RhoA在低氧条件下通过JNK通路调控胶质瘤细胞的侵袭

发布时间：2018-09-10 10:27

【摘要】：第一部分胶质瘤细胞在低氧条件下侵袭性的表现目的：探讨正常条件和低氧条件下U251胶质瘤细胞侵袭性的表现。方法：低氧条件下培养U251胶质瘤细胞,Western Blotting检测低氧条件下细胞中HIF-1α表达,明确细胞的低氧环境是否成功建立；在正常条件和低氧条件下培养U251胶质瘤细胞,Transwell实验检测正常条件和低氧条件下U251胶质瘤细胞的侵袭能力；明胶酶谱法检测正常条件和低氧条件下U251胶质瘤细胞分泌到培养液中活性MMP-2的量。采用统计学软件SPSS19.0进行数据统计学分析。结果：在低氧条件下U251胶质瘤细胞中HIF-1α的表达随着培养时间的延长而增加；在低氧条件下U251胶质瘤细胞的侵袭能力明显高于正常条件下U251胶质瘤细胞的侵袭能力(p0.05)；在低氧条件下U251胶质瘤细胞分泌到培养液中的活性MMP-2明显高于正常条件下U251胶质瘤细胞分泌到培养液中MMP-2的活性(p0.01)。结论：低氧条件能够增加U251胶质瘤细胞的侵袭能力,而且这种侵袭能力的增加是通过U251胶质瘤细胞向周围分泌活性MMP-2实现的。第二部分：低氧条件下c-Jun NH2末端酶在U251胶质瘤细胞侵袭中的作用目的：探讨在低氧条件下c-Jun NH2末端酶在U251胶质瘤细胞侵袭中的作用；方法：低氧条件下培养U251胶质瘤细胞,Western Blotting检测低氧条件下细胞中磷酸化c-Jun和磷酸化JNK的表达；Western Blotting检测低氧条件下利用JNK抑制剂处理U251胶质瘤细胞24小时p-c-Jun和p-JNK的表达；Transwell实验检测低氧条件下利用JNK抑制剂处理U251胶质瘤细胞24小时细胞侵袭性的变化；明胶酶谱法检测低氧条件下利用JNK抑制剂处理U251胶质瘤细胞24小时分泌到培养液中活性MMP-2的量。采用统计学软件SPSS19.0进行数据统计学分析。结果：在低氧条件下U251胶质瘤细胞中p-c-Jvn和p-JNK的表达随培养时间的延长而增加；在低氧条件下JNK抑制剂抑制p-c-Jun和p-JNK的表达；在低氧条件下JNK抑制剂抑制U251胶质瘤细胞的侵袭能力；在低氧条件下JNK抑制剂抑制细胞向周围分泌活性MMP-2。结论：低氧条件下JNK-c-Jun途径参与了低氧条件诱导的U251胶质瘤细胞侵袭能力增加的机制。通过抑制JNK-c-Jun途径可以抑制低氧诱导的U251胶质瘤细胞侵袭能力的增加。第三部分：低氧条件下RhoA对JNK-c-Jun途径和U251胶质瘤细胞侵袭性的影响目的：探讨在低氧条件下RhoA对JNK-c-Jun途径和U251胶质瘤细胞侵袭性的调控机制；方法：低氧条件下培养U251胶质瘤细胞,Western Blotting检测低氧条件下细胞中活性RhoA的表达；Western Blotting检测低氧条件下利用RhoA-siRNA和GGTase抑制剂处理U251胶质瘤细胞24小时活性RhoA、p-c-Jun和p-JNK的表达；Transwell实验检测低氧条件下利用RhoA-siRNA和GGTase抑制剂处理U251胶质瘤细胞24小时细胞侵袭性的变化；明胶酶谱法检测低氧条件下利用RhoA-siRNA和GGTase抑制剂处理U251胶质瘤细胞24小时分泌到培养液中活性MMP-2的量。采用统计学软件SPSS19.0进行数据统计学分析。结果：在低氧条件下U251胶质瘤细胞中活性RhoA的表达随培养时间的延长而增加；在低氧条件下RhoA-siRNA和GGTase抑制剂抑制活性RhoA、p-c-Jun和p-JNK的表达；在低氧条件下RhoA-siRNA和GGTase抑制剂抑制U251胶质瘤细胞的侵袭能力；在低氧条件下RhoA-siRNA和GGTase抑制剂抑制细胞向周围分泌活性MMP-2。结论：低氧条件下RhoA通过JNK-c-Jun途径参与了低氧条件诱导的U251胶质瘤细胞侵袭能力增加的机制。通过抑制RhoA可以抑制低氧诱导的U251胶质瘤细胞侵袭能力的增加。目的：越来越多的证据表明糖尿病可能与多种肿瘤的发病相关。然而,糖尿病与脑肿瘤的发病风险目前还不清楚。方法：我们通过PubMed和EMBASASE数据库检索了到2012年5月24日以前的相关文献,并通过相关文献的引文扩大检索范围。所有数据由两名人员独立按照数据提取标准进行提取。运用随机模型计算了总的相对危险(SRR)和95%置信区间(95%CI)。研究间的异质性通过Cochran's Q和12进行统计评估。在本篇荟萃分析中共有13个研究被纳入,包括了整个丹麦的人口和来自其它地区和国家的5107506个参与者,共有2206脑肿瘤病例。结果：通过对这13个研究分析,我们发现与非糖尿病个体相比糖尿病患者可能存在脑肿瘤发病风险(SRR,1.12;95%CI,0.89-1.42)。各研究间存在明显的异质性(P0.001；I2,93.5%)。亚组分析发现女性糖尿病患者脑肿瘤的发病风险增加了24.2%(SRR,1.242;95%CI,1.026-1.502),但未在男性糖尿病患者发现明显的发病风险。本研究中未见明显的发表偏依。结论：本荟萃分析发现糖尿病患者较非糖尿病人存在可能的发病风险。虽然我们发现在女性中发现糖尿病与脑肿瘤的发病风险成正相关,但是在男性中我们并未发现。
[Abstract]:The first part is the aggressive expression of glioma cells under hypoxic condition.
Objective: To investigate the invasiveness of U251 glioma cells under normal and hypoxic conditions.
Methods: U251 glioma cells were cultured under hypoxic conditions. The expression of HIF-1a in U251 glioma cells under hypoxic conditions was detected by Western Blotting to determine whether the hypoxic environment of U251 glioma cells was successfully established. Results: The expression of HIF-1a in U251 glioma cells increased with the prolongation of culture time under hypoxia condition. The invasiveness of glioma cells was significantly higher than that of U251 glioma cells under normal conditions (p0.05), and the activity of MMP-2 secreted by U251 glioma cells in culture medium under hypoxia was significantly higher than that secreted by U251 glioma cells under normal conditions (p0.01).
CONCLUSION: Hypoxia can increase the invasive ability of U251 glioma cells, and this increase is achieved by secreting MMP-2 from U251 glioma cells.
The second part: the role of c-Jun NH2 terminal enzyme in the invasion of U251 glioma cells under hypoxia.
Objective: To investigate the role of c-Jun NH2 terminal enzyme in the invasion of U251 glioma cells under hypoxia.
Methods: U251 glioma cells were cultured under hypoxia, and the expression of phosphorylated c-Jun and phosphorylated JNK were detected by Western Blotting, the expression of p-c-Jun and p-JNK were detected by JNK inhibitor under hypoxia, and the expression of JNK was detected by Transwell assay under hypoxia. U251 glioma cells were treated with inhibitors for 24 hours. The amount of active MMP-2 secreted by U251 glioma cells treated with JNK inhibitors for 24 hours in culture medium was detected by gelatin zymography under hypoxic conditions.
Results: The expression of p-c-Jvn and p-JNK in U251 glioma cells increased with the prolongation of culture time under hypoxic conditions; JNK inhibitors inhibited the expression of p-c-Jun and p-JNK under hypoxic conditions; JNK inhibitors inhibited the invasive ability of U251 glioma cells under hypoxic conditions; JNK inhibitors inhibited the peripheral components of U251 glioma cells under hypoxic conditions; and JNK inhibitors Secretory activity MMP-2.
CONCLUSION: The JNK-c-Jun pathway is involved in the mechanism of hypoxia-induced invasion of U251 glioma cells. Inhibition of JNK-c-Jun pathway can inhibit the increase of hypoxia-induced invasion of U251 glioma cells.
The third part: the effect of RhoA on JNK-c-Jun pathway and invasion of U251 glioma cells under hypoxia.
Objective: To investigate the regulatory mechanism of RhoA on JNK-c-Jun pathway and U251 glioma cell invasion under hypoxia.
Methods: U251 glioma cells were cultured under hypoxic conditions, and the expression of RhoA, p-c-Jun and p-JNK was detected by Western Blotting, and the expression of RhoA-siRNA and GGTase inhibitor in U251 glioma cells was detected by Western Blotting. The invasiveness of U251 glioma cells was treated with RhoA-siRNA and GGTase inhibitors for 24 hours. The amount of active MMP-2 secreted by U251 glioma cells in 24 hours was detected by gelatin zymography under hypoxia. The data were analyzed by statistical software SPSS19.0.
Results: The expression of active RhoA in U251 glioma cells increased with the prolongation of culture time under hypoxic conditions; RhoA-siRNA and GGTase inhibitors inhibited the expression of active RhoA, p-c-Jun and p-JNK under hypoxic conditions; RhoA-siRNA and GGTase inhibitors inhibited the invasion of U251 glioma cells under hypoxic conditions; Inhibitors of RhoA-siRNA and GGTase inhibit the secretion of active MMP-2. from surrounding cells.
CONCLUSION: RhoA participates in the mechanism of hypoxia-induced U251 glioma cell invasion through JNK-c-Jun pathway under hypoxia, and inhibits hypoxia-induced U251 glioma cell invasion by inhibiting RhoA.
Objective:
Increasing evidence suggests that diabetes may be associated with a variety of tumors. However, the risk of diabetes and brain tumors remains unclear.
Method:
We retrieved the relevant literature up to May 24, 2012 from PubMed and EMBASASE databases and expanded the search scope by citing relevant literature. All data were extracted independently by two people according to the data extraction criteria. The total relative risk (SRR) and 95% confidence interval (95% CI) were calculated using a stochastic model. Qualitative assessments were conducted using Cochran's Q and 12. In this meta-analysis, 13 studies were included, including the Danish population as a whole and 510,7506 participants from other regions and countries, with a total of 2206 brain tumors.
Result:
Based on these 13 studies, we found that diabetic patients may have a risk of brain tumors compared with non-diabetic individuals (SRR, 1.12; 95% CI, 0.89-1.42). There was significant heterogeneity between studies (P 0.001; I 2, 93.5%). Subgroup analysis found that the risk of brain tumors in female diabetic patients increased by 24.2% (SRR, 1.242; 95% CI, 1.026). - 1.502), but no significant risk was found in male diabetic patients.
Conclusion:
This meta-analysis found that diabetes was associated with a higher risk of developing brain tumors than non-diabetes. Although we found a positive correlation between diabetes and the risk of brain tumors in women, we did not find it in men.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

【共引文献】