hIFN-α-2b-BCG活化中性粒细胞对膀胱癌细胞TRAIL受体表达的影响
发布时间：2018-09-11 11:37
【摘要】：背景： 膀胱癌是我国泌尿外科最常见的恶性肿瘤,膀胱腔内灌注卡介苗(BCG)是防治膀胱癌的重要辅助手段,但其引起的膀胱局部与全身毒副作用影响了它的应用,本实验室成功构建了新型菌苗一重组人干扰素(hIFN)-α-2b-BCG (hIFN-α-2b-BCG),它结合了野生BCG (wBCG)和hIFN-α-2b的优势,又可以避免二者的副作用。重组hIFN-α-2b-BCG可以持续分泌hIFN-α-2b,避免了单纯膀胱灌注hIFN-α-2b作用时间短的缺点,可以使BCG和hIFN-α-2b长时间共同作用,从而产生最佳的免疫治疗效应。对于BCG的抗癌作用机制,以前多从淋巴-巨噬细胞免疫角度研究。但近年来发现wBCG膀胱灌注后,尿中出现的中性粒细胞和肿瘤坏死因子相关凋亡诱导配体(TRAIL)与膀胱癌病人的预后密切相关。 目的： 本研究主要探讨中性粒细胞在重组hIFN-α-2b-BCG抗膀胱癌中的作用。我们进行了以下研究：主要比较重组hIFN-α-2b-BCG和wBCG刺激中性粒细胞后的上清液对膀胱癌细胞表面TRAIL受体的影响并探讨其机制。 方法： 应用重组hIFN-α-2b-BCG. wBCG、wBCG+hIFN-α-2b和PBS分别与中性粒细胞共孵育,进行免疫刺激后,运用ELISA方法测各点上清液中TRAIL的表达情况。实验设PBS空白对照组,实验组分为：重组hIFN-α-2b-BCG组,wBCG组,外源hIFN-α-2b组,wBCG+外源hIFN-α-2b组；选取一时间点的上清液作用于膀胱癌细胞T24细胞,用MTT方法检测膀胱癌细胞在第1天、第2天、第3天的死亡率；PBS、TRAIL、BCG和BCG联合TRAIL作用于膀胱癌细胞,流式细胞技术检测膀胱癌细胞凋亡率；利用RT-PCR的方法检测T24细胞TRAIL受体的基因转录水平,western blot技术检测膀胱癌细胞表面TRAIL受体表达情况。实验设PBS空白对照组,实验组分为3大组： 第一实验组：单纯IFN-α组,单纯TRAIL组,IFN-α联合TRAIL组,rBCG作用组(需检测IFN-α、TRAIL浓度)。 第二实验组：rBCG+中性粒细胞上清液作用组,BCG+中性粒细胞上清液作用组,IFN-α联合BCG+中性粒细胞上清液作用组。 第三试验组：rBCG作用组,BCG组,IFN-α联合BCG组。 结果： rBCG和wBCG可以明显促进TRAIL的分泌,所有时间点与PBS组和hIFN-α-2b组相比均有显著优势(p0.05)；hIFN-α-2b组与PBS组相比无统计学意义(p0.05)。rBCG组和wBCG组相比,在刺激6小时以前无统计学意义(p0.05),12小时以后重组rBCG组和hIFN-α-2b+wBCG组相当,且两组均高于wBCG组(p0.05)。本部分实验表明rBCG更能促进中性粒细胞分泌TRAIL。 在24小时,重组hIFN-α-2b-BCG可以明显促进TRAIL的分泌,与空白对照组、wBCG组和wBCG+hIFN-α-2b组相比均有显著优势(p0.05)。重组hIFN-α-2b-BCG组的T24细胞死亡率高于wBCG组、wBCG+hIFN-α-2b组(p均0.05)；流式细胞技术检测到TRAIL和BCG混合组T24细胞的处于凋亡期的细胞数量(T=B2+B4)明显增加,且处于凋亡早期的细胞数量增加更为明显,其差异有统计学意义(P0.05)；rBCG增强膀胱癌细胞表面受体mRNA的转录水平明显高于其他实验组,rBCG上调膀胱癌细胞表面TRAIL受体蛋白的表达比其他实验组影响膀胱癌细胞表面TRAIL受体蛋白的表达更加明显。 结论： 1、rBCG可以明显促进中性粒细胞TRAIL的分泌,对rBCG、wBCG和hIFN-α-2b刺激中性粒细胞分泌TRAIL作用的研究中,我们发现,重组hIFN-α-2b-BCG既有刺激中性粒细胞分泌TRAIL的作用,又有增强中性粒细胞TRAIL转录的作用。但野生BCG只能够刺激中性粒细胞释放TRAIL,而没有明显增强中性粒细胞TRAIL转录的作用；hIFN-α-2b只能够增强中性粒细胞TRAIL转录的作用,而不能明显增强中性粒细胞释放TRAIL的作用。 2. rBCG诱导中性粒细胞后的上清液比wBCG和wBCG联合hIFN-α-2b诱导后的上清液有更强的抗膀胱肿瘤能力,一方面与rBCG活化中性粒细胞产生更多的细胞因子例如本实验研究的TRAIL因子,另一方面与rBCG可以上调膀胱肿瘤细胞表面TRAIL受体的表达,增加膀胱肿瘤细胞对TRAIL的敏感性有关。
[Abstract]:Background:
Bladder cancer is the most common malignant tumor in urology in China. Intravesical BCG is an important adjuvant method in the prevention and treatment of bladder cancer. However, the local and systemic toxic side effects of BCG on bladder affect its application. A new type of vaccine, recombinant human interferon alpha-2b-BCG (hIFN-alpha-2b-BCG), has been successfully constructed in our laboratory. The recombinant hIFN-alpha-2b-BCG can continuously secrete hIFN-alpha-2b, which avoids the shortcoming of short action time of intravesical instillation of hIFN-alpha-2b. It can make BCG and hIFN-alpha-2b work together for a long time and produce the best immunotherapeutic effect. In recent years, it has been found that neutrophils and tumor necrosis factor-related apoptosis-inducing ligands (TRAIL) in urine are closely related to the prognosis of bladder cancer patients after intravesical instillation of wBCG.
Objective:
The purpose of this study was to investigate the role of neutrophils in the antitumor effect of recombinant hIFN-a-2b-BCG on bladder cancer cells.
Method:
Recombinant hIFN-a-2b-BCG.wBCG, wBCG+hIFN-a-2b and PBS were co-incubated with neutrophils respectively. After immune stimulation, the expression of TRAIL in the supernatant of each site was detected by ELISA. PBS blank control group was set up in the experiment. The experimental groups were: recombinant hIFN-a-2b-BCG group, wBCG group, exogenous hIFN-a-2b group, wBCG + exogenous hIFN-a-2b group. MTT assay was used to detect the mortality of bladder cancer cells on day 1, day 2 and day 3. PBS, TRAIL, BCG and BCG combined with TRAIL were used to treat bladder cancer cells. The apoptosis rate of bladder cancer cells was detected by flow cytometry. The TRAIL receptor gene of T24 cells was detected by RT-PCR. The expression of TRAIL receptor on the surface of bladder cancer cells was detected by Western blot.
The first experimental group: simple IFN-alpha group, simple TRAIL group, IFN-alpha combined TRAIL group, rBCG action group (to detect the concentration of IFN-alpha, TRAIL).
The second experiment group: rBCG + neutrophil supernatant group, BCG + neutrophil supernatant group, IFN - alpha combined with BCG + neutrophil supernatant group.
Third test group: rBCG action group, BCG group, IFN- alpha combined BCG group.
Result:
RBCG and wBCG can significantly promote the secretion of TRAIL, all time points compared with PBS group and hIFN-a-2b group had significant advantages (p0.05); hIFN-a-2b group compared with PBS group had no statistical significance (p0.05). rBCG group and wBCG group had no statistical significance before stimulation 6 hours (p0.05), after 12 hours, the recombinant rBCG group and hIFN-a-2b + wBCG group were equivalent, the same. The two groups were higher than those of group wBCG (P0.05). Part of the experiments showed that rBCG could promote the secretion of TRAIL. by neutrophils.
At 24 hours, recombinant hIFN-alpha-2b-BCG could significantly promote the secretion of TRAIL, compared with the blank control group, wBCG group and wBCG+hIFN-alpha-2b group had significant advantages (p0.05). The mortality of T24 cells in the recombinant hIFN-alpha-2b-BCG group was higher than that in the wBCG group, wBCG+hIFN-alpha-2b group (p0.05); the presence of T24 cells in the mixed TRAIL and BCG groups was detected by flow cytometry. The number of apoptotic cells (T = B2 + B4) increased significantly, and the number of cells in the early stage of apoptosis increased more significantly, the difference was statistically significant (P 0.05); rBCG enhanced the transcription level of receptor mRNA on the surface of bladder cancer cells significantly higher than other experimental groups, rBCG up-regulated the expression of TRAIL receptor protein on the surface of bladder cancer cells than other experimental groups. The expression of TRAIL receptor protein on bladder cancer cells was more obvious.
Conclusion:
1. rBCG can significantly promote the secretion of TRAIL in neutrophils. In the study of the effects of rBCG, wBCG and hIFN-a-2b on the secretion of TRAIL in neutrophils, we found that recombinant hIFN-a-2b-BCG can stimulate both the secretion of TRAIL in neutrophils and the transcription of TRAIL in neutrophils. HIFN-alpha-2b could only enhance the TRAIL transcription of neutrophils, but not the TRAIL release of neutrophils.
2. The supernatant of neutrophils induced by rBCG has stronger anti-bladder tumor ability than that induced by wBCG and wBCG combined with hIFN-alpha-2b. On the one hand, the supernatant of neutrophils activated by rBCG produces more cytokines such as TRAIL factor in this experiment, on the other hand, it can up-regulate TRAIL receptor on the surface of bladder tumor cells with rBCG. The expression increased the sensitivity of bladder tumor cells to TRAIL.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14
本文编号：2236587
[Abstract]:Background:
Bladder cancer is the most common malignant tumor in urology in China. Intravesical BCG is an important adjuvant method in the prevention and treatment of bladder cancer. However, the local and systemic toxic side effects of BCG on bladder affect its application. A new type of vaccine, recombinant human interferon alpha-2b-BCG (hIFN-alpha-2b-BCG), has been successfully constructed in our laboratory. The recombinant hIFN-alpha-2b-BCG can continuously secrete hIFN-alpha-2b, which avoids the shortcoming of short action time of intravesical instillation of hIFN-alpha-2b. It can make BCG and hIFN-alpha-2b work together for a long time and produce the best immunotherapeutic effect. In recent years, it has been found that neutrophils and tumor necrosis factor-related apoptosis-inducing ligands (TRAIL) in urine are closely related to the prognosis of bladder cancer patients after intravesical instillation of wBCG.
Objective:
The purpose of this study was to investigate the role of neutrophils in the antitumor effect of recombinant hIFN-a-2b-BCG on bladder cancer cells.
Method:
Recombinant hIFN-a-2b-BCG.wBCG, wBCG+hIFN-a-2b and PBS were co-incubated with neutrophils respectively. After immune stimulation, the expression of TRAIL in the supernatant of each site was detected by ELISA. PBS blank control group was set up in the experiment. The experimental groups were: recombinant hIFN-a-2b-BCG group, wBCG group, exogenous hIFN-a-2b group, wBCG + exogenous hIFN-a-2b group. MTT assay was used to detect the mortality of bladder cancer cells on day 1, day 2 and day 3. PBS, TRAIL, BCG and BCG combined with TRAIL were used to treat bladder cancer cells. The apoptosis rate of bladder cancer cells was detected by flow cytometry. The TRAIL receptor gene of T24 cells was detected by RT-PCR. The expression of TRAIL receptor on the surface of bladder cancer cells was detected by Western blot.
The first experimental group: simple IFN-alpha group, simple TRAIL group, IFN-alpha combined TRAIL group, rBCG action group (to detect the concentration of IFN-alpha, TRAIL).
The second experiment group: rBCG + neutrophil supernatant group, BCG + neutrophil supernatant group, IFN - alpha combined with BCG + neutrophil supernatant group.
Third test group: rBCG action group, BCG group, IFN- alpha combined BCG group.
Result:
RBCG and wBCG can significantly promote the secretion of TRAIL, all time points compared with PBS group and hIFN-a-2b group had significant advantages (p0.05); hIFN-a-2b group compared with PBS group had no statistical significance (p0.05). rBCG group and wBCG group had no statistical significance before stimulation 6 hours (p0.05), after 12 hours, the recombinant rBCG group and hIFN-a-2b + wBCG group were equivalent, the same. The two groups were higher than those of group wBCG (P0.05). Part of the experiments showed that rBCG could promote the secretion of TRAIL. by neutrophils.
At 24 hours, recombinant hIFN-alpha-2b-BCG could significantly promote the secretion of TRAIL, compared with the blank control group, wBCG group and wBCG+hIFN-alpha-2b group had significant advantages (p0.05). The mortality of T24 cells in the recombinant hIFN-alpha-2b-BCG group was higher than that in the wBCG group, wBCG+hIFN-alpha-2b group (p0.05); the presence of T24 cells in the mixed TRAIL and BCG groups was detected by flow cytometry. The number of apoptotic cells (T = B2 + B4) increased significantly, and the number of cells in the early stage of apoptosis increased more significantly, the difference was statistically significant (P 0.05); rBCG enhanced the transcription level of receptor mRNA on the surface of bladder cancer cells significantly higher than other experimental groups, rBCG up-regulated the expression of TRAIL receptor protein on the surface of bladder cancer cells than other experimental groups. The expression of TRAIL receptor protein on bladder cancer cells was more obvious.
Conclusion:
1. rBCG can significantly promote the secretion of TRAIL in neutrophils. In the study of the effects of rBCG, wBCG and hIFN-a-2b on the secretion of TRAIL in neutrophils, we found that recombinant hIFN-a-2b-BCG can stimulate both the secretion of TRAIL in neutrophils and the transcription of TRAIL in neutrophils. HIFN-alpha-2b could only enhance the TRAIL transcription of neutrophils, but not the TRAIL release of neutrophils.
2. The supernatant of neutrophils induced by rBCG has stronger anti-bladder tumor ability than that induced by wBCG and wBCG combined with hIFN-alpha-2b. On the one hand, the supernatant of neutrophils activated by rBCG produces more cytokines such as TRAIL factor in this experiment, on the other hand, it can up-regulate TRAIL receptor on the surface of bladder tumor cells with rBCG. The expression increased the sensitivity of bladder tumor cells to TRAIL.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14
【参考文献】
相关期刊论文 前5条
1 孙二琳;王玉叶;韩瑞发;孙岩;;重组IFN-α-2b-BCG对人PBMC的TLR4表达调节及其抗肿瘤作用的研究[J];实用肿瘤杂志;2010年02期
2 曲晶磊;唐冰;刘云鹏;曲秀娟;侯科佐;;干扰素-α通过上调DR5和抑制Akt的活性增加胃癌细胞对TRAIL的敏感性[J];现代肿瘤医学;2012年04期
3 范晓东;韩瑞发;;重组hIFN-α-2b-BCG对人外周血免疫细胞因子表达调节及抗膀胱肿瘤作用的实验研究[J];中国肿瘤临床;2007年09期
4 张薇,项永兵,刘振伟,方茹蓉,阮志贤,孙璐,高立峰,金凡,高玉堂;1973-1999年上海市区老年人恶性肿瘤发病趋势分析[J];中华老年医学杂志;2005年09期
5 陈万青,张思维,邹小农,戴旭东,李连弟;中国1990～1992年膀胱癌死亡分布特征分析[J];中国肿瘤;2005年08期
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