MCTs在人星形细胞瘤中的表达变化及MCT抑制剂对U251细胞增殖的影响

发布时间：2018-05-14 06:08

本文选题：羧酸转运蛋白 + 星形细胞瘤　；参考：《重庆医科大学》2014年硕士论文

【摘要】：第一部分MCTs在人星形细胞瘤中的表达变化及其与病理级别之间的关系目的观察单羧酸转运蛋白（MCT）1、2、4在人不同病理级别星形细胞瘤中的表达差异，探讨星形细胞瘤增殖、生长的分子机制。材料和方法 1.标本的采集：人星形细胞瘤组织标本均取自重庆医科大学附属第一医院神经外科和第三军医大学附属大坪医院神经外科星形细胞瘤患者，共计51例。经过查询患者病史及病理检验证实，所有患者术前均未接受过免疫治疗、放疗和化疗等。 2.病理级别鉴定：应用HE染色对组织标本进行观察，确定病理级别。 3.MCTs在人不同病理级别星形细胞瘤中的表达检测：（1）应用免疫组织化学技术，观察人不同病理级别星形细胞瘤组织和对照组组织中MCTs的表达变化；（2）应用Western Blot技术，检测人不同病理级别星形细胞瘤组织和对照组组织中MCTs蛋白的表达变化。 4.统计学分析：运用图像分析软件和SPSS17.0统计软件对实验结果进行统计分析。结果免疫组织化学结果显示：对照组和星形细胞瘤组织中均有MCT1的表达。对照组织中MCT1主要表达在星形细胞和血管内皮细胞中。在不同病理级别的星形细胞瘤中，MCT1主要分布在瘤细胞的胞核和胞质中。Western Blot结果表明：各级别肿瘤组织中均可见MCT1的高表达，，与对照组相比，MCT1在低级别（WHOⅠ级和Ⅱ级）肿瘤组织中表达增强，在高级别（Ⅲ-Ⅳ级）肿瘤组织中表达进一步增强，差异具有统计学意义（P0.05）。免疫组织化学结果显示：MCT2主要表达在对照组的神经元和少量胶质细胞中。在肿瘤组织中主要分布在瘤细胞的胞质和胞膜上。Western Blot结果显示：MCT2的表达随着病理级别的升高而增强（P0.05）。免疫组织化学显示：MCT4主要分布在正常脑组织的星形胶质细胞上，低级别星形细胞瘤的胞浆，间变形星形细胞瘤、胶质母细胞瘤的瘤细胞的胞浆和胞膜上。Western Blot结果表明：与正常脑组织相比，MCT4在低级别（Ⅰ级和Ⅱ级）星形细胞瘤组织中表达增多（P0.05），在高级别（Ⅲ-Ⅳ级）肿瘤组织中表达进一步增多（P0.05），而分别在低级别肿瘤组织之间和高级别肿瘤组织之间相比较，其表达含量无统计学差异（P＞0.05）。结论（1）与正常脑组织相比，在星形细胞瘤中MCT1和MCT2表达增强；相对于恶性程度低的星形细胞瘤，恶性程度较高者表达更为强烈，并且随着病理级别的升高表达逐渐增强。MCT1、2在星形细胞瘤中表达增强，其强度变化与肿瘤的恶性程度呈正相关。（2）MCT4主要表达在不同病理级别肿瘤组织的瘤细胞胞浆和胞膜上，MCT4在低级别肿瘤组织中表达增强，在高级别肿瘤组织中表达进一步增强，提示MCT4可能在星形细胞瘤的增殖和迁移过程中具有重要的作用，可能作为星形细胞瘤的标记物和治疗靶点之一。第二部分CHC干扰对U251细胞增殖的影响目的探讨单羧酸转运蛋白（MCT）抑制剂α-氰基-4羟基苯乙烯（CHC）干扰U251细胞内乳酸代谢是否具有抗肿瘤作用。方法 1. U251细胞的常规培养。 2.实验分组：将U251细胞分为对照组、低剂量组（CHC：5mmol/L）和高剂量组（CHC：10mmol/L） 3. CHC干预后细胞增殖能力检测：MTT法检测对照组、低剂量组和高剂量组U251细胞在24、48、72h增殖能力。 4. CHC干预后细胞内MCTs表达变化检测：运用免疫细胞化学技术和Western Blot观察CHC干预48h后对照组、低剂量组和高剂量组U251细胞内MCT1、MCT2、MCT4蛋白含量的变化。 5. CHC干预后细胞内乳酸浓度检测：分光光度计分别检测CHC干预48h后对照组、低剂量组和高剂量组细胞内乳酸含量。 7. CHC干预后细胞周期检测：运用流式细胞仪观察CHC干预48h后对照组、低剂量组、高剂量组U251细胞的细胞周期（G1期、S期和G2期）变化。结果 1. CHC干预后细胞增殖能力的变化：MTT检测结果显示，，低剂量组和高剂量组U251细胞在24h、48h及72h增殖能力均受到抑制，与对照组相比，有显著性差异，上述3个时间点高剂量组抑制能力均高于低剂量组，且在48h抑制效应最强。 2. CHC干预后MCT1的表达变化：对照组中，MCT1主要呈线性完整的表达于U251细胞胞膜上，在CHC作用下，MCT1的表达位置由胞膜表达转为胞浆表达。Western Blot结果显示：与对照组相比较，MCT1在低、高剂量组中表达均减少，并且随着药物浓度的增加MCT1的表达逐渐减少，差异具有统计学意义（P0.05）。 3. CHC干预后MCT2的表达变化：MCT2主要表达在U251细胞的胞浆内，与对照组相比，低、高剂量组中MCT2表达减弱。Western Blot分析结果表明：与对照组相比较，低、高剂量组中MCT2表达减少，差异有统计学意义（P0.05）；而高剂量和低剂量两组间比较，无统计学差异（P0.05）。 4. CHC干预后MCT4的表达变化：MCT4主要表达在对照组及低、高剂量组中U251细胞的胞膜上。Western Blot分析结果显示：与对照组相比较，低、高剂量组中MCT4表达无明显变化，差异无统计学意义（P0.05）。 5. CHC干预后细胞内乳酸浓度的变化：与对照组相比较，低、高剂量组细胞内乳酸浓度升高，U251细胞内乳酸浓度在对照组中（0.189±0.012）mmol/L、低剂量组中（0.412±0.015）mmol/L、高剂量组中（0.895±0.010）mmol/L，三组细胞内乳酸浓度差异具有统计学意义（P0.05）。 6. CHC干预后细胞周期的变化：流式细胞仪检测结果显示，低剂量组及高剂量组G0/G1期的细胞分别为49.08%、56.21%，较对照组（35.1%）增多；同时低剂量组S期细胞数为41.63%，高剂量组为31.27%，均低于对照组S期细胞数（55.38%）。结论 1.应用MCT抑制剂CHC干扰U251细胞后，MCT1的表达明显受到抑制，并且其抑制程度随着药物浓度的增加逐渐增强；CHC对MCT2的抑制作用远不如MCT1，而MCT4在U251细胞上的表达基本上不受CHC的影响。 2.CHC能够增加细胞内乳酸的生成，从而影响星形细胞瘤的增殖和生长。 3.CHC能够阻止或抑制U251细胞有丝分裂的进行，S期细胞数量减少，G0/G1期细胞数量增多，使U251细胞阻滞在G1期，从而抑制其增殖。
[Abstract]:Part one: the expression of MCTs in human astrocytoma and its relationship with pathological grade.
objective
Objective To observe the difference of the expression of mono carboxylic acid transporter (MCT) 1,2,4 in astrocytomas of different pathological grades, and to explore the molecular mechanism of the proliferation and growth of astrocytomas.
Materials and methods
1. specimen collection: the tissue specimens of human astrocytoma were taken from the Department of neurosurgery in First Affiliated Hospital of Chongqing Medical University and the astrocytoma in the Department of neurosurgery in the Affiliated Hospital of Third Military Medical University, a total of 51 cases. After inquiring the patient's history and pathological examination, all the patients had not been treated with immunotherapy, radiotherapy and chemotherapy before operation. Wait.
2. pathological grade identification: HE staining was used to observe tissue specimens and determine the pathological grade.
Expression of 3.MCTs in astrocytomas of different pathological grades:
(1) immunohistochemical staining was used to observe the expression of MCTs in different pathological grades of astrocytoma and control group.
(2) using Western Blot technology to detect the expression of MCTs protein in different pathological grades of astrocytoma and control group.
4. statistical analysis: using the image analysis software and SPSS17.0 statistical software, the results of the experiment were statistically analyzed.
Result
The immunohistochemical results showed that MCT1 was expressed in the control and astrocytoma tissues. MCT1 was mainly expressed in astrocytes and vascular endothelial cells in the control tissue. In the astrocytoma of different pathological grades, MCT1 was mainly distributed in the nucleus and cytoplasm of the tumor cells and the results of.Western Blot were found in various tumor groups. The high expression of MCT1 was found in the fabric. Compared with the control group, the expression of MCT1 was enhanced in the low grade (WHO grade I and II) tumor tissues, and the expression was further enhanced in the high grade (grade III - IV) tumor tissues, and the difference was statistically significant (P0.05).
The immunohistochemical results showed that MCT2 was mainly expressed in the neurons and a small number of glial cells in the control group. The results of the.Western Blot in the cytoplasm and the membrane of the tumor cells showed that the expression of MCT2 was enhanced with the increase of pathological grade (P0.05).
Immunohistochemistry showed that MCT4 was mainly distributed in astrocytes of normal brain tissue, cytoplasm of low grade astrocytoma, intercellular astrocytoma, cytoplasm of glioblastoma cells and.Western Blot on the membrane: compared with normal brain tissue, MCT4 was at low grade (grade I and grade II) astrocytomas. The expression increased (P0.05) in the high grade (grade III - IV) tumor tissue (P0.05), and there was no significant difference in the expression level between the low level tumor tissue and the high grade tumor tissue (P > 0.05).
conclusion
(1) the expression of MCT1 and MCT2 was enhanced in astrocytoma compared with normal brain tissue; compared with low malignant astrocytoma, the expression of higher malignant degree was more intense, and the expression of.MCT1,2 in astrocytoma was enhanced gradually with the increase of pathological grade, and the intensity changes and the malignant degree of the tumor were Cheng Zhengxiang. Close.
(2) MCT4 is mainly expressed in the cytoplasm and membrane of tumor cells of different pathological grade tumor tissues. The expression of MCT4 is enhanced in low level tumor tissue and further enhanced in high grade tumor tissue, suggesting that MCT4 may play an important role in the proliferation and migration of astrocytoma, and may be used as a marker for astrocytoma. And one of the targets of treatment.
The effect of the second part of CHC interference on the proliferation of U251 cells
objective
Objective to investigate whether the mono carboxylic acid transporter (MCT) inhibitor, alpha cyanoyl -4 hydroxy styrene (CHC), interferes with lactate metabolism in U251 cells.
Method
Routine culture of 1. U251 cells.
2. experiment group: U251 cells were divided into control group, low dose group (CHC:5mmol/L) and high dose group (CHC:10mmol/L).
3. the ability of cell proliferation after CHC intervention: MTT method was used to detect the proliferation of U251 cells in control group, low dose group and high dose group U251 cells.
Detection of intracellular MCTs expression changes after 4. CHC intervention: the changes of MCT1, MCT2, MCT4 protein content in U251 cells in low dose group and high dose group were observed by immunocytochemical technique and Western Blot after CHC intervention in 48h control group.
5. the concentration of lactic acid in the cells after CHC intervention was detected by spectrophotometer. The lactate content in the control group, the low dose group and the high dose group was detected by CHC after 48h intervention.
Cell cycle detection after 7. CHC intervention: a flow cytometry was used to observe the changes in the cell cycle (G1, S and G2) of the U251 cells in the low dose group and the high dose group of the U251 cells after the intervention of 48h.
Result
The changes of cell proliferation after 1. CHC intervention: MTT test results showed that the proliferation ability of U251 cells in 24h, 48h and 72h in low dose group and high dose group were inhibited, and compared with the control group, there was significant difference. The inhibition ability of high dose group at these 3 time points was higher than that in low dose group, and the inhibitory effect of 48h was the strongest.
The expression changes of MCT1 after 2. CHC: in the control group, MCT1 was mainly expressed linearly and completely on the cell membrane of U251 cells. Under the action of CHC, the expression of MCT1 expressed from the membrane to the cytoplasm of the cytoplasm.Western Blot, which showed that the expression of MCT1 in the high dose group decreased as compared with the control group, and with the increase of the concentration of the drug. The expression of MCT1 increased gradually, and the difference was statistically significant (P0.05).
3. CHC expression changes in the expression of MCT2: MCT2 was mainly expressed in the cytoplasm of U251 cells. Compared with the control group, the expression of MCT2 expression in the high dose group was reduced by.Western Blot analysis. The results showed that the expression of MCT2 in the high dose group was lower than that in the control group, and the difference was statistically significant (P0.05), while the high and low dose groups were compared, and the comparison between the high dose group and the high dose group was statistically significant (P0.05). There was no statistical difference (P0.05).
The expression of MCT4 in 4. CHC: the expression of MCT4 was mainly expressed in the control group and low, and the results of.Western Blot analysis on the cell membrane of U251 cells in the high dose group showed that there was no significant change in the MCT4 expression in the low and high dose group compared with the control group, and the difference was not statistically significant (P0.05).
The intracellular lactate concentration changes after 5. CHC intervention: compared with the control group, the lactate concentration in the low and high dose groups increased, the lactate concentration in the U251 cell was (0.189 + 0.012) mmol/L, the low dose group was (0.412 + 0.015) mmol/L, and the high dose group was (0.895 + 0.010) mmol/L. The difference of lactate concentration in the three groups was statistically significant. Meaning (P0.05).
The cell cycle changes after 6. CHC intervention: the flow cytometry showed that the cells in the G0/G1 phase of the low dose group and the high dose group were 49.08% and 56.21%, respectively, compared with the control group (35.1%), at the same time, the number of S cells in the low dose group was 41.63% and the high dose group was 31.27%, which was lower than the number of S cells in the control group (55.38%).
conclusion
1. when MCT inhibitor CHC was used to interfere with U251 cells, the expression of MCT1 was obviously inhibited, and its inhibition was gradually enhanced with the increase of drug concentration; the inhibition of CHC to MCT2 was far less than that of MCT1, and the expression of MCT4 on U251 cells was largely unaffected by CHC.
2.CHC can increase the production of lactic acid in cells, thereby affecting the proliferation and growth of astrocytomas.
3.CHC can prevent or inhibit the progression of mitosis in U251 cells. The number of cells in phase S is reduced, the number of G0/G1 cells is increased, and U251 cells are blocked in the G1 phase, thus inhibiting the proliferation of U251 cells.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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