【摘要】：研究背景:甲狀腺癌是內分泌系統(tǒng)最常見的惡性腫瘤之一,已成為國際上發(fā)病率增長最快的實體瘤。臨床上用于甲狀腺癌早期篩查的指標極為匱乏,亟待尋找新的分子標志物。大量研究發(fā)現生物節(jié)律基因在大腸癌、乳腺癌等腫瘤的發(fā)生及進展中發(fā)揮著重要作用。其中CLOCK、BMAL1、NPAS2三種生物節(jié)律基因的蛋白產物組成正性調控元件,驅動生物節(jié)律的產生,并通過調控下游c-Myc、P53、P21等大量鐘控基因參與腫瘤細胞增殖、侵襲、轉移等重要功能,但生物節(jié)律基因在甲狀腺癌領域的研究較少。課題組前期通過免疫組化預實驗,發(fā)現甲狀腺乳頭狀癌組織中CLOCK基因表達升高。這些提示我們CLOCK基因在甲狀腺癌的發(fā)生及進展中可能發(fā)揮重要作用,但其生物學作用及分子機制尚不清楚。目的:利用CLOCK基因siRNA干涉片段,下調甲狀腺癌細胞中CLOCK基因表達水平,分析干涉后甲狀腺癌細胞的生物學功能變化。并檢測P21、P53、CyclinD1等鐘控基因的表達變化,初步探討CLOCK基因參與甲狀腺癌發(fā)生及進展的分子作用機制。方法:常規(guī)培養(yǎng)甲狀腺癌細胞株BCPAP,常規(guī)培養(yǎng)。根據CLOCK基因設計特異性的siRNA,利用Lipofecter 2000試劑轉染BCPAP細胞,將甲狀腺癌細胞株分為空白對照組(KD組),轉染試劑對照組(ZD組),單純siRNA對照組(SD),陰性對照組(YD組)和干涉組(GS組)5組。給予不同處理,利用Western Blot檢測干涉效率,CCK8實驗檢測細胞增殖能力,粘附實驗檢測細胞粘附能力,流式細胞儀檢測細胞凋亡程度,Transwell侵襲實驗評估侵襲與轉移能力。并通過Western Blot檢測P21、P53、CyclinD1等分子在干涉CLOCK基因后的表達變化。結果:1.干涉組(GS組)BCPAP細胞增殖能力較空白對照組(GD組)降低(P0.05)。ZD組、SD組、YD組與GD組比無明顯改變(P0.05)。2.干涉組(GS組)BCPAP細胞凋亡較空白對照組(GD組)增多(P0.05)。ZD組、SD組、YD組與GD組比無明顯改變(P0.05)。3.干涉組(GS組)BCPAP細胞粘附能力較空白對照組(GD組)減低(P0.05)。ZD組、SD組、YD組與GD組比無明顯改變(P0.05)。4.干涉組(GS組)BCPAP遷移及侵襲細胞數較空白對照組(GD組)減少(P0.05)。ZD組、SD組、YD組與GD組比無明顯改變(P0.05)。5.P21、P53蛋白在干涉組(GS組)較空白對照組(GD組)升高(P0.05),CyclinD1蛋白在干涉組(GS組)較空白對照組(GD組)降低(P0.05)。結論:1.下調CLOCK基因的表達可以促進甲狀腺癌細胞凋亡,抑制甲狀腺癌細胞增殖、浸潤及轉移。2.CLOCK基因可能是通過調控甲狀腺癌細胞中P21、P53和CyclinD1重要基因的表達,參與甲狀腺癌的發(fā)生及進展。
[Abstract]:Background: thyroid carcinoma is one of the most common malignant tumors in the endocrine system. Clinical indicators for early screening of thyroid cancer are extremely scarce, so it is urgent to find new molecular markers. A large number of studies have found that biological rhythm genes play an important role in the occurrence and progression of colorectal cancer and breast cancer. The protein products of the three biological rhythm genes of CLOCK,BMAL1,NPAS2 constitute positive regulatory elements, which drive the production of biological rhythms, and participate in the important functions of tumor cell proliferation, invasion and metastasis by regulating a large number of clock control genes such as c-Myche P53, P21 and so on. However, biological rhythm genes in the field of thyroid cancer are less studied. We found that the expression of CLOCK gene in papillary thyroid carcinoma was higher than that in thyroid papillary carcinoma by immunohistochemistry. These results suggest that our CLOCK gene may play an important role in the carcinogenesis and progression of thyroid carcinoma, but its biological role and molecular mechanism are still unclear. Aim: to down-regulate the expression of CLOCK gene in thyroid cancer cells by siRNA interference fragment of CLOCK gene and analyze the biological function of thyroid cancer cells after intervention. The expression of P21, P53, CyclinD1 and other clock controlled genes was detected, and the molecular mechanism of CLOCK gene involved in the carcinogenesis and progression of thyroid carcinoma was preliminarily investigated. Methods: thyroid cancer cell line BCPAP, was cultured routinely. Thyroid cancer cells were transfected into BCPAP cells by Lipofecter 2000 reagent according to CLOCK gene design. Thyroid cancer cells were divided into five groups: blank control group (KD group), transfection reagent control group (ZD group), siRNA control group (SD), negative control group (YD group) and intervention group (GS group). Different treatments were used to detect the ability of cell proliferation by Western Blot and CCK8, and the degree of cell apoptosis by flow cytometry and the ability of invasion and metastasis by transwell invasion assay. The expression of P21, P53, CyclinD1 and other molecules were detected by Western Blot after interfering with CLOCK gene. The result is 1: 1. The proliferative ability of BCPAP cells in the intervention group (GS group) was lower than that in the blank control group (GD group) (P0.05). There was no significant difference between the SD group (P0.05) and the GD group (P0.05). 2. The apoptosis of BCPAP cells in the intervention group (GS group) was higher than that in the blank control group (GD group) (P0.05). The adhesion ability of BCPAP cells in the intervention group (GS group) was lower than that in the blank control group (GD group) (P0.05). There was no significant difference between the SD group and the GD group (P0.05). 4. The number of BCPAP migration and invasion cells in the intervention group (GS group) was lower than that in the blank control group (GD group) (P0.05). There was no significant change in the number of BCPAP migration and invasion cells between the SD group and the GD group in the ZD group (P0.05). The expression of p53 protein in the intervention group (GS group) was higher than that in the blank control group (GD group) (P0.05). The expression of CyclinD1 protein in the intervention group (GS group) was higher than that in the control group (P0.05). ) compared with the blank control group (GD group) decreased (P0.05). Conclusion 1. Down-regulating the expression of CLOCK gene can promote the apoptosis of thyroid cancer cells, inhibit the proliferation, invasion and metastasis of thyroid cancer cells. 2. CLOCK gene may regulate the expression of P21, p53 and CyclinD1 genes in thyroid cancer cells. Participate in the development and progression of thyroid carcinoma.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R736.1

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