CN102980995A - Method for detecting protective effect of estrogen receptor on penis vascular endothelial cell - Google Patents
Method for detecting protective effect of estrogen receptor on penis vascular endothelial cell Download PDFInfo
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- CN102980995A CN102980995A CN2012105113416A CN201210511341A CN102980995A CN 102980995 A CN102980995 A CN 102980995A CN 2012105113416 A CN2012105113416 A CN 2012105113416A CN 201210511341 A CN201210511341 A CN 201210511341A CN 102980995 A CN102980995 A CN 102980995A
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Abstract
The invention discloses a method for detecting a protective effect of an estrogen receptor on a penis vascular endothelial cell. Firstly, the tissue is made into a paraffin section and is then dyed and colored through an immunohistochemical three-step method. The method is provided for testing the endothelial cell apoptosis functional state and damage degree of a marker protein in the endothelial cell and further detecting the protective effect of the estrogen receptor ER beta on a penis vascular endothelium.
Description
Technical field
The present invention relates to the method that histochemistry is detected, especially, the present invention relates to a kind of estrogen receptor that detects to the method for penis vessel endothelial cell protective effect.
Background technology
Blood vessel endothelium has been played the part of very important role in the erection process of penis, blood vessel endothelium dysfunction is one of pathologic basis of Erectile Dysfunction (ED), and, the generation of ED often early than other with the angiocardiopathy of obvious endothelial injuries such as coronary heart disease etc., be considered to the early warning device of this type of disease.Vascular endothelial cell is the mechanical barrier between blood circulation and vascular smooth muscle cell, be subject to easily the damage of inside and outside many factors such as hypertension, high fat of blood, hyperglycaemia, oxidative stress, hyperhomocysteinemiainjury, smoking etc., the especially endocrine function imbalance of blood vessel inner skin cell function after impaired, make the various active material of its secretion or the balance between other materials relevant with these active substances is broken, thereby cause endothelial injuries/dysfunction.
Protection penis vessel endothelium is the new way for the treatment of of erectile dysfunction.Existing studies confirm that; estrogen improves vascular function by direct effect (improve endothelial function, suppress vascular smooth muscle cell proliferation, migration) and indirect effect (regulate the fat metabolism, improve hemostatic system function and antioxidation); weaken the cell effect of destructive stimulus; it is important cardiovascular protection factor; estrogen is by its acceptor cardiovascular endothelium to be had protective effect; lack estrogen or its acceptor, the blood vessel endothelium subject to damage.Estrogen receptor (estrogenreceptor, ER) comprise 2 hypotypes of α and β, expression is all arranged in blood vessel, research in recent years is found, also there is ER in the corpora cavernosa penis, in the penis that comprises a plurality of species such as rodent, rabbit, find to have the expression of ER α and ER β, and take ER β as main.
Two kinds of marker proteins of tool are expressed in the endothelial cell: CD34 and vWF, wherein the CD34 molecule belongs to cadherin family, be that a kind of relative molecular mass is the transmembrane protein of 105000-120000 high glycosylation, optionally be expressed in the mankind and other mammal candidate stem cells, CFU-GM and vascular endothelial cell; VWF is as a kind of glycoprotein that endothelial cell synthesizes and secretes, and being released into blood when endothelial cell damage increases vWF content in the endothelium.In numerous endothelium marker proteins, CD34 and vWF have more susceptibility and specificity, so CD34 and vWF can be used as the index of reflection inner skin cell function state and degree of injury.Therefore, how according to functional status and the degree of injury of the check of the marker protein in endothelial cell endothelial cell apoptosis, become the major issue that detects the effect of estrogen receptor protection penis vessel endothelium.
Summary of the invention
The object of the invention is: provide a kind of functional status and degree of injury of checking endothelial cell apoptosis according to the marker protein in the endothelial cell, and then provide a kind of estrogen receptor ER β that detects to the method for the protective effect of penis vessel endothelium.
The technical scheme that the present invention takes is: a kind of estrogen receptor that detects comprises the steps: the method for penis vessel endothelial cell protective effect
(1), paraffin section is made
(1) fixing: as to select just dead ER β deficiency animal and intact animal, get part penis stage casing tissue, with the PBS flushing, put into fixedly 12h of immobile liquid 4% paraformaldehyde phosphate buffer;
(2) dehydration: remove immobile liquid, will organize and use distilled water flushing 3 times, with 50% alcohol flushing 2 times, dewater step by step with alcohol again: 70% alcohol 1 day, 80% alcohol spends the night, 95% alcohol 3h, anhydrous alcohol I, each 2h of anhydrous alcohol II;
(3) transparent: as to be organized in transparent 45min in anhydrous alcohol that volume ratio is 1:1 and the dimethylbenzene mixed liquor, transparent each 30min in dimethylbenzene I, the dimethylbenzene II after the dehydration;
(4) embedding: in the constant temperature oven tissue after transparent is immersed paraffin, 58 ℃ volume ratio is 1:1 dimethylbenzene and paraffin mixed liquor 45min, after paraffin I, paraffin II, paraffin III are total to 2.5h, with paraffin III investing tissue;
(5) section: the tissue block after the embedding is cut into the paraffin band of 3-5 μ m with microtome after finishing;
(6) paster: the paraffin band is opened up sheet in 50 ℃ of warm water, then the clean microslide of pre-service drags for sheet, and taeniae telarum can stick on microslide;
(7) roasting sheet: roasting microslide 2h with taeniae telarum in 68 ℃ of constant temperature ovens;
(2), SP immunohistochemical three-step approach
(1) dewaxing: should place at room temperature 60min with being stained with organized microslide before the dewaxing, or toast 20min in 60 ℃ of constant temperature ovens, rear with dimethylbenzene I, the immersion of dimethylbenzene II, altogether 25min;
(2) aquation: the microslide after the dewaxing comes downwards to 95% alcohol, 80% alcohol, 70% alcohol I and each aquation 2min of 70% alcohol II at anhydrous alcohol I, each aquation 2min of anhydrous alcohol II;
(3) microslide after the aquation washes 2-3 time with PBS, each 5min;
(4) blocking-up: 3%H
2O
2Deionized water or 80% methyl alcohol are hatched the microslide 10min after the flushing, and is active with deactivating endogenous peroxydase;
(5) the PBS flushing is 2-3 time, each 5min;
(6) antigen retrieval: microslide is put PH 6.0, temperature and is repaired 15-20min in 95 ℃ the antigen retrieval liquid, naturally more than the cooling 20min, uses the cold water flush mug again, accelerates to be cooled to room temperature;
(7) the PBS flushing is 2-3 time, each 5min;
(8) sealing: organize dropping normal goats serum confining liquid on microslide, incubated at room 20min gets rid of unnecessary liquid;
(9) drip the anti-50 μ l of I, room temperature leaves standstill 1h, perhaps 37 ℃ of 1h, perhaps 4 ℃ spend the night after at 37 ℃ of rewarming 45min;
(10) the PBS flushing is 2-3 time, each 5min;
(11) anti-40～50 μ l of the II of dropping horseradish peroxidase-labeled, room temperature leaves standstill 1h, or 37 ℃ of 1h;
(12) the PBS flushing is 2-3 time, each 5min;
(13) dripping SP is Streptavidin-peroxidase, and room temperature or 37 ℃ are hatched 30min-1h;
(14) the PBS flushing is 2-3 time, each 5min;
(15) colour developing: DAB developer 5-10min, grasp dye levels at microscopically, endochylema is brown person and is judged to be positive cell;
(16) 10 minutes cessation reactions of tap water flushing;
(17) redye: haematoxylin redyeing 2min, hydrochloride alcohol differentiation;
(18) tap water flushing 10-15min;
(19) conventional dehydration, transparent, sealing fluid-tight sheet and microscopy, wherein the mounting mode covers with cover glass for dropping in neutral gum by the tissue again.
Described PBS is the phosphate buffer of pH7.4,0.01M, by NaCl 8g, and KCl 0.2g, Na
2HPO
412H
2O 3.63g or Na
2HPO
41.44g, and KH
2PO
40.24g, be dissolved in the 900ml distilled water, with hydrochloric acid adjust pH to 7.4, adding water, to be settled to 1L formulated.
4% paraformaldehyde phosphate buffer pH is 7.4 described in the step (), and its compound method is:
(1) 0.1M phosphate buffer: take off state A liquid 400ml and B liquid 80ml mix after, transfer pH in 7.4, be settled to again 500ml;
A liquid: 0.1mol/L Na
2HPO
4, i.e. Na
2HPO
412H
2O 35.8g adds water and is settled to 1000ml;
B liquid: 0.1mol/L NaH
2PO
4, i.e. NaH
2PO
42H
2O 15.6g adds water and is settled to 1000ml;
(2) 4% paraformaldehyde phosphate buffers: get the 20g paraformaldehyde and be dissolved in the 500ml 0.1M phosphate buffer, dissolve behind heating and the stir about 2-3h.
Microslide pretreatment mode described in the step () in the paster is: a. develops a film: soak microslide with 1%HCl and spend the night, distilled water flushing is dried behind the 95% alcohol-pickled 2h; B. gluing: be coated with the anticreep agent, described anticreep agent is poly-D-lysine or APES and 3-aminopropyl-3-(ethoxymethyl) silane, and wherein the collocation method of poly-D-lysine is that poly-D-lysine is that PLL 5g is dissolved among the distilled water 1000ml.
Developer described in the step (two), its collocation method is: add 1 developer A in 1ml water, shake up, then add 1 developer B, shake up, add 1 developer C again, shake up, wherein: developer A is DAB, and developer B is H
2O
2, developer C is phosphate buffer.
Antigen retrieval liquid described in the step (two) is 0.01M citrate buffer or 0.01M citrate buffer, the compound method of wherein said 0.01M citrate buffer is: take off and add water to 400ml after stating A liquid 8ml and B liquid 42ml mixing, transfer pH in 6.0, be settled to again 500ml;
A liquid: 0.1mol/L citric acid, i.e. C
6H
8O
7H
2O 21.01g adds water and is settled to 1000ml;
B liquid: 0.1mol/L sodium citrate, i.e. Na
3C
6H
5O
72H
2O 29.41g adds water and is settled to 1000ml.
I described in the step (two) is anti-to be CD34, vWF, and concentration was respectively 1: 100 and 1: 50, antibody dilution 0.01M PBS; The anti-concentration of described II is 1: 800, antibody dilution 0.01M PBS.
Sealing liquid described in the step (two) is neutral gum, 50% buffering glycerine or whiteruss.
Among the result of the method for the invention, brown color or sepia are judged to be positive staining.The immunohistochemical staining result can carry out the sxemiquantitative scoring by staining power and scope under 100 times of visuals field: 0 minute=without positive staining, 1 minute=weak or fragmentary positive staining, 2 minutes=medium tenacity positive staining, cell namely＜50% is positive, the dyeing of 3 minutes=strong positive, namely〉50% cell is positive, and every group of sample observed 4 sections, every whole corpora cavernosa penis tangent planes of sections observation bilateral, results averaged.Method of the present invention, by detecting the situation of endothelial cell marker thing, Primary Study the protective effect of ER β to the corpora cavernosa penis blood vessel endothelium.
Description of drawings
Fig. 1 is the endothelial cell marker thing vWF ImmunohistochemistryResults Results of the intact animal of the method for the invention detection;
Fig. 2 is the endothelial cell marker thing vWF ImmunohistochemistryResults Results of the ER β deficiency animal of the method for the invention detection;
Wherein: be the positive position of vWF shown in the arrow.
Embodiment
The present invention is described in detail below in conjunction with embodiment.
Embodiment 1
1, paraffin section is made
(1) fixing: as to select just dead ER β deficiency animal and intact animal, get part penis stage casing tissue, with the PBS flushing, put into fixedly 12h of immobile liquid 4% paraformaldehyde phosphate buffer;
Described 4% paraformaldehyde phosphate buffer pH is 7.4, and its compound method is:
1) 0.1M phosphate buffer: take off state A liquid 400ml and B liquid 80ml mix after, transfer pH in 7.4, be settled to again 500ml;
A liquid: 0.1mol/L Na
2HPO
4, i.e. Na
2HPO
412H
2O 35.8g adds water and is settled to 1000ml;
B liquid: 0.1mol/L NaH
2PO
4, i.e. NaH
2PO
42H
2O 15.6g adds water and is settled to 1000ml;
2) 4% paraformaldehyde phosphate buffer: get the 20g paraformaldehyde and be dissolved in the 500ml 0.1M phosphate buffer, dissolve behind heating and the stir about 2-3h;
(2) dehydration: remove immobile liquid, will organize and use distilled water flushing 3 times, with 50% alcohol flushing 2 times, dewater step by step with alcohol again: 70% alcohol 1 day, 80% alcohol spends the night, 95% alcohol 3h, anhydrous alcohol I, each 2h of anhydrous alcohol II;
(3) transparent: as to be organized in transparent 45min in anhydrous alcohol that volume ratio is 1:1 and the dimethylbenzene mixed liquor, transparent each 30min in dimethylbenzene I, the dimethylbenzene II after the dehydration;
(4) embedding: in the constant temperature oven tissue after transparent is immersed paraffin, 58 ℃ volume ratio is 1:1 dimethylbenzene and paraffin mixed liquor 45min, after paraffin I, paraffin II, paraffin III are total to 2.5h, with paraffin III investing tissue;
(5) section: the tissue block after the embedding is cut into the paraffin band of 3-5 μ m with microtome after finishing;
(6) paster: the paraffin band is opened up sheet in 50 ℃ of warm water, then the clean microslide of pre-service drags for sheet, and taeniae telarum can stick on microslide;
Described microslide pretreatment mode is: a. develops a film: soak microslide with 1%HCl and spend the night, distilled water flushing is dried behind the 95% alcohol-pickled 2h; B. gluing: be coated with the anticreep agent, described anticreep agent is poly-D-lysine or APES and 3-aminopropyl-3-(ethoxymethyl) silane, and wherein the collocation method of poly-D-lysine is that poly-D-lysine is that PLL 5g is dissolved among the distilled water 1000ml;
(7) roasting sheet: roasting microslide 2h with taeniae telarum in 68 ℃ of constant temperature ovens;
2, SP immunohistochemical three-step approach
(1) dewaxing: should place at room temperature 60min with being stained with organized microslide before the dewaxing, or toast 20min in 60 ℃ of constant temperature ovens, rear with dimethylbenzene I, the immersion of dimethylbenzene II, altogether 25min;
(2) aquation: the microslide after the dewaxing comes downwards to 95% alcohol, 80% alcohol, 70% alcohol I and each aquation 2min of 70% alcohol II at anhydrous alcohol I, each aquation 2min of anhydrous alcohol II;
(3) microslide after the aquation washes 2-3 time with PBS, each 5min;
(4) blocking-up: 3%H
2O
2Deionized water or 80% methyl alcohol are hatched the microslide 10min after the flushing, and is active with deactivating endogenous peroxydase;
(5) the PBS flushing is 2-3 time, each 5min;
(6) antigen retrieval: microslide is put PH 6.0, temperature and is repaired 15-20min in 95 ℃ the antigen retrieval liquid, naturally more than the cooling 20min, uses the cold water flush mug again, accelerates to be cooled to room temperature;
(7) the PBS flushing is 2-3 time, each 5min;
(8) sealing: organize dropping normal goats serum confining liquid on microslide, incubated at room 20min gets rid of unnecessary liquid;
(9) drip the anti-50 μ l of I, room temperature leaves standstill 1h, perhaps 37 ℃ of 1h, perhaps 4 ℃ spend the night after at 37 ℃ of rewarming 45min;
(10) the PBS flushing is 2-3 time, each 5min;
(11) anti-40～50 μ l of the II of dropping horseradish peroxidase-labeled, room temperature leaves standstill 1h, or 37 ℃ of 1h;
(12) the PBS flushing is 2-3 time, each 5min;
(13) dripping SP is Streptavidin-peroxidase, and room temperature or 37 ℃ are hatched 30min-1h;
(14) the PBS flushing is 2-3 time, each 5min;
(15) colour developing: DAB agent colour developing 5-10min, grasp dye levels at microscopically, endochylema is brown person and is judged to be positive cell;
Described developer, its collocation method is: add 1 developer A in 1ml water, shake up, then add 1 developer B, shake up, add 1 developer C again, shake up, wherein: developer A is DAB, and developer B is H
2O
2, developer C is phosphate buffer;
(16) 10 minutes cessation reactions of tap water flushing;
(17) redye: haematoxylin redyeing 2min, hydrochloride alcohol differentiation;
(18) tap water flushing 10-15min;
(19) conventional dehydration, transparent, sealing fluid-tight sheet and microscopy, wherein the mounting mode covers with cover glass for dropping in neutral gum by the tissue again.
Described PBS is the phosphate buffer of pH7.4,0.01M, by NaCl 8g, and KCl 0.2g, Na
2HPO
412H
2O 3.63g or Na
2HPO
41.44g, and KH
2PO
40.24g, be dissolved in the 900ml distilled water, with hydrochloric acid adjust pH to 7.4, adding water, to be settled to 1L formulated.I described in the step 2 is anti-to be CD34, vWF, and concentration was respectively 1: 100 and 1: 50, antibody dilution 0.01M PBS; The anti-concentration of described II is 1: 800, antibody dilution 0.01M PBS.
The described methods and results of above-described embodiment is shown in Fig. 1-2 and table 1; the present invention can check functional status and the degree of injury of endothelial cell apoptosis; find out that from the result penis sponge sinusoidal endothelial cells mark CD34 of ER β deficiency animal and vWF protein expression reduce; prompting cavernous sinus endothelial cell reduces, and shows that estrogen receptor ER β is to the protective effect of penis vessel endothelium.
The coloration result of mark CD34 and vWF in two kinds of animal endothelial cells of table 1
| ? | The mouse number | CD34 | vWF |
| Intact animal | 6 | 3.00±0.00 | 2.75±0.50 |
| ER β deficiency animal | 6 | 2.25±0.50 | 2.00±0.00 |
Claims (9)
1. one kind is detected estrogen receptor to the method for penis vessel endothelial cell protective effect, at first tissue is made paraffin section, again by the colour developing of dyeing of SABC three-step approach.
2. described detection estrogen receptor comprises the steps: the method for penis vessel endothelial cell protective effect according to claim 1
(1), paraffin section is made
(1) fixing: as to select just dead ER β deficiency animal and intact animal, get part penis stage casing tissue, with the PBS flushing, put into fixedly 12h of immobile liquid 4% paraformaldehyde phosphate buffer;
(2) dehydration: remove immobile liquid, will organize and use distilled water flushing 3 times, with 50% alcohol flushing 2 times, dewater step by step with alcohol again: 70% alcohol 1 day, 80% alcohol spends the night, 95% alcohol 3h, anhydrous alcohol I, each 2h of anhydrous alcohol II;
(3) transparent: as to be organized in transparent 45min in anhydrous alcohol that volume ratio is 1:1 and the dimethylbenzene mixed liquor, transparent each 30min in dimethylbenzene I, the dimethylbenzene II after the dehydration;
(4) embedding: in the constant temperature oven tissue after transparent is immersed paraffin, 58 ℃ volume ratio is 1:1 dimethylbenzene and paraffin mixed liquor 45min, after paraffin I, paraffin II, paraffin III are total to 2.5h, with paraffin III investing tissue;
(5) section: the tissue block after the embedding is cut into the paraffin band of 3-5 μ m with microtome after finishing;
(6) paster: the paraffin band is opened up sheet in 50 ℃ of warm water, then the clean microslide of pre-service drags for sheet, and taeniae telarum can stick on microslide;
(7) roasting sheet: roasting microslide 2h with taeniae telarum in 68 ℃ of constant temperature ovens;
(2), SP immunohistochemical three-step approach
(1) dewaxing: should place at room temperature 60min with being stained with organized microslide before the dewaxing, or toast 20min in 60 ℃ of constant temperature ovens, rear with dimethylbenzene I, the immersion of dimethylbenzene II, altogether 25min;
(2) aquation: the microslide after the dewaxing comes downwards to 95% alcohol, 80% alcohol, 70% alcohol I and each aquation 2min of 70% alcohol II at anhydrous alcohol I, each aquation 2min of anhydrous alcohol II;
(3) microslide after the aquation washes 2-3 time with PBS, each 5min;
(4) blocking-up: 3%H
2O
2Deionized water or 80% methyl alcohol are hatched the microslide 10min after the flushing, and is active with deactivating endogenous peroxydase;
(5) the PBS flushing is 2-3 time, each 5min;
(6) antigen retrieval: microslide is put PH 6.0, temperature and is repaired 15-20min in 95 ℃ the antigen retrieval liquid, naturally more than the cooling 20min, uses the cold water flush mug again, accelerates to be cooled to room temperature;
(7) the PBS flushing is 2-3 time, each 5min;
(8) sealing: organize dropping normal goats serum confining liquid on microslide, incubated at room 20min gets rid of unnecessary liquid;
(9) drip the anti-50 μ l of I, room temperature leaves standstill 1h, perhaps 37 ℃ of 1h, perhaps 4 ℃ spend the night after at 37 ℃ of rewarming 45min;
(10) the PBS flushing is 2-3 time, each 5min;
(11) anti-40～50 μ l of the II of dropping horseradish peroxidase-labeled, room temperature leaves standstill 1h, or 37 ℃ of 1h;
(12) the PBS flushing is 2-3 time, each 5min;
(13) dripping SP is Streptavidin-peroxidase, and room temperature or 37 ℃ are hatched 30min-1h;
(14) the PBS flushing is 2-3 time, each 5min;
(15) colour developing: DAB developer 5-10min, grasp dye levels at microscopically, endochylema is brown person and is judged to be positive cell;
(16) 10 minutes cessation reactions of tap water flushing;
(17) redye: haematoxylin redyeing 2min, hydrochloride alcohol differentiation;
(18) tap water flushing 10-15min;
(19) conventional dehydration, transparent, sealing fluid-tight sheet and microscopy, wherein the mounting mode covers with cover glass for dropping in neutral gum by the tissue again.
3. described detection estrogen receptor is characterized in that the method for penis vessel endothelial cell protective effect according to claim 2: described PBS is the phosphate buffer of pH7.4,0.01M, by NaCl 8g, and KCl 0.2g, Na
2HPO
412H
2O 3.63g or Na
2HPO
41.44g, and KH
2PO
40.24g, be dissolved in the 900ml distilled water, with hydrochloric acid adjust pH to 7.4, adding water, to be settled to 1L formulated.
4. described detection estrogen receptor is characterized in that the method for penis vessel endothelial cell protective effect according to claim 3: 4% paraformaldehyde phosphate buffer pH is 7.4 described in the step 1, and its compound method is:
(1) 0.1M phosphate buffer: take off state A liquid 400ml and B liquid 80ml mix after, transfer pH in 7.4, be settled to again 500ml;
A liquid: 0.1mol/L Na
2HPO
4, i.e. Na
2HPO
412H
2O 35.8g adds water and is settled to 1000ml;
B liquid: 0.1mol/L NaH
2PO
4, i.e. NaH
2PO
42H
2O 15.6g adds water and is settled to 1000ml;
(2) 4% paraformaldehyde phosphate buffers: get the 20g paraformaldehyde and be dissolved in the 500ml 0.1M phosphate buffer, dissolve behind heating and the stir about 2-3h.
According to claim 4 described detection estrogen receptor to the method for penis vessel endothelial cell protective effect, it is characterized in that: the microslide pretreatment mode described in the step 1 in the paster is: a. develops a film: soak microslide with 1%HCl and spend the night, distilled water flushing is dried behind the 95% alcohol-pickled 2h; B. gluing: be coated with the anticreep agent, described anticreep agent is poly-D-lysine or APES and 3-aminopropyl-3-(ethoxymethyl) silane, and wherein the collocation method of poly-D-lysine is that poly-D-lysine is that PLL 5g is dissolved among the distilled water 1000ml.
According to claim 5 described detection estrogen receptor to the method for penis vessel endothelial cell protective effect; it is characterized in that: developer described in the step 2; its collocation method is: add 1 developer A in 1ml water; shake up, then add 1 developer B, shake up; add again 1 developer C; shake up, wherein: developer A is DAB, and developer B is H
2O
2, developer C is phosphate buffer.
According to claim 6 described detection estrogen receptor to the method for penis vessel endothelial cell protective effect, it is characterized in that: the liquid of antigen retrieval described in the step 2 is 0.01M citrate buffer or 0.01M citrate buffer, the compound method of wherein said 0.01M citrate buffer is: take off and add water to 400ml after stating A liquid 8ml and B liquid 42ml mixing, transfer pH in 6.0, be settled to again 500ml;
A liquid: 0.1mol/L citric acid, i.e. C
6H
8O
7H
2O 21.01g adds water and is settled to 1000ml;
B liquid: 0.1mol/L sodium citrate, i.e. Na
3C
6H
5O
72H
2O 29.41g adds water and is settled to 1000ml.
8. described detection estrogen receptor is characterized in that the method for penis vessel endothelial cell protective effect according to claim 7: I described in the step 2 is anti-to be CD34, vWF, and concentration was respectively 1: 100 and 1: 50, antibody dilution 0.01M PBS; The anti-concentration of described II is 1: 800, antibody dilution 0.01M PBS.
9. described detection estrogen receptor is characterized in that the method for penis vessel endothelial cell protective effect according to claim 8: the liquid of sealing described in the step 2 is neutral gum, 50% buffering glycerine or whiteruss.
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