Construction And Detection Of T24/ADM Orthotopic Bladder Cancer Multidrug Resistance Model

Part1Establishment of multi-drug resistant bladder cancer T24/ADM cell linesObjective:To establish drug-resistant human bladder cancer cell T24/ADM models and preliminary testing of its drug resistance.Methods:Human bladder cancer T24 cell lines was explosed to adriamycin(ADM) in the culture medium for establishing of drug resistant cell lines:concentrations of ADM was lowly stepwise increased for long explsure.There morphologic studies and Growth were performed with Inverted microscope.Drug tolerance were determined by MTT assay, The mRNA expression of mdr-1 was detected by reverse transcriptase-polymerase chain reaction(RT-PCR).Results:The establishment of drug-resistant cell line had been made by Low-concentration gradient induced by adriamycin for 3 months. T24/ADM-resistant cells were slightly larger than the parental cells in morphology, increased more tolerant to ADM, Under the action of the same concentration of ADM T24/ADM cell were survival more than the T24 cell survival, Difference between the groups was significant, P<0.01. RT-PCR showed the mRNA expression of mdr-1 was significantly higher in T24/ADM cell lines, and expression in T24 cell lines was not obvious, the difference was significant,P<0.01.Conclusion:T24/ADM cell lines established by Low-concentration adriamycin gradient method were with multi-drug resistance, The resistance mechanism is mainly with the mdr-1 gene expression.Part twoThe effection from Trypsin method and hydrochloric acid method to model building of bladder cancer in situObjective:Comparing the impact of two different methods of bladder mucosal injury by Hydrochloric acid and parenzyme in situ cell transplantation in animal model of bladder cancer, and evaluating their advantages and disadvantages.Methods:Two groups of nude mice with 10 mice in each group were anaesthetized Intraperitoneally and then the indwelling intravenous needles were put through the urethra to the mice bladder under sterile conditions. After catheterization one group mice were injected 100 ul 10% trypsin into bladder and retained 30 min,another group were injected 100ul O.lmol·L-1 Hcl and retained 15 s.Then the two groups of nude mice were perfused 1×107 L-1 in human bladder cancer T24 cell suspension into the bladder to retain 1-2h after washing twice with PBS. Separately another group of five post-catheterization nude mice were directly perfused the cells into bladder to retain the same 1-2h. Regularlly observation of the general growth of nude mice. each group of nude mice were sacrificed and anatomised after 4 weeks, observed of bladder tumor formation and metastasis, and to take bladder tissue and suspicious line of renal pathological examination.Results:In the Trypsin damaged group there were two underwent reddish intermittent hematuria and lower abdominal mass was not obvious. To 28 days more the mice were sacrificed, generally two mice suffered bladder tumor visibly and Pathologically, mostly limited to mucosa, muscular layer did not break through,accounting for more than 50% of bladder. Hydrochloric acid damage group of mice began to undergo intermittent haematuria about 12 days,4 of which can only reach the lower abdomen mung bean size of mass, mobilely touched. There are two deaths in 2days. To 28 days off the mice were sacrificed, a total of eight with bladder tumor formation in general and Pathologically, Cell structure disorder, no breakthrough in serosal layer, there are two with visible swelling of the right kidney, the surface uneven, color dark red,and the transferred cancer Pathologically of the two kidneys.The group of no Mucosal damage were no significant change.Conclusion:Trypsin destruction of the bladder mucosa is slight, but the tumor formation rate is lower; Destruction of mucosal injury by hydrochloride is serious, but the tumor formation rate of satisfaction, in the cell transplantation model established by in situ bladder cancer,it can increase tumor formation rate greatly.Part 3Construction and Initial detection of T24/ADM orthotopic bladder cancer multidrug resistance modelObjective:Establish the orthotopic bladder cancer model of multidrug resistance as the human's,and Initially detect its resistance condition. Methods:Two groups of nude rats 4-6 weeks of age were inculated with 1×107 cell of T24 or T24/ADM,following with observation and putting down their meat,drink,mental condition,urine and abdominal mass growth.Animals were sacrificed after 4 weeks later,then their bladder were weighted and measured,histopathologic assessment was performed,mdrl was detected by PCR,and cells from the bladder tumors were detected of multidrug resisence by MTT. Results:Group of nude rats inculated with T24/ADM generated tumors about 80%(8/10),the one inculated with T24 was 90%(9/10) and about 2-3 days early.The blank group had no rats emerge tumors in bladder mucosa at all.Bladder weight and volum:0.8±0.3) g, (1.0±0.5)g;(875±158)mm3,(903±192) mm3, Difference between the two groups had no significant (P> 0.05).Histopathologic detection:The two groups of bladder cancer tissue biopsies can be seen more chaotic arrangement of cell structure, cell body shape is irregular, to the depth of myometrial invasion in different without breaking the film. Between the two groups there were no significantly difference. PCR detection of mdrl expression differences between the two groups was significant (P<0.01). Cytological detection of drug-resistant cell volume is slightly larger,and no significant difference in morphology.MTT detection:cells from the inculated T24/ADM mice bladder tumor were more resistance to ADM than the ones from the inculated T24 mice bladder tumor P<0.01),and for several other drugs were also resistant.Conlusion:Cell transplantation was successfully used to establish bladder cancer model in situ of T24/ADM,and with muti-drug resistance characteristics.The model laid the foundation for further multi-drug resistance research of bladder cancer.