LXRs激动剂对人表皮鳞癌SCL-1细胞增殖作用的影响

发布时间：2018-05-27 23:26

本文选题：SCL-1细胞 + LXRs　；参考：《第三军医大学》2017年硕士论文

【摘要】：研究背景与目的皮肤鳞状细胞癌(squamous cell carcinoma,SCC)是一种常见的,发病率较高的皮肤角质形成细胞来源的恶性肿瘤。SCC的病因较复杂,其发病涉及紫外线长时间照射、环境与遗传因素、病毒感染等因素。目前的常用治疗手段主要有手术切除治疗。肝脏X受体(LXRs)作为转录因子的核激素受体,发现其参与了调控多种正常细胞和肿瘤细胞的增殖情况。其可降低包括血管平滑肌细胞,子宫内膜细胞,胰岛细胞,肝细胞,角质形成细胞和淋巴细胞的增殖,还对黑素瘤、乳腺癌、肺癌、前列腺癌等多种肿瘤都有着抑制细胞增殖的作用。前期研究也已发现LXRs在皮肤中有广泛表达,但目前尚未明确LXRs在皮肤鳞癌发生发展中的具体作用。Kruppel样因子4(KLF4)作为一种目前已知的重要的蛋白质,存在于多种组织和细胞中,如广泛表达于肺、肠、皮肤、睾丸、胸膜、心肌等器官或组织。它在体内不但参与调节不同细胞的生长、分化以及凋亡等生物学过程,还参与了炎症及多种肿瘤相关的疾病的发生发展过程,其在调节细胞增殖上既有抑制肿瘤的作用也有促进肿瘤发生的作用。目前研究发现KLF4在鳞状细胞的增殖上是作为抑癌因子存在的,但具体作用机制仍有待进一步研究。鉴于LXRs及KLF4在皮肤鳞癌发生和发展中的可能作用,本实验观察了LXRs激动剂T0901317对体外人表皮鳞癌SCL-1细胞增殖和对裸鼠异体移植肿瘤生长的影响,以探讨KLF4因子在LXRs途径抑制皮肤鳞癌SCL-1细胞增殖中的作用。方法1.裸鼠皮肤鳞癌SCL-1细胞荷瘤模型构建及肿瘤生长曲线测定:皮下注射SCL-1细胞构建裸鼠荷瘤模型,给予T0901317试剂灌胃处理后测量实验组及对照组异体移植肿瘤生长数据,对比两组肿瘤的生长曲线情况。2.SCL-1细胞增殖率检测:采用CCK-8法检测不同浓度T0901317(1、10、20μmol/L)作用SCL-1细胞24、48、72h后细胞的增殖率。3.SCL-1细胞的细胞周期检测:用流式细胞术检测不同浓度T0901317(1、10、20μmol/L)作用SCL-1细胞24h后的细胞周期分布情况。4.SCL-1细胞的细胞周期相关因子的m RNA表达:RT-PCR法检测不同浓度T0901317(1、10、20μmol/L)作用SCL-1细胞24h后细胞周期相关因子PCNA、p21、p27、CCND1的基因表达变化。5.SCL-1细胞的细胞周期相关因子的蛋白表达:用Western blot检测不同浓度T0901317(1、10、20μmol/L)作用SCL-1细胞24h后细胞周期相关因子PCNA、p21、p27、CCND1以及KLF4因子的蛋白表达变化。结果1.裸鼠荷瘤实验组与对照组生长曲线可见较明显差异,实验组肿瘤体积小于对照组肿瘤体积。在体内LXRs激动剂作用可一定程度抑制肿瘤的体积大小。2.T0901317在一定范围内呈时间和浓度依赖性地抑制体外培养的表皮鳞癌SCL-1细胞增殖。3.T0901317作用于表皮鳞癌SCL-1细胞24h后,SCL-1细胞的G1期百分比上升,而S期、G2期百分比下降。4.T0901317作用于表皮鳞癌SCL-1细胞24h后,在一定浓度范围内诱导了P21的表达增加,KLF4因子蛋白的表达随药物浓度也呈增加趋势,PCNA的表达趋势与P21呈相反的趋势,而其他细胞周期相关因子表达未见明显差异。结论LXRs激动剂可以抑制体内及体外的SCL-1细胞的增殖,其可能的机制为抑制细胞周期的进行,即通过调节细胞周期负性调节因子p21的表达,导致细胞周期静息甚至停滞于G1期,从而抑制细胞增殖。并且其在作用过程中,KLF4因子可能起重要的作用,但具体机制仍需要进一步的研究阐明。
[Abstract]:Background and objective squamous cell carcinoma (SCC) is a common cause of high incidence of cutaneous keratinocyte derived from the malignant tumor of.SCC, the cause of which is complicated, which involves long ultraviolet radiation, environmental and genetic factors, and disease and other factors. The main methods of treatment are mainly hands. The liver X receptor (LXRs), as a nuclear hormone receptor of the transcription factor, has been found to be involved in regulating the proliferation of various normal and tumor cells. It can reduce the proliferation of vascular smooth muscle cells, endometrium cells, islet cells, hepatocytes, keratinocytes and lymphocytes, and also to melanoma, breast cancer, and lung. A variety of cancers, such as cancer and prostate cancer, have the effect of inhibiting cell proliferation. Previous studies have also found that LXRs is widely expressed in the skin, but the specific role of LXRs in the development of squamous cell carcinoma of the skin is not yet clear,.Kruppel like factor 4 (KLF4), as an important known protein, exists in a variety of tissues and cells. It is widely expressed in lung, intestines, skin, testis, pleura, myocardium and other organs or tissues. It not only participates in the biological process of regulating the growth, differentiation and apoptosis of different cells, but also participates in the process of inflammation and the development of various tumor related diseases. It has the effect of regulating the proliferation of cells as well as inhibiting the tumor. The present study shows that KLF4 exists in the proliferation of squamous cells as tumor suppressor factors, but the specific mechanism remains to be further studied. In view of the possible role of LXRs and KLF4 in the development and development of squamous cell carcinoma of the skin, this experiment observed the proliferation of SCL-1 cells in human epidermal squamous cell carcinoma and the proliferation of LXRs agonist T0901317 in vitro. The effect of KLF4 factor on the proliferation of SCL-1 cells in skin squamous cell carcinoma by LXRs pathway in nude mice. Method 1. SCL-1 cell tumor model construction and tumor growth curve determination in nude mice skin squamous cell carcinoma: subcutaneous injection of SCL-1 cells to construct nude mice bearing tumor model, and to give T0901317 reagents after gavage treatment Test group and control group allograft tumor growth data, compared the growth curve of two groups of tumor growth curve.2.SCL-1 cell proliferation rate detection: CCK-8 method was used to detect the proliferation rate of SCL-1 cell 24,48,72h after 24,48,72h (1,10,20 mu mol/L) 24,48,72h cell proliferation rate.3.SCL-1 cell cycle detection: using flow cytometry to detect different concentrations of T09013 17 (1,10,20 mu mol/L) cell cycle distribution of SCL-1 cells 24h, m RNA expression of cell cycle related factors in.4.SCL-1 cells: RT-PCR method for detecting different concentrations of T0901317 (1,10,20 mu mol/L) action of cell cycle related factors after SCL-1 cells Protein expression of Western blot was used to detect the changes of cell cycle related factors PCNA, p21, p27, CCND1, and KLF4 factor expression after 24h (1,10,20 mu mol/L) of different concentrations of T0901317 (1,10,20 mu mol/L) in SCL-1 cells. Results the growth curve of the 1. nude mice bearing tumor experimental group was significantly different from that of the control group, and the tumor volume of the experimental group was less than that of the control group. The volume of LXRs agonists in the body inhibited the tumor size to a certain extent.2.T0901317 in a certain range of time and concentration dependent inhibition of the proliferation of epidermal squamous cell carcinoma SCL-1 cell proliferation.3.T0901317 in the epidermal squamous cell carcinoma SCL-1 cell 24h, the percentage of G1 phase in SCL-1 cells increased, while S phase, G2 phase percentage decreased.4. After the effect of T0901317 on the SCL-1 cell 24h of epidermal squamous cell carcinoma, the expression of P21 increased in a certain concentration range. The expression of KLF4 factor protein increased with the concentration of drug, and the tendency of PCNA expression was opposite to P21, but the expression of other cell cycle related factors was not significantly different. Conclusion LXRs agonist could inhibit the body. And the proliferation of SCL-1 cells in vitro, its possible mechanism is to inhibit the cell cycle, that is, by regulating the expression of the negative regulatory factor p21 in the cell cycle, resulting in the cell cycle resting even stagnating in the G1 phase, thus inhibiting the proliferation of cells. And in the process of action, the KLF4 may play an important role, but the specific mechanism still needs to be done. Further research clarifies.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5

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