Expression And Clinical Significance Of Heparanase In Non-small-cell Lung Cancer

Objective:To understand expression of heparanase protein in the lung in the specimens of 38 cases of lung cancer;the lung specimens of seven cases of benign tumors;10 cases of normal tissue, obtained from Anhui Medical University Affiliated Hospital of thoracic surgery.To study the relationship between the expression of heparanase and the clinical significance of lung cancer.To study the role of heparanase in tumor invasion and metastasis.Methods:To collect 38 cases of lung cancer specimens taken from Anhui Medical University Affiliated Hospital chest surgery operation.All the patients were not carried out preoperative radiotherapy or chemotherapy,and confirmed by pathological diagnosis. 22 cases of squamous cell carcinoma,adenocarcinoma in 12 cases,adenosquamous carcinoma in 4 cases.Clinical phases according to the International Union Against Cancer (UICC) 1997 Annual standard phased. I II, 20, III IV Period, the 18 cases;Highly differentiated in 26 cases,12 cases of poorly differentiated.Lymph node metastases negative 11, positive in 27 cases.The patients aged was 38 to 75 years old, male 34 cases and 4 females.The other seven cases of lung benign tumor samples were such as pneumonia cases of fake tumour 4 , two cases of pulmonary tuberculosis, chest and fiber tumour 1 case. The 10 normal lung tissue cases were confirmed by the pathology confirmation.The specimen after obtained placed promptly in -80°C ultra-low temperature refrigerators reserved. Paraffin wax produced package after planting area.Three-steps methods used SP immunohistochemistry were carried out. Rabbit anti-human HPSE antibody were purchased from Wuhan Boster immunohistochemical SP detection(testing) kit were purchased from Beijing Zhong Shan Golden Bridge Technology Co.,Ltd.China.Steps and Sequence of operation carried out according to kit instructions.To take wax block doing 4μm in serial sectioning after Xylene dewaxing,hydration gradient ethanol,and then placed in the freshly prepared 3% H2O2 solution soaked 10 min at room temperature.Hydration-xylene dewaxing,gradient ethanol,and then flushing with distilled water, To place the chips into the containers with antigen retrieval solution of 0. 01M citrate buffer liquid(pH6. 0) containers. To set at the microwave carried on the microwave to repair oven for 30 minutes . Cooling at room temperature , Rinsing in PBS (phosphate buffer liquid). Dropping an anti– (Adds by drops one anti-),and incubating the sections for 30 min at the 37 degrees C Electric Water Tank Temperature ,4℃refrigerator overnight. Rinsing three times in PBS, droping the goat anti-human.Placing at the 37℃constant temperature water tank electric and incubation(incubating) 10 min.Rinsing three times in PBS (phosphate buffer liquid),droping the goat anti- . Placing at the 37℃constant temperature water tank electric and incubation(incubating) 10 min; Rinsing in PBS,The chips in DAB-H 2 O 2 color were displayed.To terminate the response after the show brown granules. Hematoxylin stained and division in the hydrochloric acid alcohol. To response into blue in ammonia water.Conventional dehydration and xylene transparent.To seal and mount the slice with Neutral gums.Negative control for each batch of tes(ttrial)after PBS was replaced by one anti- resistance.In the cytoplasm cells containing brown granules were considered as positive staining cells.Under the high power microscope each slice choosed 4 fields of vision.The number of positive cells were counted separately. Carried on the semi-quantitative graduation(classification),then averaged.Using semi-quantitative grading method to determine staining results(dyeing result). The determination standard is as follows:Under high power microscope the staining situation in cytoblastema are①No coloring is 0, 1 in light yellow, brown-yellow 2, brown for 3.②Positive range(scope): 5% 0 points, 5% ~ 25% of 1, 26% ~ 50% for 2, 51% to 75% of the three, 75% to 4 pm.Two results (the combined result) add together <2 can divide into the negative. 2 ~ 3 is divided into weak positive.4 ~ 5 is divided into moderately positive (+ +).6 ~ 7 is divided into strongly positive (+++).Results:Immunohistochemical staining showed that heparanase protein which major position in the cells was located in different organizations situation had the difference ones.The normal lung organization does not express, in the benign lung tumor expresses 1 example or case. Non-small cell lung cancer heparanase expression was 28 cases. The positive rate of three kinds of specimens were 00.00%, 14.28%, 73 . 68%.Non-small cell lung cancer Heparanase protein expression rate was significantly higher than normal lung tissue and benign lung tumor tissue, there is statistical significance. Heparanase expression level was related to the tumor lymph shift and TNM by stages, and not obvious relevance(significant correlation) with degree of tumor differentiation in patients.Conclusion:Research has shown that a variety of human malignancies (breast cancer, liver cancer, malignant melanoma, etc.) can be detected in the expression of heparanase,and is associated with poor after the tumor clinical surgery technique.Our research findings show in the non-cellule lung cancer organization's positive expression rate is higher than the control group by far. The results also showed: Heparanase was mainly located in the membrane and cytoplasm. Cell membrane stained deeply.Staining outside the cancer edges of heparanase is stronger than the central spot.So we speculate that heparanase is a synthetic binding on intracellular membrane.In cancer invasion and metastasis occured that heparanase secreted out of cell and dissolved in BM and ECM of HPSE.Thereby it contributed to cancer invasion and metastasis,promoted cancer of the attacks and transfer or shift.The positive rate of lymph node metastasis was significantly higher than tumors without lymph node metastasis.(or The lymph node metastases positive rate no higher than the lymph node metastases tumor.) And the lymph node metastasis assumed strongly positive expression of Duocheng.Also it indicated that HPSE was closely related to the transfer of lung cancer.