曲古菌素A调控Survivin干预神经胶质瘤凋亡的研究

发布时间：2018-04-22 21:12

本文选题：神经胶质瘤 + 曲古菌素A　；参考：《吉林大学》2014年硕士论文

【摘要】：神经胶质瘤作为目前临床上的高发性脑部肿瘤，由于死亡率高，复发率高的特点，严重威胁着人类的健康。但是由于其发病部位的特殊性，常规的手术治疗被极大的限制，在治疗时得不到很好的效果。而目前常规的抗肿瘤药在治疗神经胶质瘤时，虽然可以对肿瘤细胞有一定的杀伤效果，但也对正常的神经组织和细胞产生较大的损伤，并且在预后过程中，神经胶质瘤细胞能够产生极强的耐药性，从而限制了常规的抗肿瘤药在神经胶质瘤治疗中的使用。因此，寻找治疗神经胶质瘤的新靶点以及开发治疗神经胶质瘤的新药物具有极大的临床参考价值和实用意义。 Survivin作为目前已发现的最强抑制凋亡蛋白之一，可以通过直接抑制凋亡终端效应蛋白Caspase-3，达到抑制凋亡，促进增殖的作用，对肿瘤的发生发展有十分重要的意义。已有研究报道，Survivin在神经胶质瘤中有较高表达，与此同时在正常的神经组织和细胞中Survivin的表达较低。这种在肿瘤和正常组织中表达的特异性使其极有可能成为神经胶质瘤治疗领域的新靶点。但目前，对于神经胶质瘤中Survivin的调控机制尚不明确。曲古菌素A（TSA）作为组蛋白去乙酰化酶抑制剂中的代表，可以通过抑制肿瘤细胞中过高表达的去乙酰化酶的活性，调节肿瘤中去乙酰化水平，调节相关基因的转录达到有效杀伤肿瘤细胞的作用。在抗肿瘤的同时，TSA还具有广谱、高效，低毒的特点。已有研究表明，TSA在有效杀伤肿瘤细胞的同时，对神经组织和细胞有一定的保护作用。TSA这种对肿瘤和正常组织凋亡调控的特异性可能与Survivin在肿瘤和正常组织中表达的特异性存在一定的相关性。实验目的：本实验拟研究TSA对神经胶质瘤细胞凋亡作用的影响，并进一步的探讨TSA通过调控Survivin介导神经胶质瘤凋亡的相关机制。实验方法：分别对PC12细胞系及C6细胞系进行培养，通过CCK-8实验检测TSA在不同剂量浓度下对PC12细胞及C6细胞存活率的影响，确定TSA是否对神经胶质瘤细胞杀伤的同时对正常神经细胞无损伤并确定TSA的最佳工作浓度；采用细胞形态学实验和流式细胞术，观察TSA给药前后，PC12细胞及C6细胞形态的变化以及凋亡率的变化，进一步确定TSA对PC12细胞及C6细胞凋亡的影响；HDACs活性试剂盒检测TSA给药前后，第Ⅰ类HDAC活性的变化；免疫荧光染色观察，在TSA给药前以及TSA给药后使第Ⅰ类HDAC活性发生变化时，PC12细胞及C6细胞中Survivin及被其直接抑制的Caspase-3表达的变化，研究TSA对神经胶质瘤细胞及正常神经细胞中Survivin表达的影响以及神经胶质瘤细胞细胞中第Ⅰ类HDAC活性与Survivin表达的关系；采用Western blot法观察不同剂量TSA作用下，神经胶质瘤细胞及正常神经细胞中Survivin和Caspase-3的表达变化，通过上述指标来评价TSA对Survivin的调控作用以及TSA通过调控Survivin对神经胶质瘤细胞凋亡的介导作用。并选用丙戊酸钠为工具药，使用HDACs活性试剂盒和western blot法进一步验证实验的结论。实验结果： 1. CCK-8实验结果显示：与Control组相比较，C6细胞TSA100nM组存活率为：81.33%±4.15；200nM组的存活率为：65.33%±2.15；400nM组存活率为：37.14%±2.66。与Control组相比较， PC12细胞TSA100nM组、200nM组存活率无明显差异，在400nM时，存活率为84.67%±4.33。 2.细胞形态学实验结果显示：与Control组比较，发现PC12的密度无明显变化，细胞形态也无明显改变；而C6细胞200nM给药组与Control组比较，细胞密度明显减少，细胞间距变大，细胞轮廓不清。 3.流式细胞术实验结果表明： TSA在200nM浓度下，PC12细胞的凋亡率（5.42%±0.23）与Control组的凋亡率（3.37%±0.36）相比较，无明显差异。而在200nM浓度下C6细胞的凋亡有明显增加，其结果与毒性试验的结果相一致，200nM组凋亡率为：30.11%±1.97，Control组的凋亡率为3.12%±1.31，存在明显差异（P＜0.01）。 4.第Ⅰ类HDAC活性检测实验结果表明：C6细胞的Control组中HDAC1、HDAC2、HDAC3、HDAC8活性，与PC12细胞的Control组比较，均有显著性差异（P＜0.01），活性明显高于PC12细胞的Control组；而在C6细胞200nMTSA给药处理24h后，与C6细胞Control组相比较，HDAC1、HDAC2、HDAC3、HDAC8的活性均有明显下降（P＜0.01），且下降的比例巨大。而PC12细胞200nM给药组与PC12细胞Control组比较，HDAC1、HDAC2、HDAC3、HDAC8的活性无明显变化。 5.免疫荧光染色结果显示：C6细胞Control组Survivin的荧光强度明显高于C6细胞给药组。而PC12细胞Control组Survivin的荧光强度与PC12细胞给药组无明显差别。同时，C6细胞Control组Survivin的荧光强度也明显高于PC12细胞Control组Survivin的荧光强度；而在TSA给药后，C6细胞中的Caspase-3活性的荧光强度明显高于C6细胞Control组。而PC12细胞Control组Caspase-3的荧光强度与PC12细胞给药组无明显差别。 6. Western blot实验结果显示：TSA给药剂量为100nM、200nM、400nM。Survivin在C6细胞中的表达随着TSA剂量的增加，呈现逐步下降的趋势，而PC12细胞中的Survivin无明显变化；C6细胞中Caspase-3的表达随着TSA剂量的增加而增加，在100nM和200nM给药时，PC12细胞中的Caspase-3与PC12细胞Control组比较无明显差异，在400nM剂量时有明显上升（P＜0.01）。 7.在验证性实验中，选用丙戊酸钠为工具药，采用HDAC活性检测实验和Western blot实验，实验结果显示与TSA的相关实验结果基本一致。实验结论： 1.在一定剂量范围内，TSA可以有效杀伤神经胶质瘤细胞而对正常神经细胞无损伤； 2.神经胶质瘤中第Ⅰ类HDACs的活性明显高于正常神经细胞，TSA可以有效降低神经胶质瘤细胞第Ⅰ类HDACs的活性； 3.神经胶质瘤中Survivin的表达明显高于正常神经细胞，抑制Survivin蛋白水平的表达是TSA杀伤神经胶质瘤细胞的机制之一； 4. TSA可能通过降低神经胶质瘤中第Ⅰ类HDACs的活性，，改变神经胶质瘤中组蛋白去乙酰化的水平，调控Survivin的表达，进而促进Caspase-3的活性增加，达到促进神经胶质瘤细胞凋亡的作用。
[Abstract]:As a clinical high incidence brain tumor, neuroglioma is a serious threat to human health because of its high mortality and high recurrence rate. However, conventional surgical treatment has been greatly restricted because of its specific location. While the tumor cells can have a certain killing effect on the tumor cells, it also produces greater damage to normal nerve tissue and cells, and in the process of prognosis, the neuroglioma cells can produce very strong resistance, which restricts the use of conventional antitumor drugs in the treatment of glioma. The new targets of glioma and the development of new drugs for glioma are of great clinical value and practical significance.
As one of the strongest inhibitory apoptotic proteins that have been found at present, Survivin can inhibit apoptosis and promote proliferation by inhibiting apoptotic terminal effect protein Caspase-3 directly, and it is of great significance for the development of tumor. It has been reported that Survivin has high expression in glioma and is at the same time in normal. The expression of Survivin in neural tissue and cells is low. The specificity expressed in tumors and normal tissues makes it possible to be a new target in the field of neuroglioma treatment. However, the regulation mechanism of Survivin in glioma is not yet clear.
As a representative of histone deacetylase inhibitors, curacicotin A (TSA) can regulate the level of deacetylation in tumor by inhibiting the activity of exorbitant deacetylase in tumor cells, regulating the transcription of related genes to effectively kill tumor cells. At the same time, TSA also has broad spectrum, high efficiency and low toxicity. Some studies have shown that TSA has a certain protective effect on the tissue and cells of the tumor cells while it is effective in killing tumor cells. The specificity of.TSA on the regulation of tumor and normal tissue apoptosis may be related to the specificity of Survivin expression in the tumor and normal tissues.
Objective:
This study aims to investigate the effect of TSA on the apoptosis of glioma cells, and to further explore the mechanism of TSA mediated apoptosis of glioma by regulating Survivin.
Experimental methods:
The PC12 cell line and the C6 cell line were cultured, and the effects of TSA on the survival rate of PC12 cells and C6 cells at different doses were tested by CCK-8. It was determined whether TSA had no damage to glioma cells and no damage to normal neurons and determined the optimum working concentration of TSA, and the cell morphology experiment and flow formula were used. The changes in the morphology and apoptosis rate of PC12 cells and C6 cells before and after TSA were observed, and the effect of TSA on the apoptosis of PC12 cells and C6 cells was further determined. The HDACs activity kit was used to detect the changes of class I HDAC activity before and after TSA administration, immunofluorescence staining, and the first class HDA before and after TSA administration and TSA. The effects of TSA on the expression of Survivin in glioma cells and normal neurons, as well as the relationship between the expression of Survivin in glioma cells and normal neurons, and the relationship between the activity of class I HDAC and the expression of Survivin in glioma cells, as well as the changes in the expression of Survivin in PC12 and C6 cells and the expression of Caspase-3 in C6 cells, and the relationship between the activity of class I HDAC and the expression of Survivin in glioma cells, were observed by Western blot. The expression of Survivin and Caspase-3 in glioma cells and normal nerve cells under the same dose of TSA was used to evaluate the regulatory role of TSA on Survivin and the mediating effect of TSA on the apoptosis of glioma cells by regulating Survivin. The use of sodium valproate as a tool, the use of HDACs active kits and we The stern blot method further verified the conclusion of the experiment.
Experimental results:
1. CCK-8 experimental results showed that compared with group Control, the survival rate of C6 cell TSA100nM group was 81.33% + 4.15, and the survival rate of 200nM group was 65.33% + 2.15, and the survival rate of group 400nM was compared with that of Control group, PC12 cell TSA100nM group, 200nM group survival rate had no obvious difference, and the survival rate was 84.67% + at 400nM.
The experimental results of 2. cell morphology showed that compared with the Control group, the density of PC12 was not obviously changed, and the cell morphology did not change obviously, but the cell density of the 200nM administration group of C6 cells decreased obviously, the cell spacing became larger, and the cell profile was not clear.
The results of 3. flow cytometry showed that the apoptosis rate (5.42% + 0.23) of PC12 cells (5.42% + 0.23) was not significantly different from that of Control group (3.37% + 0.36) at the concentration of TSA, but the apoptosis of C6 cells increased significantly at 200nM concentration, and the result was consistent with the results of toxicity test. The apoptosis rate of 200nM group was 30.11% + 1.97, Control. The apoptosis rate of the group was 3.12% + 1.31, and there was a significant difference (P < 0.01).
4. the test results of class I HDAC activity test showed that the activity of HDAC1, HDAC2, HDAC3 and HDAC8 in the Control group of C6 cells was significantly different from the Control group of PC12 cells (P < 0.01), and the activity was significantly higher than that of PC12 cells. 3, the activity of HDAC8 decreased significantly (P < 0.01), and the proportion of the decrease was huge, but the activity of HDAC1, HDAC2, HDAC3 and HDAC8 was not significantly changed in the 200nM administration group of PC12 cells and the Control group of PC12 cells.
5. the results of immunofluorescence staining showed that the fluorescence intensity of Survivin in the Control group of C6 cells was significantly higher than that in the C6 cell administration group, but the fluorescence intensity of Survivin in the Control group of PC12 cells was not significantly different from that of the PC12 cell administration group. Meanwhile, the fluorescence intensity of Survivin in the C6 cell Control group was significantly higher than that of the PC12 cells. After TSA administration, the fluorescence intensity of Caspase-3 activity in C6 cells was significantly higher than that in the Control group of C6 cells, but the fluorescence intensity of Caspase-3 in the Control group of PC12 cells was not significantly different from that in the PC12 cell group.
The results of 6. Western blot showed that the dosage of TSA was 100nM, the expression of 200nM and 400nM.Survivin in C6 cells was gradually decreasing with the increase of TSA dose, while Survivin in PC12 cells did not change obviously, and the expression of Caspase-3 in C6 cells increased with the increase of dosage. There was no significant difference in Caspase-3 between cells and PC12 cells in Control group, but increased significantly at 400nM dose (P < 0.01).
7. in the confirmatory experiment, the sodium valproate was used as the tool, the HDAC activity test and the Western blot experiment were used. The experimental results showed that the related experimental results of TSA were basically the same.
Experimental conclusions:
1. in a certain dose range, TSA can effectively kill glioma cells without damage to normal neurons.
2. the activity of type HDACs of glioma is significantly higher than that of normal nerve cells. TSA can effectively reduce the activity of type HDACs of glioma cells.
3. the expression of Survivin in neuroglioma is obviously higher than that of normal nerve cells, and the inhibition of the expression of Survivin protein is one of the mechanisms of TSA killing neuroglioma cells.
4. TSA may reduce the activity of class I HDACs in glioma, change the level of histone deacetylation in glioma, regulate the expression of Survivin, and then promote the increase of Caspase-3 activity, so as to promote the apoptosis of glioma cells.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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