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  本文選題：大腸癌　切入點：多藥耐藥　出處：《承德醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：大腸癌(colorectal cancer,CRC)是人類最常見的惡性腫瘤之一,嚴(yán)重危害著人類的健康。化療是治療中晚期大腸癌的一種重要策略,但多種原因?qū)е碌亩嗨幠退?multidrug resistance,MDR)使得臨床上化療效果并不理想。MDR的機制非常復(fù)雜,但其中一個主要的原因是MDR基因MDR1過度表達P-糖蛋白(P-gp)。P-gp過表達可作為腫瘤化療耐藥的一個預(yù)測指標(biāo)。任何能抑制或減少P-gp表達的因子均有助于提高化療效果。張力蛋白同源的10號染色體缺失的磷酸酶(PTEN)基因是一個抑癌基因,促進野生型PTEN基因及蛋白的表達可以增加傳統(tǒng)化療藥物的化療效果,逆轉(zhuǎn)腫瘤細(xì)胞多藥耐藥。檢索Uni Hi數(shù)據(jù)庫發(fā)現(xiàn)PTEN蛋白和P-gp之間存在相互關(guān)系。目前,已證實mi R-21是PTEN的調(diào)控基因,且多項研究報導(dǎo)mi R-21表達水平與細(xì)胞凋亡、增殖與遷移、預(yù)后、化療耐藥等密切相關(guān),但mi R-21參與腫瘤耐藥的機制目前尚不清楚。檢索RNA 22發(fā)現(xiàn),miR-21與MDR1基因有部分堿基序列互補配對,預(yù)測miR-21也可能是MDR1的調(diào)控基因,進而調(diào)控P-gp的表達。另外,福爾馬林固定石蠟包埋是臨床病理組織最常規(guī)的儲存方式,石蠟標(biāo)本包含有豐富的臨床信息和病理資料,且石蠟組織中mi RNAs的分子結(jié)構(gòu)和化學(xué)性質(zhì)穩(wěn)定,因此石蠟標(biāo)本是進行分子生物學(xué)實驗及人類疾病回顧性研究的現(xiàn)成資源。目的:本實驗通過檢測PTEN與P-gp在大腸癌組織及癌旁正常大腸黏膜組織中的表達,分析PTEN、P-gp與大腸癌臨床病理學(xué)特征的關(guān)系及二者的相關(guān)性,通過石蠟組織提取mi R-21,分析mi R-21與大腸癌分化程度的關(guān)系及mi R-21與P-gp的相關(guān)性,探討mi R-21是否與大腸癌多藥耐藥的形成有關(guān),從而為評估大腸癌患者化療耐藥提供新的分子標(biāo)志物和潛在的治療靶點方法：收集2015年1月至2016年1月承德醫(yī)學(xué)院附屬醫(yī)院術(shù)前均未行放化療的大腸癌與癌旁正常黏膜石蠟標(biāo)本各65例、新鮮標(biāo)本各20例。采用免疫組化SP法檢測65例石蠟組織和癌旁正常組織中PTEN蛋白、P-gp的表達,結(jié)合光學(xué)顯微鏡與MiVnt纖維圖像分析系統(tǒng)對實驗結(jié)果進行分析;采用Western Blot方法檢測新鮮組織中PTEN蛋白、P-gp表達,運用Quantity One軟件對條帶灰度值進行量化分析;采用實時熒光定量PCR(q RT-PCR)檢測并分析40例大腸癌石蠟組織與相應(yīng)癌旁正常黏膜組織中mi R-21的表達。應(yīng)用SPSS 19.0軟件對實驗數(shù)據(jù)進行統(tǒng)計分析。結(jié)果:1免疫組化實驗結(jié)果1.1 PTEN蛋白與P-gp在大腸癌組織和癌旁正常組織中的表達PTEN蛋白在大腸癌組織中的陽性表達率(52.3%)顯著低于癌旁正常組織(76.9%),差異具有統(tǒng)計學(xué)意義(χ2=8.613,P=0.003)。P-gp在大腸癌組織中陽性表達率(89.2%)顯著高于癌旁正常組織(75.4%),差異具有統(tǒng)計學(xué)意義(χ2=4.279,P=0.039)1.2大腸癌組織中PTEN蛋白、P-gp表達與臨床病理學(xué)特征的關(guān)系大腸癌組織中PTEN蛋白表達與分化程度、浸潤深度、局部淋巴結(jié)轉(zhuǎn)移有關(guān),差異具有統(tǒng)計學(xué)意義(P0.05)。大腸癌組織中P-gp表達與分化程度和浸潤深度有關(guān),差異具有統(tǒng)計學(xué)意義(P0.05)。1.3大腸癌中PTEN蛋白表達與P-gp表達的相關(guān)性34例PTEN蛋白陽性表達的大腸癌組織中P-gp陽性表達占27例(79.4%),P-gp陰性表達占7例(20.6%);31例PTEN蛋白陰性表達的大腸癌組織中P-gp陽性表達占31例(100%)。Spearman相關(guān)性分析顯示大腸癌組織中PTEN蛋白和P-gp的表達呈負(fù)相關(guān)(r=-0.332,P=0.007)。2 Western Blot實驗結(jié)果大腸癌組織中PTEN蛋白的表達顯著低于癌旁正常組織,差異具有統(tǒng)計學(xué)意義(t=-1.178,P=0.000);癌組織中P-gp表達顯著高于癌旁正常組織,差異具有統(tǒng)計學(xué)意義(t=-3.275,P=0.004),且P-gp在中低分化組中的表達較高分化組中表達更高,差異具有統(tǒng)計學(xué)意義(t=7.905,P=0.000)。mi R-21在大腸癌組織中平均相對表達量較癌旁正常黏膜明顯升高,差異具有統(tǒng)計學(xué)意義(P=0.000),且中、低分化組與各自相應(yīng)的正常黏膜組織相比較,差異具有統(tǒng)計學(xué)意義(P中=0.030、P低=0.000)。在高、中、低分化組中,mi R-21的平均相對表達量分別是相應(yīng)正常黏膜組織的1.25、1.69、2.04倍;組間比較:低分化組mi R-21表達較高分化組明顯升高,差異有統(tǒng)計學(xué)意義(P0.05)。3.2大腸癌組織中mi R-21表達與P-gp表達的相關(guān)性Spearman相關(guān)分析顯示miR-21表達與P-gp表達在大腸癌組織中呈正相關(guān)(r=0.444,P=0.004)。結(jié)論:1大腸癌組織中PTEN蛋白表達降低與分化程度、浸潤深度、局部淋巴結(jié)轉(zhuǎn)移密切相關(guān)。2大腸癌組織中P-gp表達升高與分化程度和浸潤深度密切相關(guān)。3大腸癌組織中PTEN蛋白表達與P-gp表達呈負(fù)相關(guān),PTEN與大腸癌多藥耐藥的關(guān)系可能與P-gp高表達有關(guān)。4大腸癌組織中miR-21表達與分化程度密切相關(guān),與P-gp表達呈正相關(guān),miR-21與大腸癌多藥耐藥的關(guān)系也可能與P-gp高表達有關(guān)。
[Abstract]:Colorectal cancer (colorectal cancer CRC) is one of the most common malignant tumor that seriously endangers human health. The chemotherapy is an important strategy in the treatment of advanced colorectal cancer, but multidrug resistance caused by various reasons (multidrug, resistance, MDR) the clinical treatment effect is not ideal.MDR mechanism is very complex however, one of the main reasons is the MDR gene overexpression of MDR1 glycoprotein P- (P-gp).P-gp expression as a predictor of tumor resistance to chemotherapy. Any can inhibit or reduce the expression of P-gp factor are helpful to enhance chemotherapy effect. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene is a tumor suppressor gene, promote the expression of wild type PTEN gene and protein can increase the chemotherapeutic effect of traditional chemotherapy drugs, reversing the multidrug resistance of tumor cells. PTEN protein and P-gp Uni Hi database retrieval There is a correlation between. At present, it has been confirmed that MI R-21 is the PTEN gene, and the expression of a number of studies reported that MI R-21 level and cell apoptosis, proliferation and migration, prognosis, drug resistance is closely related, but the mechanism of MI R-21 in tumor resistance is unclear. The retrieval of RNA 22 found that miR-21 and MDR1 gene partial nucleotide sequence complementary, prediction of miR-21 may also be MDR1 gene, expression and regulation of P-gp. In addition, formalin fixed and paraffin embedded tissue is the most conventional clinical pathological storage, paraffin contains the clinical information and pathological data rich, molecular structure and chemical properties and stability of RNAs MI in paraffin embedded tissues therefore, is paraffin specimens of the molecular biology study and retrospective study of human diseases existing resources. Objective: through the detection of PTEN and P-gp in colorectal cancer tissue and normal The expression of intestinal mucosa in the analysis of PTEN, correlation between P-gp and clinicopathological features of colorectal cancer and two patients, through the paraffin tissue extraction of MI R-21 and MI R-21, the correlation analysis between P-gp MI and R-21 and differentiation degree of colorectal cancer, to investigate whether mi R-21 associated with the formation of multidrug resistance of colorectal cancer. In order to provide, and potential therapeutic targets for the assessment of new molecular markers in patients with colorectal cancer chemotherapy: from January 2015 to January 2016 in Affiliated Hospital of Chengde Medical College were treated without preoperative chemotherapy of colorectal cancer and adjacent normal mucosa paraffin specimens of 65 cases, 20 cases of fresh specimens. PTEN protein was detected by immunohistochemistry SP methods 65 cases of paraffin embedded tissues and adjacent normal tissues, the expression of P-gp and MiVnt fiber by optical microscope image analysis system was used to analyze the experimental results; using Western Blot method to detect the fresh group The fabric in the PTEN protein, the expression of P-gp, using the Quantity software of One band gray value quantitative analysis; by using real-time quantitative PCR (Q RT-PCR) to detect the expression and analysis of 40 cases of colorectal cancer tissue and adjacent normal mucosa tissues in MI R-21. SPSS 19 software was used for statistical analysis of experimental data. 1 immunohistochemistry results: the positive expression of PTEN protein in colorectal cancer tissues the expression rate of PTEN protein and P-gp in 1.1 colorectal cancer tissues and adjacent normal tissues (52.3%) was significantly lower than that in adjacent normal tissues (76.9%), the difference was statistically significant (2=8.613, P=0.003).P-gp in colorectal carcinoma the positive expression rate (89.2%) was significantly higher than that in adjacent normal tissues (75.4%), the difference was statistically significant (2=4.279, P=0.039) PTEN protein in 1.2 colorectal cancer tissues, P-gp expression of PTEN and clinicopathological features in colorectal cancer organization Protein expression and degree of differentiation, depth of invasion, lymph node metastasis, the difference was statistically significant (P0.05). The expression of P-gp and differentiation and depth of infiltration of colorectal carcinoma, the difference was statistically significant (P0.05) P-gp associated with the expression of P-gp in 34 cases of the positive expression of PTEN protein in colorectal carcinoma and the positive expression for in 27 cases the expression of PTEN protein in colorectal cancer.1.3 (79.4%), negative expression of P-gp accounted for 7 cases (20.6%); the positive expression of P-gp in colorectal carcinoma and 31 cases of negative expression of PTEN protein in 31 cases (100%) were negatively related to the expression of.Spearman protein and correlation analysis showed PTEN P-gp in colorectal cancer tissue (r=-0.332. The experimental results of.2 Western Blot P=0.007) expression of PTEN protein in colorectal carcinoma was significantly lower than that in normal tissues, the difference was statistically significant (t=-1.178, P=0.000); P-gp in cancer tissue was significantly higher than that in adjacent normal tissues, The difference was statistically significant (t=-3.275, P=0.004), and P-gp in low differentiation group the expression of high differentiation group was higher, the difference was statistically significant (t=7.905, P=0.000).Mi R-21 in colorectal cancer with average relative expression compared with adjacent normal mucosa was significantly increased, the difference was statistically significant (P=0.000). And in low differentiation group compared with their corresponding normal tissues, the difference was statistically significant (P =0.030, P low =0.000). In the high and low differentiation group, the average Mi of the relative expression of R-21 were 1.25,1.69,2.04 times that of normal mucosal tissues; comparison between groups: low differentiation group mi R-21 expression of high differentiation group was significantly increased, the difference was statistically significant (P0.05) between Spearman and P-gp expression correlation analysis showed that the expression of miR-21 in colorectal cancer tissues was positively correlated with the P-gp mi R-21 expression of.3.2 in colorectal carcinoma ( R=0.444, P=0.004). Conclusion: to reduce the depth of invasion and differentiation, expression of PTEN protein in 1 colorectal cancer tissues, the expression of P-gp was negatively correlated with P-gp protein expression of PTEN increased with the degree of differentiation and infiltration depth of.3 is closely related to colorectal carcinoma metastasis of.2 in colorectal carcinoma and regional lymph nodes, high expression of relationship drug resistant PTEN and colorectal cancer may be related to the expression of miR-21 and P-gp.4 in colorectal cancer tissue is closely related with the degree of differentiation was positively correlated with the expression of P-gp, the relationship of miR-21 with multidrug resistance of colorectal cancer may also be associated with high expression of P-gp.

【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34

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