TrKB、VEGF在鼻咽癌中的表达及其调控肿瘤侵袭转移的体内体外研究

发布时间：2018-02-16 22:31

本文关键词： 鼻咽癌酪氨酸激酶B 脑源性神经生长因子微血管密度血管形成　出处：《中南大学》2012年博士论文　论文类型：学位论文

【摘要】：目的鼻咽癌(Nasopharyngeal carcinoma, NPC)是来源于鼻咽上皮组织的高度恶性肿瘤,其预后与肿瘤的局部复发、淋巴结转移和远处转移密切相关。有研究发现肿瘤血管形成与恶性肿瘤生长、侵袭转移与及预后密切相关。血管内皮生长因子(vascular endothelial growth factor, VEGF)是人们发现的重要的血管生成因子,通过促肿瘤血管生成而在肿瘤发生发展中发挥重要作用。酪氨酸激酶B(Tyrosine kinase B, TrKB)在肿瘤血管生成及肿瘤生长,浸润转移及预后密切相关。本部分研究意在探讨VEGF及TrKB在NPC组织中的表达并比较两者表达的相关性及分析与肿瘤临床特征的关系。方法采用免疫组化方法检测55例鼻咽癌组织和30例正常鼻咽组织中VEGF及TrKB蛋白表达水平,比较二者表达差异。根据患者年龄、肿瘤临床分型、原发灶大小、淋巴结转移和有无远处转移将病例分组,应用双变量相关pearson检验分析研究VEGF及TrKB表达与肿瘤临床病理特征之间的关系结果 1.VEGF蛋白于肿瘤细胞浆及血管内皮细胞浆中均表达,TrKB蛋白阳性表达绝大多数位于细胞浆内。在55例鼻咽癌中,45例表达TrKB阳性,表达率为82.1%,47例表达VEGF阳性,表达率为85.7%。30例正常鼻咽组织中,只有3例表达TrKB阳性,5例表达VEGF阳性,肿瘤组织中TrKB、VEGF阳性表达率明显高于正常鼻咽组织(P0.05),鼻咽癌组织中VEGF与TrKB表达有明显相关性(相关系数R=0.586,P0.05)。 2.VEGF蛋白表达与NPC的临床分期(P=0.002)、肿瘤的直径大小(P=0.009)及有无淋巴结转移(P=0.001)明显相关,与患者性别、年龄、有无远处转移无关(P0.05)。TrKB蛋白表达与临床分期(P=0.00)、肿瘤的直径大小(P=0.00)、有无淋巴结转移(P=0.006)明显相关,与患者性别、年龄、有无远处转移无关(P0.05)。结论 1.VEGF, TrKB在NPC组织中表达增加,可能与鼻咽癌的发生发展有关； 2.TrKB可能与鼻咽癌血管形成有关。目的探讨通过酪氨酸激酶受体抑制剂K252a抑制TrKB表达,观察抑制TrKB表达对体外培养的鼻咽癌CNE-1细胞增殖、运动迁移、细胞周期、细胞凋亡及对VEGF表达的影响。方法分别用不同浓度K252a干预鼻咽癌CNE-1细胞,MTT实验比较不同浓度K252a对鼻咽癌CNE-1细胞生长的影响,并计算细胞生存(增殖)率,筛选出K252a最佳干预浓度；通过划痕愈合试验观察抑制TrKB表达后对鼻咽癌CNE-1细胞运动迁移能力的影响；流式细胞术分析抑制TrKB表达对鼻咽癌CNE-1细胞迁移、细胞周期和凋亡的影响。分别用RT-PCR、Western-blot检测K252a干预鼻咽癌CNE-1细胞后VEGF、TrkB在mRNA以及蛋白层面表达的改变并与对照组比较差异。结果 MTT实验：K252a能明显抑制鼻咽癌CNE-1细胞的增殖,使细胞数量减少、密度减低、细胞生存率下降,其对鼻咽癌CNE-1细胞生长的抑制作用与K252a干预时间、浓度呈正相关。K252a作用48h使鼻咽癌CNE-1细胞增殖率抑制最明显的浓度为400nmol/L,细胞生存减少50%(与对照组相比),故选取此浓度为K252a最佳干预浓度。划痕愈合实验：抑制TrKB表达可使划痕愈合延迟,使12h鼻咽癌CNE-1细胞迁移距离明显小于对照组,但无明显统计学差异(P=0.18),使24h小时细胞迁移距离细胞迁移距离明显少于对照组(P=0.013),说明抑制TrKB表达能一定程度上抑制鼻咽癌CNE-1细胞的运动迁移能力。流式细胞仪分析：K252a干预鼻咽癌CNE-1细胞的细胞周期分布,与对照组相比,鼻咽癌CNE-1细胞中处于G0/G1期的细胞显著增多(57.67士7.03vs73.86±4.22, P=0.018),而处于S期(30.67±3.68vs15.37±2.16,P=0.025)的细胞显著减少,比较有统计学意义(P0.05)；流式细胞仪技术检测K252a干预鼻咽癌CNE-1细胞后细胞凋亡情况,K252a与对照组细胞的凋亡率比较有统计学意义(22.27±3.09vs3.42±2.56,P=0.01)。 Western blot:K252a干预鼻咽癌CNE-1细胞后,与对照组相比,VEGF蛋白表达水平下调(0.6883±0.19vs0.378±0.44),TrKB蛋白表达水平下调(0.631±0.15vs0.313±0.28),差别均有统计学意义(P0.05)。 RT-PCR:K252a干预鼻咽癌CNE-1细胞后,与对照组相比,细胞内TrkB mRNA表达下调(0.67±0.37vs0.435±0.63)；VEGF mRNA表达下调(0.6883±0.49vs0.425±0.17),差异有统计学意义(P0.05)。结论 1.TrKB表达抑制可降低鼻咽癌CNE-1细胞生长迁移能力； 2.TrKB表达抑制可通过促进鼻咽癌CNE-1细胞凋亡、细胞周期改变和降低VEGF表达,从而减弱生长与迁移能力； 3.TrKB通过调控VEGF影响鼻咽癌血管形成。目的体内肿瘤生长依赖肿瘤血管生成。近年来发现VEGF通过诱导血管生成及促进血管内皮细胞分裂从而促进血管形成。研究证实血管生成、微血管与肿瘤侵袭转移之间的关系密切,微血管密度(microvessel density, MVD)与肿瘤的转移和预后有关,且VEGF表达与MVD的表达有良好的相关性。前部分体外实验研究已经发现K252a能抑制VEGF蛋白和基因水平的表达,本部分实验通过建立裸小鼠移植瘤模型,探讨K252a干预鼻咽癌CNE-1细胞对人鼻咽癌血管生成抑制作用的体内研究,为针对鼻咽癌抗血管生成基因治疗寻找新的思路。方法使用4-6周龄雄性BALB/C-nu/nu裸鼠,体重在16-20g,将人鼻咽癌CNE-1细胞于裸鼠右侧季肋部皮下注射,建立荷人鼻咽癌细胞裸小鼠皮下移植瘤模型。随机将实验动物分为2组,A组：K252a干预组,7只；B组：PBS组,7只；于接种肿瘤细胞后第6、8、10、12、14天用微量注射器于A组裸鼠肿瘤内多点注射K252a药物,400nmol/L200ul/次,对两组移植瘤模型进行为期21天的实验观察,白第一次注射药物后每隔2天测量1次裸鼠肿瘤体积并绘制肿瘤生长曲线。于接种后第21天处死,剥离肿瘤标本,测量最后体积,观察标本整体特征。取出部分组织的标本于固定于10%福尔马林溶液中,于我院病理科常规石蜡包埋后行连续切片,进行常规HE染色、VEGF、 TrKB和CD31相关抗原免疫组化染色。结果 1.肿瘤标本大体形态及HE染色观察：2组实验动物成瘤均于接种后第4天。两组肿瘤与周围组织分界清楚,包膜基本完整。K252a干预组治疗组皮下肿瘤瘤体较小,呈不规则球形,也有的呈分叶状,表面灰白色,质地较硬。剖面呈白色,剖面渗血较少。对照组肿瘤瘤体较K252a组明显增大,表面多不平,有分叶状结节状,表面丰富血管形成,剖面渗血较多； 2.实验动物瘤体体积观察：K252a干预组从给药开始,肿瘤生长缓慢,第21天肿瘤瘤体体积为(328±326.7)mm3,同期对照组为(1044±818)mm3； 3.两组CD31标记的MVD计数与VEGF、TrKB染色评分对比及相关性分析 K252a干预组和对照组阳性血管计数分别为22.39±4.72、49.79±11.12,方差分析各组间差别具有显著性(P0.05)。K252a干预组VEGF表达明显比对照组下降,并有统计学差异(7.57±2.82vs9.86±2.79,P=0.007),K252a干预组明显比对照组TrKB表达下降,并有统计学差异(5.8571±2.79vs7.43±2.64,P=0.005),各组VEGF表达与MVD明显相关(对照组：r=0.88,P=0.009,K252a组：r=0.96,P=0.01),MVD与TrKB表达明显正相关(对照组：r=0.79,P=0.03,K252a组：r=0.85,P=0.01)。对照组与K252a组VEGF表达与TrKB无明显相关性。结论 1.抑制TrKB的表达能抑制鼻咽癌在动物体内的生长；并能通过减少VEGF表达明显减少鼻咽癌组织内的肿瘤新生血管； 2.TrKB有可能成为抗鼻咽癌新生血管治疗的新靶点。
[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 卤 7.03 vs 73.86 卤 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 卤 0.19 vs 0.378 卤 0.44 ) , and the level of TrKB protein expression was down regulated ( 0.631 卤 0.15 vs 0.313 卤 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 卤 0.37 vs 0.435 卤 0.63 ) and VEGF mRNA expression was down - regulated ( 0.6883 卤 0.49 vs 0.425 卤 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 卤 326.7 ) mm3 on day 21 , and the control group was ( 1044 卤 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 .

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.63

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