Expression Of P16 And VEGF In Breast Cancer Tissue And Their Clinical Significance

PrefaceThe breast cancer of the preface is one of woman's common malignant tumour. In our country, the incidence of disease of the breast cancer is rising, is second only to the stomach cancer and lung cancer in recent years. Though has made certain progress in diagnose in early days , treat in early daysing, but its death rate has not still obviously dropped. Discover , the transformation of the malignant tumour spreads a series of interaction with the aid of cancer organization and cell's outer room quality and other cells, among them P16 restrains cancer gene and VEGF and plays an important role.Human between emergence and cell hyperplasia of malignant tumour cycle out of control to closely related with. Cell cycle whether growth that cell go round and begin again split course is sure periodicity. Studies have shown that only receives the activation of the cell cycle albumen dependence kinase ( CDK) , could finish cell cycle . CDK is it is it activate to combine by cycle albumen with cell to want, CDK at the same time can combine and suppress with a series of protein. P16 lies in people chromosome 9p21, show the son and 2 implicit sons to make up outside 3, protein PI 6 of one 16kD of code. Prove MTSl gene result P16 albumen whether cell cycle albumen dependence 4 kinase (CDK4) suppress the factor already now, participate in the regulation in cycle of cell's hyperplasia directly, so this gene structure or the disappearance functionally will lead to the fact that the cell unusually grows. P16 forfeiture of albumen can make cell cycle albumen dependence kinase CDK4 activate , cause Rb albumen lasting phosphoric acid and cells unrestricted hype rplasia, thus causethe emergence of the tumour. P16 albumen can combine with CDK4 , suppress CDK4/CD compound catalysis nuclear cell very of thing by G1 —S a activation changed, thus suppress cells hyperplasia and transform malignantly. So, when P16 gene is lost alive because of lack , sudden change or unusual methyl of 5' CpG island , it can cause P16 have the scarce without being expressed the albumens like, suppress tumour the ability of the hyperplasiases not to reduce , lead to the fact there arent cell. P16 albumen express malignant degree, clinical by stages in breast cancer, lymph gland is it is it shoulder relevantly to present to shift.Though cancer gene activate and restrain cancer mistake of gene living to have important function in emergence and prognosis of the cancer, but tumour function that blood vessel turn into cant ignore still more. The invasion and attack of the malignant tumour is shifted there are remarkable relations with the forming of the blood capillary in the tumour, the new bom blood vessel in the tumour is organized is the foundation that the tumour cell grows , attacks and shifts . Regulate factor that blood vessel turn into a lot of , among them the most important thing is the leather growth factor in the blood vessel. The growth factor of endothelial cell of blood vessel ( VEGF) is playing an important role among them . VEGF can promote the leather cell division , hyperplasia in the blood vessel , migrate , it is penetrating to improve the blood vessel, change the host outside cells, thus promote the blood vessel to take shape . Research verifies that not only VEGF not only relates to the blood vessel takes place, hyperplasia , but also directly correlated with transformation of the tumour. VEGF whether one heparin is it gather body candy albumen with 2 exocrine to kiss, its molecular weight is 34.42KD, urge to have silks to split function to endothelial cells, and can hydrolysis the basis membrane , cause the blood vessel to increase penetratingly . At present, known VEGF high to express among intestines and stomach cancer, kidney cancer, bladder cancer. VEGF is it is it closely related with to shift by lymph gland with breast cancer to express, this hydrolysis basis membrane with VEGF, cause the blood vessel to increase and have something to do penetratingly . In the capillary vein end, the cancer cell swimming out of can enter the lymphatic vessel , cause the lymph gland to shift . It is one of the in-dexes of the judgement prognosis with generally acknowledged breast cancer that the lymph gland is shifted . So, VEGF expresses the emergence with the breast cancer and lymph gland and is shifted and closely related with, and can be regarded as the independent index of judging the prognosis.In this experiment, immunohistochemistry method was used to discuss the relationship between the represent of P16 and VEGF and cancerometastasis. The deletion of P16 and excess of VEGF occurred correlation significantly. The deletion and mutation of gene MTS1 irritated the production, regulation and strengthening of VEGF, while the excessive presentation of VEGF promoted the deletion of P16, and increased its carcinogenic effect. It was aimed at clarifying the effect of P16 and VEGF on the carcinogenesis, metastasis and prognosis in mammary cancer. Combined determination of P16 and VEGF had significant reference value to judge the infiltration degree, lymphatic metastasis and prognosis in mammary cancer. It offered basic accordance and effective way for early diagnosis , prediction, and effective treatment for mammary cancer.Material and method1. Clinical materialsChoose liaoning Province 2000 - 01 ~ 2002 - 06 tumour hospital by pathology verify is it walk abreast for breast cancer breast cancer effect a radical cure 58 cases of skill to make a definite diagnosis of, and the clinical pathology materials are complete, is women , 32 ~74 ages (average 49.91) years old. Have not done any chemotherapy or radiotherapy before the skill . Reply and read HE cutting into slices and clinical medical history materials. Each file wax piece cut three into slices in succession , thick 4 μm , is it used in routine HE dye to have 1 among more them, two walk immune group dye. ER , PR,C-erbB-2 dye the result and come from the clinical case history materials.2. Main reagent and instrumentreagent resist the people P16 albumen antibody with the type rabbit promptly ( BA0266) , namely resist people VEGF antibody with the type rabbit (BA0407) , and DAB develop the color reagent box (ED1022 ) buy from Fu-jian step new bioengineering Co. ,Ltd. with reagent box, SP of type, (SA1022) promptly.3. MethodAll patient cancer organization do immunity organize chemistry to be dyed , P16 and VEGF dye and adopt SP law. According to age, patient of breast cancer,, tumour size , lymph gland shift or not , TNM by stages, ER , PR ,C-erbB-2 divide into groups, combine clinical pathology and do statistics analysis.[ 1 ] Organize the adopting of the material . Paraffin wax, patient of breast cancer , which bury 58 samples, each wax piece disconnected to is it fetch 3 4jjun thick section to cut in succession, choose first make routine HE dye, the others make P16 and VEGF immune group dye respectively among them.[2] Deparaffinization with slices and dewaxes with slices. Each 15 minutes ofXyolI , 11,1.[3] Hydration in the gradient alcohol. 100% of the I , II , 95% , 90% , 80% , 70% each 5 minutes of alcohols.[4] Distilled water washed the slices for5 minutes, PBS washed the slices 5 minutes for 3 times.[5] Deactivation of Endogenous peroxidase. Put in the 3% H2O2 PBS (135mlPBS +30% H2O215ml ) with room temperature for 15 minutes, or drip and add 1 drop of Endogenous peroxidase block with room temperature for 15 minutes.[6] PBS washed the slices 5 minutes for 3 times.[7] Add Non-immune goat serum in the box immune serum for 15 minutes.[8] Add 1:50 dilute resist with type mouse people P16 and VEGF each of monoclonal antibody cut into slices and drip and add 100ml promptly to drip, protect and hatch and breed for 30 minutes under 37 degrees Centigrade in the box .[9] PBS washed the slices 5 minutes for 3 times.[10] Dripped the Biotinylated goat anti-rabbt IgG antibody, mouse resist sheep IgG antibodies, each 1 drop for 20mins in room temperature.[ 11 ] PBS washed the slices 5 minutes for 3 times.[12] Dripped the SP ( Streptavidin-conjugated peroxidase ) reagent offered with the reagent box ,-each 1 drop for 20mins in room temperature.[ 13 ] PBS washed the slices 5 minutes for 3 times.[14] DAB (3 , 3-diaminobenzidine ) develops the color. 150mlPBS + 25mgDAB + 25ul30% H2O2 in room temperature for 3-10 minutes. Or dripped fresh DAB (each 1 drop fof A, B,and C, add distilled water to lml) lOOul for 3-10 minutes under microscope.[15] Washed the slices for 5 minutes in the running water and distilled water washed the slices for 2 minutes.[16] Dyed for one minute to reply , 1% hydrochloric acid and alcohol split up several seconds usually.[17] Split up for 10 minutes of cell nucleus in the running water and distilled water washed for 5 minutes.[18] Dehydrated, transparent and sealed slice. Dehydrated in the gradient alcohol (70% , 80% , 90% , 95% , 100% I , 100% II ) each for 3 minutes , the xyol I , II each for 3 minutes, sealed with neutral gum.4. Immune Group Dye Result JudgeEach of standard cut into slices by 2 a pair of blind 5 representative high power visions of observation , doctor of pathology , each vision count 200 tumour cell, according to mirror being positive to dye cell account for cancer cell percentage of total amount judge synthetically. The afterbirth thick liquid of cancer cell or the cell nucleus is yellow or the pale brown is positive. Negative ( - ) : There is no pale brown to dye or the positive cell counts ^ 10% on average ; Positive ( + ) : The positive cell counts on average > 10%.5. Analysis statisticsExpression of P16and VEGFwith breast cancer clinical pathology characteristic organize credit type, relation that grade adopt card side examine and not relevant to analyse. Carry on statistical analysis in SPSS10.0 software, with P < 0.05 regarded as the difference standard of the significance.Results1. Expression of PI 6 albumenP16 albumen express 43 cases of masculine gender in 58 cases of breast cancer are organized, express 15 negativly, total positive rate is 74.14%. P16 albumen positive to is it lie in cell nucleus mainly to express, lie in cytoplasm on a small quantity , dye yellow or pale brown.2. Expression of VEGF albumenVEGF albumen express 44 cases of masculine gender in 58 cases of breast cancer are organized, express 14 negativly, total positive rate is 75. 86%. VEGF is expressed positively for being yellow or pale brown, orientate as the cytoplasm.3. PI6,VEGF and clinical pathology factor of breast cancerPI6 expresses age, size of tumour, menopause state, expression of ER , PR ,C-erbB-2 with the patient and has nothing to do ( P > 0.05) , and with organization credit grade of breast cancer, TNM by stages, lymph gland shift relevant (P <0.05).VEGF does not have remarkable dependence ( P > 0. 05 ) to express the age , size of tumour , menopause state , expression of ER , PR , C-erbB-2, TNM with the patient by stages, and is shifted and closely related with the lymph gland (P<0.05).4. In the correlation analysis of the representation of protein PI 6 and VEGF in mammary cancer, 36 out of 58 cases showed positive representation of protein PI6 and VEGF,8 cases showed positive representation of VEGF and negative representation of protein P16, 7 cases showed positive representation of protein P16 and negative representation of VEGF, and 7 cases showed negative representation of both protein PI 6 and VEGF. Chi-square test for the paired data showed that there had correlation of protein PI 6 and VEGF in mammary cancer.