肿瘤坏死因子-α介导缓激肽开放血脑屏障的作用

发布时间：2018-02-25 22:04

本文关键词： 肿瘤坏死因子-α 缓激肽血脑屏障热休克因子-1 热休克蛋白70 紧密连接相关蛋白神经胶质瘤　出处：《中国医科大学》2007年博士论文　论文类型：学位论文

【摘要】： 目的一直以来，人们认为缓激肽(bradykinin，BK)开放血脑屏障(blood-brain barrier，BBB)的作用是由于血管内皮细胞上存在B2受体，缓激肽与其结合后引起血脑屏障结构和功能改变，导致血脑屏障开放。最近有研究发现，在正常脑组织和肿瘤的血管内皮细胞上未见缓激肽B2受体的表达，而在肿瘤细胞上发现了高水平表达的缓激肽B2受体，且B2受体表达程度与血脑屏障开放程度密切相关。由此可以推测缓激肽不是直接作用于血管内皮细胞引起血脑屏障变化，，而可能是缓激肽首先与肿瘤细胞上的B2受体结合，诱发肿瘤细胞释放某种细胞因子或其他介质，进而作用于血脑屏障引起其结构和功能改变，导致血脑屏障开放。但其确切机制尚待探讨。在研究缓激肽开放血肿瘤屏障(blood-tumor barrier，BTB)的过程中，Inamura等经颈内动脉向脑胶质瘤大鼠缓慢小剂量灌注缓激肽15min后，血肿瘤屏障开放达最大，此后虽继续灌注，但效应呈减弱趋势，即缓激肽开放血肿瘤屏障过程中诱发了快速耐受性。有关缓激肽诱发肿瘤屏障开放产生快速耐受性的机制，目前尚不清楚。本研究的目的就是为了明确，缓激肽与肿瘤细胞上的B2受体结合后到底引起何种细胞因子和／或递质释放，进而如何影响血脑屏障的开放的，以及缓激肽开放血肿瘤屏障过程中诱发快速耐受性的可能机制。方法 1．复制大鼠C6胶质瘤模型按照Nicolau等报道的方法，收集培养的C6胶质瘤细胞1×10~5／5μL通过立体定位仪接种于动物大脑右侧尾状核，约15天形成荷瘤鼠。 2．采用伊文思兰(evans blue，EB)和透射电镜技术，检测应用缓激肽前后荷瘤鼠血脑屏障渗透性及紧密连接改变。 3．采用伊文思兰和透射电镜技术，检测应用肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)前后荷瘤鼠血脑屏障渗透性及紧密连接改变。 4．采用放射免疫法检测应用缓激肽前后C6胶质瘤细胞培养液中TNF-α的含量。 5．采用Western blotting法检测应用缓激肽前后C6胶质瘤细胞内热休克因子1(heat shock factor 1，HSF1)和热休克蛋白70(heat shock protein 70，HSP70)的蛋白质表达水平。 6．采用RT-PCR技术检测应用缓激肽前后C6胶质瘤细胞内TNF-αmRNA的转录水平。 7．免疫组织化学法检测C6动物模型的脑肿瘤微血管于TNF-α作用前后的紧密连接相关蛋白occludin表达水平。结果 1．成功复制了大鼠C6胶质瘤模型。 2．应用缓激肽后，C6胶质瘤大鼠血肿瘤屏障渗透性及紧密连接开放增加，于15min时出现高峰，60min时接近对照组水平。 3．应用TNF-α后，C6胶质瘤大鼠血肿瘤屏障渗透性及紧密连接开放增加，于15min时出现高峰，60min时接近对照组水平。 4．培养的C6胶质瘤细胞经缓激肽处理后，培养液中的TNF-α浓度增加，于15min时达高峰，60min时仍高于对照组。 5．培养的C6胶质瘤细胞经缓激肽处理后，HSF1和HSP70蛋白质的表达增加，且分别于缓激肽处理后5min和30min时表达最多，60min时仍高于对照组。 6．C6胶质瘤细胞经缓激肽处理后，细胞内TNF-αmRNA水平增加，于缓激肽处理后10min时出现高峰，其后逐渐减少，60min时达最低。 7．TNF-α作用于C6胶质瘤大鼠后，胶质瘤微血管上的紧密连接相关蛋白occludin表达减少。结论 1．单独应用缓激肽及TNF-α均可显著增加C6胶质瘤大鼠血肿瘤屏障通透性及紧密连接开放程度，二者均于给药15min后出现高峰，60min时接近对照组水平。 2．缓激肽促进了胶质瘤细胞内HSF1蛋白质和TNF-αmRNA的表达，并促进了TNF-α的释放。三者的时程变化不同，HSF1的表达高峰于给予缓激肽5min后出现，TNF-αmRNA的表达高峰于给药10min后出现，TNF-α释放高峰于给药15min后出现。提示增加的HSF1的表达可能促进了TNF-αmRNA的表达和TNF-α的释放，这可能是缓激肽选择性开放血肿瘤屏障的机制之一。 3．缓激肽引起胶质瘤组织内TNF-α含量达高峰与紧密连接相关蛋白occludin表达达低峰的时间点一致，相同时间点的血肿瘤屏障开放达最大。提示缓激肽开放血肿瘤屏障可能是通过TNF-α影响紧密连接相关蛋白occludin的表达而实现的。
[Abstract]:objective
All along, people think that bradykinin (bradykinin, BK) to open the blood brain barrier (blood-brain barrier, BBB) the effect is due to the presence of B2 receptors on vascular endothelial cells, combines with bradykinin induced changes of blood brain barrier function, leading to the opening of blood brain barrier. Recent studies have found that no bradykinin B2 receptor expression in the the normal brain tissue and tumor vascular endothelial cells, and the high level expression of bradykinin B2 receptor was found in tumor cells, and the expression of B2 is closely related with the blood brain barrier opening degree. It can be speculated that bradykinin is not a direct effect on vascular endothelial cells caused by changes of blood brain barrier, and may be the first with bradykinin B2 receptor on the tumor cells, tumor cells induced release of some cytokines or other media, and the effect on blood brain barrier caused by its structure and function change, blood lead The brain barrier is open, but its exact mechanism remains to be discussed.
In the tumor of bradykinin (blood-tumor barrier, open the blood-brain barrier BTB) in the process of Inamura via the carotid artery to the brain glioma in rats after 15min infusion of small doses of slow, blood tumor barrier opened to the maximum, then although continued perfusion, but the effect was decreased and that of bradykinin in opening blood tumor barrier in the process of inducing tachyphylaxis. Mechanism of bradykinin induced tumor barrier tachyphylaxis, is unclear.
The purpose of this study is to clear the combination of bradykinin and B2 receptor on the tumor cells after cause any cytokine and / or neurotransmitter release, and then how to affect the BBB, the possible mechanism of tachyphylaxis and bradykinin opening blood tumor barrier in the process.
Method
1., the rat C6 glioma model was duplicated. According to the method reported by Nicolau, the C6 glioma cells (1 x 10~5 / 5 L) were collected and inoculated on the right caudate nucleus of the animal brain by stereotactic apparatus. The tumor bearing mice were formed for 15 days.
2. Evans Lan (Evans blue, EB) and transmission electron microscopy were used to detect the permeability and close connection of blood brain barrier in the mice with bradykinin before and after the application of bradykinin.
3. by Evans blue and transmission electron microscopy, detection of tumor necrosis factor alpha (tumor alpha factor- alpha necrosis, TNF-) and the changes of tight junction tumor after the blood brain barrier permeability.
4. the content of TNF- alpha in the culture fluid of C6 glioma cells was detected by radioimmunoassay before and after the application of bradykinin.
5. the expression level of heat shock factor 1 (heat shock factor 1, HSF1) and heat shock protein 70 (heat shock protein 70, HSP70) in C6 glioma cells before and after bradykinin treatment were detected by Western blotting.
6. the transcriptional level of TNF- alpha mRNA in C6 glioma cells was detected by RT-PCR technique before and after the application of bradykinin.
7. immunohistochemical method was used to detect the expression of close linked protein occludin in the microvascular of brain tumor of C6 animal model before and after the action of TNF- alpha.
Result
1. the rat model of C6 glioma was successfully replicated.
2. after the application of bradykinin, the permeability and close connection of blood tumor barrier in C6 glioma rats increased, the peak was at 15min, and the level of the control group was close to the control group at 60min.
3. after the use of TNF- alpha, the permeability and close connection of blood tumor barrier in C6 glioma rats increased, the peak was at 15min, and the level of 60min was close to the control group.
4. the cultured C6 glioma cells were treated with bradykinin, the concentration of TNF- alpha in the culture medium increased and reached the peak at 15min, and was still higher than that of the control group at 60min.
5., after treated with bradykinin, the expression of HSF1 and HSP70 protein increased in C6 cultured glioma cells. The expression of HSF1 and HSP70 protein was the most in bradykinin treated 30min and 60min, respectively.
After treatment with bradykinin, the level of TNF- alpha mRNA in 6.C6 glioma cells increased, and the peak appeared at 10min after bradykinin treatment, then decreased gradually and reached the lowest level at 60min.
After the action of 7.TNF- alpha on C6 glioma rats, the expression of tight junction related protein occludin on the microvasculature of glioma was reduced.
conclusion
1. alone application of bradykinin and TNF- alpha significantly increased the permeability of blood tumor barrier and the degree of tight junction opening in the C6 glioma rats. Both of them showed a peak after the administration of 15min, and 60min approached the level of the control group at the time of 60min.
2. bradykinin promoted the expression of HSF1 protein in glioma cells and TNF- alpha mRNA, and promoted the release of TNF- alpha. Variation of the difference between the three, the peak expression of HSF1 in 5min after administration of bradykinin, the peak expression of TNF- alpha mRNA in 10min after administration, TNF- was released to high peak after treatment of 15min. Expression increase of HSF1 may promote the expression of mRNA and TNF- alpha TNF- alpha release, which may be one of the mechanisms of bradykinin selective opening of blood tumor barrier.
3. bradykinin TNF- alpha in glioma tissues reached the peak and tight junction protein occludin expression reached the peak of the low consistent point in time, the opening of the blood tumor barrier at the same time as that of bradykinin. The opening of blood tumor barrier may be through the TNF- alpha on the expression of TJ associated protein occludin and achieve.

【学位授予单位】：中国医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R33

【相似文献】