| Objective: Osteosarcoma is the primary malignant neoplasms,Theincidence of osteosarcoma in the primary bone tumors is second after plasmacell myeloma, which accounts for about one-third of primary malignant of thebone tumor. The Osteosarcoma has a poorly prognosis。 Although theapplication of neoadjuvant chemotherapy has made no lung metastases occurin the patients with osteosarcoma when they see a doctor from the original20%to70%now, But for nearly30years,The treatments of osteosarcomahave no obvious breakthrough。For the visit the patients with osteosarcomahas appeared pulmonary metastasis, the5year's survival rates are still below20%。At present for the clear or control to pulmonary metastatic lesions thechemotherapy drug,still have a poor results。For a single focus of pulmonarymetastatic lesions we can remove it with the way of surgery, but for themultiple focus of pulmonary metastasis the only way we can rely on is thedrugs。So how we can have a early diagnosis for the osteosarcoma, especiallyhow we can have a early diagnosis for the osteosarcoma pulmonary metastasiswhich has become a bottleneck。we improve the survival rates of patients withosteosarcoma。Now the research about the gene related the osteosarcomapulmonary metastasis has become a hot, because these genes for the earlydiagnosis of osteosarcoma lung metastases can provide a prognostic markers,and can become a new treatment targets of the chemotherapy drug。Forimprove the survival rates of the patients with osteosarcoma it has a especiallyimportant clinical significance。This experiment through the method ofimmunohistochemistry to test the expression of Cyr61in the osteosarcomasamples, to compare and discuss the expression and clinical significance ofCyr61in patients with osteosarcoma。Materials and Methods:The specimens were taken from42 osteosarcoma operation,embedding blocks from osteosarcoma samples ofsurgical intervention in the bone disease offices of The Third AffiliatedHospital of Hebei University from july of2008to January of2012, including19cases of patients with early osteosarcoma lung metastases。According to thebone tumors clinical stages of the Enneking,Ⅱstages18cases, Ⅲstages24cases。on the side18cases of osteofibrous dysplasia surgical specimensparaffin were selected。 Cyr61Rabbit Anti-Human polyclonal antibo-dy (bs-1290) PV Two-step method rabbit immunohistochemical testkit(PV-6001)Procedure:1, dewaxing, hydration biopsy,2, according to thespecial requirements of primary antibody application of the, we made apretreatment to the tissue。3, Block endogenous peroxidases by3%H2O2for20min, Then wash3x2min with PBS,4,Adding primary antibody, throughnight in fridge at4°wash3x2min with PBS。5, Adding PV-6001rbbit IgGantibody-HRP polymeric, incubation30minutes at37degrees,then wash3x2min with PBS。6, Dyeing by DAB,7, Flushing with distilled water8,Counterstain dehydration, hyaline9, seal coverslip。We by PV two-stepimmunohistochemical method detect Cyr61protein's expression in the tissuesamples of osteosarcoma and osteofibrous dysplasia。Seting the knownpositive results as the positive control and the primary antibody is replaced byPBS buffer solution in the negative control。Judgement standard: The dyeing results from specimen of patients withosteosarcoma and osteofibrous dysplasia were compared。Then comparingwith the dyeing results of specimen of the patients with osteosarcoma lungmetastases and no lung metastases。 Grade by two scale of gradesemiquantitative analysis:(1) Dyeing strength score: colorless0points, onepiont for light brown, two points for claybank, three points for dark brown。(2)The positive percentage rating of target cells score: the percentage of positivetarget cells <10%for1point,11%-50%for2points,50%to75%for thethree points,>75%for4points。Two score multiplication≥3is positive,the score take account of both the intensity and number of positive。We shouldpay attention to several problems in the procedure of positive results judement: (1), Dyeing of section has a better contrast, background clearly,(2) Thepositive cells must have an accurate location, bleeding necrosis and suffersextruding tissues and the slice edge should not be used as a judgementstandard。The statistical software spss17.0were use to statistics the data weobtained。Result: Cyr61expresses in both osteosarcoma organization and osteo-fibrous dysplasia organization。The positive rate in42tissue of osteosarcomawas88.09%(37/42), and the positive percentage in pulmonary metastasistissue was89.47%(17/19), The positive percentage in unpulmonarymetastasis tissue was86.96%(20/23)。In18cases of osteofibrous dysplasiatissue the positive expression rate was38.89%(7/18)。 The differentiallyexpression which the positive expression rate88.09%(37/42)in the tissue ofosteosarcoma and the positive expression rate38.89%(7/18) in thesteofibrous dysplasia in statistics have statistical meaning after the statisticaltreatment diference(P <0.05),The positive expression results which occur inpulmonary metastasis of osteosarcoma and no pulmonary metastasisosteosarcoma have no obviously difference in statistical, it was no statisticalmeaning (P>0.05), and According to Enneking, by stagesⅡwere22casespositive expression rate86.36%(19/22), by stages Ⅲ were20cases90.00%(18/20), two groups of expression rate were have unobvious difference asstatistical, it had no statistical meaning. And according to the patient's age,gender, and alkaline phosphatase (ALP) level all the case were divide intodifferent。between groups the expression was no statistical meaning (P>0.05)。Conclusion: Through the detection to Cyr61expression in samples tissueof the40osteosarcoma cases and16osteofibrous dysplasia cases, at the sametime research and analysis the results, the conclusion we reached is:1, Cyr61has a obvious expression in osteosarcoma cell。2, Cyr61high expression may promote the development and occurrenceof osteosarcoma。3, Cyr61high or low expression,which has no significant correlation to the distant metastasis of osteosarcoma。... |
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