熊果酸对人皮肤鳞癌的抑制作用及机制研究

发布时间：2018-03-03 02:33

本文选题：A431细胞　切入点：熊果酸　出处：《南京医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 背景和目的: 近年来皮肤癌特别是鳞状细胞癌(以下简称鳞癌)的发生率明显上升,是一种侵袭性较强的肿瘤,预后较差。熊果酸(Ursolic acid,UA)是存在于天然植物中的一种三萜类化合物,是多种抗癌中草药的主要抗肿瘤活性成分之一。研究表明,UA对多种肿瘤具有明显的抑制作用,但其抗肿瘤的确切机制和途径尚未完全明确。目前有关UA对人皮肤鳞癌是否具有抗癌作用及其机制尚报道较少。核转录因子кB(uclear factor—kappa B,NF-кB)是近年来发现的转录调控因子NF-кB, NF-кB可通过调控Bcl-2、IAPs、TRAF等抗凋亡基因的表达,抑制肿瘤细胞凋亡。本文将观察UA能否减少裸鼠荷瘤的生长体积和质量,并探讨UA是否能通过NF-кB通路诱导皮肤鳞癌细胞凋亡,为皮肤鳞癌的临床用药提供基础证据。方法: 1.细胞培养:培养人皮肤鳞癌细胞株A431细胞和人皮肤角质形成细胞株HaCaT细胞。 2.肿瘤接种及给药:取A431细胞悬液接种于雄性BALB/c裸鼠裸鼠左腋下,接种后10天给药,以腹腔注射方式给予UA,以氟尿嘧啶(5-FU)做对照,隔日注射一次,共10次。 3.肿瘤鉴定:取肿瘤组织HE切片进行组织鉴定。 4.药物安全性观察:观察给药后小鼠的身体状况,包括皮肤色泽、饮食,大小便、精神状态等并取肝、脾病理切片观察细胞坏死程度。 5. UA抑癌作用观察:隔日一次测量肿瘤长径a(mm),及相垂直短径b(mm),并用公式V(mm3)=(a×b~2)×1/2计算体积,20日后剥离肿瘤称重.。抑制率(%)=(对照组平均瘤重—实验组平均瘤重)/对照组平均瘤重×100% 6. UA对皮肤鳞癌细胞增殖活性影响:以MTT法检测不同浓度UA及5-FU对A431细胞增殖活性变化并筛选合适药物浓度。 7. UA对皮肤鳞癌细胞凋亡率影响:以流式细胞仪和Hoechst染色检测细胞凋亡水平变化。 8. UA对NF-кB信号通路相关蛋白表达影响:分别提取用药后不同时间细胞蛋白,以Western blot法检测pIкBα、Bcl-2、c-IAP-2和IкBα蛋白表达水平。结果 1.接种肿瘤组织鉴定:经病理组织学鉴定荷瘤鼠皮肤内所取组织为为皮肤鳞癌。 2.药物安全性:UA组药物生理副作用小于5-FU组。 3. UA抑癌作用: UA能明显抑制裸鼠皮肤鳞癌组织生长,并呈浓度依赖性。 4. UA对皮肤鳞癌细胞增殖活性影响:UA对A431细胞增殖活性有显著抑制作用,且呈时间-剂量依赖性。UA对HaCaT细胞的增殖的抑制作用却远低于5-FU。 5. UA对皮肤鳞癌细胞凋亡率影响: UA能诱导A431细胞凋亡,并呈浓度依赖性,但对HaCaT细胞凋亡率低于5-FU。 6. NF-кB信号通路相关蛋白表达变化:pIкBα、Bcl-2、c-IAP-2的表达水平逐渐下降,IкBα蛋白的表达水平逐渐增高。结论 UA对裸鼠皮肤鳞癌有明显的抑制作用,并呈浓度依赖性。UA对A431细胞增殖活性具有明显的抑制作用。UA能明显诱导A431细胞凋亡,且有较特异的杀伤作用。UA可能通过阻断NF-кB通路,进而引起Bcl-2和c-IAP2等抗凋亡基因的表达减少,从而诱导皮肤鳞癌细胞凋亡。
[Abstract]:Background and purpose:
In recent years, skin cancer especially squamous cell carcinoma (hereinafter referred to as squamous cell carcinoma) were significantly increased, a strong aggressive tumor with poor prognosis. Ursolic acid (Ursolic acid UA) is a kind of three terpene compounds found in natural plants, is one of the main antitumor constituents of various anti-cancer herbs in. The study shows that UA has obvious inhibitory effects on many kinds of cancer, but the exact mechanism and method of anti tumor has not yet completely clear. At present the UA on human skin squamous cell carcinoma has anticancer effect and mechanism is rarely reported. The nuclear transcription factor kappa B (uclear factor kappa B, NF- K B) is in recent years, found that the transcription factor NF- kappa B, NF- kappa B can be regulated by Bcl-2, IAPs, anti apoptosis gene expression of TRAF, inhibit the apoptosis of tumor cell. This paper will investigate whether UA can reduce the volume and weight of tumor growth in nude mice, and to explore whether UA can Induced apoptosis of skin squamous cell carcinoma NF- cells through NF kappa B pathway, and provide the basis for clinical evidence of squamous cell carcinoma.
Method:
1. cell culture: cultured human skin squamous cell carcinoma cell line A431 cells and human skin keratinocyte cell line HaCaT cells.
2. tumor inoculation and administration: A431 cell suspension was inoculated into the left axilla of BALB/c nude mice. After 10 days inoculation, UA was administered by intraperitoneal injection, with fluorouracil (5-FU) as a control, and injected 10 times every other day.
3. identification of tumor: tissue identification was carried out by HE section of tumor tissue.
4. drug safety observation: observe the physical condition of mice after the administration, including skin color, diet, urine and urine, mental state and so on, and take the liver and spleen pathological section to observe the degree of cell necrosis.
5. observe the anti-tumor effect of UA: the next day a measurement of tumor size a (mm), and vertical short diameter B (mm), using the formula V (mm3) = (a * b~2 * 1/2) to calculate the volume, 20 days after the tumor inhibition rate. Weighing (%) = (control group average the tumor weight of the experimental group, the average tumor weight) / control group the average tumor weight x 100%
The effect of 6. UA on the proliferation activity of skin squamous cell carcinoma cells: MTT assay was used to detect the proliferation of A431 cells with different concentrations of UA and 5-FU and to screen the appropriate drug concentration.
The effect of 7. UA on the apoptosis rate of skin squamous cell carcinoma cells: the change of cell apoptosis was detected by flow cytometry and Hoechst staining.
8. UA of NF- kappa B signaling pathway related protein expression: effects of different time after administration of cell protein was extracted to detect pI kappa B alpha, Western blot Bcl-2, c-IAP-2 and I kappa B alpha level protein expression results.
1. inoculated tumor tissue identification: histologically, the tissues of the tumor bearing mice were identified as skin squamous cell carcinoma.
2. drug safety: the physiological side effects of group UA were less than that in group 5-FU.
3. UA tumor suppressor effect: UA can obviously inhibit the growth of squamous cell carcinoma tissue in nude mice, and it is concentration dependent.
4., the effect of UA on the proliferation of skin squamous cell carcinoma: UA has a significant inhibitory effect on the proliferation activity of A431 cells, and in a dose time dependent manner, the inhibitory effect of.UA on the proliferation of HaCaT cells is much lower than that of 5-FU..
The effect of 5. UA on the apoptosis rate of skin squamous cell carcinoma cells: UA can induce apoptosis of A431 cells in a concentration dependent manner, but the apoptosis rate of HaCaT cells is lower than that of 5-FU.
6. expression of NF- kappa B signaling pathway related protein: pI kappa B alpha, Bcl-2, the expression level of c-IAP-2 decreased gradually, the expression level of I kappa B alpha protein increased gradually.
conclusion
UA has an obvious inhibitory effect on nude mouse skin squamous cell carcinoma, in a dose-dependent manner..UA has obvious inhibitory effect on.UA could induce apoptosis of A431 cells on A431 cell proliferation and killing effect of.UA may be more specific by blocking NF- kappa B pathway, resulting in reduced expression of anti apoptosis gene Bcl-2 and c-IAP2. In order to induce apoptosis of skin squamous cell carcinoma cells.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5

【参考文献】