| PrefacePKA is a kind of protein kinase of serin/ threonine, which is located in cell. PKA can phosphorylate many kinds of residues of serin and threonine lying in target protein upon cAMP, thus it can activate a series of substrate to play a role of signal conduction. During metabolism, proliferation, differentiation, ap-optosis and canceration of cell, PKA play an important role. The molecule of PKA is a isotetramer composed of two regulatory subunits and two catalytic sub-units. According to the difference between RI and RII which belong to regulatory subunits, PKA can be divided into two subtypes - PKAI andPKAII. PKAI is expressed much in the tumor cells and the cells exposed to stimulation from mito-gen and participates and regulate signal conduction pathway of mitosis mediated by EGF and TGFα. PKAII is expressed in the normal cells in the non multiplication period and growth inhibiting period and can inhibit signal conduction of mitosis and growth of tumor cells. Many tests have proved that there are overex-pression of RIα in ovarian cancer, lung cancer, colon cancer and pancreatic cancer. Thereby in order to investigate the relation RIαand RII with transitional cell carcinoma of the bladder, we detect RIaand RII in carcinoma of bladder by immunohistochemical method.Material and Method1. Sample selectionWe studied paraffin samples from 48 patients(35 males and 13 females) whose initial diagnosis are transitional cell carcinoma of bladder from 2003. The age of patients ranged from 23years to 81 years (the mean age is 54 years. ).Depending on the standard of WHO, the grade of tumor cells were divided into Gl(21) , G2(19) , G3(8). According to the standard of UICC -TNM, the clinical stage included Tl(16), T2(19), T3(13). Else 12 normal mucous membrane of urinary bladder used for control were collected from the patients who had hyperplasia of prostate gland and had been operated.2. Major agentsAnti - RIa antibody was bought from Merck company of Germany. Anti -RII antibody was bought from Epitomics company of America. SP immunohisto-chemical kit and DAB developer were bought from maixin biotechnological company of Fuzhou.3. Experimental methodWe took S — P method to make immunohistochemical stain. In the course of stain, we severely controlled temperature and time.1. The paraffin sections were deparaffinaged by dimethyl benzene routinely.2. The sections were hydrated by 100% , 95% , 85% and 75% alcohlo.3. Antigens were repaired through high temperature and high pressure. 4.3% hydrogen dioxide (A liquid) was dropped and it existed on sectionfor 10 minutes.5. The sections were washed in PBS three times, 5 minutes every time.6. The sections were incubated with 10% normal goat serum(B liquid) for 20 minutes at 37 °C.7. One antibody was dropped after serum was threw away, then stay overnight at 4t.8. The sections were washed in PBS three times, 5 minutes every time.9. The sections were incubated with biotin labeled two antibody(C liquid) for 30 minutes 37 T!.10. The sections were washed in PBS three times, 5 minutes every time.11. The sections were incubated with streptomycete avidin - peroxidase solution ( D liquid) for 30 minutes 31%.12. The sections were washed in PBS three times, 5 minutes every time.13. Use DAB developer for colouration.14. The sections were counterstained by hematoxylin, dehydrated by gradi-ent alcohol, then hyaliniz by dimethyl benzene.15. The sections were sealed by neutral gummi.4. Statistical treatmentStatistical treatment was done by SPSS11. 5 software. The relation of RIa and RII expression with clinical pathology was analyzed by x test , the absor-bance was analyzed by SNK method, the correlation betweem the above two indexes and the comparison between absorbance method and traditional semiquan-titative method were analyzed by Spearman correlation analytical method.Results1. RIa immunoreactivity ( IR) in the cytoplasm of transitional epithelial cells and tumor cells presented brown - yellow or dark brown, but there was no RIa IR in interstitial substance. RII IR could not be observed in carcinoma of bladder cells, but could de observed in interstitial substance ( eg. cytoplasm of vascular smooth muscle cytoplasm, presenting pallideflavens. The positive expression rate of RIa subunit in normal bladder cells (16. 67% , 2/12) is markedly lower than that in transitional cell carcinoma of the bladder cells ( 95. 83% , 46/48). The positive expression rate of RII subunit in normal bladder cells is 75% (9/12) , but that in transitional cell carcinoma of the bladder cells is 50% (24/48).2. According to the ratio of positive cells expression, the results could be divided into 5 grades ( -- + + + +). - represents 0, + represents <25% ,+ + represents <25% -50% , + + + represents 50% -75% , + + + + represents >75%. - - + + was called low expression group and + + - + + ++ was called high expression group. The percentage of high expression group showed 88. 89% in G2 ~ G3 , 38.10% in Gl, 81.25% in T2 - T3, 31.25% in Tl.3. The absorbance of RIa in every grade and every stage of control group and tumor group was measured and the mean value of absorbance was caculated. The absorbance was 10. 10 6. 57 in control group, 19. 00 10. 03 in Tl group, 26. 82 8. 65 in T2 group, 33. 87 6. 05 in T3 group;it was 20. 70 9. 63 in Glgroup, 27. 85 8. 89 in G2 group, 36.24 4. 20 in G3 group.Conclusion1. The RIa subunit of has the PKA can promote occurrence and development in transitional cell carcinoma of the bladder. It is an important sign of poorly differentiated invasive transitional cell carcinoma of the bladder.2. The pathway that the RIa subunit promote carcinoma is irrelevant with RII subunit.3. There is no relation between the expression of RII and carcinoma of bladder.
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