The Expressions Of Polygene Proteins Of Esophageal Precancerous Lesions And Esophageal Carcinoma

Background and objectEsophageal carcinoma is a common malignant tumor of digestive tract in china. Shantou is a high incidence area of esophageal carcinoma. Patients of preliminary diagnosis mostly are in meta-late. At present, surgery, radiotherapy and chemotherapy have been widespread developed, but the five-year survival rate of esophageal carcinoma is low. Nowdays, the true etiological factor of esophageal carcinoma is not clear, so it is difficult to do the" three earlier" work. And early diagnosis is very important. Mass population survey and experimental pathology investigation suggest carcinogenesis of esophageal carcinoma is a slow process and may last a very long time. There have been some studies about single or double gene expression in the every stage of esophagus carcinogenesis. But systemic and polygene investigation is lack. So, we collected randomly forty-seven cases chronic esophagitis and eight-five cases esophageal carcinoma specimen to perform immunohistochemistry study for oncogene protein MDM2, C-myc,anti-oncogene protein P53, P16 and cell cycle protein P27 and P21. So we can explore law and character of these proteins in the process of normal esophageal epithelia to chronic esophagitis and/or atypical hyperplasia to carcinoma and metastasis. And explore the relationship of the change of these proteins and the occurrenc and development. Applying flow cytometer, we detect the DNA content of esophageal carcinoma cell and investigate quantity the content of the six proteins in normal esophageal epithelia, para-cancer mucosa and esophageal carcinoma. So that we can understand the correlation of these proteins and esophagus carcinomatous change and make a relative analysis with immunohistochemistry study. This may provid an objective mark for early dignosis and prognosis of esophageal carcinoma. Methods 1 Study Objects?Collectting eighty-five waxes with cancer and para-cancer mucosa of esophageal carcinoma from July 2002 to Dec. 2002 in pathological research room of medical college of Shantou university. Forty-seven cases chronic esophagitis conservated waxes come from preserved waxes in our pathological room from Oct. 1995 to July 2003. (D Collectting fresh specimens of esophageal carcinoma, para-cancer epithelia and relatively normal cutting edge epithelia from Dec. 2003 to April 2004 in our room. In these specimens, esophageal carcinoma and para-cancer mucosa is 16 cases respectively, and normal mucosa 12 cases. (3) HE staining: making histopathologic dignosis for tissuesections, And single cell smear staining for defining cellular form and integrity. (4)Immunohistochemistry method: Detecting the expressions of MDM2, C-myc, P53, P16, P27 and P21 protein for forty-seven cases chronic esophagitis and eighty-five cases esophageal carcinoma and para-cancer epithlia by immunohistochemistry EnVision two-step method.2 Flow cytometer detecting method: Cutting tissues to make fragments by eye scissors and making single cell suspension and staining by PI. Detecting cell DNA content, cell proliferation activity marker PI for twelve cases normal esophageal epithelia, sixteen cases para-cancer and sixteen cases esophageal carcinoma cell. And quantitively analyzing the expression of MDM2, C-myc, P53, P16, P27 and P21 proteins.Result1 Histopathology: Under the light microscope, the dignosisforty-seven cases chronic esophagitis are coincidence with Liufusheng' s the dignosis standard for chronic esophagitis. Fifty-two cases with para-cancer tissue in eighty-five cases esophageal carcinoma, the mucosa epithelia of para-cancer are classified with mild, moderate and severe atypical, and esophageal squamous cell carcinoma are classified with grade I,II and III, among them, grade I is thirty cases, grade II thirty-nine and grade III sixteen cases.2 Innnunohistochemistry: CD Immunohistochemistry stainingcondition: The MDM2 protein is located in nuclei andcytoplasm or only cytoplasm. The C-myc protein is locatedin cytoplasm. The P53, P27 and P21 proteins are located innuclei. The P16 protein is located in nuclei and cytoplasmor only nuclei. ?The expressions of the above six proteinsin esophageal different lesions : P53 protein has noexpression and the others proteins have different degreeexpressions in normal epithelia of esophagus. From normalepithelia of esophagus to chronic esophagitis to mildatypical hyperplasia to moderate atypical hyperplasia tosevere atypical hyperplasia to carcinoma in situ toinfiltration carcinoma, the positive rates of MDM2, P53, P27and P21 proteins expression have a rising tendency gradually(7-0. 118^0. 161 > 7=0. 227 and 7=0. 274, KQ. 05). The positiverate of P16 protein expression is decreasing gradually(t=-0. 207, P=0. 004). Generally, the positive rate of C-mycprotein have no significant difference in every group. Butthe positive rate of C-myc protein is higher in esophagealcarcinoma than para-cancer tissue (x2=5. 233, P=0. 022).In addition, the positive rates of P53 protein are similar in three groups of para-carcer atypical epithelia,carcinoma in situ and esophageal invasive carcinoma (x2=1.460, P=0. 482), and is higher than normal esophageal epithelia( x 2=22. 768, P=0. 000); The positive rates of MDM2 protein are similar in three groups of normal esophageal epithelia, para-carcer atypical epithelia and carcinoma in situ (x2=3.302, /M). 220), and lower than esophageal invasion carcinoma (x2=21.739, P=0.000); Compared with normal mucosa, there are striking low-expression of P27 protein (/K0.05). ?The expressions of the above six proteins in chronic esophagitis and para-cancer atypical hyperplasia epithelia of esophagus: In forty-seven cases chronic esophagitis, twenty-three cases with simply hyperplasia and twenty-four cases with atypical hyperplasia of epithelia. The expressions of the above six proteins are very similar in mucosa epithelia of chronic esophagitis and para-cancer atypical hyperplasia epithlia ( .P >0.05). (D The relationship of the six proteins expression and clinical pathology character of esophageal carcinoma: The positive expression of MDM2 protein is negative correlated with tumor differentiative degree Cr=-0. 323, P=0. 004), but no correlation with tumor invasive depth and lymph node metastasis (P>0. 05). The expressions ofC-myc, P53, P16, P27 and P21 proteins have no correlation with pathologic grade, invasive depth and lymph node metastasis of esophageal carcinoma. ?The relationship of the expression intensity among the above six proteins: Making correlation analysis according to the positive expression intensity of the above six proteins, the resultsdisplay the expression of MDM2 protein is negative correlation with P16 and P21 protein, and positive correlation with P27 protein. The expression of P53 protein is negative correlation with C-myc, P16, P27 and P21 protein. The expression of P16 protein is correlation with P21protein. And there have no relation among the rest protein. Flow cytometer detecting result1 The expressions of the above six proteins in different lesions of esophagus and esophageal carcinoma.P53 protein has no expression and the others proteins have different degree expressions in normal esophageal mucosa. The expression rates and quantities of MDM2, C-myc, P53 and P21 proteins all in esophageal carcinoma are higher than normal esophageal mucosa and para-cancer tissue (P <0.05). The expression quantitiy of P16 protein in esophageal carcinoma is lower than normal esophageal mucosa and para-cancer tissue (P<0. 05). And from normal mucosa to para-cancer tissue to esophageal carcinoma tissue, the expression quantity of P16 protein has a linearity decreasing tendency (/s=24. 478 , P=0. 000). The quantitive expression of P27 protein has also a linearity decreasing tendency (F=9.132, P=Q. 002). In the carcinogenesis process, the positive rates of P27 protein expression have no statistically significance in the three groups.2 DNA ploidy detecting result?DNA index of esophageal carcinoma (1. 62±0. 38) is markedly higher than normal mucosa of esophagus (DI=1. 00±0. 06)andpara-cancer tissue (DI= 1. 00±0.10) . Both their lvalue are 0. 007. ?According to DNA ploidy judgement standard: Twelve cases normal esophageal mucosa are diploid. In sixteen cases esophageal carcinoma, fourteen cases are heteroploid (87.5%) and two cases are diploid (12.5%). Heteroploid rate of esophageal carcinoma is striking higher than normal mucosa of esophagus and para-cancer tissue (K0. 01).? By-analyzing cell cycle, we discovery: the three groups, normal mucosa, para-cancer tissue and esophageal carcinoma, PI values have a linearity rising tendency(P=8. 861, P=0. 009). Both normal mucosa and para-cancer tissue, para-cancer tissue and esophageal carcinoma, PI values have no statistically significance (P=0. 287, /M). 380), but PI value of esophageal carcinoma is higher than normal mucosa (/*=0. 038) Conclusion(Dlmmunohistochemistry studies of six proteins display the changes of P53 and P16 proteins expression occurrenc in earlier period of carcinomatous change. The expression of MDM2 protein is correlated with biology behavior of esophageal squamous cell carcinoma. ?The expression condition of six proteins in mucosa epithelia of chronic esophagitis is similar to para-cancer mucosa by immuhistochemistry method, this shows that chronic esophagitis may be a precancerous lesion. ?Applying flow cytometer and quantitively studying the expressions of the six proteins in normal mucosa of esophagus, para-cancer mucosa and esophageal carcinoma cell help to investigatethe change of protein quantity in the process of esophageal carcinoma occurrenc. (D Associating flow cytometer quantitive detecting protein content with analyzing DNA ploidy and cell proliferation activity help to know the relationship of the change of protein expression and cell proliferation in the cell carcinomatous change process of esophageal epithelia.