本文關(guān)鍵詞： 鼻咽癌 酪氨酸激酶B 腦源性神經(jīng)生長因子 微血管密度 血管形成　出處：《中南大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：目的 鼻咽癌(Nasopharyngeal carcinoma, NPC)是來源于鼻咽上皮組織的高度惡性腫瘤,其預(yù)后與腫瘤的局部復(fù)發(fā)、淋巴結(jié)轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移密切相關(guān)。有研究發(fā)現(xiàn)腫瘤血管形成與惡性腫瘤生長、侵襲轉(zhuǎn)移與及預(yù)后密切相關(guān)。血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)是人們發(fā)現(xiàn)的重要的血管生成因子,通過促腫瘤血管生成而在腫瘤發(fā)生發(fā)展中發(fā)揮重要作用。酪氨酸激酶B(Tyrosine kinase B, TrKB)在腫瘤血管生成及腫瘤生長,浸潤轉(zhuǎn)移及預(yù)后密切相關(guān)。本部分研究意在探討VEGF及TrKB在NPC組織中的表達(dá)并比較兩者表達(dá)的相關(guān)性及分析與腫瘤臨床特征的關(guān)系。 方法 采用免疫組化方法檢測55例鼻咽癌組織和30例正常鼻咽組織中VEGF及TrKB蛋白表達(dá)水平,比較二者表達(dá)差異。根據(jù)患者年齡、腫瘤臨床分型、原發(fā)灶大小、淋巴結(jié)轉(zhuǎn)移和有無遠(yuǎn)處轉(zhuǎn)移將病例分組,應(yīng)用雙變量相關(guān)pearson檢驗分析研究VEGF及TrKB表達(dá)與腫瘤臨床病理特征之間的關(guān)系 結(jié)果 1.VEGF蛋白于腫瘤細(xì)胞漿及血管內(nèi)皮細(xì)胞漿中均表達(dá),TrKB蛋白陽性表達(dá)絕大多數(shù)位于細(xì)胞漿內(nèi)。在55例鼻咽癌中,45例表達(dá)TrKB陽性,表達(dá)率為82.1%,47例表達(dá)VEGF陽性,表達(dá)率為85.7%。30例正常鼻咽組織中,只有3例表達(dá)TrKB陽性,5例表達(dá)VEGF陽性,腫瘤組織中TrKB、VEGF陽性表達(dá)率明顯高于正常鼻咽組織(P0.05),鼻咽癌組織中VEGF與TrKB表達(dá)有明顯相關(guān)性(相關(guān)系數(shù)R=0.586,P0.05)。 2.VEGF蛋白表達(dá)與NPC的臨床分期(P=0.002)、腫瘤的直徑大小(P=0.009)及有無淋巴結(jié)轉(zhuǎn)移(P=0.001)明顯相關(guān),與患者性別、年齡、有無遠(yuǎn)處轉(zhuǎn)移無關(guān)(P0.05)。TrKB蛋白表達(dá)與臨床分期(P=0.00)、腫瘤的直徑大小(P=0.00)、有無淋巴結(jié)轉(zhuǎn)移(P=0.006)明顯相關(guān),與患者性別、年齡、有無遠(yuǎn)處轉(zhuǎn)移無關(guān)(P0.05)。 結(jié)論 1.VEGF, TrKB在NPC組織中表達(dá)增加,可能與鼻咽癌的發(fā)生發(fā)展有關(guān)； 2.TrKB可能與鼻咽癌血管形成有關(guān)。 目的 探討通過酪氨酸激酶受體抑制劑K252a抑制TrKB表達(dá),觀察抑制TrKB表達(dá)對體外培養(yǎng)的鼻咽癌CNE-1細(xì)胞增殖、運(yùn)動遷移、細(xì)胞周期、細(xì)胞凋亡及對VEGF表達(dá)的影響。 方法 分別用不同濃度K252a干預(yù)鼻咽癌CNE-1細(xì)胞,MTT實驗比較不同濃度K252a對鼻咽癌CNE-1細(xì)胞生長的影響,并計算細(xì)胞生存(增殖)率,篩選出K252a最佳干預(yù)濃度；通過劃痕愈合試驗觀察抑制TrKB表達(dá)后對鼻咽癌CNE-1細(xì)胞運(yùn)動遷移能力的影響；流式細(xì)胞術(shù)分析抑制TrKB表達(dá)對鼻咽癌CNE-1細(xì)胞遷移、細(xì)胞周期和凋亡的影響。 分別用RT-PCR、Western-blot檢測K252a干預(yù)鼻咽癌CNE-1細(xì)胞后VEGF、TrkB在mRNA以及蛋白層面表達(dá)的改變并與對照組比較差異。 結(jié)果 MTT實驗：K252a能明顯抑制鼻咽癌CNE-1細(xì)胞的增殖,使細(xì)胞數(shù)量減少、密度減低、細(xì)胞生存率下降,其對鼻咽癌CNE-1細(xì)胞生長的抑制作用與K252a干預(yù)時間、濃度呈正相關(guān)。K252a作用48h使鼻咽癌CNE-1細(xì)胞增殖率抑制最明顯的濃度為400nmol/L,細(xì)胞生存減少50%(與對照組相比),故選取此濃度為K252a最佳干預(yù)濃度。 劃痕愈合實驗：抑制TrKB表達(dá)可使劃痕愈合延遲,使12h鼻咽癌CNE-1細(xì)胞遷移距離明顯小于對照組,但無明顯統(tǒng)計學(xué)差異(P=0.18),使24h小時細(xì)胞遷移距離細(xì)胞遷移距離明顯少于對照組(P=0.013),說明抑制TrKB表達(dá)能一定程度上抑制鼻咽癌CNE-1細(xì)胞的運(yùn)動遷移能力。 流式細(xì)胞儀分析：K252a干預(yù)鼻咽癌CNE-1細(xì)胞的細(xì)胞周期分布,與對照組相比,鼻咽癌CNE-1細(xì)胞中處于G0/G1期的細(xì)胞顯著增多(57.67士7.03vs73.86±4.22, P=0.018),而處于S期(30.67±3.68vs15.37±2.16,P=0.025)的細(xì)胞顯著減少,比較有統(tǒng)計學(xué)意義(P0.05)；流式細(xì)胞儀技術(shù)檢測K252a干預(yù)鼻咽癌CNE-1細(xì)胞后細(xì)胞凋亡情況,K252a與對照組細(xì)胞的凋亡率比較有統(tǒng)計學(xué)意義(22.27±3.09vs3.42±2.56,P=0.01)。 Western blot:K252a干預(yù)鼻咽癌CNE-1細(xì)胞后,與對照組相比,VEGF蛋白表達(dá)水平下調(diào)(0.6883±0.19vs0.378±0.44),TrKB蛋白表達(dá)水平下調(diào)(0.631±0.15vs0.313±0.28),差別均有統(tǒng)計學(xué)意義(P0.05)。 RT-PCR:K252a干預(yù)鼻咽癌CNE-1細(xì)胞后,與對照組相比,細(xì)胞內(nèi)TrkB mRNA表達(dá)下調(diào)(0.67±0.37vs0.435±0.63)；VEGF mRNA表達(dá)下調(diào)(0.6883±0.49vs0.425±0.17),差異有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 1.TrKB表達(dá)抑制可降低鼻咽癌CNE-1細(xì)胞生長遷移能力； 2.TrKB表達(dá)抑制可通過促進(jìn)鼻咽癌CNE-1細(xì)胞凋亡、細(xì)胞周期改變和降低VEGF表達(dá),從而減弱生長與遷移能力； 3.TrKB通過調(diào)控VEGF影響鼻咽癌血管形成。 目的 體內(nèi)腫瘤生長依賴腫瘤血管生成。近年來發(fā)現(xiàn)VEGF通過誘導(dǎo)血管生成及促進(jìn)血管內(nèi)皮細(xì)胞分裂從而促進(jìn)血管形成。研究證實血管生成、微血管與腫瘤侵襲轉(zhuǎn)移之間的關(guān)系密切,微血管密度(microvessel density, MVD)與腫瘤的轉(zhuǎn)移和預(yù)后有關(guān),且VEGF表達(dá)與MVD的表達(dá)有良好的相關(guān)性。前部分體外實驗研究已經(jīng)發(fā)現(xiàn)K252a能抑制VEGF蛋白和基因水平的表達(dá),本部分實驗通過建立裸小鼠移植瘤模型,探討K252a干預(yù)鼻咽癌CNE-1細(xì)胞對人鼻咽癌血管生成抑制作用的體內(nèi)研究,為針對鼻咽癌抗血管生成基因治療尋找新的思路。 方法 使用4-6周齡雄性BALB/C-nu/nu裸鼠,體重在16-20g,將人鼻咽癌CNE-1細(xì)胞于裸鼠右側(cè)季肋部皮下注射,建立荷人鼻咽癌細(xì)胞裸小鼠皮下移植瘤模型。隨機(jī)將實驗動物分為2組,A組：K252a干預(yù)組,7只；B組：PBS組,7只；于接種腫瘤細(xì)胞后第6、8、10、12、14天用微量注射器于A組裸鼠腫瘤內(nèi)多點注射K252a藥物,400nmol/L200ul/次,對兩組移植瘤模型進(jìn)行為期21天的實驗觀察,白第一次注射藥物后每隔2天測量1次裸鼠腫瘤體積并繪制腫瘤生長曲線。 于接種后第21天處死,剝離腫瘤標(biāo)本,測量最后體積,觀察標(biāo)本整體特征。取出部分組織的標(biāo)本于固定于10%福爾馬林溶液中,于我院病理科常規(guī)石蠟包埋后行連續(xù)切片,進(jìn)行常規(guī)HE染色、VEGF、 TrKB和CD31相關(guān)抗原免疫組化染色。 結(jié)果 1.腫瘤標(biāo)本大體形態(tài)及HE染色觀察：2組實驗動物成瘤均于接種后第4天。兩組腫瘤與周圍組織分界清楚,包膜基本完整。K252a干預(yù)組治療組皮下腫瘤瘤體較小,呈不規(guī)則球形,也有的呈分葉狀,表面灰白色,質(zhì)地較硬。剖面呈白色,剖面滲血較少。對照組腫瘤瘤體較K252a組明顯增大,表面多不平,有分葉狀結(jié)節(jié)狀,表面豐富血管形成,剖面滲血較多； 2.實驗動物瘤體體積觀察：K252a干預(yù)組從給藥開始,腫瘤生長緩慢,第21天腫瘤瘤體體積為(328±326.7)mm3,同期對照組為(1044±818)mm3； 3.兩組CD31標(biāo)記的MVD計數(shù)與VEGF、TrKB染色評分對比及相關(guān)性分析 K252a干預(yù)組和對照組陽性血管計數(shù)分別為22.39±4.72、49.79±11.12,方差分析各組間差別具有顯著性(P0.05)。K252a干預(yù)組VEGF表達(dá)明顯比對照組下降,并有統(tǒng)計學(xué)差異(7.57±2.82vs9.86±2.79,P=0.007),K252a干預(yù)組明顯比對照組TrKB表達(dá)下降,并有統(tǒng)計學(xué)差異(5.8571±2.79vs7.43±2.64,P=0.005),各組VEGF表達(dá)與MVD明顯相關(guān)(對照組：r=0.88,P=0.009,K252a組：r=0.96,P=0.01),MVD與TrKB表達(dá)明顯正相關(guān)(對照組：r=0.79,P=0.03,K252a組：r=0.85,P=0.01)。對照組與K252a組VEGF表達(dá)與TrKB無明顯相關(guān)性。 結(jié)論 1.抑制TrKB的表達(dá)能抑制鼻咽癌在動物體內(nèi)的生長；并能通過減少VEGF表達(dá)明顯減少鼻咽癌組織內(nèi)的腫瘤新生血管； 2.TrKB有可能成為抗鼻咽癌新生血管治療的新靶點。
[Abstract]:Purpose Nasopharyngeal carcinoma ( NPC ) is a highly malignant tumor derived from nasopharyngeal epithelial tissue . Its prognosis is closely related to local recurrence , lymph node metastasis and distant metastasis of tumor . It is found that vascular endothelial growth factor ( VEGF ) plays an important role in tumor angiogenesis and tumor growth , invasion and metastasis and prognosis . method The expression of VEGF and TrKB in 55 nasopharyngeal carcinoma tissues and 30 normal nasopharyngeal tissues was detected by immunohistochemistry . The relationship between VEGF and TrKB expression and clinical pathological characteristics of tumor was studied by means of double variable correlation pearson test . Results 1 . The expression of VEGF protein in tumor cell plasm and vascular endothelial cell plasm and TrKB protein positive expression were mostly in cytoplasm . In 55 cases of nasopharyngeal carcinoma , 45 cases expressed TrKB positive , the expression rate was 82.1 % , and the expression rate was 85.7 % . In 30 normal nasopharyngeal tissues , only 3 cases were expressed in TrKB positive , 5 cases were positive for VEGF , the expression of VEGF in tumor tissues was significantly higher than that in normal nasopharyngeal tissues ( P0.05 ) . 2 . The expression of VEGF was significantly correlated with the clinical stage of NPC ( P = 0.002 ) , the diameter of the tumor ( P = 0 . 009 ) and the lymph node metastasis ( P = 0 . 001 ) . The expression of TrKB was significantly correlated with the clinical stage ( P = 0.00 ) , the diameter of the tumor ( P = 0.00 ) , and the absence of lymph node metastasis ( P = 0.006 ) . Conclusion 1 . The expression of VEGF and TrKB in NPC tissues may be related to the development of NPC ; 2 . TrKB may be related to the formation of nasopharyngeal carcinoma . Purpose To investigate the inhibitory effect of TrKB expression on proliferation , movement , cell cycle , cell apoptosis and the expression of VEGF in nasopharyngeal carcinoma CNE - 1 cells cultured in vitro by inhibiting the expression of TrKB by tyrosine kinase receptor inhibitor . method Cell survival ( proliferation ) rate of CNE - 1 cells was determined by MTT assay , and the effect of TrKB expression on the migration of CNE - 1 cells in NPC CNE - 1 cells was observed . The effects of TrKB expression on the migration , cell cycle and apoptosis of CNE - 1 cells were observed by flow cytometry . The expression of VEGF and TrkB in nasopharyngeal carcinoma CNE - 1 cells was detected by RT - PCR and Western - blot , respectively . Results The proliferation of CNE - 1 cells in NPC was significantly inhibited by MTT assay . The inhibition of proliferation of CNE - 1 cells and the decrease of cell survival rate in NPC CNE - 1 cells were significantly inhibited . The results showed that the migration distance of CNE - 1 cells was significantly lower than that in the control group ( P = 0 . 013 ) , and that the inhibition of TrKB expression could inhibit the movement of nasopharyngeal carcinoma CNE - 1 cells to some extent . Flow cytometry showed that the cell cycle distribution of CNE - 1 cells was significantly increased ( 57.67 鹵 7.03 vs 73.86 鹵 4.22 , P = 0 . 018 ) in NPC CNE - 1 cells compared with control group . Western blot : Compared with the control group , the level of VEGF protein expression was down regulated ( 0.6883 鹵 0.19 vs 0.378 鹵 0.44 ) , and the level of TrKB protein expression was down regulated ( 0.631 鹵 0.15 vs 0.313 鹵 0.28 ) , and the difference was statistically significant ( P0.05 ) . Compared with the control group , the expression of TrkB mRNA was down - regulated ( 0.67 鹵 0.37 vs 0.435 鹵 0.63 ) and VEGF mRNA expression was down - regulated ( 0.6883 鹵 0.49 vs 0.425 鹵 0.17 ) , which was statistically significant ( P0.05 ) . Conclusion 1 . TrKB expression inhibition can decrease the growth and migration ability of nasopharyngeal carcinoma CNE - 1 cells . 2 . TrKB expression inhibition can decrease the growth and migration ability by promoting apoptosis , cell cycle change and VEGF expression in nasopharyngeal carcinoma CNE - 1 cells . 3 . TrKB affects the angiogenesis of nasopharyngeal carcinoma by regulating VEGF . Purpose In recent years , it has been found that the relationship between angiogenesis , angiogenesis , invasion and metastasis of vascular endothelial cells is closely related , and the expression of VEGF and MVD is closely related to the metastasis and prognosis of tumor . method BALB / C - nu / nu nude mice 4 - 6 weeks old were injected subcutaneously in nude mice at 16 - 20g . The nude mice were randomly divided into two groups : group A : group two , group A : group A , group B : PBS group , group A : group A : 6 , 8 , 10 , 12 , 14 days after inoculation of tumor cells , the tumor volume of nude mice was measured once every 2 days , and tumor growth curve was drawn . The specimens were taken out on the 21st day after inoculation , the final volume was measured and the overall characteristics of the specimens were observed . The specimens from some tissues were fixed in 10 % formalin solution , and the specimens were sectioned after paraffin embedding in the normal paraffin embedded in our hospital , and then stained with HE staining , VEGF , TrKB and CD31 - related antigens . Results 1 . The gross appearance of tumor specimen and HE staining were observed : 2 groups of experimental animals were treated with tumor in the 4th day after inoculation . The tumor of the two groups was more clear than that of surrounding tissues , and the capsule was basically intact . 2 . The volume of tumor volume in experimental animals was observed : the tumor growth was slow , the volume of tumor body was ( 328 鹵 326.7 ) mm3 on day 21 , and the control group was ( 1044 鹵 818 ) mm3 in the same period . 3 . The MVD counts of CD31 markers in both groups were compared with those of VEGF and TrKB , and their correlation was analyzed . There was a significant difference between the expression of VEGF and the expression of VEGF in the control group ( r = 0 . 88 , P = 0 . 009 , K - group : r = 0 . 96 , P = 0 . 01 ) . Conclusion 1 . The inhibition of TrKB expression can inhibit the growth of nasopharyngeal carcinoma in animals . 2 . TrKB may become a new target for the treatment of nasopharyngeal carcinoma .

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R739.63

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 胡偉漢,張國義,劉立志,吳湖炳,李立,高遠(yuǎn)紅,潘q，

本文編號：1516583