發(fā)布時(shí)間：2018-09-11 11:37

【摘要】：背景： 膀胱癌是我國(guó)泌尿外科最常見(jiàn)的惡性腫瘤,膀胱腔內(nèi)灌注卡介苗(BCG)是防治膀胱癌的重要輔助手段,但其引起的膀胱局部與全身毒副作用影響了它的應(yīng)用,本實(shí)驗(yàn)室成功構(gòu)建了新型菌苗一重組人干擾素(hIFN)-α-2b-BCG (hIFN-α-2b-BCG),它結(jié)合了野生BCG (wBCG)和hIFN-α-2b的優(yōu)勢(shì),又可以避免二者的副作用。重組hIFN-α-2b-BCG可以持續(xù)分泌hIFN-α-2b,避免了單純膀胱灌注hIFN-α-2b作用時(shí)間短的缺點(diǎn),可以使BCG和hIFN-α-2b長(zhǎng)時(shí)間共同作用,從而產(chǎn)生最佳的免疫治療效應(yīng)。對(duì)于BCG的抗癌作用機(jī)制,以前多從淋巴-巨噬細(xì)胞免疫角度研究。但近年來(lái)發(fā)現(xiàn)wBCG膀胱灌注后,尿中出現(xiàn)的中性粒細(xì)胞和腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(TRAIL)與膀胱癌病人的預(yù)后密切相關(guān)。 目的： 本研究主要探討中性粒細(xì)胞在重組hIFN-α-2b-BCG抗膀胱癌中的作用。我們進(jìn)行了以下研究：主要比較重組hIFN-α-2b-BCG和wBCG刺激中性粒細(xì)胞后的上清液對(duì)膀胱癌細(xì)胞表面TRAIL受體的影響并探討其機(jī)制。 方法： 應(yīng)用重組hIFN-α-2b-BCG. wBCG、wBCG+hIFN-α-2b和PBS分別與中性粒細(xì)胞共孵育,進(jìn)行免疫刺激后,運(yùn)用ELISA方法測(cè)各點(diǎn)上清液中TRAIL的表達(dá)情況。實(shí)驗(yàn)設(shè)PBS空白對(duì)照組,實(shí)驗(yàn)組分為：重組hIFN-α-2b-BCG組,wBCG組,外源hIFN-α-2b組,wBCG+外源hIFN-α-2b組；選取一時(shí)間點(diǎn)的上清液作用于膀胱癌細(xì)胞T24細(xì)胞,用MTT方法檢測(cè)膀胱癌細(xì)胞在第1天、第2天、第3天的死亡率；PBS、TRAIL、BCG和BCG聯(lián)合TRAIL作用于膀胱癌細(xì)胞,流式細(xì)胞技術(shù)檢測(cè)膀胱癌細(xì)胞凋亡率；利用RT-PCR的方法檢測(cè)T24細(xì)胞TRAIL受體的基因轉(zhuǎn)錄水平,western blot技術(shù)檢測(cè)膀胱癌細(xì)胞表面TRAIL受體表達(dá)情況。實(shí)驗(yàn)設(shè)PBS空白對(duì)照組,實(shí)驗(yàn)組分為3大組： 第一實(shí)驗(yàn)組：?jiǎn)渭僆FN-α組,單純TRAIL組,IFN-α聯(lián)合TRAIL組,rBCG作用組(需檢測(cè)IFN-α、TRAIL濃度)。 第二實(shí)驗(yàn)組：rBCG+中性粒細(xì)胞上清液作用組,BCG+中性粒細(xì)胞上清液作用組,IFN-α聯(lián)合BCG+中性粒細(xì)胞上清液作用組。 第三試驗(yàn)組：rBCG作用組,BCG組,IFN-α聯(lián)合BCG組。 結(jié)果： rBCG和wBCG可以明顯促進(jìn)TRAIL的分泌,所有時(shí)間點(diǎn)與PBS組和hIFN-α-2b組相比均有顯著優(yōu)勢(shì)(p0.05)；hIFN-α-2b組與PBS組相比無(wú)統(tǒng)計(jì)學(xué)意義(p0.05)。rBCG組和wBCG組相比,在刺激6小時(shí)以前無(wú)統(tǒng)計(jì)學(xué)意義(p0.05),12小時(shí)以后重組rBCG組和hIFN-α-2b+wBCG組相當(dāng),且兩組均高于wBCG組(p0.05)。本部分實(shí)驗(yàn)表明rBCG更能促進(jìn)中性粒細(xì)胞分泌TRAIL。 在24小時(shí),重組hIFN-α-2b-BCG可以明顯促進(jìn)TRAIL的分泌,與空白對(duì)照組、wBCG組和wBCG+hIFN-α-2b組相比均有顯著優(yōu)勢(shì)(p0.05)。重組hIFN-α-2b-BCG組的T24細(xì)胞死亡率高于wBCG組、wBCG+hIFN-α-2b組(p均0.05)；流式細(xì)胞技術(shù)檢測(cè)到TRAIL和BCG混合組T24細(xì)胞的處于凋亡期的細(xì)胞數(shù)量(T=B2+B4)明顯增加,且處于凋亡早期的細(xì)胞數(shù)量增加更為明顯,其差異有統(tǒng)計(jì)學(xué)意義(P0.05)；rBCG增強(qiáng)膀胱癌細(xì)胞表面受體mRNA的轉(zhuǎn)錄水平明顯高于其他實(shí)驗(yàn)組,rBCG上調(diào)膀胱癌細(xì)胞表面TRAIL受體蛋白的表達(dá)比其他實(shí)驗(yàn)組影響膀胱癌細(xì)胞表面TRAIL受體蛋白的表達(dá)更加明顯。 結(jié)論： 1、rBCG可以明顯促進(jìn)中性粒細(xì)胞TRAIL的分泌,對(duì)rBCG、wBCG和hIFN-α-2b刺激中性粒細(xì)胞分泌TRAIL作用的研究中,我們發(fā)現(xiàn),重組hIFN-α-2b-BCG既有刺激中性粒細(xì)胞分泌TRAIL的作用,又有增強(qiáng)中性粒細(xì)胞TRAIL轉(zhuǎn)錄的作用。但野生BCG只能夠刺激中性粒細(xì)胞釋放TRAIL,而沒(méi)有明顯增強(qiáng)中性粒細(xì)胞TRAIL轉(zhuǎn)錄的作用；hIFN-α-2b只能夠增強(qiáng)中性粒細(xì)胞TRAIL轉(zhuǎn)錄的作用,而不能明顯增強(qiáng)中性粒細(xì)胞釋放TRAIL的作用。 2. rBCG誘導(dǎo)中性粒細(xì)胞后的上清液比wBCG和wBCG聯(lián)合hIFN-α-2b誘導(dǎo)后的上清液有更強(qiáng)的抗膀胱腫瘤能力,一方面與rBCG活化中性粒細(xì)胞產(chǎn)生更多的細(xì)胞因子例如本實(shí)驗(yàn)研究的TRAIL因子,另一方面與rBCG可以上調(diào)膀胱腫瘤細(xì)胞表面TRAIL受體的表達(dá),增加膀胱腫瘤細(xì)胞對(duì)TRAIL的敏感性有關(guān)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.14

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