阿維A酸聯(lián)合TNF-a對人皮膚鱗狀細(xì)胞癌A431增殖和Caspase-3表達(dá)的影響
發(fā)布時(shí)間：2018-08-22 09:57
【摘要】：目的：皮膚鱗狀細(xì)胞癌(squamous cell carcinoma，SCC,簡稱鱗癌)是起源于表皮的一種惡性腫瘤，其發(fā)病率逐年上升。對常規(guī)治療效果不佳，外科手術(shù)是首選的治療方法，然而皮膚鱗癌轉(zhuǎn)移后的治療是期待解決的問題。因此，尋求有效的藥物進(jìn)行治療具有重要意義。 維甲酸類（retinoids）屬于維生素A的一類衍生物，包含多種同分異構(gòu)體：13-順式維甲酸，全反式維甲酸，9-順式維甲酸等。以往研究證明維甲酸類化合物在細(xì)胞的增生、分化，皮膚炎癥過程中發(fā)揮重要作用，對腫瘤細(xì)胞具有很強(qiáng)的誘導(dǎo)分化抑制增殖的作用。 腫瘤壞死因子（Tumor Necrosis Factor，TNF）由巨噬細(xì)胞產(chǎn)生及釋放，同時(shí)，B淋巴細(xì)胞產(chǎn)生一種與TNF類似的淋巴毒素,其與TNF享有共同受體。為了區(qū)分,巨噬細(xì)胞產(chǎn)生的毒素稱為TNF-α，淋巴細(xì)胞產(chǎn)生的毒素稱為TNF-β，通過與細(xì)胞膜表面的對應(yīng)受體結(jié)合，激活細(xì)胞信號轉(zhuǎn)導(dǎo)系統(tǒng)可引起腫瘤細(xì)胞的凋亡，TNF-α可使腫瘤局部產(chǎn)生凝血，腫瘤血源阻斷引起局部缺血壞死，可抑制腫瘤細(xì)胞生長。 本研究以人表皮鱗癌細(xì)胞株A431為研究對象，探討阿維A酸TNF-α在不同濃度、不同作用時(shí)間上對鱗狀細(xì)胞癌的作用及形態(tài)分化的影響，探討兩藥聯(lián)合應(yīng)用對人A431細(xì)胞表達(dá)Caspase-3的影響。 方法： 1細(xì)胞培養(yǎng)：在DMEM培養(yǎng)基中培養(yǎng)A431細(xì)胞，置于飽和濕度、37℃、5%CO2的培養(yǎng)箱內(nèi)做常規(guī)傳代培養(yǎng)。 2用四甲基偶氮唑藍(lán)（MTT）比色法檢測 2.1TNF-α對A431細(xì)胞增殖抑制率的影響 設(shè)空白對照組陰性對照組和實(shí)驗(yàn)組，實(shí)驗(yàn)組分為濃度為1U／L、2U／L.、5U/L的TNF-α組分別于加藥后培養(yǎng)12h24h48h進(jìn)行測定。 2.2測定阿維A、阿維A聯(lián)合TNF-α對A431細(xì)胞增殖抑制率的影響 設(shè)空白對照組陰性對照組和實(shí)驗(yàn)組，實(shí)驗(yàn)組又分為濃度各為10μmol／L阿維A酸組，10μmol／L阿維A酸組與5U/L TNF-α組聯(lián)合用藥組，分別于加藥后培養(yǎng)24h48h72h進(jìn)行測定。 3倒置相差顯微鏡及HE染色法觀察經(jīng)過不同濃度藥物及聯(lián)合藥物處理后細(xì)胞的分化及形態(tài)變化。 4應(yīng)用免疫細(xì)胞化學(xué)法測定對Caspase-3蛋白表達(dá)的影響。 5流式細(xì)胞儀（FCM）檢測不同濃度藥物及聯(lián)合藥物處理的A431細(xì)胞Caspase-3蛋白表達(dá)狀況。 6運(yùn)用統(tǒng)計(jì)軟件SPSS13.0對數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析。 結(jié)果： 1MTT比色分析法檢測顯示 1.1TNF-α對人表皮鱗癌細(xì)胞A431增殖抑制的影響 TNF-α組與陰性對照組相比，TNF-α在1U/L至5U/L濃度范圍內(nèi)對具有明顯的增殖抑制作用，呈現(xiàn)明顯的劑量和時(shí)間效應(yīng)關(guān)系。在12h、24h、48h時(shí)間段內(nèi)，TNF-α隨著濃度的增加對A431細(xì)胞的抑制也出現(xiàn)加強(qiáng)，且以濃度為5U/L組作用在48h的抑制率為最高，抑制率相比差異（P0.05）具有統(tǒng)計(jì)學(xué)意義。 1.2將阿維A按不同濃度進(jìn)行組合，細(xì)胞培養(yǎng)48小時(shí)后采用MTT比色法檢測各組細(xì)胞的抑制率，根據(jù)試驗(yàn)結(jié)果，我們選擇了10μmol／L阿維A進(jìn)行后續(xù)試驗(yàn)。阿維A聯(lián)合TNF-α組：將實(shí)驗(yàn)分為3組，陰性對照組10μmol／L阿維A組10μmol／L阿維A聯(lián)合5U/L TNF-α組。上述各組藥物作用細(xì)胞48h后，各實(shí)驗(yàn)組細(xì)胞的抑制率分別是12.35%23.46%、47.56%，可見聯(lián)合用藥組對細(xì)胞的抑制率明顯高于單獨(dú)用藥組，差別具有統(tǒng)計(jì)學(xué)意義（P0.05） 2藥物對A431細(xì)胞形態(tài)及分化的影響 2.1加入TNF-α細(xì)胞變化主要表現(xiàn)在兩方面 2.1.1細(xì)胞貼壁能力的變化：經(jīng)24h的處理后，實(shí)驗(yàn)組貼壁能力下降，部分細(xì)胞呈漂浮狀態(tài)，而對照組細(xì)胞貼壁能力無明顯變化。隨著對處理時(shí)間的加長及增加藥物的濃度，細(xì)胞貼壁能力比之前明顯下降。 2.1.2細(xì)胞大小和形態(tài)的變化：處理24h后，實(shí)驗(yàn)組僅少部分細(xì)胞變小、變圓，而對照組未有明顯變化。繼續(xù)延長處理時(shí)間及加大藥物的濃度，細(xì)胞的分布出現(xiàn)分散，呈顯著皺縮，甚至破裂，失去原有折光性，生長緩慢。 2.2聯(lián)合藥物后細(xì)胞變化主要表現(xiàn)在兩方面。 2.2.1細(xì)胞貼壁能力的變化：處理24h后，實(shí)驗(yàn)組貼壁能力明顯下降，部分細(xì)胞呈漂浮生長狀態(tài)，而陰性對照組細(xì)胞貼壁能力未有明顯變化，貼壁良好。 2.2.2細(xì)胞大小和形態(tài)的變化：處理24h后，實(shí)驗(yàn)組少數(shù)細(xì)胞變小、變圓，而陰性對照組細(xì)胞形態(tài)和大小未有明顯變化。隨著處理時(shí)間的增長，藥物濃度的增加，細(xì)胞散在分布，呈顯著皺縮，甚至破裂，失去原有折光性，生長緩慢。而陰性對照組細(xì)胞數(shù)目增多貼壁生長，多角形，可見偽足，胞漿飽滿，折光性強(qiáng)，相鄰細(xì)胞生長匯合成片，逐漸形成細(xì)胞單層，無細(xì)胞脫落。 3免疫細(xì)胞化學(xué)s-p法檢測 3.1TNF對A431細(xì)胞Caspase-3蛋白表達(dá)的影響：陰性對照組細(xì)胞經(jīng)過免疫細(xì)胞化學(xué)染色后Caspase-3蛋白表達(dá)較少，其胞漿中有少量深棕色顆粒。經(jīng)過濃度為1U/L、2U/L、5U/L的TNF-α作用48h后，Caspase-3蛋白的表達(dá)就有明顯增高，并且隨著作用時(shí)間的增長，作用藥物濃度的增加，Caspase-3蛋白的表達(dá)逐漸增高。 3.2測定阿維A酸及阿維A聯(lián)合TNF-α對A431細(xì)胞Caspase-3蛋白表達(dá)影響：陰性對照組細(xì)胞經(jīng)過免疫細(xì)胞化學(xué)染色后Caspase-3蛋白表達(dá)較少，其胞漿中有少量深棕色顆粒。經(jīng)過濃度為10μmol／L的阿維A酸，10μmol／L阿維A聯(lián)合TNF-α作用48h后，10μmol／L阿維A聯(lián)合TNF-α組Caspase-3蛋白的表達(dá)就有明顯增高，且較單獨(dú)10μmol／L阿維A組及TNF-α組增高明顯。 4流式細(xì)胞術(shù)檢測不同濃度藥物作用后，細(xì)胞在G1前停滯。而TNF-α可以誘導(dǎo)細(xì)胞凋亡。 結(jié)論： 1TNF-α有效地抑制人表皮鱗癌細(xì)胞A431的增殖，促使人表皮鱗癌細(xì)胞A431的凋亡。 2TNF-α促使人表皮鱗癌細(xì)胞A431凋亡主要是通過激活Caspase-3途徑導(dǎo)致的。 3阿維A酸與TNF-α聯(lián)合應(yīng)用可以促使人表皮鱗癌細(xì)胞A431凋亡，，并且比單一阿維A酸或TNF-α效果好。 4阿維A酸與TNF-α聯(lián)合應(yīng)用促使人表皮鱗癌細(xì)胞A431凋亡是通過激活Caspase-3蛋白的表達(dá)。
[Abstract]:Objective: Squamous cell carcinoma (SCC) is a malignant tumor originating from epidermis. The incidence of SCC is increasing year by year. Surgical treatment is the preferred treatment for patients with poor conventional treatment. However, the treatment of skin squamous cell carcinoma after metastasis is a problem to be solved. Treatment is of great significance.
Retinoic acids (retinoids) are a class of derivatives of vitamin A. They contain a variety of isomers: 13-cis-retinoic acid, all-trans-retinoic acid, 9-cis-retinoic acid, etc. Previous studies have shown that retinoids play an important role in cell proliferation, differentiation, skin inflammation process, and strongly induce differentiation of tumor cells. The role of proliferation.
Tumor Necrosis Factor (TNF) is produced and released by macrophages. At the same time, B lymphocytes produce a lymphotoxin similar to TNF, which shares a common receptor with TNF. The activation of cell signal transduction system can induce apoptosis of tumor cells. TNF-alpha can induce local coagulation, blockade of tumor blood source can cause local ischemia and necrosis, and inhibit the growth of tumor cells.
In this study, human epidermal squamous cell carcinoma cell line A431 was used to investigate the effect of TNF-a on the morphological differentiation and the expression of Caspase-3 in human A431 cells.
Method:
1 Cell culture: A431 cells were cultured in DMEM medium and subcultured in a saturated humidity incubator at 37 C and 5% CO2.
2 detection with four methyl azolium blue (MTT) colorimetric method.
Effect of 2.1TNF- alpha on proliferation inhibition rate of A431 cells
A blank control group, a negative control group and an experimental group were set up. The experimental group was divided into 1 U/L, 2 U/L., and 5 U/L TNF-a groups, which were cultured for 12 hours, 24 hours and 48 hours respectively.
2.2 effect of AVI A, avi A combined with TNF- alpha on the inhibition rate of A431 cell proliferation
A blank control group, a negative control group and an experimental group were set up. The experimental group was divided into 10-micromol/L Averacic acid group, 10-micromol/L Averacic acid group and 5-U/L TNF-alpha group, and cultured for 24 hours, 48 hours and 72 hours respectively.
3 Inverted phase contrast microscope and HE staining were used to observe the cell differentiation and morphological changes after different concentrations of drugs and combination of drugs.
4 immunocytochemistry was used to determine the expression of Caspase-3 protein.
Caspase-3 protein expression in A431 cells was detected by flow cytometry (FCM).
6 using statistical software SPSS13.0 to conduct statistical analysis of the data.
Result:
1MTT colorimetric analysis showed that
Effect of 1.1TNF- alpha on proliferation inhibition of human epidermoid carcinoma cell line A431
Compared with the negative control group, TNF-alpha had a significant inhibitory effect on the proliferation of A431 cells in the range of 1U/L to 5U/L, showing a dose-and time-dependent relationship. The inhibition rate was statistically significant (P0.05).
1.2 After 48 hours of cell culture, the inhibition rate of each group was detected by MTT colorimetric method. According to the results, we chose 10 micromol/L acitretin for follow-up test. The combination of acitretin and TNF-alpha group: the experiment was divided into three groups, the negative control group? 10 micromol/L acitretin group? 10 micromol/L acitretin combined with 5 U/L TNF. After 48 hours of treatment, the inhibition rates of cells in each experimental group were 12.35%? 23.46% and 47.56% respectively. The inhibition rates of cells in the combined group were significantly higher than those in the single group (P 0.05).
2 Effects of drugs on the morphology and differentiation of A431 cells
2.1 the changes of TNF- alpha cells were mainly manifested in two aspects.
2.1.1 Cell adherence ability: After 24 hours treatment, the adherence ability of the experimental group decreased, some cells were floating, but the adherence ability of the control group did not change significantly.
2.1.2 Cell size and morphology: After 24 hours of treatment, only a few cells in the experimental group became smaller and rounded, but there was no significant change in the control group.
2.2 the cell changes after drug combination were mainly manifested in two aspects.
2.2.1 Cell adherence ability: After 24 hours of treatment, the adherence ability of the experimental group decreased significantly, some cells showed floating growth, while the negative control group had no significant change in adherence ability, adherence was good.
2.2.2 Cell Size and Morphology Changes: After 24 hours of treatment, a few cells in the experimental group became smaller and rounded, while the cell morphology and size in the negative control group did not change significantly. The number of cells increased and adhered to the wall, polygonal, visible pseudopodia, cytoplasm plump, refractive, adjacent cell growth sink synthesized slices, gradually formed a single layer of cells, no cell shedding.
3 immunocytochemistry S-P assay
3.1 Effect of TNF on the expression of Caspase-3 protein in A431 cells: After immunocytochemical staining, the expression of Caspase-3 protein in the negative control group was low, and there were a few dark brown granules in the cytoplasm. With the increase of drug concentration, the expression of Caspase-3 protein was gradually increased.
3.2 To determine the effect of Averatric acid and Averatric acid combined with TNF-alpha on the expression of Caspase-3 protein in A431 cells: The expression of Caspase-3 protein in A431 cells of negative control group was less after immunocytochemical staining, and there were a few dark brown granules in their cytoplasm. The expression of Caspase-3 protein in Averatrol A combined with TNF-alpha group was significantly higher than that in 10 micromol/L Averatrol alone and TNF-alpha group.
Flow cytometry showed that the cells stopped before G1 and TNF-a could induce apoptosis.
Conclusion:
1TNF- alpha can effectively inhibit the proliferation of A431 and promote the apoptosis of A431 cells.
2TNF- alpha promotes A431 apoptosis in human epidermoid carcinoma cells mainly through activation of Caspase-3 pathway.
The combination of Averatric acid and TNF-alpha can induce apoptosis of human epidermal squamous cell carcinoma cell line A431, and the effect is better than that of single Averatric acid or TNF-alpha.
Averatric acid combined with TNF-a induces apoptosis of human epidermal squamous cell carcinoma cell line A431 by activating the expression of Caspase-3 protein.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.5
[Abstract]:Objective: Squamous cell carcinoma (SCC) is a malignant tumor originating from epidermis. The incidence of SCC is increasing year by year. Surgical treatment is the preferred treatment for patients with poor conventional treatment. However, the treatment of skin squamous cell carcinoma after metastasis is a problem to be solved. Treatment is of great significance.
Retinoic acids (retinoids) are a class of derivatives of vitamin A. They contain a variety of isomers: 13-cis-retinoic acid, all-trans-retinoic acid, 9-cis-retinoic acid, etc. Previous studies have shown that retinoids play an important role in cell proliferation, differentiation, skin inflammation process, and strongly induce differentiation of tumor cells. The role of proliferation.
Tumor Necrosis Factor (TNF) is produced and released by macrophages. At the same time, B lymphocytes produce a lymphotoxin similar to TNF, which shares a common receptor with TNF. The activation of cell signal transduction system can induce apoptosis of tumor cells. TNF-alpha can induce local coagulation, blockade of tumor blood source can cause local ischemia and necrosis, and inhibit the growth of tumor cells.
In this study, human epidermal squamous cell carcinoma cell line A431 was used to investigate the effect of TNF-a on the morphological differentiation and the expression of Caspase-3 in human A431 cells.
Method:
1 Cell culture: A431 cells were cultured in DMEM medium and subcultured in a saturated humidity incubator at 37 C and 5% CO2.
2 detection with four methyl azolium blue (MTT) colorimetric method.
Effect of 2.1TNF- alpha on proliferation inhibition rate of A431 cells
A blank control group, a negative control group and an experimental group were set up. The experimental group was divided into 1 U/L, 2 U/L., and 5 U/L TNF-a groups, which were cultured for 12 hours, 24 hours and 48 hours respectively.
2.2 effect of AVI A, avi A combined with TNF- alpha on the inhibition rate of A431 cell proliferation
A blank control group, a negative control group and an experimental group were set up. The experimental group was divided into 10-micromol/L Averacic acid group, 10-micromol/L Averacic acid group and 5-U/L TNF-alpha group, and cultured for 24 hours, 48 hours and 72 hours respectively.
3 Inverted phase contrast microscope and HE staining were used to observe the cell differentiation and morphological changes after different concentrations of drugs and combination of drugs.
4 immunocytochemistry was used to determine the expression of Caspase-3 protein.
Caspase-3 protein expression in A431 cells was detected by flow cytometry (FCM).
6 using statistical software SPSS13.0 to conduct statistical analysis of the data.
Result:
1MTT colorimetric analysis showed that
Effect of 1.1TNF- alpha on proliferation inhibition of human epidermoid carcinoma cell line A431
Compared with the negative control group, TNF-alpha had a significant inhibitory effect on the proliferation of A431 cells in the range of 1U/L to 5U/L, showing a dose-and time-dependent relationship. The inhibition rate was statistically significant (P0.05).
1.2 After 48 hours of cell culture, the inhibition rate of each group was detected by MTT colorimetric method. According to the results, we chose 10 micromol/L acitretin for follow-up test. The combination of acitretin and TNF-alpha group: the experiment was divided into three groups, the negative control group? 10 micromol/L acitretin group? 10 micromol/L acitretin combined with 5 U/L TNF. After 48 hours of treatment, the inhibition rates of cells in each experimental group were 12.35%? 23.46% and 47.56% respectively. The inhibition rates of cells in the combined group were significantly higher than those in the single group (P 0.05).
2 Effects of drugs on the morphology and differentiation of A431 cells
2.1 the changes of TNF- alpha cells were mainly manifested in two aspects.
2.1.1 Cell adherence ability: After 24 hours treatment, the adherence ability of the experimental group decreased, some cells were floating, but the adherence ability of the control group did not change significantly.
2.1.2 Cell size and morphology: After 24 hours of treatment, only a few cells in the experimental group became smaller and rounded, but there was no significant change in the control group.
2.2 the cell changes after drug combination were mainly manifested in two aspects.
2.2.1 Cell adherence ability: After 24 hours of treatment, the adherence ability of the experimental group decreased significantly, some cells showed floating growth, while the negative control group had no significant change in adherence ability, adherence was good.
2.2.2 Cell Size and Morphology Changes: After 24 hours of treatment, a few cells in the experimental group became smaller and rounded, while the cell morphology and size in the negative control group did not change significantly. The number of cells increased and adhered to the wall, polygonal, visible pseudopodia, cytoplasm plump, refractive, adjacent cell growth sink synthesized slices, gradually formed a single layer of cells, no cell shedding.
3 immunocytochemistry S-P assay
3.1 Effect of TNF on the expression of Caspase-3 protein in A431 cells: After immunocytochemical staining, the expression of Caspase-3 protein in the negative control group was low, and there were a few dark brown granules in the cytoplasm. With the increase of drug concentration, the expression of Caspase-3 protein was gradually increased.
3.2 To determine the effect of Averatric acid and Averatric acid combined with TNF-alpha on the expression of Caspase-3 protein in A431 cells: The expression of Caspase-3 protein in A431 cells of negative control group was less after immunocytochemical staining, and there were a few dark brown granules in their cytoplasm. The expression of Caspase-3 protein in Averatrol A combined with TNF-alpha group was significantly higher than that in 10 micromol/L Averatrol alone and TNF-alpha group.
Flow cytometry showed that the cells stopped before G1 and TNF-a could induce apoptosis.
Conclusion:
1TNF- alpha can effectively inhibit the proliferation of A431 and promote the apoptosis of A431 cells.
2TNF- alpha promotes A431 apoptosis in human epidermoid carcinoma cells mainly through activation of Caspase-3 pathway.
The combination of Averatric acid and TNF-alpha can induce apoptosis of human epidermal squamous cell carcinoma cell line A431, and the effect is better than that of single Averatric acid or TNF-alpha.
Averatric acid combined with TNF-a induces apoptosis of human epidermal squamous cell carcinoma cell line A431 by activating the expression of Caspase-3 protein.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.5
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