Relationship Between Expression Of C-erbB-2 And CD44V6 Gene And Biological In Breast Cancer

PrefaceThe emergence mechanism of the tumour is very complicated. Many scholars have proposed the hypothesis that functions of many genes are in coordination in the study of cancer gene with the developing of molecular biology. They think that in each stage of tumor's development there are two or more unusually activate or inactivate cancer genes with different functions playing a different role, promoting the canceration of the cell and cooperating each other on the time and space.C - erbB -2 gene is one of the most close genes with breast cancer, which lies in long arm of 17 chromosome. Its code molecular weight is 185KD and it is participated in adjusting and controlling cell growing, hyperplasia and splitting up. It can be activated and have transforming nature of tumour, when receive some informations from some factors in or out the body. C - erbB - 2 cancer gene increasing and the excessive expression of its albumen result present positive correlation, which often hints high malignant degree, poor reaction to the endocrine treatment, apt to recur and shorter life cycle. So C - erbB - 2 can be regarded as an independent index of judging the breast cancer prognosis.Tumour transfering process involves that tumour cell transfer the protective screen of cell, basis membrane of blood vessel, blood vessel and enter the surrounding tissue to blood. Liotta etc put forward three - step theory that the tumour soaks into , degrade and move. Obviously, the invasion and transformation of the tumour cell need to transfer through the quality or other organizations, cells and finish it by arranging interaction mainly. CD44 family is a kind of cell 's surface albumen with highly heterogeneity, seize molecules. Human CD44gene is orientated as 11P13, which is composed of 20 highly conservative outside factors. CD44V6 is a kind of concatenation of CD44 turned into the allo-some. Its express can influence tumour sport abiliy of cell by influence tumour cell literary composition of skeleton distributing, helping the tumour cell to be shifted latent energy. Promotion of CD44V6 shift function mechanism might express tumour between cell and between lymphatic vessel and blood vessel that far apart of CD44V6 a certain mix body combine, make it lodge and grow steady effectively to tumour cell not to shift. Research verifies with duplicate CD44V6 molecule excessive expression is closely related with duplicate tumour developments.This research uses immune method to study C - erbB - 2 and CD44V6 gene albumen relation with prognosis and shifting of breast cancer. Combined the clinical pathology factor and followed up a case by regular visits to analysis, whether the two genes could be regarded as the biological index of the breast cancer with reliable prognosis.Material and method1. Clinical materials1520 person with breast cancer were in 3 years regular visits after the operation follow up a case to originally by what pathology verify in Liaoning tumour hospital mammary gland department from January 1998 - January 2000. 78 samples among them ( mainly recur) are buried and wrapped up with paraffin wax, 24 -74 years old, average 47. 69 years old. Skill make chemotherapy and radiotherapy any, all sample accept WHO (1981 ) the criteria for classification go on the pathology dividing into type, among them pipe 51 of cancer, little leaf 8 of cancer of soaking into etc. little to in charge of 11 of cancer , nipple form 1 of cancer , marrow kinds 3 of cancer , mucus gland 4 of cancer. And follow up a case by regular visits to one year to go on to all case, it shifts to recur to happen to have 51 among more them, recur 27 of shifting etc. 43 patients survived for more than 3 years.2. Main reagent and instrumentMouse resist people C - erbB - 2, monoclonal antibody of CD44V6, Namely SP reagent box , EDTA antigen liquid and DAB color reagent box purchase from Fuzhou maixing biotechnology Co. , Ltd.3. Method[ 1 ] Organize the adopting of the material . Paraffin wax, patient of breast cancer , which bury 78 samples, each wax piece disconnected to is it fetch 3 4|xm thick section to cut in succession, choose first make routine HE dye , the others make C - erbB - 2 , CD44V6 immune group dye respectively among them.[2 ] Deparaffinization with slices and dewaxes with slices. Each 15 minutesofxyoii, n,n.[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 for 5 minutes, PBS washed the slices 5 minutes for 3 times.[5] Deactivation of Endogenous peroxidase. Put in the 3% H2 O2 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: 100 dilute resist with type mouse people C - erbB - 2 , CD44V6 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% H2O2in 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 one 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. Result judgeEach slice cut by 2 pathology doctors and a pair of blind 5 representative high power visions of observation, each vision count 200 tumour cell, according to mirror being positive to dye cell account for cancer cell percentage of total a-mount judge synthetically. The afterbirth thick liquid of cancer cell or the yellow or comprehensive yellow of cell membrane is positive. Negative ( - ) : Have not dyed comprehensivly and yellowly or the positive cell counts 10% on average ; Positive ( + ) : The positive cell counts on average > 10%.Contrast the experiment ; it makes negative to contrast to resist to substitute by PBS , with known masculine slices as positive contrast.5. Analysis statisticsExpression of C - erbB -2 and CD44V6 with 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 SPSS 11.0 software, with P <0.05 regarded as the difference standard of the significance.Results1. Expression of C - erbB - 2 albumenThere are 28 examples that express C - erbB - 2 in 78 cases of breast cancer .The positive rate is 35. 9% and the positive particle is issued on the cell membrane of the cancer cell mainly, also issued in the cell afterbirth thick liquid of the cancer cell partly.2. Expression of CD44V6 albumenThere are 60 examples that express CD44V6 in 78 cases of breast cancer. The positive rate is 76. 9% and the positive particle is issued on the cell membrane of the cancer cell mainly, also issue in the cell afterbirth thick liquid of the cancer cell partly.3. C - erbB - 2 , CD44 V6 and clinical pathology factor of breast cancer High expression of C - erbB - 2 and CD44V6 is unrelated to age, tumoursize , pathology type , ER and PR and it presents positive correlation with organization credit magnitude and lymph gland in breast cancer is ( P <0.05).4. C - erbB - 2 and CD44V6 albumen express relation with breast cancer prognosisThis group of 78 breast cancer follow up a case by regular visits to3 years. 51 of them recurred in one year but 27 of them didnt. 35 of them died in 3 years. 43 of them survived for more than 3 years. The shifting rate and the 3 -year survival rate is low with those who express C - erbB - 2 and CD44V6 (P < 0.05).5. The relation between C -erbB -2 and CD44V6 expressThere are 24 patients who express CD44 V6 in those C - erbB - 2 positive, analyse relevantly , expresses of CD44V6 is in positive correlation with expresses of C -erbB -2 (X2 > 5. 81 , P <0.05).Conclusions1. The expressions of C - erbB - 2 and CD44V6 have obvious correlation...