阿维A酸联合TNF-a对人皮肤鳞状细胞癌A431增殖和Caspase-3表达的影响
发布时间：2018-08-22 09:57
【摘要】：目的：皮肤鳞状细胞癌(squamous cell carcinoma，SCC,简称鳞癌)是起源于表皮的一种恶性肿瘤，其发病率逐年上升。对常规治疗效果不佳，外科手术是首选的治疗方法，然而皮肤鳞癌转移后的治疗是期待解决的问题。因此，寻求有效的药物进行治疗具有重要意义。 维甲酸类（retinoids）属于维生素A的一类衍生物，包含多种同分异构体：13-顺式维甲酸，全反式维甲酸，9-顺式维甲酸等。以往研究证明维甲酸类化合物在细胞的增生、分化，皮肤炎症过程中发挥重要作用，对肿瘤细胞具有很强的诱导分化抑制增殖的作用。 肿瘤坏死因子（Tumor Necrosis Factor，TNF）由巨噬细胞产生及释放，同时，B淋巴细胞产生一种与TNF类似的淋巴毒素,其与TNF享有共同受体。为了区分,巨噬细胞产生的毒素称为TNF-α，淋巴细胞产生的毒素称为TNF-β，通过与细胞膜表面的对应受体结合，激活细胞信号转导系统可引起肿瘤细胞的凋亡，TNF-α可使肿瘤局部产生凝血，肿瘤血源阻断引起局部缺血坏死，可抑制肿瘤细胞生长。 本研究以人表皮鳞癌细胞株A431为研究对象，探讨阿维A酸TNF-α在不同浓度、不同作用时间上对鳞状细胞癌的作用及形态分化的影响，探讨两药联合应用对人A431细胞表达Caspase-3的影响。 方法： 1细胞培养：在DMEM培养基中培养A431细胞，置于饱和湿度、37℃、5%CO2的培养箱内做常规传代培养。 2用四甲基偶氮唑蓝（MTT）比色法检测 2.1TNF-α对A431细胞增殖抑制率的影响 设空白对照组阴性对照组和实验组，实验组分为浓度为1U／L、2U／L.、5U/L的TNF-α组分别于加药后培养12h24h48h进行测定。 2.2测定阿维A、阿维A联合TNF-α对A431细胞增殖抑制率的影响 设空白对照组阴性对照组和实验组，实验组又分为浓度各为10μmol／L阿维A酸组，10μmol／L阿维A酸组与5U/L TNF-α组联合用药组，分别于加药后培养24h48h72h进行测定。 3倒置相差显微镜及HE染色法观察经过不同浓度药物及联合药物处理后细胞的分化及形态变化。 4应用免疫细胞化学法测定对Caspase-3蛋白表达的影响。 5流式细胞仪（FCM）检测不同浓度药物及联合药物处理的A431细胞Caspase-3蛋白表达状况。 6运用统计软件SPSS13.0对数据进行统计分析。 结果： 1MTT比色分析法检测显示 1.1TNF-α对人表皮鳞癌细胞A431增殖抑制的影响 TNF-α组与阴性对照组相比，TNF-α在1U/L至5U/L浓度范围内对具有明显的增殖抑制作用，呈现明显的剂量和时间效应关系。在12h、24h、48h时间段内，TNF-α随着浓度的增加对A431细胞的抑制也出现加强，且以浓度为5U/L组作用在48h的抑制率为最高，抑制率相比差异（P0.05）具有统计学意义。 1.2将阿维A按不同浓度进行组合，细胞培养48小时后采用MTT比色法检测各组细胞的抑制率，根据试验结果，我们选择了10μmol／L阿维A进行后续试验。阿维A联合TNF-α组：将实验分为3组，阴性对照组10μmol／L阿维A组10μmol／L阿维A联合5U/L TNF-α组。上述各组药物作用细胞48h后，各实验组细胞的抑制率分别是12.35%23.46%、47.56%，可见联合用药组对细胞的抑制率明显高于单独用药组，差别具有统计学意义（P0.05） 2药物对A431细胞形态及分化的影响 2.1加入TNF-α细胞变化主要表现在两方面 2.1.1细胞贴壁能力的变化：经24h的处理后，实验组贴壁能力下降，部分细胞呈漂浮状态，而对照组细胞贴壁能力无明显变化。随着对处理时间的加长及增加药物的浓度，细胞贴壁能力比之前明显下降。 2.1.2细胞大小和形态的变化：处理24h后，实验组仅少部分细胞变小、变圆，而对照组未有明显变化。继续延长处理时间及加大药物的浓度，细胞的分布出现分散，呈显著皱缩，甚至破裂，失去原有折光性，生长缓慢。 2.2联合药物后细胞变化主要表现在两方面。 2.2.1细胞贴壁能力的变化：处理24h后，实验组贴壁能力明显下降，部分细胞呈漂浮生长状态，而阴性对照组细胞贴壁能力未有明显变化，贴壁良好。 2.2.2细胞大小和形态的变化：处理24h后，实验组少数细胞变小、变圆，而阴性对照组细胞形态和大小未有明显变化。随着处理时间的增长，药物浓度的增加，细胞散在分布，呈显著皱缩，甚至破裂，失去原有折光性，生长缓慢。而阴性对照组细胞数目增多贴壁生长，多角形，可见伪足，胞浆饱满，折光性强，相邻细胞生长汇合成片，逐渐形成细胞单层，无细胞脱落。 3免疫细胞化学s-p法检测 3.1TNF对A431细胞Caspase-3蛋白表达的影响：阴性对照组细胞经过免疫细胞化学染色后Caspase-3蛋白表达较少，其胞浆中有少量深棕色颗粒。经过浓度为1U/L、2U/L、5U/L的TNF-α作用48h后，Caspase-3蛋白的表达就有明显增高，并且随着作用时间的增长，作用药物浓度的增加，Caspase-3蛋白的表达逐渐增高。 3.2测定阿维A酸及阿维A联合TNF-α对A431细胞Caspase-3蛋白表达影响：阴性对照组细胞经过免疫细胞化学染色后Caspase-3蛋白表达较少，其胞浆中有少量深棕色颗粒。经过浓度为10μmol／L的阿维A酸，10μmol／L阿维A联合TNF-α作用48h后，10μmol／L阿维A联合TNF-α组Caspase-3蛋白的表达就有明显增高，且较单独10μmol／L阿维A组及TNF-α组增高明显。 4流式细胞术检测不同浓度药物作用后，细胞在G1前停滞。而TNF-α可以诱导细胞凋亡。 结论： 1TNF-α有效地抑制人表皮鳞癌细胞A431的增殖，促使人表皮鳞癌细胞A431的凋亡。 2TNF-α促使人表皮鳞癌细胞A431凋亡主要是通过激活Caspase-3途径导致的。 3阿维A酸与TNF-α联合应用可以促使人表皮鳞癌细胞A431凋亡，，并且比单一阿维A酸或TNF-α效果好。 4阿维A酸与TNF-α联合应用促使人表皮鳞癌细胞A431凋亡是通过激活Caspase-3蛋白的表达。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5
【相似文献】
相关期刊论文 前10条
1 Gottschling M.;Roewert J.;I. Nindl;周少娜;;日光角化病和皮肤鳞状细胞癌的Tenascin-C模式和剪切变异体[J];世界核心医学期刊文摘(皮肤病学分册);2006年12期
2 黄培根,莫梅英,沈淑静;裸小鼠自发性皮肤鳞状细胞癌的裸鼠间移植及其生物学初探[J];广东医学院学报;1993年Z1期
3 孙桂珍，张宗琴，朱晓琳，蓝毓滨，李春梅，宁晓明;皮肤鳞状细胞癌、基底细胞癌、恶性黑素瘤449例统计分析[J];临床皮肤科杂志;1995年01期
4 李强，国桂松;无水酒精局部注射治愈多原发皮肤鳞状细胞癌一例[J];河南肿瘤学杂志;1996年04期
5 杨海平;皮肤鳞状细胞癌中Ki-67和p53免疫反应性对预后的意义[J];国外医学.皮肤性病学分册;1998年01期
6 刘金晶;颗粒酶杀细胞的机制[J];中国医学科学院学报;2002年04期
7 张春玲,吴立军,田代真一,小野寺敏,池岛乔;冬凌草甲素通过激活caspase诱导HeLa细胞凋亡[J];中国药理学通报;2003年05期
8 亓麟;陈振雨;冷向锋;王彦进;朱月华;;RECK及MMP-9蛋白在皮肤鳞状细胞癌中的表达及临床病理意义(英文)[J];现代生物医学进展;2009年20期
9 郭丰富;金光庭;顾润国;;肾移植术后并发皮肤鳞状细胞癌一例[J];中华器官移植杂志;1996年02期
10 曾建新,王文亮;细胞凋亡信号转导的分子研究新进展[J];临床与实验病理学杂志;2000年06期
相关会议论文 前10条
1 刘林]
本文编号：2196757
本文链接：https://www.wllwen.com/yixuelunwen/pifb/2196757.html
最近更新
教材专著
