【作者】 沈瑞林；

【导师】 温端改；

【作者基本信息】 苏州大学 ， 泌尿外科学， 2009， 博士

【摘要】 第一部分肿瘤坏死因子相关诱导凋亡配体以及联合丝裂霉素C对人膀胱癌细胞作用的体外实验目的检测肿瘤坏死因子相关诱导凋亡配体(Tumor necrosis factor relatedapoptosis-inducing ligand,TRAIL)对人膀胱癌细胞株体外生长的抑制作用和凋亡作用,以及TRAIL联合丝裂霉素C(MMC)治疗膀胱癌的协同作用。方法人膀胱癌细胞株T24、5637体外培养。将T24、5637膀胱癌细胞接种至96孔培养板,培养24h后分别加入浓度为1、10、100μg/L的TRAIL,0.1、1.0、10.0mg/L的MMC,不同浓度TRAIL和MMC作用24h,采用噻唑蓝比色(4,5-dimethy thiazoleor 2,5-diphenyl tetrazolium bromide,MTT)法分别检测膀胱癌细胞的生存率,并将单独用TRAIL、MMC与TRAIL联合MMC对膀胱癌细胞株T24、5637的生长抑制情况进行比较;将T24及5637膀胱癌细胞接种至12孔板,培育24小时后加入不同浓度的TRAIL、MMC、TRAIL联合MMC并作用12h,用流式细胞术检测不同处理组膀胱癌细胞的凋亡率和死亡率,并将单独用TRAIL、MMC与TRAIL联合MMC组进行比较。结果TRAIL能有效抑制人膀胱癌细胞株T24、5637的生长。①MTT实验结果:1、10、100μg/L的TRAIL对膀胱癌T24细胞的抑制率分别为26.4%、29.3%和37.7%,与无药物组比较差异有显著性(P＜0.01)。三种浓度的TRAIL对膀胱癌5637细胞的抑制率分别为13.7%、21.9%和59.1%,1μg/L TRAIL组与无药物组比较差异无显著性(P＞0.05),10、100μg/L TRAIL组与无药物组比较差异有显著性(P＜0.01)。10.0mg/LMMC对膀胱癌T24、5637细胞的抑制率分别为36.0%、26.7%;而100μg/L TRAIL联合10.0 mg/L MMC组对膀胱癌T24、5637细胞的抑制率分别达到58.4%、90.7%,联合用药有明显的协同作用,与单纯100μg/L TRAIL组、单纯10.0 mg/L MMC组相比差异有显著性(P＜0.05)。②流式细胞术检测结果:100μg/L TRAIL引起膀胱癌T24、5637细胞的凋亡,凋亡率分别为20.1%、47.5%,与无药物组1.9%、5.1%的凋亡率相比差异有显著性(P＜0.01);10.0mg/L MMC对膀胱癌T24、5637细胞的凋亡率分别为13.1%、18.1%;而100μg/L TRAIL联合10.0 mg/L MMC组对膀胱癌T24、5637细胞的凋亡率分别达到41.5%、61.5%,两者有明显的协同作用,与单纯100μg/L TRAIL组、单纯10.0 mg/L MMC组相比差异有显著性(P＜0.05)。结论在体外实验中,①TRAIL能有效地抑制人膀胱癌细胞株T24、5637的生长,诱导人膀胱癌细胞株T24、5637凋亡;②TRAIL联合MMC抑制人膀胱癌细胞株T24、5637生长和诱导人膀胱癌细胞株T24、5637凋亡具有协同作用。第二部分Ad-TRAIL以及联合MMC对膀胱癌作用的在体实验目的:建立人膀胱癌细胞株T24 BALB/c-nu小鼠皮下移植瘤模型;在体研究真核表达载体Ad-TRAIL转染T24膀胱癌组织的效果及其对膀胱癌的治疗作用,以及联合MMC治疗膀胱癌的协同作用。方法:①人膀胱癌细胞株T24体外培养。6周龄免疫缺陷小鼠(BALB/c-nu)共5只。在超净台内将小鼠右侧前肢根部背侧皮肤用75%酒精消毒后皮下接种0.2ml浓度为1×10~7个/ml的T24人膀胱癌细胞株悬液,等上述接种小鼠的肿瘤长至直径1cm大小时,处死小鼠,切取肿瘤。再切开肿瘤,取质地均匀处组织,切成2mm~3,10～15mg大小,分别移植至40只6周龄BALB/c-nu小鼠右前肢根部背侧皮下,每只小鼠移植1块。移植方法:先用75%酒精消毒移植处皮肤,切一0.4cm大小的皮肤切口,分离皮下组织,将组织块植入皮下,0号线缝合皮肤切口。继续按常规饲养小鼠并观察注射部位的变化。②上述BALB/c-nu小鼠模型40只,肿瘤长到0.4～0.6cm后随机分成四组,分别为单用MMC组(A组):腹腔注射0.1g/L MMC(10μl/g体重),连续注射7d;瘤内注射5×10~8pfu/ml Ad空载体0.1ml×3处,分别注射于肿瘤的4、8、12点,1次/2d,共3次;单用Ad-TRAIL组(B组):腹腔注射PBS(10μl/g体重)×7d,瘤内注射5×10~8pfu/mlAd-TRAIL 0.1ml×3处,1次/2d,共3次;联合Ad-TRAIL和MMC组(C组):瘤内注射Ad-TRAIL同B组,腹腔注射MMC同A组;对照组(D组):腹腔注射PBS(10μl/g体重)×7d,瘤内注射5×10~8pfu/ml Ad空载体0.1ml×3处,1次/2d,共3次。给药结束2周后脱颈处死荷瘤鼠,测量小鼠体重,所荷肿瘤的直径和重量。取出肿瘤制成常规病理切片观察。用RT-PCR和Western blot测量肿瘤组织内TRAIL mRNA及TRAIL蛋白的表达水平。结果:①第一批5只小鼠,有3只荷瘤模型建立成功,至细胞注射后50d,3只小鼠荷瘤直径达0.6～1.0cm,并经病理组织学证实;第二批40只小鼠肿瘤模型建立全部成功,成瘤率达100%,移植后4周所有小鼠肿瘤直径在0.4～0.6cm。②四组小鼠给药前体重及所荷肿瘤平均直径差异无统计学意义(P＞0.05);给药结束2周后,A、B、C组所荷肿瘤直径和重量均显著小于D组(P＜0.001),而C组又显著小于A、B组(P＜0.01),B组又显著小于A组(P＜0.001);A、C组体重均显著小于D组(P＜0.01),而B组和D组差异无统计学意义(P＞0.05);肿瘤组织病理切片镜下观察,组织坏死由多到少依次为C组、B组、A组和D组;B、C组肿瘤组织内均有TRAIL mRNA和TRAIL蛋白的表达,且两组间表达相对量差异无统计学意义(P＞0.05),而A、D组则无TRAIL mRNA和TRAIL蛋白的表达。结论:①成功构建裸鼠人膀胱癌细胞株T24皮下移植瘤模型;②在体实验中,Ad-TRAIL能成功转染T24膀胱癌组织且表达TRAIL蛋白;③TRAIL对T24人膀胱癌裸鼠移植瘤具有明显的抑制作用,且TRAIL联合MMC具有协同抗膀胱癌作用。更多还原

【Abstract】 PartⅠExperimental study of a combination of TRAIL and MMC on treatment of bladder cancer cell strains in vitroObjective:To explore the effect of TRAIL(Tumor necrosis factor-related apoptosis-inducing ligand) on inhibition and apoptosis of bladder cancer cell strains T24 and 5637 in vitro,and to explore the anti-tumor synergistic effects of TRAIL and MMC on bladder cancer cell strains.Methods:T24 and 5637 bladder cancer cell strains were employed in our study, 100μl of target cell suspension were added to each well of 96-well plates,by adding a concentration of 1,10,100μg/L TRAIL,0.1,1.0,10.0 mg/L mitomycin C(MMC) respectively.The bladder cancer cells were incubated for 24 hours with TRAIL,MMC and TRAIL combined with MMC at different concentration.MTT working solution was added to each culture well and calculated the survival rates of bladder cancer cells and compared the deference.Bladder cancer cell suspension was added to 12-well plates and incubated for 24h,and then incubated for 12 hours with TRAIL,MMC and TRAIL combined with MMC at different concentration.The rates of apoptosis and death of bladder cancer cells were detected by flow cytometry,and compared the deference within TRAIL group,MMC group and the group of TRAIL combined with MMC.Results:TRAIL inhibited efficiently the growth of human bladder cancer cell strains T24,5637 significantly.①MTT experiment results:the growth inhibition rate(GIR) of 1,10,100μg/L TRAIL on bladder cancer T24 cell strain were 26.4%,29.3%and 37.7%, there was a significant difference(P＜0.01) to the non-drug group.The GIR of three concentrations of TRAIL on bladder tumor 5637 cell strain were 13.7%,21.9%and 59.1%, respectively.Compare to the group of non-drug,there was no significant difference to the group of 1μg/L TRAIL(P＞0.05),whereas there was a significant difference to the group 10,100μg/L TRAIL(P＜0.01).10.0mg/L MMC on bladder cancer T24,5637 cell strains, the GIR were 36.0%,26.7%,whereas 100μg/L TRAIL combined with 10.0mg/L MMC on the bladder tumor cell strains T24,5637,the GIR reached 58.4%,90.7%,respectively, There was significant difference to 100μg/L TRAIL and 10.0mg/L MMC,both have obvious synergy(P＜0.05).②the results of detecting by flow cytometry:100μg/L TRAIL induced bladder tumor cell strains T24,5637 apoptosis,apoptosis rates were 20.1%,47.5%, and there was a significant differences(P＜0.01) to the apoptosis rate 1.9%,5.1%of the group of non-drug;The apoptosis rates were 13.1%,18.1%,respectively.when use individually 10.0mg/L MMC on bladder cancer cell strains T24,5637;whereas 100μg/L TRAIL combined with 10.0mg/L MMC on the bladder tumor cell strains T24,5637,the apoptosis rate reached 41.5%,61.5%,respectively.There was significant difference to 100μg/L TRAIL and 10.0mg/L MMC,both have obvious synergy in two cell strains (p＜0.05).Conclusions:In vitro,①TRAIL can effectively inhibit the growth of bladder cancer cell strains T24,5637,and it also can induce apoptosis in human bladder tumor cell strains T24,5637.②TRAIL can enhance the effect of MMC on inhibiting the growth of human bladder cancer cell strains T24,5637,and they also have the synergistic effect on induction of human bladder cancer cell strains apoptosis.PartⅡThe effect of Ad-TRAIL and MMC on bladder tumors in vivoObjective:To study the therapeutic effects of eukaryotic expression vector coding human tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) combined with Mitomycin C(MMC) on bladder carcinoma in vivo.Methods:Methods:①T24 human bladder cancer cell strain cultivate in vitro.Five 6-week-old immune-deficient mice(BALB/c-nu),the roots of the right forelimb dorsal skin with 75%alcohol disinfection,then inoculated subcutaneously with 0.2ml concentration of 1×10~7/ml of the T24 human bladder cancer cell line suspension.Up to the tumor size of 1cm in diameter,the mice were sacrificed and tumors excised,check the texture uniformity of department organizations,and cut into about 2 mm~3,10～15mg size, 40 mice of 6-week-old BALB/c-nu were transplanted to subcutaneous dorsal roots of the right forelimb.Transplantation methods:using 75%alcohol disinfection of transplanted skin,cut the size of 0.4cm skin incision,separation of subcutaneous tissue,the subcutaneous tissue implants,0~# suture the skin incision line.Continue to conventional feeding mice and to observe changes in the injection site.②These 40 BALB/c-nu mice, tumor grow to 0.4～0.6cm were randomly divided into four groups,namely group MMC(A group):intraperitoneal injection of 0.1g/L MMC(10μl/g body weight) keep on 7d; intratumoral injection of 5×10~8 pfu/ml Ad empty vector 0.1ml×3,the tumors were injected in 4,8,12 points,1/2d,a total of 3 times;Group Ad-TRAIL(B group):abdominal injection of PB S(10μl/g)×7d,intratumoral injection of 5×10~8 pfu/ml Ad-TRAIL 0.1ml×3, 1/2d,a total of 3 times;Group MMC and Ad-TRAIL(C group):abdominal injection of MMC as the group A,intratumoral injection of Ad-TRAIL as the group B;group control (group D):intraperitoneal injection of PBS(10μl/g)×7d,intratumoral injection of 5×10~8 pfu/ml Ad empty vector 0.1ml×3 sites,1/2d,a total of 3 times.Two weeks after drug delivery,to measure mouse body weight,diameter and weight of tumors,observe the tumor cells under the microscope(×400),and measure the TRAIL mRNA and TRAIL protein expression within tumor tissue by using RT-PCR and western blot.Results:①the first batch of five mice,has three tumor-bearing model of success,to cells after injection of 50d,3 tumor-beating mice diameter reach 0.6～1.0cm,and confirmed by histopathology;the second batch of 40 mice tumor model for all of the success of tumor rate was 100%,4 weeks after transplantation all mice tumor diameter are 0.4～0.6cm.②pre-delivery weight and the load average diameter of the tumor was no significant difference in four groups of mice(P＞0.05);Tumor diameter and weight of group A,B,C were significantly smaller than group D(P＜0.001)2 weeks after delivery, while the group C was significantly smaller than group A and B(P＜0.01),group B was significantly smaller than group A(P＜0.001);Weight of group A and C were significantly less than group D(P＜0.01),while there was no significant difference between group B and group D(P＞0.05);Under the microscope,the necrosis of tumor cells, group C＞B＞A＞D;TRAIL mRNA and TRAIL protein expressed in tumor cell within Group B and C,and the difference of expression of the relative volume of Group B and C was not statistically significance(P＞0.05),while group A and D did not express the TRAIL mRNA and TRAIL protein in tumor cells.Conclusion:①Constructed successfully in nude mice of human bladder cancer cell strain T24 nude model;②Experiments in vivo,Ad-TRAIL was successful transfected into T24 bladder cancer and expressed TRAIL protein;③MMC and Ad-TRAIL have obvious inhibitory effect to T24 human bladder cancer in nude mice transplanted tumor,and both has synergistic effect with combination.更多还原