Immunohistochemistry Anti-off Piece Of Several Methods Of Comparison And Pcna, Mmp-7 And Vegf In The Cath-d And Hsp70 Expression In Esophageal Cancer And Its Clinical Significance

Objective: In today's immunolaboratorial researches andpathological dignoses, immunohistochemistry is one of thelabeld immunoassays used widely. Although Immuno-histochemistry has so many advantages that its operation issimple and convenient, that its results can be easily observedand that its specificity is high, the results ofimmunohistochemical staining is not very stable and can beaffected by a lot of factors. Some reasearchers suggest that thereare many factors affecting immunohistochemical staining, suchas the cleaning degree of glass slides, different methods ofantigen repairing(including using microwave,autoclave,boilingwater or digesting with enzymes ), disposing glass slides withhigh pressure and reagent types of different companies. A greatmany scholars are devoting themselves to exploring the factorsaffecting immunohistochemical staining and to establishinguniform operational criterions and assessing standards in orderto guarantee the correct use of immunohistochemistry. It hasbeen a long time that paraffin slice escapes from glass slideduring the course of immunohistochemical staining. The slicetends to escape from glass slide partly or entirely after antigenrepairing and being dipped in buffer for a long time. This willhave influence on the judgement of the staining results.Different agents of adhibiting tissue slice and differentgelatinizing methods cause different effect on preventing sliceescaped from glass slide. In our work, we usually meet with theproblem that paraffine slice escapes from glass slide. Therefore,in this research, we compared the immunohistochemicalstaining intensity and the effect on preventing slice escapedfrom glass slide of three different agents of adhibibiting tissueslice, APES, Poly-L-Lysine and white latex. Furthermore, thegelatinizing methods of Poly-L-Lysine are compared andimproved. A method of gelatinizing with Poly-L-Lysine that canbe repeated very well is summarized successfully.Methods: In this research, three kinds of agents ofadhibiting tissue slice (APES, Poly-L-Lysine and white latex)are gelatinized. The gelatinizing method of APES is accordingto the instruction. The gelatinizing method of white latexconsults a literature. Excepting for the gelatinizing method ofdipping, the condition of Poly-L-Lysine's gelatinizing method isexplored. Different volume of 0.01% Poly-L-Lysine wasgelatinized once (40μL,80μL,120μL,160μL); the same quantitydifferent concentration of Poly-L-Lysine was gelatinizedonce(0.02% Poly-L-Lysine 40μL, 0.0133% Poly-L-Lysine60μL,0.01% Poly-L-Lysine 80μL); 0.01% Poly-L-Lysine 80μLwas gelatinized once or twice. The influence placed in 37℃for4 weeks was observed (adhering tissue slice on the day or afterbeing placed in 37℃for 4 weeks). After antigen repairing withcitric acid buffer, immunohistochemical staining is performed.Each slice's staining intensity was measured by HPIAS colorfulpathology system of high definition. The situation of slicesescaping from glass slide was observed by naked eye.Results:1 Comparison of the effect of different agents of adhibitingtissue sliceThe specific staining average OD value of APES group is0.1980±0.0120, Poly-L-Lysine dipping group is 0.2020±0.0082,and White Latex group is 0.2580±0.0164. The non-specificstaining average OD: APES group is 0.0105±0.0010,Poly-L-Lysine dipping group is 0.0106±0.0009,White Latexgroup is 0.0298±0.0027. There is no significant differencebetween the APES group and the Poly-L-Lysine dipping groupin both specific staining and non-specific staining (P>0.05). Thespecific staining and non-specific staining of White Latex groupare significantly higher than both the APES group (P<0.01) andthe Poly-L-Lysine dipping group (P<0.01). Specific stainingaverage OD value divided by non-specific staining average OD:APES group is 18.81,Poly-L-Lysine dipping group is 19.08 andWhite Latex group is 8.66. The intact slices percentage of thethree kind agents of adhibiting tissue slice: APES group is 55%,Poly-L-Lysine dipping group is 57.5% and White Latex group is50%. The three groups cannot get high percentage of intactslices all.2 Comparison of the effect using different gelatinizing methodof Poly-L-Lysine1) Comparison of Poly-L-Lysine of same concentration anddifferent volume 0.01% Poly-L-Lysine was gelatinized oncein different volume: 40μL, 80μL, 120μL, and 160μL. The intactslice percentages of the four groups are respectively 57.1%,78.6%, 60.3% and 50%. The best volume of Poly-L-Lysine is80μL.2) Comparison of Poly-L-Lysine of same quantity and differentconcentration 0.02% Poly-L-Lysine 40μL, 0.0133%Poly-L-Lysine 60μL, 0.01% Poly-L-Lysine 80μL are gelatinizedonce. The intact slice percentages of the three groups arerespectively 58.3%, 50% and 78.1%. Gelatinizing using 0.01%Poly-L-Lysine 80μL can have better effect on preventing sliceescaped from glass slide.3) Comparison of gelatinizing once and twice 0.01%Poly-L-Lysine 80μL gelatinized once or twice. The intact slicepercentage of the group gelatinizing once is 74.6%. The intactslice percentage of the group gelatinizing twice is 90.2%. Theintact slice percentage of the group gelatinizing twice issignificantly higher than the group gelatinizing once (P<0.01).In the group gelatinizing twice, there is no slice escapingmoderately and severely and the percentage of slices escapingslightly is lower than the group gelatinizing once. The specificstaining average OD value: the group gelatinizing once is0.1244±0.0054, the group gelatinizing twice is 0.1252±0.0032.There is no significant difference between the two groups(P>0.05). The coefficient of variation of the staining intensity ofthe group gelatinizing twice (CV=2.56%) is lower than that ofthe group gelatinizing once (CV=4.34%).4) Comparison of adhering slice on the day and being placed in37℃for 4 weeks The glass slides gelatinized with 0.01%Poly-L-Lysine 80μL adhibited tissue slice on the day or afterbeing placed in 37℃for 4 weeks. The intact slice percentagesof the two groups are the same (75%).Conclusion:1 Although the positive-staining intensity of APES andPoly-L-Lysine is lower than White Latex, the positive-stainingaverage OD values divided by the negative-staining average ODin APES and Poly-L-Lysine are higher than White Latex. Theformer two excel White Latex in the specificity ofimmunodetecting.2 The effects on preventing slice escaped from glass slide usingAPES, White Latex and dipping Poly-L-Lysine are all notsatisfactory.3 The intact slice percentage of gelatinizing twice using 0.01%Poly-L-Lysine 80μL is the highest and it doesnot have slicesescaping moderately and severely. There is no significantdifference between the group of gelatinizing once and twice instaining intensity. The staining using the method of gelatinizingtwice is more symmetrical than using the method of gelatinizingonce.4 The method of preventing slice escaped from glass slide thatwe summarized is gelatinizing twice with Poly-L-Lysine. Itrefers that two glass slides are gelatinized each time using0.01% Poly-L-Lysine 80μL and are gelatinized twice totally.Objective: Esophageal carcinoma is one of the commonalimentary canal malignant tumors. The death rate of esophagealcarcinoma in China is high. The patients are most men ratherthan women. Most of the patients are over 40 years old,especially over 60 years old. The incidence rate of esophagealcarcinoma is high in Hebei Province.The histogenesis, morphological classification and thejudgement of biological behaviour of esophageal carcinomacontribute to the pathology. With the development of the theoryand technique of molecular biology, the understanding ofesophageal carcinoma comes into microcosmic world. Thestudy of common morphorlogical pathology is not adapt to therequirement of oncology. Investigation of esophageal carcinomain molecular level and index for the molecular pathologicdiagnosis and the clinical judgement of prognosis and theselecting of treatmental blueprint have made the study ofoncology pathology going into a new developmental phases.Reasearches indicate that there are a great deal of proteinsassociated with the biological behaviour and prognosis ofesophageal carcinoma, including oncogene, tumor suppressorgene, growth factor and proteins regulating apoptosis.Proliferating cell nuclear antigen (PCNA) is a kind of 36KDnuclear protein associated with cell proliferation. It can be usedas a good index estimating the situation of cell proliferation.Matrix metalloproteinase-7(MMP-7) can degrade the importantcomponents of cellular matrix and floor membrane. It plays animportant sole in the progression of many malignant tumors.Vascular endotheial growth factor (VEGF) can promote theproliferation of vascular endothelium. It not only providesnutrition for tumor cells, but also helps tumor metatases.Cathepsin D (Cath-D) exsists widely in all kinds of tissue cellsand tumor cells. Reasearches indicate that Cath-D exhibitssignificant correlation with the invasion, metastases andprognosis of gastric-intestinal cancer and mammary cancer. Themain function of heat shock protein 70(HSP 70) is to protectproteins that are necessary for cells'life in order to maintain thesurvival of cells when cells meet an emergency. The recentresearch suggests that HSP 70 over expresses in many malignanttumors.In order to understand the expression and the significanceof the 5 kind proteins, PCNA, MMP-7, VEGF, Cath-D and HSP70 in esophageal carcinoma, in this study, immunohistochemicalstaining is performed in order to observe their expression inprotein level in all the 42 cases and to provide a better index fordignosis, prognosis judgement and treatment of esophagealcarcinoma.Methods: 42 cases of esophageal carcinoma which weredemonstrated by pathologic HE staining are made tissueparaffine masses. Glass slides are gelatinized twice with 0.01%Poly-L-Lysine 80μL. Immunohistochemical staining isperformed using antibodies against PCNA, MMP-7, VEGF,Cath-D and HSP 70. By microscope we observed thepositive-expression position of each antibody and calculated thepositive –expression percentage of tumor cells .If the positive–expression percentage of tumor cells was >50%, it wasregarded as over expression. If the positive–expressionpercentage of tumor cells <50% or negative, it was regarded aslow expression .The patients were divided into two groupsaccording to the depth of invasion: the depth of invasion wassubmucosa and the depth of invasion is muscle layer. Accordingto the situation of lymph node metastases, the patients weredivided into two groups: absence of lymph node metastases andpresence of lymph node metastases. According to the situationof prognosis, the patients are divided into two groups: patientssurvived longer than 5 year and patients survived less than 2year. The over expression of the 5 kind proteins'associationwith invasion, lymph node metastases and prognosis areexamined. Compare the multivariate analysis with the univariateanalysis.Results:1 The expression of PCNA, MMP-7, VEGF, Cath-D andHSP 70 PCNA stains tumor cell's nucleolus brown-yellowgranule or stains tumor cell's nucleolus brown-yellowsymmetrically. MMP-7, VEGF and HSP 70 all stain tumor cell'scytoplasma brown-yellow. Cath-D stains tumor cell'scytoplasma or cytoplasmic membrane brown-yellow and stainsother cells brown-yellow.2 Positive rate of the expression of PCNA, MMP-7, VEGF,Cath-D and HSP 70 Positive rate of the expression ofPCNA, MMP-7, VEGF, Cath-D and HSP 70 is separately 83.3%,80.9%, 100%, 61.9% and 64.3%.3 The expression of PCNA, MMP-7, VEGF, Cath-D and HSP70 and clinicalpathologic features and prognosis1) The over expression rates of PCNA in the group of invadingsubmucosa and the group of invading muscle layer areseparately 20.00% and 81.48%. There is significant differencebetween the two groups (P<0.01). The over expression rate ofPCNA in the group of invading muscle layer is significantlyhigher than the group of invading submucosa .The overexpression rate of PCNA in the group of absence of lymph nodemetastases and the group of presence of lymph node metastasesis separately 30.00% and 86.36%. There is significant differencebetween the two groups (P<0.01). The over expression rate ofPCNA in the group of presence of lymph node metastases issignificantly higher than the group of absence of lymph nodemetastases. The over expression rate of PCNA in the groupsurvived longer than 5 year and the group survived less than 2year is separately 38.89%, 78.95%. There is significantdifference between the two groups (P<0.01). The overexpression rate of PCNA in the group survived less than 2 yearis significantly higher than the group survived longer than 5year.2) The over expression rate of MMP-7 in the group of invadingsubmucosa and the group of invading muscle layer and extoblastis separately 26.67, 66.67%. There is significant differencebetween the two groups (P<0.05). The over expression rate ofMMP-7 in the group of invading muscle layer is significantlyhigher than the group of invading submucosa. The overexpression rate of MMP-7 in the group of absence of lymphnode metastases and the group of presence of lymph nodemetastases is separately 35.00%, 68.18%. There is significantdifference between the two groups (P<0.05). The overexpression rate of MMP-7 in the group of presence of lymphnode metastases is significantly higher than the group ofabsence of lymph node metastases. The over expression rate ofMMP-7 in the group survived longer than 5 year and the groupsurvived less than 2 year is separately 38.89%,52.63%. There isno significant difference between the two groups (P>0.05).3) The over expression rate of VEGF in the group of invadingsubmucosa and the group of invading muscle layer is separately13.33%, 81.48%. There is significant difference between thetwo groups (P<0.01). The over expression rate of VEGF in thegroup of invading muscle layer is significantly higher than thegroup of invading submucosa. The over expression rate ofVEGF in the group of absence of lymph node metastases andthe group of presence of lymph node metastases is separately25.00%, 86.36%. There is significant difference between thetwo groups (P<0.01). The over expression rate of VEGF in thegroup of presence of lymph node metastases is significantlyhigher than the group of absence of lymph node metastases. Theover expression rate of VEGF in the group survived longer than5 year and the group survived less than 2 year is separately33.33 %,78.95%. There is significant difference between thetwo groups (P<0.01). The over expression rate of VEGF in thegroup survived less than 2 year is significantly higher than thegroup survived longer than 5 year.4) The over expression rate of Cath-D in the group of invadingsubmucosa and the group of invading muscle layer is separately26.67%, 59.26%. There is significant difference between thetwo groups (P<0.05). The over expression rate of Cath-D in thegroup of invading muscle layer is significantly higher than thegroup of invading submucosa. The over expression rate ofCath-D in the group of absence of lymph node metastases andthe group of presence of lymph node metastases is separately30.00%, 63.64%. There is significant difference between thetwo groups (P<0.05). The over expression rate of Cath-D in thegroup of presence of lymph node metastases is significantlyhigher than the group of absence of lymph node metastases. Theover expression rate of Cath-D in the group survived longer than5 year and the group survived less than 2 year is separately28.57%,63.16%. There is significant difference between thetwo groups (P<0.05). The over expression rate of Cath-D in thegroup survived less than 2 year is significantly higher than thegroup survived longer than 5 year.5) The over expression rate of HSP 70 in the group of invadingsubmucosa and the group of invading muscle layer is separately53.33%, 44.44%. There is no significant difference between thetwo groups (P>0.05). The over expression rate of HSP 70 in thegroup of absence of lymph node metastases and the group ofpresence of lymph node metastases is separately 55.00%,40.91%. There is no significant difference between the twogroups (P>0.05). The over expression rate of HSP 70 in thegroup survived longer than 5 year and the group survived lessthan 2 year is separately 66.67%,31.58%. There is significantdifference between the two groups (P<0.01). The overexpression rate of HSP 70 in the group survived longer than 5year is significantly higher than the group survived less than 2year.4 Multivariate analysis of patient's prognosisAccording to the result 3, PCNA, VEGF, Cath-D, HSP70are all independent prognosis indexs. The over expression ofPCNA, VEGF, Cath-D and low expression of HSP70 exist inpatients of poor prognosis. The low expression of PCNA, VEGF,...