OK-432对膀胱肿瘤抑制作用及其机制研究

发布时间：2018-03-21 14:49

本文选题：OK-432　切入点：膀胱肿瘤　出处：《华中科技大学》2015年博士论文　论文类型：学位论文

【摘要】：研究背景及目的膀胱癌是泌尿系统最常见的恶性肿瘤之一,在世界范围内排在所有恶性肿瘤的第九位。据报道,全球每年大约有330,000新发病例。膀胱癌的新发病例中70%为非肌层浸润性膀胱癌(Non-Muscle-Invasive bladder cancer, NMIBC),目前在临床上对于非肌层浸润性膀胱癌的治疗,经尿道膀胱肿瘤切除术(Transurethral Resection Of Bladder Tumor, TURBt)是治疗膀胱肿瘤的金标准,术后再辅助其他的药物进行膀胱灌注治疗。膀胱灌注治疗是非肌层浸润性膀胱癌治疗的重要组成部分,对预防膀胱肿瘤的复发和进展显得尤为重要。因此,膀胱肿瘤术后灌注的药物选择就显得非常关键。研究一种既可以预防膀胱肿瘤复发,而且对机体的副作用又小的药物就显得迫在眉睫。 OK-432为溶血性链球菌A3或SIPI722低毒变异株经青霉素灭活、冷冻干燥等过程处理得到的灭活菌体制剂。作为一种灭活的菌体,进入体内能诱发机体的免疫功能,主要包括刺激巨噬细胞、NK细胞等免疫细胞来而发挥抗肿瘤作用,但是其具体的作用机制还不是很明确。本研究将OK-432与膀胱癌细胞系在体外共培养,了解OK-432对膀胱癌细胞系增殖、凋亡、细胞周期、侵袭及转移的影响,以及应用OK-432进行大鼠膀胱灌注了解OK-432在体内对膀胱肿瘤的作用,探讨OK-432对膀胱癌的抑制作用及相关的作用机制。研究方法 1、将膀胱癌细胞系T24和EJ细胞与不同浓度OK-432(0,0.125,0.25,0.5,1,2,4ke/ml,1ke OK-432相当于100ug冻干的链球菌株)共培养24,48,72小时后,分别应用MTS法检测其OD值,来研究OK-432对T24和EJ细胞增殖的影响。 2、将不同浓度OK-432(0,0.1,0.5,1ke/m1)与T24和EJ细胞作用2周后,在倒置显微镜下观察其对T24和EJ细胞克隆形成的影响。 3、将1,2,4ke/ml OK-432与T24和EJ细胞作用24小时后,应用EDU法检测OK-432对T24和EJ细胞增殖的影响。 4、将1,2,4ke/ml OK-432与T24和EJ细胞作用24小时后,应用流式细胞术检测OK-432对T24和EJ细胞凋亡的影响,并应用western-blot检测相关凋亡蛋白的表达情况。 5、将1,2,4ke/ml OK-432与T24和EJ细胞作用24小时后,应用流式细胞术检测OK-432对T24和EJ细胞周期的影响,并应用western-blot检测相关周期蛋白的表达情况。 6、将1,2,4ke/ml OK-432与T24和EJ细胞作用6时后,应用Transwell检测OK-432对T24和EJ细胞侵袭和迁移的影响,并应用western-blot检测EMT相关蛋白的表达情况。 7、将1,2,4ke/ml OK-432与T24和EJ细胞作用24小时后,应用western-blot检测TLR4/NF-κb信号通路相关蛋白的表达。 8、应用MNU把大鼠诱导形成膀胱肿瘤后,将OK-432进行大鼠膀胱灌注,16周后处死大鼠并取出膀胱,进行石蜡包埋、切片、免疫组化检测。结果 1、MTS果发现,0.5,1,2,4ke/ml OK-432对T24和EJ细胞抑制作用明显,且呈现浓度依赖性；克隆形成实验研究表明,0.1,0.5,1ke/ml OK-432对T24和EJ细胞的克隆形成有抑制作用,而且呈浓度依赖性的抑制;EDU实验结果表明,1,2,4ke/ml OK-432对T24和EJ细胞的增殖有抑制作用,也呈浓度依赖性。 2、应用流式细胞术检测OK-432对T24和EJ细胞的凋亡实验研究结果显示,1,2,4ke/ml OK-432对T24和EJ细胞的促凋亡作用与正常对照组相比要显著,且相关的凋亡蛋白cleaved caspase3, cleaved caspase7, caspase9, Bax和cleaved PARP的蛋白表达量增加,Bc1-2的蛋白表达量减少。 3、应用流式细胞术检测OK-432对T24和EJ细胞周期研究结果显示,OK-432可以使T24和FJ细胞的细胞周期停滞在G0/G1,而且周期蛋白P15、P16的表达量F降,而Cyclin D1和Cyclin D3的表达量升高。 4、Transwell实验结果表明,1,2,4ke/ml OK-432对T24和EJ细胞的侵袭和迁移有明显的抑制作用,且EMT相关的蛋白E-cadherin表达上调,N-cadherin, MMP9, Snail, Vimentin的蛋白表达量下调。 5、Western-blot检测TLR4/NF-κ b信号通路研究结果显示,OK-432作用于T24和EJ细胞后,TLR4的蛋白表达量上调,而p-NF-κ b和p-I κ Ba表达上调。 6、在体内实验中,MNU对大鼠进行膀胱灌注8周后,大鼠膀胱肿瘤模型建立；后进行OK-432膀胱灌注,16周后处死大鼠并取出膀胱组织行免疫组化检查。结果显示,MNU组PCNA的表达较OK-432组和正常对照组要高；TUNEL的结果显示OK-432组细胞的凋亡阳性率比MNU组和正常对照组要高。结论 OK-432能够抑制膀胱肿瘤细胞系T24和EJ细胞的增殖,并促进其凋亡,而且能够使T24和EJ细胞的细胞周期停滞在G0/G1； OK-432还能抑制T24和EJ细胞的侵袭和迁移;另外,研究发现OK-432可以通过TLR4/NF-κ b抑制膀胱肿瘤细胞的增殖和促进其凋亡；而且在体内实验中也证实OK-432能够抑制肿瘤细胞增殖和促进其凋亡。
[Abstract]:Background and purpose of research
Bladder cancer is one of the most common malignant tumor of urinary system in the world in all malignant tumors in ninth. According to reports, there are about 330000 new cases annually. New cases of bladder cancer in 70% for non muscle invasive bladder cancer (Non-Muscle-Invasive bladder, cancer, NMIBC), currently in clinical for non muscle invasive bladder cancer treated by transurethral resection of bladder tumor (Transurethral Resection Of Bladder Tumor, TURBt) is the gold standard for the treatment of bladder tumor, postoperative auxiliary other drugs intravesical therapy. Intravesical therapy is an important part of non muscle invasive bladder cancer. Is very important for the prevention of bladder cancer recurrence and progression of bladder cancer is. Therefore, the postoperative infusion drug selection is very important. A study can prevent the recurrence of bladder cancer, and The drug that has a small side effect on the body seems imminent.
OK-432 hemolytic streptococcus A3 or mutant SIPI722 toxicity by penicillin inactivation, freeze-drying treatment process by inactivated bacteria preparation. As a kind of inactivated bacteria can enter the body by the body's immune function, including macrophages, NK cells and other immune cells to exert antitumor effect, but its the specific mechanism is not clear.
This study will be co cultured in vitro with OK-432 and bladder cancer cell lines, OK-432 proliferation of bladder cancer cell line apoptosis, cell cycle, invasion and metastasis, and the application of OK-432 to rat bladder perfusion in understanding OK-432 effect in vivo of bladder tumors, to investigate the inhibitory effect of OK-432 on bladder cancer and the related role the mechanism.
research method
1, the bladder cancer cell lines T24 and EJ cells with different concentrations of OK-432 (0,0.125,0.25,0.5,1,2,4ke/ml, 1ke OK-432 is equivalent to 100ug freeze-dried Streptococcus strains) were cultured after 24,48,72 hours, to detect the OD respectively using MTS method, to research on the influence of OK-432 on T24 and EJ cell proliferation.
2, after the action of different concentrations of OK-432 (0,0.1,0.5,1ke/m1) and T24 and EJ cells for 2 weeks, the effects on the formation of T24 and EJ cells were observed under the inverted microscope.
3, after the action of 1,2,4ke/ml OK-432 with T24 and EJ cells for 24 hours, the EDU assay was used to detect the effect of OK-432 on the proliferation of T24 and EJ cells.
4, after 24 hours of 1,2,4ke/ml OK-432 interaction with T24 and EJ cells, the effects of OK-432 on T24 and EJ cell apoptosis were detected by flow cytometry, and the expression of related apoptosis proteins was detected by Western-blot.
5, after 24 hours of 1,2,4ke/ml OK-432 interaction with T24 and EJ cells, the effects of OK-432 on T24 and EJ cell cycle were detected by flow cytometry, and the expression of related cyclin was detected by Western-blot.
6, after 6 hours of 1,2,4ke/ml OK-432 interaction with T24 and EJ cells, Transwell was used to detect the effect of OK-432 on invasion and migration of T24 and EJ cells, and Western-blot was used to detect the expression of EMT related proteins.
7, after the action of 1,2,4ke/ml OK-432 with T24 and EJ cells for 24 hours, the expression of TLR4/NF- kappa B signaling pathway related proteins was detected by Western-blot.
8, after MNU was used to induce bladder tumor in rats, OK-432 was perfused into the bladder of rats. After 16 weeks, the rats were killed and the bladder was removed. Paraffin embedded and sections were taken for immunohistochemical examination.
Result
1, MTS found 0.5,1,2,4ke/ml OK-432 on T24, and EJ cells were significantly inhibited, and in a concentration dependent manner; clone formation experiment, 0.1,0.5,1ke/ml OK-432 T24 and cloning of EJ cells can inhibit the formation, but also showed a concentration dependent inhibition; experimental results show that the EDU can inhibit the proliferation of T24 1,2,4ke/ml OK-432 and EJ cells, but also in a concentration dependent manner.
2, detection of OK-432 on T24 and EJ cell apoptosis by flow cytometry showed that 1,2,4ke/ml, T24 and OK-432 on EJ cell apoptosis compared with normal control group significantly, and the apoptosis protein cleaved Caspase3, cleaved caspase7, caspase9, Bax and cleaved expression and PARP protein expression increased the Bc1-2 protein decreased.
3, flow cytometry was used to detect the cell cycle of OK-432 and T24 and EJ. The results showed that OK-432 could arrest the cell cycle of T24 and FJ cells in G0/G1, and the expression of cyclin P15 and P16 decreased, while the expression of Cyclin and P15 increased.
4, Transwell results showed that 1,2,4ke/ml OK-432 significantly inhibited the invasion and migration of T24 and EJ cells, and EMT related protein E-cadherin expression was upregulated, and N-cadherin, MMP9, Snail, and Vimentin protein expression was downregulated.
5, Western-blot detected the TLR4/NF- kappa B signaling pathway. The results showed that after OK-432 acted on T24 and EJ cells, the TLR4 protein expression increased, while p-NF- kappa B and p-I kappa Ba expression increased.
6, in vivo, MNU on rat bladder perfusion 8 weeks after the establishment of rat model of bladder tumors; after OK-432 instillation, the rats were killed after 16 weeks and remove the bladder tissue by immunohistochemical examination. The results showed that the expression of PCNA in MNU group than in OK-432 group and normal control group to high TUNEL; the results showed that the positive rate of cell apoptosis in OK-432 group was higher than MNU group and normal control group.
conclusion
OK-432 can inhibit bladder cancer cell line T24 and EJ cell proliferation and promote apoptosis, but also can make the cell cycle of T24 cells and EJ cells arrest in G0/G1; OK-432 can inhibit the invasion and migration of T24 cells and EJ cells; in addition, it was found that OK-432 could inhibit TLR4/NF- by kappa B bladder tumor cell proliferation and promote apoptosis; but also confirmed that OK-432 can inhibit the proliferation and promote the apoptosis of tumor cells in vivo.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.14

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