本文選題：羧酸轉(zhuǎn)運(yùn)蛋白 + 星形細(xì)胞瘤��； 參考：《重慶醫(yī)科大學(xué)》2014年碩士論文

【摘要】：第一部分MCTs在人星形細(xì)胞瘤中的表達(dá)變化及其與病理級(jí)別之間的關(guān)系 目的 觀察單羧酸轉(zhuǎn)運(yùn)蛋白（MCT）1、2、4在人不同病理級(jí)別星形細(xì)胞瘤中的表達(dá)差異，探討星形細(xì)胞瘤增殖、生長(zhǎng)的分子機(jī)制。 材料和方法 1.標(biāo)本的采集：人星形細(xì)胞瘤組織標(biāo)本均取自重慶醫(yī)科大學(xué)附屬第一醫(yī)院神經(jīng)外科和第三軍醫(yī)大學(xué)附屬大坪醫(yī)院神經(jīng)外科星形細(xì)胞瘤患者，共計(jì)51例。經(jīng)過(guò)查詢(xún)患者病史及病理檢驗(yàn)證實(shí)，所有患者術(shù)前均未接受過(guò)免疫治療、放療和化療等。 2.病理級(jí)別鑒定：應(yīng)用HE染色對(duì)組織標(biāo)本進(jìn)行觀察，確定病理級(jí)別。 3.MCTs在人不同病理級(jí)別星形細(xì)胞瘤中的表達(dá)檢測(cè)： （1）應(yīng)用免疫組織化學(xué)技術(shù)，觀察人不同病理級(jí)別星形細(xì)胞瘤組織和對(duì)照組組織中MCTs的表達(dá)變化； （2）應(yīng)用Western Blot技術(shù)，檢測(cè)人不同病理級(jí)別星形細(xì)胞瘤組織和對(duì)照組組織中MCTs蛋白的表達(dá)變化。 4.統(tǒng)計(jì)學(xué)分析：運(yùn)用圖像分析軟件和SPSS17.0統(tǒng)計(jì)軟件對(duì)實(shí)驗(yàn)結(jié)果進(jìn)行統(tǒng)計(jì)分析。 結(jié)果 免疫組織化學(xué)結(jié)果顯示：對(duì)照組和星形細(xì)胞瘤組織中均有MCT1的表達(dá)。對(duì)照組織中MCT1主要表達(dá)在星形細(xì)胞和血管內(nèi)皮細(xì)胞中。在不同病理級(jí)別的星形細(xì)胞瘤中，MCT1主要分布在瘤細(xì)胞的胞核和胞質(zhì)中。Western Blot結(jié)果表明：各級(jí)別腫瘤組織中均可見(jiàn)MCT1的高表達(dá)，，與對(duì)照組相比，MCT1在低級(jí)別（WHOⅠ級(jí)和Ⅱ級(jí)）腫瘤組織中表達(dá)增強(qiáng)，在高級(jí)別（Ⅲ-Ⅳ級(jí)）腫瘤組織中表達(dá)進(jìn)一步增強(qiáng)，差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 免疫組織化學(xué)結(jié)果顯示：MCT2主要表達(dá)在對(duì)照組的神經(jīng)元和少量膠質(zhì)細(xì)胞中。在腫瘤組織中主要分布在瘤細(xì)胞的胞質(zhì)和胞膜上。Western Blot結(jié)果顯示：MCT2的表達(dá)隨著病理級(jí)別的升高而增強(qiáng)（P0.05）。 免疫組織化學(xué)顯示：MCT4主要分布在正常腦組織的星形膠質(zhì)細(xì)胞上，低級(jí)別星形細(xì)胞瘤的胞漿，間變形星形細(xì)胞瘤、膠質(zhì)母細(xì)胞瘤的瘤細(xì)胞的胞漿和胞膜上。Western Blot結(jié)果表明：與正常腦組織相比，MCT4在低級(jí)別（Ⅰ級(jí)和Ⅱ級(jí)）星形細(xì)胞瘤組織中表達(dá)增多（P0.05），在高級(jí)別（Ⅲ-Ⅳ級(jí)）腫瘤組織中表達(dá)進(jìn)一步增多（P0.05），而分別在低級(jí)別腫瘤組織之間和高級(jí)別腫瘤組織之間相比較，其表達(dá)含量無(wú)統(tǒng)計(jì)學(xué)差異（P＞0.05）。 結(jié)論 （1）與正常腦組織相比，在星形細(xì)胞瘤中MCT1和MCT2表達(dá)增強(qiáng)；相對(duì)于惡性程度低的星形細(xì)胞瘤，惡性程度較高者表達(dá)更為強(qiáng)烈，并且隨著病理級(jí)別的升高表達(dá)逐漸增強(qiáng)。MCT1、2在星形細(xì)胞瘤中表達(dá)增強(qiáng)，其強(qiáng)度變化與腫瘤的惡性程度呈正相關(guān)。 （2）MCT4主要表達(dá)在不同病理級(jí)別腫瘤組織的瘤細(xì)胞胞漿和胞膜上，MCT4在低級(jí)別腫瘤組織中表達(dá)增強(qiáng)，在高級(jí)別腫瘤組織中表達(dá)進(jìn)一步增強(qiáng)，提示MCT4可能在星形細(xì)胞瘤的增殖和遷移過(guò)程中具有重要的作用，可能作為星形細(xì)胞瘤的標(biāo)記物和治療靶點(diǎn)之一。 第二部分CHC干擾對(duì)U251細(xì)胞增殖的影響 目的 探討單羧酸轉(zhuǎn)運(yùn)蛋白（MCT）抑制劑α-氰基-4羥基苯乙烯（CHC）干擾U251細(xì)胞內(nèi)乳酸代謝是否具有抗腫瘤作用。 方法 1. U251細(xì)胞的常規(guī)培養(yǎng)。 2.實(shí)驗(yàn)分組：將U251細(xì)胞分為對(duì)照組、低劑量組（CHC：5mmol/L）和高劑量組（CHC：10mmol/L） 3. CHC干預(yù)后細(xì)胞增殖能力檢測(cè)：MTT法檢測(cè)對(duì)照組、低劑量組和高劑量組U251細(xì)胞在24、48、72h增殖能力。 4. CHC干預(yù)后細(xì)胞內(nèi)MCTs表達(dá)變化檢測(cè)：運(yùn)用免疫細(xì)胞化學(xué)技術(shù)和Western Blot觀察CHC干預(yù)48h后對(duì)照組、低劑量組和高劑量組U251細(xì)胞內(nèi)MCT1、MCT2、MCT4蛋白含量的變化。 5. CHC干預(yù)后細(xì)胞內(nèi)乳酸濃度檢測(cè)：分光光度計(jì)分別檢測(cè)CHC干預(yù)48h后對(duì)照組、低劑量組和高劑量組細(xì)胞內(nèi)乳酸含量。 7. CHC干預(yù)后細(xì)胞周期檢測(cè)：運(yùn)用流式細(xì)胞儀觀察CHC干預(yù)48h后對(duì)照組、低劑量組、高劑量組U251細(xì)胞的細(xì)胞周期（G1期、S期和G2期）變化。 結(jié)果 1. CHC干預(yù)后細(xì)胞增殖能力的變化：MTT檢測(cè)結(jié)果顯示，，低劑量組和高劑量組U251細(xì)胞在24h、48h及72h增殖能力均受到抑制，與對(duì)照組相比，有顯著性差異，上述3個(gè)時(shí)間點(diǎn)高劑量組抑制能力均高于低劑量組，且在48h抑制效應(yīng)最強(qiáng)。 2. CHC干預(yù)后MCT1的表達(dá)變化：對(duì)照組中，MCT1主要呈線(xiàn)性完整的表達(dá)于U251細(xì)胞胞膜上，在CHC作用下，MCT1的表達(dá)位置由胞膜表達(dá)轉(zhuǎn)為胞漿表達(dá)。Western Blot結(jié)果顯示：與對(duì)照組相比較，MCT1在低、高劑量組中表達(dá)均減少，并且隨著藥物濃度的增加MCT1的表達(dá)逐漸減少，差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 3. CHC干預(yù)后MCT2的表達(dá)變化：MCT2主要表達(dá)在U251細(xì)胞的胞漿內(nèi)，與對(duì)照組相比，低、高劑量組中MCT2表達(dá)減弱。Western Blot分析結(jié)果表明：與對(duì)照組相比較，低、高劑量組中MCT2表達(dá)減少，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；而高劑量和低劑量?jī)山M間比較，無(wú)統(tǒng)計(jì)學(xué)差異（P0.05）。 4. CHC干預(yù)后MCT4的表達(dá)變化：MCT4主要表達(dá)在對(duì)照組及低、高劑量組中U251細(xì)胞的胞膜上。Western Blot分析結(jié)果顯示：與對(duì)照組相比較，低、高劑量組中MCT4表達(dá)無(wú)明顯變化，差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。 5. CHC干預(yù)后細(xì)胞內(nèi)乳酸濃度的變化：與對(duì)照組相比較，低、高劑量組細(xì)胞內(nèi)乳酸濃度升高，U251細(xì)胞內(nèi)乳酸濃度在對(duì)照組中（0.189±0.012）mmol/L、低劑量組中（0.412±0.015）mmol/L、高劑量組中（0.895±0.010）mmol/L，三組細(xì)胞內(nèi)乳酸濃度差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。 6. CHC干預(yù)后細(xì)胞周期的變化：流式細(xì)胞儀檢測(cè)結(jié)果顯示，低劑量組及高劑量組G0/G1期的細(xì)胞分別為49.08%、56.21%，較對(duì)照組（35.1%）增多；同時(shí)低劑量組S期細(xì)胞數(shù)為41.63%，高劑量組為31.27%，均低于對(duì)照組S期細(xì)胞數(shù)（55.38%）。 結(jié)論 1.應(yīng)用MCT抑制劑CHC干擾U251細(xì)胞后，MCT1的表達(dá)明顯受到抑制，并且其抑制程度隨著藥物濃度的增加逐漸增強(qiáng)；CHC對(duì)MCT2的抑制作用遠(yuǎn)不如MCT1，而MCT4在U251細(xì)胞上的表達(dá)基本上不受CHC的影響。 2.CHC能夠增加細(xì)胞內(nèi)乳酸的生成，從而影響星形細(xì)胞瘤的增殖和生長(zhǎng)。 3.CHC能夠阻止或抑制U251細(xì)胞有絲分裂的進(jìn)行，S期細(xì)胞數(shù)量減少，G0/G1期細(xì)胞數(shù)量增多，使U251細(xì)胞阻滯在G1期，從而抑制其增殖。
[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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R739.41

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1 李文娟;MCTs在人星形細(xì)胞瘤中的表達(dá)變化及MCT抑制劑對(duì)U251細(xì)胞增殖的影響[D];重慶醫(yī)科大學(xué);2014年

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