The Research Of Chromosomal Genomic Imbalance In High Recurrent Transitional Cell Carcinoma Of Urinary Bladder

Transitional cell carcinoma (TCC) is one of the most common human malignant tumors in China, it was the 8^th malignant tumor in male, 10^th in female, and the post-operative recurrence is the major obstacle to improve the prognosis of TCC patients. After recurrence, the malignant degree increase, and often lead to tumor metastasis, so that, the recurrence of TCC has become the most important problem in clinical and research works.Many specialists have done their research works on TCC by using the methods of comparative genomic hybridization (CGH) and Microsatellite analysis (MA), but got some different results, and there were a few articles on the molecular mechanisms of the high recurrent TCC. In order to make clear the mechanism, the formalin-fixed paraffin-embedded bladder cancer tissues were selected, which were obtained from the inpatients during the period of 1996 to 2003, and then divided into non-recurrence group and high-recurrence group by following up. Every specimen was detected by CGH technique, and the aberrations of chromosome segments or loci were got by statistics analysis. And then, the appropriate microsatellite loci on these chromosome segments or loci were selected to be amplified by Polymerase chain reaction(PCR), lastly, the definite information were obtained by electrophoresis.The CGH technological platform had been established in Liver Cancer Institute of Fudan University in the research of human hepatocellular carcinoma cell model with different metastatic potentials. What we performed was the indirect CGH technique in the formalin-fixed, paraffin-embedded bladder cancer tissues, many conditions needed investigating. Firstly, we improved the method of isolation of genomic DNA from paraffin-embedded tissues. Secondly, we used the DNase plus Universal Linkage System to label the tumor probes, used the same amount of probes with that of labeled by Nick Translation Labeling method to do the contrast test in order to adjust the quantity of the probes.In the 2^nd part of the experiment, we performed the MA to theparaffin-embedded tissues based on the PCR technique, then used the capillary electrophoresis (CE) and correlated annalistic software to analyze the aberration of every locus, finally the approximate positions of the aberrations of chromosomes were obtained. The present study can do the early works for the later whole genomic scan to find the "recurrence related genes" of bladder cancer.Part oneComparative Genomic Hybridization Research on High Recurrence Transitional Carcinoma of Urinary BladderThere were many scholars performed researches on the recurrence of bladder cancer, but they did not do that on the whole genomic level. What we performed in this part was to discuss the change of genomic imbalance in high recurrence bladder cancer, find the aberration of chromosome segments or loci correlated with the recurrence of bladder cancer.Of the inpatients in our hospital from the year of 1996 to 2003, they were divided into high-recurrence (HR) group and non-recurrence (NR) group by following up. A total of 20 cases of formalin-fixed, paraffin-embedded bladder cancer specimens were selected, 11 cases were HR group, and 9 cases were NR group. We improved the method of isolation of genomic DNA from formalin-fixed, paraffin-embedded bladder cancer specimens, and used the DNase plus ULS to label the tumor probes. Same amount of probes were used to perform CGH with that of labeled by Nick Translation Labeling method to do the contrast test in order to adjust the quantity of the probes. And then, every specimen was detected by CGH technique, and the aberrations of chromosome segments or loci were got by statistics analysis. After that, count the probability of the aberrations by chi-square exact probabilities test.After improving the DNA isolation protocol, the quantity and quality of the genomic DNA were better, but the fragments were different, and the degradation ones were eliminated. Among all the bladder cancer tissues, the most frequent chromosomal gains were lp> \q* 3p> 5p> 5q^ 8q^ 17q. However, the most frequent chromosomal loss were 1 lq.. 1 lp> 17p. Compared with the NR group, in HR group, the probability of 9q loss was 0.032; lip loss was 0.024; 9q plus1 lp losses were 0.54, respectively.We got the conclusion that the ULS labeling system could be used in formalin-fixed, paraffin-embedded tissues to perform CGH. The chromosomal sections or loci losses on 9q and lip had relationship with bladder cancer recurrence. There were likely to harbor "recurrence-related genes" of bladder cancer on the 2 arms. The losses or inactivation of the genes on the arms might result in the recurrence of transitional cell carcinoma of urinary bladder. However, owing to the disadvantages of CGH technique, additional methods should be used to identify the creditability of the results. Despite that, the results of the research in this part gave us the primary information of the chromosome aberrations on the recurrence of transitional cell carcinoma of urinary bladder.Part twoMicrosatellite Analysis of High Recurrence Transitional Cell Carcinomaof Urinary BladderThe results of the first part demonstrated that the loss of chromosomal sections or loci on 9q and lip had relationship with bladder cancer recurrence. The aim of this part of the research was to discuss the aberration of microsatellite DNA on the 2 arms in high recurrence transitional cell carcinoma of bladder, and validate the previous results of CGH.Based on the fact of the chromosome segments or loci loss in bladder cancer tissues, 8 microsatellite loci were selected on the 9q and lip, they were D9S1862^ D9S1847> D9S153> D9S53, D11S9O4> D11S92K D11S922, D11S899. A total of 20 cases of formalin-fixed, paraffin-embedded bladder cancer specimens were selected, 10 cases were HR group, and 10 cases were NR group. The microsatellite primers were designed according to the reference reports and NCBI website. A kind of fluorescent dye, FAM, was labeled on the 5' end in one strand of the primers. The microsatellite DNAs were amplified by PCR, after dilution and denaturalization of the products, they were performed capillary electrophoresis on the 3700 Genetic Analyzer, the data were automatically collected and analyzed by the software of GeneScan Analysis3.7.The aberration of all of the 8 microsatellite loci were observed in the 20...