membrane that one deck is translucent, cyst membrane is torn and expose fallopian tube, zygote transplation in the ovum shifting tube is expanded portion to fallopian tube.Fallopian tube is carefully put back to, sewed up.3 repetitions are established in experiment.

Three, the screening of transgenic mice

The evaluation of positive mice and breeding: utilization PCR and Southern Blot method are screened positive transgenic mice;

(1) PCR screening technique:

Template DNA is the Mus tail DNA of the young Mus of the female Mus of the pseudo-fetus product Founder of institute; (transgenic animal that the female Mus of pseudo-fetus produces are called Founder) forward primer is seen the artificial sequence 5 in the sequence table; Reverse primer is seen the artificial sequence 6 in the sequence table; Amplification 572bp fragment can not amplify that this is segmental negative, can amplify this fragment and through correct positive of order-checking.The result is as shown in Figure 2, and No. 6 are positive, and the male PCR product of PCR is checked order in+positive contrast, the sequence that records as the artificial sequence in the sequence table 4 from 5 ' terminal 853-1424 position nucleotide shown in, show that PCR product result is correct.

(2) Southern Blot screening technique:

1, the preparation of genomic DNA: the method for extracting DNA by conventional Mus tail prepares DNA;

2, the restriction enzyme digestion of genomic DNA:

In the 1.5ml centrifuge tube, add 20 μ g DNA (1 μ g/ μ l) successively, 4.0 μ l, 10 * enzyme action buffer, 5.0 μ l EcoRI (10U/ μ l) add ddH ₂O to 500 μ l, 37 ℃ of digestion are spent the night.

3, the agarose gel electrophoresis of genomic DNA digestion product

Prepare 0.8% gel: the running gel that generally is used for Southern hybridization gets 0.8%.Electrophoresis: add 10 * Loading buffer in the electrophoresis sample, go up appearance behind the mixing, stay one or two swimming lanes to add DNA Marker.1-2V/cm, DNA swims to positive pole from negative pole.Electrophoresis to bromophenol blue indicator stops electrophoresis during near the gel other end.Normal electrophoresis pattern presents a successive Coating tape.

4, DNA transfers to solid support (capillary tube method) from agarose gel

1) reagent is prepared: degeneration liquid: 0.5M NaOH; 1.5M NaCl; Neutralizer: 1M Tris-HCl (pH 7.4); 1.5M NaCl; Shift liquid (20 * SSC): 175.3g NaCl; 82.2g trisodium citrate, NaOH transfers pH to 7.0, adds ddH ₂O to 1000ml.

2) operating procedure:

Alkaline denaturation: under the room temperature gel was immersed in the degeneration liquid of several times volume 30 minutes.

Neutralization: gel was transferred to neutralizer 15 minutes.

Shift: cut out the positively charged nylon membrane and cut off one jiao by the size of gel and serve as a mark, after the water-soaked, immerse and shifted in the liquid 5 minutes.Cut one than the wide slightly rectangular Whatman 3mm filter paper of film as salt bridge, cut by the size of gel again that 3-5 opens filter paper and a large amount of napkin is subsequent use.Transfer process generally needs 8-24 hour, every napkin that has wet of changing at a distance from several hours.Shift liquid with 20 * SSC.Attention can not have bubble between film and glue.To prevent to be infected with on the film other dirt in the whole operation process.

Shift to finish the back and take out film, immerse 6 * SSC solution number minute, the gel particle of being infected with on the flush away film places between two filter paper, and 80 ℃ of bakings 2 hours are clipped in the NC film between two layers of filter paper then, are stored in dry place.

5, probe mark: the probe that carries out Southern hybridization is generally used the radioactive substance labelling

1) get 25-50ng template DNA (plasmid vector that the EcoRI enzyme action make up to be accomplished reclaims the 2.7K fragment) in the 0.5ml centrifuge tube, ice bath is put in 100 ℃ of degeneration 5 minutes immediately.

2) in another 0.5ml centrifuge tube, add: 10 μ l Labeling, 5 * buffer (contains random primer; Random primer is little nucleotide sequence); 2 μ l dNTPmix (containing dATP, dGTP, each 0.5mM of dTTP), 2 μ l BSA (bSA), 3 μ l [α- ³²P] dCTP, 5U Klenow enzyme.

3) the degeneration template DNA in the step 1) is joined step 2) in centrifuge tube in, add ddH ₂O to 50 μ l, mixing was placed 90 minutes in the room temperature.

4) add 50 μ l stop buffer cessation reactions.

6, hybridization

Prehybridization: film immersed among 2 * SSC 5 minutes, in the hybridization bottle, added hybridization solution (film of 8cm * 8cm adds 5ml and get final product), and the back side of film is adjacent to a hybridization bottle wall, and the front is towards hybridization solution.Put into 42 ℃ of hybrid heaters, make the hybridization system be raised to 42 ℃, obtain the hybridization solution of prehybridization.

Hybridization: pour out the hybridization solution of prehybridization, change to equivalent new be warming up to 42 ℃ hybridization solution.With 100 ℃ of heating of probe 5 minutes, make its degeneration, be added to rapidly in the hybridization bottle, 42 ℃ of hybridization are spent the night.

7, wash film and detection:

Take out film, rinsing is 5 minutes in 2 * SSC solution, washes film according to following condition then:

2 * SSC/0.1%SDS, 42 ℃, 10 minutes;

1 * SSC/0.1%SDS, 42 ℃, 10 minutes;

0.5 * SSC/0.1%SDS, 42 ℃, 10 minutes;

0.2 * SSC/0.1%SDS, 56 ℃, 10 minutes;

0.1 * SSC/0.1%SDS, 56 ℃, 10 minutes.

In washing the process of film, the activity on the radiacmeter detection membrane is constantly used in constantly jolting.

Facts have proved, when activity is indicated numerical value than the high 1-2 of environmental background times, be the terminating point of washing film.No matter the above-mentioned membrane process of washing reaches terminal point in which in step, all must stop to wash film.The film that washes immersed among 2 * SSC 2 minutes, took out film, blotted the moisture content on film surface with filter paper, and with the preservative film parcel, noted between preservative film and the film bubble being arranged.Film is faced up, put into the phosphorus plate, scanning phosphorus plate after three days, result such as Fig. 3 (1 positive contrast is the probe of the aromatase gene of recovery, the 6 positive Mus that change the aromatase gene that contain for southern blot analysis).

Above result shows that the positive transgenic mice that screening obtains is correct.

Four, hereditary stability detects

With positive female Mus and the public Mus pairing of wild type C57BL/6 that the step 3 screening obtains, breed and obtain F1 generation; F1 is screened for mice, and method is said with step 3.By that analogy, breed F2 generation, in F3 generation,, in F4 generation,, in each generation, all carried out positive-selecting, all can detect the gene of this insertion in the positive transgenic mouse.

Five, the phenotypic evaluation of positive transgenic mouse and histologic analysis and gene expression analysis

Carry out urogenital system phenotypic evaluation, histopathological analysis, gene expression analysis after the F1 generation that obtains according to the method described above and F2 grown up for the public Mus of positive transgenic.

(1) phenotypic evaluation

The result: the public Mus of positive transgenic grows up, and urogenital system phenotypic evaluation result is as shown in Figure 4 in the back; The left side two Mus are respectively 1 positive genetically modified urine retention mice; (2) wild-type mice of expression negative control group, the anatomy analysis that contains the positive Mus that changes the aromatase gene that the right one Mus is analyzed for Fig. 3 Southern blot are the morphological change of the bladder that causes of urine retention.

(2) histologic analysis

The bladder and the urethra of said transgenic mice obtained in dissection, puts in 4% the paraformaldehyde solution fixing 48 hours, wash and carries out organization embedding after 4 hours, and HE dyeing and collagen Van Giesan dye, and concrete steps are following:

The tissue that 1, will fix was with PBS washing 2 times, each 10 minutes.Dewater then and waxdip, method is following: 70% ethanol (1 hour), 80% ethanol (1 hour); 90% ethanol (1 hour), 95% ethanol (1 hour), dehydrated alcohol (1 hour * 2); Xylene (15 minutes are once, and 5 minutes are once), paraffin one (30 minutes); Paraffin two (30 minutes), paraffin three (30 minutes);

2, the tissue that will handle is placed on the embedded box bottom central, pours the wax of thawing into, cools off in the room temperature;

3, section:

1) paraffin mass that will fix and fix is contained on the folder thing platform of microtome;

2) will protect the cutter lid and be pushed into open position;

3) shake the fast advance and retreat handwheel, the paraffin mass and the edge of a knife are pressed close to, but be no more than the edge of a knife;

4) angle and the position between the adjustment paraffin mass and the edge of a knife, blade becomes the 15-30 degree approximately with paraffin section;

5) the adjustment caliper profiler is to required slice thickness, general 5 μ m;

6) all are adjusted the back and just can cut into slices, and at this moment the right hand shakes big handwheel, and left hand is held brush pen the wax band is mentioned, and rocking-turn speed transfers to suitable with per minute 40-50.

When the wax that 7) is cut into took 20-30 centimetre to, the right hand was provoked the wax band with another brush pen, lay in (note: the one side by knife face is down more smooth) on the wax tep reel.

4, paster: the wax band that cuts places and drags for sheet machine warm water (note wax disk(-sc) bright finish downward), treat that it launches fully after, the microscope slide of handling with the bonding die agent of getting a cleaning is fished for middle part to the sheet.And perform labelling.

5, roasting sheet: place slide sample on the horse, 37 ℃ of temperature were baked sheet 24 hours.

6, HE dyeing:

1) organization chip places xylene to soak 15 minutes;

2) soaked again 7 minutes behind the replacing xylene;

3) soaked 5 minutes among the dehydrated alcohol I;

4) soaked 5 minutes among the dehydrated alcohol II;

5) soaked 5 minutes in 95% ethanol;

6) soaked 5 minutes in 90% ethanol;

7) soaked 5 minutes in 80% ethanol;

8) soaked 5 minutes in 70% ethanol;

9) soaked 15 minutes in the hematoxylin;

10) washing is 2 minutes;

11) the hydrochloride alcohol differentiation is 10 seconds;

12) in 80% ethanol 2 minutes;

13) in 70% ethanol 2 minutes;

14) in Yihong 79 seconds;

15) soaked 3 minutes in 90% ethanol;

16) soaked 3 minutes in 95% ethanol;

17) soaked 3 minutes among the dehydrated alcohol II;

18) soaked 5 minutes among the dehydrated alcohol I;

19) among the xylene II 3 minutes;

20) among the xylene I 3 minutes;

21) neutral gum mounting, microscopy.

7, Van Gieson dyeing:

1) preparation of reagent:

WeigertShi haematoxylin: A liquid: 1g haematoxylin (haematoxylin), 100ml anhydrous alcohol; B liquid: 4ml 30% ferric chloride (ferric chloride), 95ml distilled water, 1ml hydrochloric acid.

Van GiesonShi liquid: A liquid: 1g acid fuchsin (acid fuchsin), 100ml distilled water.B liquid: 1.22% picric acid saturated aqueous solution (picric acid).

The WeigertShi haematoxylin faces with before getting A liquid and B liquid equal portions and mixes, and uses up in the several hrs, and length must not be above one day.

Haematoxylin is joined after more than 20 day ability maturation must be prepared in advance.This fluid power opposing faintly acid dye liquor, to the effect of the nucleus that dyes at acid dye liquor, difficult quilt is sloughed color, if special engrain then need not break up.

Van GiesonShi liquid is got B liquid 9ml, adds A liquid 1ml, can use.

2) operational approach:

Section dewaxes to water;

With WeigertShi Lignum Sappan uniformly dyeing 20min; Washing; Can break up in case of necessity; Flowing water flushing 10min; Dye Van GiesonShi liquid 1min; Break up fast with 95% ethanol; The anhydrous alcohol dehydration, xylene is transparent, sealing.

Coloration result is as shown in Figure 5, and (A representes the HE coloration result, and B representes collagen fiber dyeing (Van Gieson dyeing) result, and wherein, 1 representes wild-type mice, the positive transgenic mice of 2 expressions; Pink part is represented collagen fiber, and the orange part of b is represented muscle fiber, and the result shows, positive transgenic mice bladder retention of urine, and lamina propria thickens in the bladder, the detrusor arrangement disorder.The atrophy of detrusor layer.The sphincter of urethra atrophy.Van Gieson dyeing shows that collagen increases.

(3) gene expression analysis:

Dissect bladder quick-freezing in liquid nitrogen of obtaining transgenic mice fast ,-80 ℃ of preservations are waited until and are extracted RNA and protein, do gene expression through RT-PCR and Western Blot method and detect, and concrete grammar is following:

1、RT-PCR：

1) extraction and the detection of total RNA

A takes out bladder rapidly from liquid nitrogen, about clip 50mg, shred and put into the homogenate cup, adds 1ml Trizol, with homogenizer homogenate 1-2min, tissue homogenate is changed in the 1.5ml centrifuge tube, and room temperature is placed 5min.

It is isoamyl alcohol/chloroform of 1: 24 that B adds 200 μ l volume ratios, and fully mixing must become milky, places 10min in the ice.

4 ℃ of C are with the centrifugal 10min of 12000 commentaries on classics/min.

D gets supernatant, adds equal-volume isopropyl alcohol (pre-cooling) mixing.

Behind the E-20 ℃ of placement 1h, 4 ℃, the centrifugal 10min of 10000 commentaries on classics/min.

F abandons supernatant, and deposition is dissolved in the ethanol of 800 μ l 75%, and room temperature is placed 10min, 4 ℃, the centrifugal 10min of 10000 commentaries on classics/min.

G repeating step 6) once.

H carefully abandons supernatant, 65 ℃ of oven dry in calorstat; The foranalysis of nucleic acids appearance is measured the OD of total RNA ₂₆₀/ OD ₂₈₀, according to A ₂₆₀Value is calculated the RNA sample concentration: RNA sample concentration (ng/ μ l)=A ₂₆₀* DNA extension rate * 50.

The digestion of trace amount DNA:

A adds the 10xDNase-1 buffer of 10 μ l in the RNA aqueous solution, 0.5 μ lRnsin (40-200U/ μ l), and 2.0 μ l DNase-1 (5U/ μ l), 4.0 μ l0.1MDTT transfer to 100ml with DEPC water, 37 ℃ of insulation 15min.

B adds isopyknic phenol: chloroform (1: 1) mixing.

4 ℃ of C, the centrifugal 10min of 12000r/min.

D gets supernatant, adds NaAc and the precooled ethanol mixing of 2.5 times of volumes of the 3mol/l of 1/10 volume.

More than the E-20 ℃ of deposition 30min, 10000r/min, 4 ℃ of centrifugal 15min.

F deposition is dried with ethanol drip washing twice, is dissolved in the nuclease free water, be stored in-70 ℃ subsequent use.

The content of G RNA that UV spectrophotometer measuring is carried.

2) the synthetic cDNA article one chain of reverse transcription

A reverse transcription system is as shown in table 1

Table 1: reverse transcription system

Reactive component	Reaction volume μ l
		M-MLV?5xbuffer	4.0μl
dNTPs(10mM)	1.0μl
		RNasin(40-200U/μl)	0.5μl
Oligo?dT(20uM)	4.0μl
		RNA	1.0μg
DEPC water	Complement to 20 μ L

B reverse transcription operation sequence:

1. with behind the above composition mixing, of short duration centrifugal, 75 ℃ of incubation 5min on the PCR appearance.

2. place 5min on ice immediately.

3. add M-MLV (200U/ μ l) 1.0 μ l mixings, of short duration centrifugal.

4. on the PCR appearance, carry out following program: 37 ℃ of 1h, 95 ℃ of 5min, reverse transcription product is subsequent use in-20 ℃.

3) sxemiquantitative PCR detects the Expression of Related Genes situation, and (Cyp19a1) is example with the aromatase gene, and method is following:

PCR reaction system: 2 μ L 10xbuffer, 1.6 μ L dNTPs (2.5uM each), 0.4 μ Lprimer F (10uM), 0.4 μ L primer R (10uM), 1 μ L cDNA, 0.3 μ L Taq enzyme (10U/ μ L), ddH ₂O complements to 20 μ L.

Response procedures is following:

The primers F of aromatase gene is seen the artificial sequence 7 in the sequence table, and R sees the artificial sequence 8 in the sequence table.

Result's (A1, A2 are the aromatase transgenic mice, and WT1, WT2 are wild-type mice) as shown in Figure 6, Fig. 6 show that the aromatase transgenic mice express to change the gene expression dose of aromatase, and wild mouse this expression of gene not.

2、Western?Bloting

1) preparation of protein example:

By requirement of experiment bladder is taken out (the unsuitable multigelation of sample) in animal body; Take a morsel (about 1-2g) put into the even oar device of glass and grind to form homogenate with RIPAbuffer, the abundant cracking of ice bath relief half an hour cell, 4 ℃; The centrifugal 10min of 12000rpm/min gets supernatant.Add and go up an appearance Buffer about 1/4 volume, be placed on then that heating 3-4min makes albuminous degeneration in the boiling water bath, can be as the sample point sample.

2) SDS-poly amic acid gel electrophoresis (SDS-PAGE)

1. prepare SDS-poly amic acid gel (generally adopting 10% SDS-PAGE) as requested.

The gel that 2. will prepare is packed in the electrophresis apparatus.

3. design the application of sample order, perform experimental record, press the predefined procedure application of sample.General lysate is pressed 10-20ul/ road point sample.

4. connect electrophoretic apparatus and power supply, voltage is transferred to 100V electrophoresis 10-20min, treat that bromophenol blue moves out of the spacer gel position and use 200V again instead, powered-down behind the 30-40min.

5. unload the gel glass plate from electrophoretic apparatus, water is rinsed well, prepares to change film.

3) change film (the wet commentaries on classics)

1. the gel glass plate is placed the container that fills the electrophoretic transfer buffer, soak a few minutes.

2. be with glove, (83mm * 75mm), as far as possible avoid polluting filter paper and film with reducing in immersion of good filter paper and film and the electrophoretic transfer buffer, drives away and stays the bubble on film to be ready to filter paper and NC film.

3. open transfer box and place in the tray, place it on the transfer box wall after with transfering buffering liquid foam-rubber cushion being soaked into fully, place a wetted filter paper on the sponge again.

4. press the sequence unit good (attention can not have bubble and device electrode groove not to put back) of " sponge-filter paper-gel-pvdf membrane-filter paper-sponge "

5. with the ice chest buffering liquid groove of packing into, fill with the transfering buffering liquid of 4 ℃ of pre-coolings.

6. whole device is placed on and uses magnetic stirrer in the ice bath, connect transfer electrode constant current 100mA and shifted 3 hours.Electricity change finish after, pvdf membrane is performed mark places and contain 2% defatted milk powder (TBST preparation) and debita spissitudo (1: 500-1: one resisting and hatched 1 hour 1000).Discard one and resist, the film bar still places loading slot, and the film in each groove washs 15 minutes once with TBST in the shaking table washing, and 5 minutes once.

4) adding two resists and anti-combining

1. according to experiment needs and suitable ELIAS secondary antibody and the diluted concentration (TBST dilution, 1: 2000) of design alternative, add under the two anti-20mL left and right sides room temperatures in each loading slot and hatch 1h, notice that all parts that guarantee film contact with solution in shaking table.

2. discard two and resist, the film bar still places loading slot, and each groove adds TBST shaking table washing washing about 5 minutes, changes liquid 4-5 time repeatedly.

One anti-for aromatase (sc-14244, Santa Cruz Biotechnology, INC); Two anti-are the anti-goat IgG of Radix Cochleariae officinalis enzyme labelling rabbit (ZB-2306, middle China fir Golden Bridge).

5) chromogenic reaction

Substrate luminescence method: make it even with after the equal-volume mixing in 1: 1 of two kinds of chromogenic substrates it being covered the film surface, wrap film, in the darkroom, the X-ray sheet is covered (time is weighed according to the brightness of light) above the film, development, photographic fixing at once with the glass film.Result's (the positive transgenic mice of A1 and A2, WT are wild-type mice) as shown in Figure 7, Fig. 7 show the protein expression level that the aromatase transgenic mice is expressed changes aromatase, and not this proteic expression of wild mouse.

Prove that more than the transgenic mice that the present invention obtains is correct, can be used as animal model with bladder outlet obstructed.

The experiment repetition of structure transgenic mice and screening and evaluation 3 times is an experimental subject with 30 mices at every turn, obtains 20 positive transgenic mices altogether.Inguinal hernia appearred when big in all positive transgenic mices at 2 months, 20 have merely hit 18 had urine retention when big in 8 months, and FBOO shows that the sickness rate of animal model of the present invention is high, can reach 90%.

Judge the standard of morbidity: mice occurred inguinal hernia at two months when big, and it is slow relatively take action, and when picking up mice, wild-type mice is understood and stress be urinated, and this transgenic mouse can or seldom stress not be urinated, and pushes abdominal part artificially and also can only get rid of a little urine slightly.Can judge it has thus fallen ill.

Embodiment 2: aromatase inhibitor is to the influence of animal model with bladder outlet obstructed

1, Preparation of model method: the method for reference implementation example 1 makes up model, and the screening of positive mouse model: the back 15 days newborn mice of will being born is cut tail, extracts genomic DNA, and PCR filters out male aromatase overexpression Mus;

2, medicine:

The matched group of positive mice, Semen Maydis oil is available from sigma company;

The drug treating group, letrozole (Letrozo1) is available from Novartis Pharma Schweiz AG; Anastrozole is available from AstraZeneca drugmaker; Finrozole is from Finland Hormos drugmaker.

3, test method:

1) 2 monthly age inguinal hernias and FBOO mouse model are divided into totally 4 groups of positive controls, letrozole group, Anastrozole and finrozole groups at random.

2) medicine injection:

The letrozole powder is made in its uniform dissolution Semen Maydis oil with shaking to suspend, according to the 0.4mg/Kg/d administration, whenever at a distance from 24 hours injection 0.1ml.Continuous use 30 days.

The Anastrozole powder is made in its uniform dissolution Semen Maydis oil with shaking to suspend, according to the 0.4mg/Kg/d administration, whenever at a distance from 24 hours injection 0.1ml, continuous use 30 days);

The finrozole powder is made in its uniform dissolution Semen Maydis oil with shaking to suspend, according to the 0.4mg/Kg/d administration, whenever at a distance from 24 hours injection 0.1ml, continuous use 30 days);

Positive controls: inject the Fructus Maydis oil that 0.1ml does not have gonadal hormone every day, give 30 days continuously.

4, test item:

With the mice that aromatase inhibitor was handled, carry out the therapeutical effect of method research aromatase inhibitors such as histopathological analysis, protein science analysis, changes in gene expression analysis to inguinal hernia and bladder outlet.

5, result of the test:

1) shown in the accompanying drawing 8: compare with positive controls, (left side WT is a wild mouse for transgenic mouse that letrozole is handled and positive control Mus; The positive contrast of middle AROM, the right are the AROM group that aromatase inhibitor is handled), the hernia transference cure is (hernia often is accompanied by urethral obstruction) obviously.The transgenic mouse that accompanying drawing 8A letrozole is handled; The transgenic mouse that accompanying drawing 8B Anastrozole is handled; The transgenic mouse that accompanying drawing 8C finrozole is handled.

2) histopathological analysis:

Shown in Figure 9:

A: the sphincter of wild-type mice urethral orifice;

B: the sphincter of transgenic mouse urethral orifice;

C: the sphincter of the transgenic mouse urethral orifice that letrozole is handled;

D: the sphincter of the transgenic mouse urethral orifice that Anastrozole is handled;

E: the sphincter of the transgenic mouse urethral orifice that finrozole is handled;

Compare with positive controls, the sphincter of the transgenic mouse urethral orifice that aromatase inhibitor is handled returns to the width of contrast Mus.(black lines are represented the sphincteral cross section width of urethral orifice).

The result shows: adopt letrozole, Anastrozole and finrozole can treat the FBOO mice, and best with the therapeutic outcome of letrozole.

Though, used general explanation, the specific embodiment and test in the preceding text, the present invention has been done detailed description, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.

Claims (9)

1. the application of aromatase inhibitor in the medicine of the syndrome of the lower urinary tract for preparing treatment male's FBOO and cause.

2. application according to claim 1 is characterized in that, the symptom of said male's FBOO is: frequent micturition, urgent micturition, dysuria, dripping not to the utmost, urination time prolongs.

3. application according to claim 1 is characterized in that, said male's FBOO causes by female/androgen is out of proportion.

4. according to each described application of claim 1-3, it is characterized in that said aromatase inhibitor is the tripyrrole derivant.

5. application according to claim 4 is characterized in that, said aromatase inhibitor is Anastrozole, letrozole, finrozole and vorozole.

6. application according to claim 4 is characterized in that, said aromatase inhibitor is the preparation that contains aromatase inhibitor, is made up of aromatase inhibitor and pharmaceutically acceptable carrier or diluent.

7. application according to claim 6 is characterized in that, said preparation is tablet, capsule, granule, pill, oral liquid or injection.

8. according to each described application of claim 1-3, it is characterized in that the consumption of said aromatase inhibitor class is 1-50mg/Kg/d.

9. application according to claim 8 is characterized in that, the consumption of said aromatase inhibitor class is 5-25mg/Kg/d.

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