發(fā)布時(shí)間：2018-07-26 12:20

【摘要】：背景結(jié)直腸癌是常見(jiàn)的消化道惡性腫瘤,其發(fā)病率居所有惡性腫瘤第三位,死亡率居所有惡性腫瘤第二位。人參皂苷Rg3是由人參中提取的活性成分,其可抑制腫瘤細(xì)胞的增殖、遷移和侵襲,并促進(jìn)腫瘤細(xì)胞凋亡。WEE1蛋白是調(diào)控細(xì)胞周期G2檢查點(diǎn)的重要基因,其表達(dá)變化可影響DNA損傷藥物順鉑、紫杉醇、5-氟尿嘧啶等藥物對(duì)細(xì)胞的殺傷作用。在胃癌和卵巢癌的研究中發(fā)現(xiàn),人參皂苷可協(xié)同增敏順鉑和5-氟尿嘧啶對(duì)細(xì)胞的增殖抑制效應(yīng)。然而,人參皂苷Rg3、DNA損傷藥物和WEE1蛋白之間的作用并未見(jiàn)相關(guān)報(bào)道,本研究主要探討三者之間的相互關(guān)系,探討人參皂苷Rg3增敏DNA損傷藥物的相關(guān)分子機(jī)制。第一部分人參皂苷Rg3通過(guò)抑制WEE1激酶表達(dá)增敏結(jié)腸癌對(duì)DNA損傷藥物敏感性的機(jī)制研究研究目的探討人參皂苷Rg3是否可影響結(jié)腸癌細(xì)胞對(duì)DNA損傷藥物順鉑和5-氟尿嘧啶的增殖抑制效應(yīng)及其機(jī)制。研究方法1.為了探討人參皂苷Rg3聯(lián)合DNA損傷藥物順鉑或5-氟尿嘧啶對(duì)Wi Dr、SW948和COLO205等結(jié)腸癌細(xì)胞增殖活性的影響。應(yīng)用CCK-8細(xì)胞增殖活性檢測(cè)法,檢測(cè)不同濃度人參皂苷Rg3聯(lián)合不同濃度DNA損傷藥物順鉑或5-氟尿嘧啶干預(yù)Wi Dr、SW948和COLO205三株結(jié)腸癌細(xì)胞后對(duì)其細(xì)胞增殖活性的影響。2.為了探討人參皂苷Rg3影響結(jié)腸癌細(xì)胞增殖活性的潛在機(jī)制。應(yīng)用q RT-PCR法檢測(cè)不同濃度人參皂苷Rg3干預(yù)相同時(shí)間和相同濃度人參皂苷Rg3干預(yù)不同時(shí)間后結(jié)腸癌細(xì)胞內(nèi)WEE1的表達(dá)水平變化。3.為了探討WEE1過(guò)表達(dá)在人參皂苷Rg3影響結(jié)腸癌細(xì)胞對(duì)DNA損傷藥物順鉑和5-氟尿嘧啶敏感性過(guò)程中的作用。首先應(yīng)用質(zhì)粒過(guò)表達(dá)技術(shù)對(duì)Wi Dr,SW948和COLO205等結(jié)腸癌細(xì)胞進(jìn)行WEE1過(guò)表達(dá),并應(yīng)用q RT-PCR法檢測(cè)過(guò)表達(dá)程度;其次應(yīng)用CCK-8細(xì)胞增殖活性檢測(cè)法檢測(cè)10μM/L順鉑+75μM/L人參皂苷Rg3+不同pc DNA-WEE1濃度和3μM/L 5-氟尿嘧啶+50μM/L人參皂苷Rg3+不同pc DNA-WEE1濃度等處理對(duì)于結(jié)腸癌細(xì)胞增殖活力的影響。結(jié)果1.順鉑和5-氟尿嘧啶對(duì)于結(jié)腸癌細(xì)胞Wi Dr、SW948和COLO205等增殖活力的抑制成濃度依賴性增加;隨著應(yīng)用人參皂苷Rg3濃度(0μM/L、25μM/L、50μM/L、75μM/L)的增加,在應(yīng)用相同濃度順鉑或5-氟尿嘧啶的前提下,Wi Dr,SW948和COLO205等結(jié)腸癌細(xì)胞的增殖活力逐漸降低;2.應(yīng)用人參皂苷Rg3干預(yù)三株結(jié)腸癌細(xì)胞,隨著人參皂苷Rg3干預(yù)濃度(0μM/L、25μM/L、50μM/L、75μM/L和100μM/L)的增加,Wi Dr、SW948和COLO205等結(jié)腸癌細(xì)胞中WEE1的表達(dá)明顯降低;應(yīng)用50μM/L人參皂苷Rg3干預(yù)三株結(jié)腸癌細(xì)胞,隨著人參皂苷Rg3干預(yù)時(shí)間(0 h、6 h、12 h、18 h和24h)的增加,Wi Dr,SW948和COLO205等結(jié)腸癌細(xì)胞中WEE1的表達(dá)逐漸降低;3.應(yīng)用pc DNA-WEE1質(zhì)粒可在三株結(jié)腸癌細(xì)胞中成功上調(diào)WEE1的表達(dá);在相同濃度順鉑(10μM/L)+人參皂苷Rg3(75μM/L)干預(yù)的前提下,隨著pc DNA-WEE1質(zhì)粒轉(zhuǎn)染濃度(10 ng、50 ng、100 ng、150 ng)的增加,Wi Dr、SW948和COLO205等結(jié)腸癌細(xì)胞的增殖活力逐漸增加;在相同濃度5-氟尿嘧啶(3μM/L)+人參皂苷Rg3(50μM/L)干預(yù)的前提下,隨著pc DNA-WEE1質(zhì)粒轉(zhuǎn)染濃度(10ng、50 ng、100 ng、150 ng)的增加,Wi Dr、SW948和COLO205等結(jié)腸癌細(xì)胞的增殖活力逐漸增加。結(jié)論1.人參皂苷Rg3可增敏DNA損傷藥物(順鉑和5-氟尿嘧啶)對(duì)結(jié)腸癌細(xì)胞增殖活力的抑制效應(yīng);2.人參皂苷Rg3對(duì)WEE1表達(dá)的抑制效應(yīng)呈濃度依賴性和時(shí)間依賴性增加;3.過(guò)表達(dá)WEE1可減弱人參皂苷Rg3增敏DNA損傷藥物(順鉑和5-氟尿嘧啶)對(duì)結(jié)腸癌細(xì)胞增殖活力的抑制效應(yīng)。第二部分WEE1在結(jié)直腸癌組織中的表達(dá)及臨床病理意義分析研究目的探討WEE1蛋白在結(jié)直腸癌中的表達(dá)與結(jié)直腸癌患者臨床病理參數(shù)和預(yù)后的相關(guān)性。研究方法1.為了探討WEE1在結(jié)直腸癌組織和癌旁組織中的表達(dá),應(yīng)用Western blot和q RT-PCR法檢測(cè)新鮮結(jié)直腸癌組織和癌旁組織中WEE1的表達(dá);2.為了明確WEE1蛋白在結(jié)直腸癌組織中的表達(dá)與結(jié)直腸癌患者臨床病理資料的相關(guān)性。應(yīng)用卡方檢驗(yàn)分析WEE1蛋白的陰性表達(dá)和陽(yáng)性表達(dá)與結(jié)直腸癌患者的年齡、性別、腫瘤部位、腫瘤直徑、腫瘤分化程度、有無(wú)淋巴結(jié)轉(zhuǎn)移、有無(wú)遠(yuǎn)處轉(zhuǎn)移、血清CEA水平、有無(wú)腹膜轉(zhuǎn)移、T分期、N分期、M分期及TNM分期等臨床病理參數(shù)的相關(guān)性;3.為了探討WEE1蛋白在結(jié)直腸癌組織中的表達(dá)與結(jié)直腸癌患者預(yù)后的相關(guān)性,應(yīng)用Kaplan-Meier法繪制生存曲線,以Log-rank檢驗(yàn)比較WEE1蛋白的陰性表達(dá)和陽(yáng)性表達(dá)與患者預(yù)后的相關(guān)性;應(yīng)用單因素和多因素Cox回歸分析影響結(jié)直腸癌患者預(yù)后的危險(xiǎn)因素。研究結(jié)果1.在33例結(jié)直腸癌患者病理標(biāo)本中,18例患者的結(jié)直腸癌組織中,WEE1蛋白和m RNA呈高表達(dá);而15例患者的結(jié)直腸癌組織中,WEE1蛋白和m RNA呈低表達(dá);2.免疫組織化學(xué)結(jié)果示,在142例患者的石蠟包埋結(jié)直腸癌組織中,WEE1蛋白在86例中呈陽(yáng)性表達(dá),在56例中呈陰性表達(dá);3.WEE1陽(yáng)性表達(dá)與結(jié)直腸癌患者的腫瘤分化程度(P=0.033)、N分期(P=0.005)、TNM分期(P=0.009)、Duke’s分期(P=0.001)和淋巴結(jié)轉(zhuǎn)移(P=0.006)等參數(shù)有明顯相關(guān)性,結(jié)果具有統(tǒng)計(jì)學(xué)意義;4.根據(jù)WEE1蛋白表達(dá)水平,將所有結(jié)直腸癌患者分為陽(yáng)性表達(dá)組和陰性表達(dá)組,WEE1陽(yáng)性表達(dá)組患者的平均生存時(shí)間明顯短于WEE1陰性表達(dá)組;單因素分析結(jié)果示,腫瘤大小(HR=0.5,95%置信區(qū)間0.263-0.949,P=0.034)、TNM分期(HR=1.402,95%置信區(qū)間0.934-6.176,P=0.012)、M分期(HR=1.562,95%置信區(qū)間0.231-4.685,P=0.002)、肝轉(zhuǎn)移(HR=3.462,95%置信區(qū)間1.265-10.256,P=0.014)和WEE1表達(dá)(HR=7.608,95%置信區(qū)間3.019-19.185,P0.001)是影響結(jié)直腸癌患者預(yù)后的危險(xiǎn)因素;多因素Cox比例風(fēng)險(xiǎn)回歸模型結(jié)果示,TNM分期(HR=2.899,95%置信區(qū)間2.072-8.056,P0.001)和WEE1表達(dá)(HR=6.533,95%置信區(qū)間2.93-14.566,P0.001)是影響結(jié)直腸癌患者預(yù)后的獨(dú)立危險(xiǎn)因素。5.在I期結(jié)直腸癌患者中,WEE1的表達(dá)與患者OS和DFS并無(wú)明顯相關(guān)性,而在II期和III期結(jié)直腸癌患者患者中,WEE1陽(yáng)性表達(dá)提示患者OS和DFS較差。研究結(jié)論WEE1在結(jié)直腸癌組織中呈高表達(dá);其陽(yáng)性表達(dá)提示結(jié)直腸癌患者預(yù)后較差;WEE1陽(yáng)性表達(dá)和TNM分期是影響結(jié)直腸癌患者預(yù)后的獨(dú)立危險(xiǎn)因素。
[Abstract]:Background colorectal cancer (CRC) is a common malignant tumor of the digestive tract, with the incidence of third of all malignant tumors and second of all malignant tumors. Ginsenoside Rg3 is the active ingredient extracted from ginseng, which can inhibit the proliferation, migration and invasion of tumor cells, and promote the apoptosis of tumor cell.WEE1 protein to regulate cell cycle G2 An important gene of the checkpoint, its expression changes can affect the killing effect of DNA damage drugs cisplatin, paclitaxel, 5- fluorouracil and other drugs on cells. In the study of gastric and ovarian cancer, ginsenoside can synergistically sensitized cisplatin and 5- fluorouracil to inhibit the proliferation of cells. However, ginsenoside Rg3, DNA damage drugs and WEE1 eggs The role of white is not reported. This study mainly discusses the relationship between the three and probes into the molecular mechanism of ginsenoside Rg3 sensitized DNA damage drug. Part 1. The mechanism of ginsenoside Rg3 by inhibiting the expression of WEE1 kinase to sensitist colon cancer to DNA damage drug sensitivity and study the objective of ginsenoside Rg3 Whether it can affect the proliferation inhibition effect of colon cancer cells on DNA damaged cisplatin and 5- fluorouracil and its mechanism. 1. in order to investigate the effect of ginsenoside Rg3 combined with DNA damaged cisplatin or 5- fluorouracil on the proliferation of Wi Dr, SW948 and COLO205 and other colon cancer cells. Detection of CCK-8 cell proliferation activity, detection method, detection Different concentrations of ginsenoside Rg3 combined with different concentrations of DNA injury drugs cisplatin or 5- fluorouracil in Wi Dr, SW948 and COLO205 three colon cancer cells effect on the cell proliferation activity.2. in order to explore the potential mechanism of ginsenoside Rg3 to influence the proliferation of colon cancer cells. The Q RT-PCR method was used to detect different concentrations of ginsenoside Rg3 dry Changes in the expression level of WEE1 in colon cancer cells after the intervention of the same time and the same concentration of ginsenoside Rg3 in order to explore the effect of WEE1 overexpression on Ginsenoside Rg3 effect on the sensitivity of colon cancer cells to DNA damaged cisplatin and 5- fluorouracil. First, the plasmid overexpression technique should be used for Wi Dr, SW948 and COLO. 205 of the colon cancer cells were overexpressed by WEE1, and the degree of overexpression was detected by Q RT-PCR method. Secondly, the CCK-8 cell proliferation activity detection method was used to detect the concentration of different PC DNA-WEE1 of cisplatin +75 mu M/L and 3 mu M/L 5- fluorouracil (3 mu M/L 5- fluorouracil). Results 1. cisplatin and 5- fluorouracil increased the inhibitory effect on the proliferation of Wi Dr, SW948 and COLO205 in colon cancer cells. With the increase of the concentration of ginsenoside Rg3 (0 M/L, 25 u M/L, 50 u M/L, 75 micron M/L), under the premise of the application of the same concentration of cisplatin or 5- fluorouracil, Wi The proliferation activity of colon cancer cells decreased gradually; 2. the expression of ginsenoside Rg3 in three colon cancer cells was significantly reduced with the increase of ginsenoside Rg3 intervention concentration (0 mu M/L, 25 mu M/L, 50 mu M/L, 75 mu M/L and 100 mu M/L), and the expression of WEE1 was significantly reduced in Wi Dr, SW948 and COLO205 colon cancer cells, and three colon cancer was interfered with 50 micronosides of ginsenoside. Cells, with the increase of ginsenoside Rg3 intervention time (0 h, 6 h, 12 h, 18 h and 24h), the WEE1 expression in Wi Dr, SW948, COLO205 and other colon cancer cells gradually decreased; 3. application of PC plasmid could successfully increase the expression in three colon cancer cells, under the premise of the intervention of the same concentration of cisplatin (10 mu) + ginsenoside (75 mu). With the increase of the transfection concentration of PC DNA-WEE1 plasmids (10 ng, 50 ng, 100 ng, 150 ng), the proliferation activity of Wi Dr, SW948, COLO205 and other colon cancer cells gradually increased. The proliferation activity of colon cancer cells such as 948 and COLO205 increased gradually. Conclusion 1. ginsenoside Rg3 can increase the inhibitory effect of DNA injury drugs (cisplatin and 5- fluorouracil) on the proliferation of colon cancer cells; 2. the inhibitory effect of ginsenoside Rg3 on WEE1 expression is concentration dependent and time dependent; 3. over expression of WEE1 can weaken ginseng soap. The inhibitory effect of glucoside Rg3 sensitized DNA damage drugs (cisplatin and 5- fluorouracil) on the proliferation of colon cancer cells. Second the expression and clinicopathological significance of part WEE1 in colorectal cancer tissue and its clinicopathological significance study on the correlation between the expression of WEE1 protein in colorectal cancer and the clinicopathological parameters and prognosis of colorectal cancer patients. Method 1. in order to investigate the expression of WEE1 in colorectal cancer tissue and para cancer tissue, the expression of WEE1 in fresh colorectal cancer tissues and adjacent tissues was detected by Western blot and Q RT-PCR. 2. in order to clarify the correlation between the expression of WEE1 protein in colorectal cancer tissue and the clinical data of colorectal cancer patients, chi square test analyzed WE. The negative expression and positive expression of E1 protein were related to the age, sex, tumor site, tumor location, tumor diameter, tumor differentiation, lymph node metastasis, distant metastasis, serum CEA level, T stage, N staging, M staging, TNM staging and other clinicopathological parameters; 3. The correlation between the expression of colorectal cancer and the prognosis of colorectal cancer patients. The survival curve was drawn by Kaplan-Meier method. The correlation between the negative expression and positive expression of WEE1 protein and the prognosis of the patients was compared with the Log-rank test. The risk factors affecting the prognosis of the patients with colorectal cancer were analyzed by single factor and multiple factor Cox regression analysis. 1. In 33 cases of colorectal cancer, 18 cases of colorectal cancer tissues, WEE1 protein and m RNA were highly expressed, while 15 cases of colorectal cancer tissues, WEE1 protein and m RNA were low expression; 2. immunohistochemical results showed that in 142 cases of paraffin embedded colorectal cancer tissues, the WEE1 protein was positive in 86 cases. Negative expression was found in 56 cases, and 3.WEE1 positive expression was significantly correlated with tumor differentiation (P=0.033), N staging (P=0.005), TNM staging (P=0.009), Duke 's staging (P=0.001) and lymph node metastasis (P=0.006), and the results were statistically significant. 4. according to the level of WEE1 protein expression, all patients with colorectal cancer were expressed. The average survival time of the WEE1 positive group was significantly shorter than that of the WEE1 negative expression group, and the single factor analysis showed that the tumor size (HR=0.5,95% confidence interval 0.263-0.949, P=0.034), TNM staging (HR=1.402,95% confidence interval 0.934-6.176, P=0.012), M stages (HR=1.562,95% confidence interval 0.231-4.685, HR=1.562,95%) =0.002), liver metastases (HR=3.462,95% confidence interval 1.265-10.256, P=0.014) and WEE1 expression (HR=7.608,95% confidence interval 3.019-19.185, P0.001) are the risk factors affecting the prognosis of colorectal cancer patients. The multifactor Cox proportional risk regression model results show that TNM stages (HR= 2.899,95% confidence interval) and expressions are expressed. 2.93-14.566, P0.001) is an independent risk factor affecting the prognosis of colorectal cancer patients.5. in I patients with colorectal cancer, the expression of WEE1 is not associated with the patients' OS and DFS, but in patients with II and III, WEE1 positive expression suggests that OS and DFS are poor. The positive expression indicates that the prognosis of patients with colorectal cancer is poor. WEE1 positive expression and TNM stage are independent risk factors for prognosis of colorectal cancer.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.35

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