The Clone And Identification Of Genes Specially Associated With The Generation And Development Of Bladder Cancer

Objective:(1) To determine a feasible technical routine for capturing pure nor mal bladder transitional cells and bladder transitional cancer cells by remodifyi ng the reported protocols of LCM;(2) To obtain Total RNA from homogeneou s bladder transitional cells and bladder transitional cancer cells and high-qualif y, ample aRNA for the further research of genes associated with bladder canc er;(3) To make sure of the corresponding relation between the shootings and quantity of RNA from captured bladder transitional cells. Methods:(1) Set up contral experiment I to make certain of the feasibility of the remodifrrd protoc ols of LCM;Set up contral experiment II to detect the corresponding relatio n between the shootings and quantity of RNA from captured bladder transition al cells;Obtain pure objective cells using LCM;(2) Extract, purify and concentr ate the tiny-amount Total RNA;(3)Procure high-qualify, ample aRNA applying RNA in vitro linear amplification;(4)Using RT-PCR to detect the expression le vels of β -actin in primary Total RNA and aRNA. Results:(l)RNA integrity k eeped well after remodified LCM confirmed by contral experiment I;(2) Cont ral experiment II showed that 485ng RNA could be procured by captured 40,00 0 shootings to bladder transitional cells;(3) Procured homogeneous Total RNA (ng) of purebladder transitional cells and bladder cancer cells;(4)T he size of aRNA distributed from 0.5 to 2.5kb(ug);(5)The expressionlevel of 3 -actin was integral. Conclusion:LCM combined with RNA in vitro linear amplification can be applied to procure homogeneous, sufficient, integral RNA of objective cells for the further research of gen es associated with bladder cancer.Section two: The study of genie differential expression betweenbladder cancer cells and bladder transitional cellsObjective:(l)Optimize the reaction conditions of DDRT-PCR, make certain of the optimal conditions adapted to tiny-amount RNA;(2)Screen the differentially-expressed genes between bladder cancer cells and bladder transitional cells, fur thermore, determine them as the genes associated with bladder cancer or not Methods:(l)Using fluorescence DDRT-PCR, Screen the differentially-expressed genes;(2)Clone and sequence the bands of Screen the differentially-expressed g enes;(3)Homology searches are performed against Genebank entries using NCB I BLAST programs;(4)Validate the authenticity of the bands using Reverse No rthern Blot;(5)By means of the Quantitative real-time PCR, further Validate th e authenticity and detect the relative intensity of expression of the genes;(6)Atgene,level determinate the differentially-expressed genes as ones or novel one s associated with bladder cancer. Results:(l) Named 27 differentially-expressedgenes'sfragments as BCRGl-27;(2)Results of Homology searches against Gene bankrsome fragments were homologous to known genes;some to uncharacteriz ed genes;and the others to uncharacterized human clones on chromosomes;(3)T he authenticity of differences of genie expression was 100% validated by Reverse Northern Blot and Quantitative real-time PCR. Conclusion: Determined at gene level:(l)For the first time, determined HMGB1 as the novel correlati ve gene with bladder cancer;(2) For the first time, determined TopBPl, AN GPTL3, AGA, SCAR1, M0N2 homolog, SETX, bromodomain PHD finger transc ription factor as the novel correlative genes with tumor or/and blad der cancer;(3) For the first time, determined BCRG5* 7* 8* 10* IK 12* 14* 16* 19* 26 as novel characterized genes, furthermore correlative with turn or or/and bladder cancer;(4) For the first time, extrapolated BCRG2* 3* 4% 13* 18* 20* 23* 24 as the novel correlative genes with tumor or/and b ladder cancer.Section three: The study of protein expressions of Cu> Zn-SODTopBPl HMGB1 in bladder cancerObjective: On the one hand, detect whether corresponding protein expression di fferences of Cu* Zn-SOD* TopBPl* HMGB1 exists or not;on the other hand, at protein level, further make sure that the three genes are associated with bl adder cancer. Methods: Applying immunohistochemistry in tissue microarray, d etect the protein expression of Cu* Zn-SOD* TopBPl* HMGB1 in bladder ca ncer(respective 30cases of pathological grade I * II * III) and bladder transition al cells(30cases),and analyse the correlation between the protein expressions an d pathological grade or invasive depth. Result: (l)The protein expression of C u* Zn—SOD is up-regulated in cancer cells, and that negatively correlated wit h pathological grade, positively correlated with invasive depth;(2) The proteinexpression of TopBPl is down-regulated in cancer cells;No significant differen ces of TopBPl expression were found between pathological grade, invasive de pth;(3) The protein expression of HMGB1 is up-regulated in cancer cells, mor eover negatively correlated with pathological grade, positively correlated with i nvasive depth.Comclusion:(l)Determined Cu> Zn-SOD as the correlative gene with bladder cancer at both of gene and protein level;(2)For the first time, at both of gene and protein level determined TopBPl as a novel tumor suppress or gene correlative with tumor or/and bladder cancer;(3) For the first time, at both of gene and protein level determined HMGB1 as a novel oncogene corre lative with bladder cancer.