本文選題：A431細胞　切入點：熊果酸　出處：《南京醫(yī)科大學》2010年碩士論文　論文類型：學位論文

【摘要】： 背景和目的: 近年來皮膚癌特別是鱗狀細胞癌(以下簡稱鱗癌)的發(fā)生率明顯上升,是一種侵襲性較強的腫瘤,預后較差。熊果酸(Ursolic acid,UA)是存在于天然植物中的一種三萜類化合物,是多種抗癌中草藥的主要抗腫瘤活性成分之一。研究表明,UA對多種腫瘤具有明顯的抑制作用,但其抗腫瘤的確切機制和途徑尚未完全明確。目前有關UA對人皮膚鱗癌是否具有抗癌作用及其機制尚報道較少。核轉錄因子кB(uclear factor—kappa B,NF-кB)是近年來發(fā)現(xiàn)的轉錄調控因子NF-кB, NF-кB可通過調控Bcl-2、IAPs、TRAF等抗凋亡基因的表達,抑制腫瘤細胞凋亡。本文將觀察UA能否減少裸鼠荷瘤的生長體積和質量,并探討UA是否能通過NF-кB通路誘導皮膚鱗癌細胞凋亡,為皮膚鱗癌的臨床用藥提供基礎證據。 方法: 1.細胞培養(yǎng):培養(yǎng)人皮膚鱗癌細胞株A431細胞和人皮膚角質形成細胞株HaCaT細胞。 2.腫瘤接種及給藥:取A431細胞懸液接種于雄性BALB/c裸鼠裸鼠左腋下,接種后10天給藥,以腹腔注射方式給予UA,以氟尿嘧啶(5-FU)做對照,隔日注射一次,共10次。 3.腫瘤鑒定:取腫瘤組織HE切片進行組織鑒定。 4.藥物安全性觀察:觀察給藥后小鼠的身體狀況,包括皮膚色澤、飲食,大小便、精神狀態(tài)等并取肝、脾病理切片觀察細胞壞死程度。 5. UA抑癌作用觀察:隔日一次測量腫瘤長徑a(mm),及相垂直短徑b(mm),并用公式V(mm3)=(a×b~2)×1/2計算體積,20日后剝離腫瘤稱重.。抑制率(%)=(對照組平均瘤重—實驗組平均瘤重)/對照組平均瘤重×100% 6. UA對皮膚鱗癌細胞增殖活性影響:以MTT法檢測不同濃度UA及5-FU對A431細胞增殖活性變化并篩選合適藥物濃度。 7. UA對皮膚鱗癌細胞凋亡率影響:以流式細胞儀和Hoechst染色檢測細胞凋亡水平變化。 8. UA對NF-кB信號通路相關蛋白表達影響:分別提取用藥后不同時間細胞蛋白,以Western blot法檢測pIкBα、Bcl-2、c-IAP-2和IкBα蛋白表達水平。結果 1.接種腫瘤組織鑒定:經病理組織學鑒定荷瘤鼠皮膚內所取組織為為皮膚鱗癌。 2.藥物安全性:UA組藥物生理副作用小于5-FU組。 3. UA抑癌作用: UA能明顯抑制裸鼠皮膚鱗癌組織生長,并呈濃度依賴性。 4. UA對皮膚鱗癌細胞增殖活性影響:UA對A431細胞增殖活性有顯著抑制作用,且呈時間-劑量依賴性。UA對HaCaT細胞的增殖的抑制作用卻遠低于5-FU。 5. UA對皮膚鱗癌細胞凋亡率影響: UA能誘導A431細胞凋亡,并呈濃度依賴性,但對HaCaT細胞凋亡率低于5-FU。 6. NF-кB信號通路相關蛋白表達變化:pIкBα、Bcl-2、c-IAP-2的表達水平逐漸下降,IкBα蛋白的表達水平逐漸增高。 結論 UA對裸鼠皮膚鱗癌有明顯的抑制作用,并呈濃度依賴性。UA對A431細胞增殖活性具有明顯的抑制作用。UA能明顯誘導A431細胞凋亡,且有較特異的殺傷作用。UA可能通過阻斷NF-кB通路,進而引起B(yǎng)cl-2和c-IAP2等抗凋亡基因的表達減少,從而誘導皮膚鱗癌細胞凋亡。
[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.

【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R739.5

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本文編號：1559199