本文選題：食管癌 + 環(huán)氧化酶-2�。� 參考：《河北醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:食管癌是我國最常見的惡性腫瘤之一,具有很明顯的地域性分布性。由于食管癌早期癥狀不典型,人們?nèi)狈Ρ＝∫庾R等原因,確診時多已發(fā)展到晚期,所以預(yù)后差。近年來研究表明,環(huán)氧化酶-2(cyclooxygenase-2,COX-2)在惡性腫瘤的形成過程中有重要作用,它可促進腫瘤細(xì)胞增殖,抑制腫瘤細(xì)胞凋亡,促進腫瘤的侵襲、轉(zhuǎn)移等。動物實驗證明,對腫瘤的治療選擇性COX-2抑制劑有顯著作用。因此針對腫瘤的COX-2靶點,可以選用選擇性COX-2抑制劑來治療。尼美舒利是新型選擇性COX-2抑制劑,動物實驗證實,尼美舒利不僅可以抑制腫瘤生長與轉(zhuǎn)移、預(yù)防腫瘤形成,還可與化療、放療聯(lián)合應(yīng)用,具有增強化療、放療效果的作用。奧沙利鉑是第三代化療藥,通過產(chǎn)生水化衍生物,使DNA鏈內(nèi)和鏈間形成交聯(lián),從而抑制DNA的復(fù)制及合成。本實驗從食管癌手術(shù)治療的標(biāo)本、人食管癌小鼠移植瘤兩方面,采用多種實驗方法研究COX-2在食管癌發(fā)生過程中的作用,觀察COX-2抑制劑尼美舒利、化療藥物奧沙利鉑的聯(lián)合應(yīng)用對人食管癌小鼠移植瘤生長的抑制作用,進一步探討這兩種藥物聯(lián)合應(yīng)用可能產(chǎn)生的協(xié)同作用。方法:1食管癌中COX-2的表達及意義手術(shù)切除食管癌新鮮標(biāo)本63例,術(shù)后立即取材,分別于10%福爾馬林固定,常規(guī)包埋、切片,HE染色,由兩名有經(jīng)驗病理科醫(yī)師進行組織病理學(xué)診斷。采用免疫組織化學(xué)方法檢測COX-2蛋白的表達。新鮮標(biāo)本經(jīng)70%乙醇處理后,采用流式細(xì)胞術(shù)(Flow cytometry,FCM)定量檢測標(biāo)本中的COX-2蛋白表達。2尼美舒利、奧沙利鉑聯(lián)合應(yīng)用對人食管癌小鼠移植瘤生長的影響選取C57BL/6小鼠6-7周齡,雄性,體重25-30g,在IVC環(huán)境中以滅菌處理過的飼料和水飼養(yǎng),環(huán)境溫度、濕度控制在適宜范圍。將人食管癌Eca-109細(xì)胞使用含10%胎牛血清、青霉素、鏈霉素各100U.m L-1的DMEM/F12培養(yǎng)基,在37℃的恒溫下,用含5%的二氧化碳培養(yǎng)箱中常規(guī)培養(yǎng)。觀察細(xì)胞至70%-80%融合時,用2.5g.L-1胰蛋白酶消化傳代。取生長狀態(tài)良好的對數(shù)生長期的人食管癌Eca-109細(xì)胞,PBS洗滌,用2.5g.L-1胰蛋白酶消化下來,制成細(xì)胞懸液。用不含胎牛血清的DMEM/F12培養(yǎng)基稀釋,用細(xì)胞計數(shù)板進行計數(shù),調(diào)整細(xì)胞懸液濃度為5×106.m L-1。小鼠皮膚備皮、消毒后用1ml注射器在右側(cè)后臀部注射上述單細(xì)胞懸液1ml,建立人食管癌小鼠移植瘤模型。26只全部成瘤,定期測量腫瘤長徑、短徑,待腫瘤平均直徑為4mm后,剔除腫瘤偏小的2只小鼠。將24只小鼠隨機分為4組,每組6只,組別為對照組,尼美舒利組,奧沙利鉑組,尼美舒利/奧沙利鉑組。尼美舒利組小鼠給予灌胃口服尼美舒利20mg/Kg/次,每天給藥一次,同時腹腔注射等量滅菌生理鹽水;奧沙利鉑組小鼠給予腹腔注射奧沙利鉑甘露醇注射液10mg/Kg/次,每4天給藥一次,同時灌胃口服等量滅菌生理鹽水;尼美舒利/奧沙利鉑組小鼠,同時灌胃口服尼美舒利,腹腔注射奧沙利鉑甘露醇注射液,劑量與單用組相同;對照組小鼠,灌胃口服滅菌生理鹽水,腹腔注射等量滅菌生理鹽水。定期觀察小鼠生長情況,每5天測量1次腫瘤的體積。30天后,處死小鼠切除腫瘤組織,測量4組小鼠移植瘤長徑、短徑及瘤重,估測瘤體體積和腫瘤生長抑制率并取材。FCM法定量檢測移植瘤中COX-2蛋白的表達,FCM法檢測移植瘤中細(xì)胞的凋亡率。結(jié)果:1 COX-2在食管癌變過程中的表達免疫組織化學(xué)結(jié)果顯示,食管癌細(xì)胞COX-2基因表達產(chǎn)物主要定位于細(xì)胞漿,胞漿內(nèi)出現(xiàn)棕黃色顆粒為陽性。在癌旁組織中表達率為20%,食管癌組織中表達率為90%。FCM定量檢測COX-2含量結(jié)果顯示:正常食管鱗狀上皮中COX-2的FI值為1.00±0.12,隨著食管鱗狀上皮異型性增高,COX-2的表達升高,在高分化癌中表達最高,FI值為2.89±0.22。在食管鱗狀細(xì)胞癌組織中隨著癌組織的分化程度降低而COX-2的表達減少。2尼美舒利聯(lián)合奧沙利鉑對人食管癌小鼠移植瘤生長的影響及機制接種小鼠26只,全部成瘤,成瘤率100%,成瘤時間為細(xì)胞接種后5-8天。腫瘤細(xì)胞種植后13天,腫瘤平均直徑達4mm。治療結(jié)束后,各組體積分別為:對照組:移植瘤體積2047.42±976.55 mm3,瘤重1.41±0.49g;尼美舒利組:移植瘤體積1267.14±476.38mm3,瘤重0.97±0.51g;奧沙利鉑組:移植瘤體積1054.28±102.82mm3,瘤重0.89±0.20g;尼美舒利/奧沙利鉑聯(lián)合用藥組:移植瘤體積532.66±220.11mm3,瘤重0.45±0.31g。尼美舒利組、奧沙利鉑組和尼美舒利/奧沙利鉑聯(lián)合用藥組的抑瘤率分別為46.22%、51.93%和72.88%。方差分析組間差異顯示:尼美舒利組、奧沙利鉑組和尼美舒利/奧沙利鉑聯(lián)合用藥組移植瘤生長明顯較對照組緩慢(P0.05)。尼美舒利/奧沙利鉑聯(lián)合用藥組抑瘤率明顯較尼美舒利組、奧沙利鉑組高(P0.05)。尼美舒利組和奧沙利鉑組對移植瘤的生長的抑制作用未見明顯差異(P0.05)。FCM定量檢測移植瘤的細(xì)胞凋亡,對照組食管癌細(xì)胞凋亡率為(12.12±0.47)%,尼美舒利組凋亡率為(22.16±5.24)%,奧沙利鉑組凋亡率為(27.33±7.25)%,尼美舒利/奧沙利鉑聯(lián)合用藥組凋亡率為(47.26±6.73)%。方差分析組間差異顯示:尼美舒利組、奧沙利鉑組和尼美舒利/奧沙利鉑聯(lián)合用藥組凋亡率較對照組升高,有顯著性差異(P0.05)。尼美舒利/奧沙利鉑聯(lián)合用藥組凋亡率明顯較尼美舒利組、奧沙利鉑組高,有顯著性差異(P0.05)。尼美舒利組和奧沙利鉑組對凋亡率的影響未見明顯差異(P0.05)。FCM定量檢測移植瘤中COX-2蛋白表達,尼美舒利組FI=0.598±0.090,奧沙利鉑組FI=1.390±0.192,尼美舒利/奧沙利鉑組FI=0.791±0.121。方差分析結(jié)果顯示,尼美舒利及尼美舒利/奧沙利鉑聯(lián)合用藥組COX-2蛋白表達水平低于對照組,并有顯著性差異(P0.05)。奧沙利鉑組COX-2蛋白表達水平高于對照組,并有顯著性差異(P0.05)。結(jié)論:1在癌旁組織和食管癌組織中,COX-2的表達增高,表明在食管上皮癌變過程中COX-2可能起重要作用。食管鱗狀細(xì)胞癌組織中,隨食管癌組織分化程度降低,COX-2的表達降低。2尼美舒利及奧沙利鉑均能抑制腫瘤的生長,尼美舒利/奧沙利鉑聯(lián)合用藥組抑瘤率明顯高于單用組。尼美舒利能提高了奧沙利鉑的對腫瘤的抑制效果,這種效果是通過誘導(dǎo)腫瘤細(xì)胞凋亡來抑制腫瘤的。綜上所述,本實驗從食管癌手術(shù)標(biāo)本,小鼠移植瘤兩個方面進行研究,結(jié)果表明:COX-2在食管癌變過程中起重要作用,選擇性COX-2抑制劑尼美舒利或聯(lián)合奧沙利鉑,均可通過誘導(dǎo)細(xì)胞凋亡來抑制腫瘤。尼美舒利與奧沙利鉑聯(lián)合應(yīng)用,可提高奧沙利鉑的腫瘤抑制作用。
[Abstract]:Objective: esophageal cancer is one of the most common malignant tumors in China. It has a distinct regional distribution. Due to the untypical early symptoms of esophageal cancer and the lack of health awareness, it has developed to a late stage, so the prognosis is poor. In recent years, the study showed that the epoxide -2 (cyclooxygenase-2, COX-2) was formed in the formation of malignant tumor. It plays an important role in promoting tumor cell proliferation, inhibiting tumor cell apoptosis, promoting tumor invasion, metastasis and so on. Animal experiments have proved to have significant effect on the selective COX-2 inhibitors for tumor treatment. Therefore, selective COX-2 inhibitors can be used to treat tumor COX-2 targets. Ni Mei Shug Leigh is a new type of selective COX-2. The inhibitor, animal experiments confirmed that Ni Mei Shug Leigh not only can inhibit tumor growth and metastasis, prevent tumor formation, but also can be combined with chemotherapy and radiotherapy, and has the effect of enhancing chemotherapy and radiotherapy. Oxaliplatin is the third generation of chemotherapeutic agents. By producing hydrated derivatives, the interchain and interchain of DNA can be formed to inhibit the replication of DNA. In this experiment, we studied the role of COX-2 in the carcinogenesis of esophageal cancer in two aspects of the surgical treatment of esophageal cancer and the transplanted tumor of human esophageal cancer in mice. The effects of COX-2 inhibitor nimesulide and the combination of oxaliplatin, a chemotherapeutic agent, on the growth of human esophageal cancer in mice were investigated. Synergetic effects of the combined application of these two drugs. Methods: the expression of COX-2 in 1 esophageal carcinoma and the significance of surgical resection of 63 fresh esophageal carcinoma specimens were taken immediately after operation, respectively, in 10% formalin fixation, routine embedding, slicing, HE staining, and histopathological diagnosis by two experienced pathologists. Immunohistochemistry was used. The expression of COX-2 protein was detected by the method. After 70% ethanol treatment, Flow cytometry (FCM) was used to detect the COX-2 protein expression of.2 nimesulide in the specimens. The effects of oxaliplatin on the growth of transplanted tumor in human esophageal cancer mice were selected for the selection of C57BL/6 mice for 6-7 weeks, male, body weight 25-30g, and IVC environment. Eca-109 cells with 10% fetal bovine serum, penicillin and streptomycin 100U.m L-1 were used as the DMEM/F12 medium containing 10% fetal bovine serum, penicillin and streptomycin at a constant temperature of 37. The cells were cultured in a 5% carbon dioxide incubator and used 2.5G. to observe the cells to 70%-80% fusion. L-1 trypsin digested the human esophageal cancer Eca-109 cells with good growth state during the logarithmic growth period, PBS washed and digested with 2.5g.L-1 trypsin to make the cell suspension. The cell suspension was diluted with the DMEM/F12 medium without fetal bovine serum, and the cell suspension concentration was 5 * 106.m L-1. mouse skin. After disinfection, the 1ml syringe was injected with the above single cell suspension 1ml on the right rear buttocks, and the mouse model of human esophageal cancer transplanted tumor model.26 was all tumor, and the length and diameter of the tumor were measured regularly. After the average diameter of the tumor was 4mm, 2 mice were eliminated. The 24 mice were divided into 4 groups, each group was 6 and the group was the control group, Ni Mei Shug Leigh Group, oxaliplatin group, Ni Mei Shug Leigh / oxaliplatin group. The mice in the Ni Mei Shug Leigh group were given the oral administration of Ni Mei Shug Leigh 20mg/Kg/ times, once a day, and the same dose of normal saline was injected into the abdominal cavity. The oxaliplatin group was given an intraperitoneal injection of Oxaliplatin and Mannitol Injection 10mg/Kg/ times, once every 4 days, at the same time. The nimesulide / oxaliplatin group, nimesulide / oxaliplatin group, was given the same dose of nimesulide and intraperitoneal injection of Oxaliplatin and Mannitol Injection, and the dose was the same as that in the single group. The control mice were given the normal saline by intraperitoneal injection, and the abdominal injection was used to sterilize the physiological saline. The growth of the mice was observed and the body was measured every 5 days, and the body was measured every 5 days. After.30 days, the tumor tissues were excised and the long diameter, short diameter and tumor weight of the 4 groups of mice were measured. The volume of the tumor and the inhibition rate of tumor growth were measured and the expression of COX-2 protein in the transplanted tumor was measured by.FCM. The apoptosis rate of the cells in the transplanted tumor was detected by FCM. Results: the expression of immune tissues in the process of esophageal carcinogenesis was expressed by the FCM method. The chemical results showed that the COX-2 gene expression products of esophageal cancer cells were mainly located in the cytoplasm, and the brown yellow granules were positive in the cytoplasm. The expression rate in the para cancerous tissues was 20%. The expression rate of the esophageal carcinoma tissue was 90%.FCM quantitative detection COX-2 content: the FI value of COX-2 in the normal esophageal squamous epithelium was 1 + 0.12, with the esophageal scale. The high expression of COX-2, the highest expression of COX-2, the highest expression in highly differentiated carcinoma, FI value is 2.89 + 0.22. in the squamous cell carcinoma of the esophagus with the decrease of the differentiation degree of cancer tissue, and the expression of COX-2 reduces the effect of.2 combined with oxaliplatin on the growth of the transplanted tumor of human esophageal cancer and the mechanism of the inoculation of 26 mice. The tumor formation rate was 100% and the time of tumor formation was 5-8 days after the cell inoculation. After 13 days after the tumor cells were planted, the average diameter of the tumor reached the end of 4mm. treatment. The volume of each group was as follows: the volume of the transplanted tumor was 2047.42 + 976.55 mm3 and the tumor weight was 1.41 0.49g; the nimesulide group was 1267.14 + 476.38mm3, and the tumor weight was 0.97 + 0.51g; the oxaliplatin group was moved. The tumor volume was 1054.28 + 102.82mm3 and the tumor weight was 0.89 + 0.20g; Ni Mei Shug Leigh / oxaliplatin combined group: the tumor volume was 532.66 + 220.11mm3, the tumor weight was 0.45 + 0.31g., the tumor inhibition rate of the oxaliplatin group and the Ni Mei Shug Leigh / oxaliplatin group was 46.22%, 51.93% and 72.88%. variance analysis group showed: neimego The growth of transplanted tumor in group, oxaliplatin group and Ni Mei Shug Leigh / oxaliplatin group was significantly slower than that of the control group (P0.05). The tumor inhibition rate of Ni Mei Shug Leigh / oxaliplatin group was significantly higher than that of the Ni Mei Shug Leigh group and the oxaliplatin group (P0.05). The inhibitory effect of the Ni Mei Shug Leigh group and oxaliplatin group on the growth of the transplanted tumor was not significantly different. (P0.05).FCM quantitative detection of the apoptosis of the transplanted tumor cells, the apoptosis rate of the control group was (12.12 + 0.47)%, the apoptosis rate of the Ni Mei Shug Leigh group was (22.16 + 5.24)%, the apoptosis rate of the oxaliplatin group was (27.33 + 7.25)%, and the rate of apoptosis in the combination group of Ni Mei Shug Leigh / oxaliplatin was (47.26 + 6.73)%. The difference between the groups of variance analysis showed that in the Ni Mei Shug Leigh group, The apoptosis rate of oxaliplatin group and Ni Mei Shug Leigh / oxaliplatin group was significantly higher than that of the control group (P0.05). The apoptosis rate of the combination group of Ni Mei Shug Leigh / oxaliplatin was significantly higher than that of the Ni Mei Shug Leigh group and the oxaliplatin group (P0.05). The effect of the nimisulli group and oxaliplatin group on the apoptosis rate was not obvious. Differential (P0.05).FCM quantitative detection of COX-2 protein expression in transplanted tumor, Ni Mei Shug Leigh group FI=0.598 + 0.090, oxaliplatin group FI=1.390 + 0.192, Ni Mei Shug Leigh / oxaliplatin group FI=0.791 + 0.121. variance analysis results showed that the expression level of COX-2 protein in the combination group of Ni Mei Shug Leigh and Ni Mei Shug Leigh / oxaliplatin was lower than that of the control group, and it was significant. The difference (P0.05). The expression of COX-2 protein in the oxaliplatin group was higher than that in the control group, and there was a significant difference (P0.05). Conclusion: 1 the expression of COX-2 is increased in the para cancerous and esophageal cancer tissues, indicating that COX-2 may play an important role in the carcinogenesis of the esophagus. The degree of differentiation of esophageal squamous cell carcinoma is reduced, COX-2 The expression of.2 Ni Mei Shug Leigh and oxaliplatin inhibited the growth of the tumor. The tumor inhibition rate of the combination of Ni Mei Shug Leigh / oxaliplatin was significantly higher than that in the single use group. Ni Mei Shug Leigh could improve the inhibitory effect of oxaliplatin on the tumor. This effect is to inhibit tumor by inducing tumor cells to wither. Two aspects of tumor operation specimens and mice transplanted tumor were studied. The results showed that COX-2 played an important role in the carcinogenesis of the esophagus. The selective COX-2 inhibitor Ni Mei Shug Leigh or oxaliplatin could inhibit the tumor by inducing apoptosis. The combination of Ni Mei Shug Leigh and oxaliplatin could improve the tumor suppression of oxaliplatin. Use.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.1

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5 侯建葉;;食管癌手術(shù)病人住院期間的健康教育[A];全國外科護理學(xué)術(shù)會議暨專題講座論文匯編[C];2000年

6 陳作敏;楊健;;手術(shù)治療69例食管癌體會[A];2001'全國腫瘤外科學(xué)術(shù)會議論文匯編[C];2001年

7 夏繼國;駱紅蕾;;食管癌氣管內(nèi)轉(zhuǎn)移的臨床特點[A];2004年全國危重病急救醫(yī)學(xué)學(xué)術(shù)會議論文集[C];2004年

8 高宗人;;河南省食管癌外科治療的進展及評價[A];第三屆中國腫瘤學(xué)術(shù)大會教育論文集[C];2004年

9 ;食管癌[A];第三屆中國腫瘤學(xué)術(shù)大會論文集[C];2004年

10 王中林;張蕾;劉寧;陸一民;高克柔;張曉膺;;電視胸腔鏡下手術(shù)治療食管癌臨床探索[A];第八屆華東六省一市胸心血管外科學(xué)術(shù)會議論文匯編[C];2005年

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