| Background and ObjectivesRenal carcinoma is3%of the systemic-malignant tumor, Its morbidity is only lower than bladder carcinoma in urologic tumor, the age of onset is almost.40, Its morbidity is 3.7 per one hundred million,the characteristics of renal tumor is insidious onset, and earlier transform than other tumor ,lose specificity.Its early diagnosis is rather difficult, especially the characterof iconography is not obvious,the authentication between Renal carcinoma with benign phyma is hard.so, in search of specificity antigen is a big task in advancing the therapy effection of renal tumor.In research of the tumor associated antigen (TAA),we find carbonic anhydrase-9 (CAIX) and cytokeratin19 (CK19Mrna) have a specific expression in renal tumor. Anhydrase-9 which make an important position as a biomarker we know for renal tumor is one of the new carbonic anhydrase family (CAs) isoenzymes was founded and express high in renal tumor, but the research about renal tumor diagnoseand micrometastases after the operation is only. Cytokeratin19 (CK19mRNA) is one of the tumor marker which was found in recent,it play a big role in maintaining continuity of cell and stabilizing cell form. Cytokeratin19 have a higher specificity and conservatism in tissue and cell differentiation, as well, have an important role in cell signaling,multiplication,differentiation and apoptosis.In present,there are a lot of study in thyroid carcinoma,gastric cancer,cancer of the cervix in respect to cytokeratinl9,but less in micrometastases after the operation. The aim of this experiment is that explore the application of Cytokeratin19 in renal tumor. anhydrase-9 and Cytokeratin19 have a important significance to renal tumor if it has a micrometastases in preoperative diagnosis and detection after the operation in our clinical work.We tried to detect the expression of peripheral blood samples of anhydrase-9 (CAIX) after the operation and Cytokeratin19 Pre and after operative form the level of RNA in renal tumor by reverse transcription polymerase chain reaction (RT-PCR) assay,Meanwhile we will Analysis the mechanism on renal tumor and explore the application on anhydrase-9and Cytokeratinl9 as micrometastasis-marker.Both of data were statistically analyzed by SPSS 17.0 software.α=0.05 was considered the level of significant.Materials and Methods40 peripheral blood cases of renal tumor on anhydrase-9 (CA IX) were collected form The Second Affiliated Hospital of Zhengzhou University,Henan Tomor Hospital,Henan People Hospital during Sept,2008 to Jan,2011 containing 28 males and 16 females,whose peripheral blood were taken from in morning at 6 months,12months,18 months,24 months,30 months,36 months after nephrectomy respectively,At the same time,we can select some patient doing the examination of physical/type-B ultrasonic/CT/chest X-rays.If we found some cases occuring metastasis or G250mRNA arising,we would follow up twice per months. We collecte 3ml blood in EDTAK2 tubes,and was processed to RNA extraction rapidly, then the RNA specimens were stored in-80℃refrigerator. GAPDH was used as the internal contrast, RT-PCR assay was performed to detect the CAIX mRNA in the tissue and in peripheral blood samples by semi-quantitative RT-PCR analysis.32 peripheral blood cases of renal tumor on Cytokeratinl9 were collected form The Second Affiliated Hospital of Zhengzhou University,Henan Tomor Hospital,Henan People Hospital during Jan,2010 to Jan,2011,containing 19males and 13females. Clinical stages were consisted of I 8cases,â…¡11 cases,â…¢-â…£13 cases. Histopathologic types:22cases were clear-cell carcinoma,8 cases were chromophilic, and 2 cases were chromophobe cell type, moreover, as control groups, we select 10 normal renal tissues and 10 adjacent normal tissues which were obtained more than 3 centimeters away from the edge of tumor lesions. During collecting all the tissue samples, none of the patients under medical treatment,and all samples were pathologically confirmed. Fresh renal tissue specimens taken out during the operation were put into the RNAse-Free EP tubes immediately,then stored in-80℃refrigerator to prevent mRNAdegradation.16 healthy volunteers on an empty stomach whose peripheral blood specimens were obtained in moring; The volume of blood specimen is 3ml;We will draw peripheral blood specimens in moring on an empty stomach from renal tumor patients at 2h before and 1st week,2nd week,3rd week,4th week after nephrectomy respectively,the volume of blood specimen is 3ml too, put into EDTAK2 tubes,and processed to RNA extraction rapidly, then the RNA specimens were stored in-80℃refrigerator. GAPDH was used as the internal contrast, RT-PCR assay was performed to detect the CK19mRNA in the tissue and in peripheral blood samples by semi-quantitative RT-PCR analysis.ResultsI period in the Kidney radical surger in peripheral blood samples CAIX mRNA levels atSix months to postoperative 36 months Kidney cancer patients at 18 months and 24 months time expression to have two cases of shade,â…¡period in the Kidney radical surger in peripheral blood samples CAIX mRNA levels at atSix months to postoperative 36 months Kidney cancer patients at 18 months and 24 months and 30 months time expression to have four cases of shade,â…¢-â…£period in patients with kidney cancer without shade, CAIXmRNA express, atSix months to postoperative 36 months Kidney cancer patients have seven patients died, atSix months to postoperative 36 months Kidney cancer patients, Different time pointsâ… period,â…¡period,â…¢-â…£period are statistically significant differences.According to detecting the peripheral blood of renal tumor patient,the semi-quantitative level of 6 months after surgery is 0.313±0.078,12 months is 0.275±0.064,18 months is 0.178±0.055,24 months is 0.144±0.041,30 months is 0.129±0.023,36 months is 0.120±0.018.After nephrectomy, peripheral blood CAIX mRNA of renal tumor patient have a enormous reduction with the time(F=38.644, P>0.01), but there was no significance within groups (0.54±0.13 vs 0.56±0.15, P>0.05)In the 32 renal tumor tissues, the Cytokeratin19 (CK19) total positive rate was 76% (24/32), adjacent normal tissues and normal tissues were 5.6%å'Œ0% respectively,so, renal tumor tissues was Obviously higher than that both of (P<0.01),meanwhile, there was no significant difference between adjacent normal tissues and normal renal tissues (P>0.05). In terms of histopathologic types,the positive rate of clear-cell carcinoma is 85.7%(19/22), the positive rate of chromophilic is 39.6%(3/8),the positive rate of chromophobe cell type is 0%, by comparing, the positive rate of clear-cell carcinoma is significant higher than chromophilic and chromophobe cell type,but there is no difference between chromophilic and chromophobe cell type.In the 32 peripheral blood of renal tumor patient, the Cytokeratin19 (CK19) total positive rate was 56.2%(18/32);in the control groups which conclude 16 heathy person, the Cytokeratin 19 (CK19) total positive rate was3.4%(4/16), there was difference in both. The positive rate is 28%(3/8) in stage I, stageâ…¡is 41%(5/11), and III-IV is 79%(10/13), there was differences were founded during stage I, stage II and III-IV (P<0.05),but no significant difference between stage I and stage II, nevertheless, stage III-IV was higher than both of groups above,and highly significant.In the 18 peripheral blood positive renal tumor patient,the level of CK19mRN is that,two days before operation is 0.49±0.11,one week after operation is 0.41±0.13, two weeks after operation is 0.29±0.11, three weeks after operation is 0.24±0.16, four weeks after operation is 0.17±0.09, as a whole, CK19mRNA presented decrease graduately with the time(P<0.05).Conclusions1,In renal tumor patient, CAIX mRNA of which the expression present a decrease trend with the time can be known as a good tumor marker,it paly a important role in renal carcinoma,and can guide diagnosis to us. 2,In my experiment,we can see that, as a tumor marker, the expression of CK19MRNA have a significantly differences in all kinds of histopathologic types and nor-tumor, In addition, different stage,too. These phenomenon demonstrate that CK19MRNA maybe have some relation with the stage of renal carcinoma.3,The detection of CK19MRNA and CAIX mRNA in renal tumor tissues which can preferably judge micrometastasis of patient after operation, judge prognosis, provide reasonable treatment prescription have an important significance. |
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