Survivin对神经母细胞瘤细胞生物学影响及其抑制剂YM155化疗增敏作用研究

发布时间：2018-06-13 12:42

本文选题：神经母细胞瘤 + Survivin　；参考：《山东大学》2014年博士论文

【摘要】：研究背景：神经母细胞瘤(Neuroblastoma, NB)是发生在儿童和婴儿中最常见的恶性颅外实体肿瘤之一,其起病部位隐匿,临床治疗困难,尤其对于进展期NB患儿,生存率仍然维持在20%-30%,复发率居高不下。因此,对神经母细胞瘤的生物学、遗传学特性及具体发病机制进行深入研究,对寻找新的肿瘤基因治疗方法、提高NB患儿的治愈率和改善患儿的远期预后具有重要的临床价值。 Survivin蛋白属于凋亡抑制蛋白家族,在多种恶性肿瘤中均被发现高表达,提示其可能在肿瘤发病机制中起重要作用。Survivin可能参与NB的发生、发展、演变的过程,它不仅可能是判断NB预后的重要参考指标,也可能成为NB基因治疗中的一个理想靶点。YM155是水溶survivi特异抑制剂,体内外均可特异性地抑制survivin的表达,具有强大的抗肿瘤功效,同时可增加常见肿瘤对化疗、放疗的敏感性。Survivin在NB肿瘤细胞的增殖、凋亡、迁移和转移等过程中究竟扮演着什么调控角色?而YM155作用于NB肿瘤细胞的效果如何呢?在体内、体外研究应用中YM155是否能发挥对NB肿瘤细胞的顺铂化疗增敏作用呢?本课题通过体内外实验相互结合,进一步研究Survivin和其抑制剂YM155与NB的关系,以期为NB靶向治疗寻找新的靶点。第一部分Survivin ASONDN对神经母细胞瘤细胞迁移和侵袭的影响目的将Survivin的反义寡核苷酸以不同浓度转染入人神经母细胞瘤细胞株SK-N-SH,观察Survivin对SK-N-SH增殖、凋亡、迁移和侵袭的影响。方法设计并合成Survivin的反义寡核苷酸(ASODN),以脂质体为载体,以不同浓度转染入体外培养的人神经母细胞瘤细胞株SK-N-SH;采用RT-PCR和Westernblot分析经过不同浓度Survivin ASODN干扰后SK-N-SH细胞中Survivin mRNA和蛋白表达水平；应用MTT法检测不同浓度Survivin ASODN对细胞的生长抑制作用,流式细胞仪检测转染后的细胞凋亡率；用Transwell实验分析Survivin ASODN对SK-N-SH细胞体外迁移力和侵袭力的影响。结果不同浓度Survivin ASODN作用于SK-N-SH细胞48h后,Survivin mRNA表达均明显下调,与空白对照组、脂质体转染对照组及正义寡核苷酸SODN组比较,有显著的差异(P0.05),且抑制作用呈现剂量依赖性,以浓度为800nmol/L时的作用最明显。SK-N-SH细胞经200、400、800lmol/L Survivin ASODN转染48h后,Survivin蛋白表达较空白对照组、脂质体转染对照组及正义寡核苷酸SODN组明显下降(P0.05),且抑制作用呈现剂量依赖性,以浓度为800nmol/L时的作用最明显。不同浓度Survivin ASODN转染入SK-N-SH细胞后,可不同程度地抑制该肿瘤细胞的生长增殖,相同作用时间下各浓度ASODN转染组细胞抑制率均显著高于空白对照组、脂质体转染对照组和正义寡核苷酸SODN组(P0.05)。相同浓度(800nmol/L)下Survivin ASODN随着时间的增加其抑制作用明显增强,有着较为明显的时效关系,48h抑制率升至最高。各Survivin ASODN转染组细胞凋亡率均明显高于空白对照组、脂质体转染对照组和SODN转染组(P0.05),且细胞的凋亡率随着Survivin ASODN浓度增加而增加,呈剂量依赖关系,以800nmol/L时,其促进细胞调亡的作用最强。相同浓度(800nmol/L) Survivin ASODN转染SK-N-SH细胞后,细胞凋亡率48h达高峰,72h后开始下降,表明Survivin ASODN对SK-N-SH细胞凋亡的促进作用呈一定的时间依赖性,最佳作用时间48h。细胞迁移和侵袭实验中,转染作用24、48、72h后,各Survivin ASODN转染组迁移穿膜细胞数和侵袭穿膜细胞数均明显减少,与空白对照组、脂质体转染对照组、SODN对照组间比较,有显著的差异(P0.05),且呈现一定的剂量和时间依赖性,以浓度为800nmol/L时的作用最明显,最佳作用时间为48h。结论 Survivin ASODN能通过特异性抑制SK-N-SH细胞中Survivin mRN及蛋白的表达,诱导SK-N-SH细胞凋亡,抑制肿瘤增殖,并降低SK-N-SH细胞的侵袭和迁移能力。第二部分YM155对神经母细胞瘤细胞迁移和侵袭的影响目的探讨Survivin抑制剂YM155对人神经母细胞瘤细胞株SK-N-SH增殖、凋亡、迁移和侵袭的影响及其作用机制。方法用不同浓度YM155处理SK-N-SH,采用RT-PCR和Western blot分析经处理后SK-N-SH细胞中Survivin mRNA和蛋白表达水平；应用MTT法检测处理后细胞的生长抑制作用,流式细胞仪检测处理后的细胞凋亡率,用Transwell实验分析YM155对SK-N-SH细胞体外迁移力和侵袭力的影响。结果 SK-N-SH细胞经不同浓度YM155处理后,10-500nmol/LYM155处理组SK-N-SH细胞中Survivin mRNA、蛋白表达均明显下降(P0.05),且该作用呈剂量依赖性,随YM155浓度增加,细胞中Survivin mRNA、蛋白表达随之下降。以100nmol/L YM155处理SK-N-SH细胞12、24、48、72h后,SK-N-SH细胞中Survivin蛋白表达均明显下降(P0.05),且此抑制作用呈现时间依赖性。经YM155处理72h后,SK-N-SH细胞增殖受到不同程度的抑制,抑制率均明显高于空白对照组(P0.05),且抑制率随着YM155浓度(10-500nmol/L)增加而增加,呈剂量依赖关系。100nmol/L YM155作用12、24、48、72h后,SK-N-SH细胞增殖受到不同程度的抑制,YM155组细胞抑制率均明显高于空白对照组(P0.05),且细胞的抑制率随着YM155作用时间延长而增加,呈时间依赖关系。经YM155处理72h后,SK-N-SH细胞凋亡率受到不同程度影响,凋亡率均明显高于空白对照组(P0.05),且细胞的凋亡率随着YM155浓度(10-500nmol/L)增加而增加,呈剂量依赖关系。100nmol/L YM155作用12、24、48、72h后,SK-N-SH细胞凋亡率逐渐增加,明显高于空白对照组,呈时间依赖关系。细胞迁移和侵袭实验中,10-500nmol/L YM155作用于SK-N-SH细胞12、24、48、72h后,肿瘤细胞迁移穿膜细胞数和侵袭穿膜细胞数明显减少,与空白对照组比较,有显著的差异(P0.05),亦呈现剂量和时间依赖性。结论 YM155作为特异性的Survivin抑制剂,可能通过下调NB肿瘤细胞中Survivin的表达,抑制肿瘤细胞的增殖,并可诱导肿瘤细胞凋亡,降低其迁移和侵袭能力。第三部分YM155对神经母细胞瘤细胞的顺铂化疗增敏作用研究目的研究联合应用YM155和顺铂对人神经母细胞瘤细胞细胞株SK-N-SH增殖、凋亡的影响,探讨YM155对顺铂化疗敏感性的影响及其作用机制。方法分别用顺铂、YM155、YM155联合顺铂处理SK-N-SH,应用MTT法检测处理后细胞的生长抑制作用,流式细胞仪检测处理后的细胞凋亡率,采用Western blot分析经过处理后SK-N-SH细胞中Survivin蛋白表达水平。结果 YM155联合顺铂(0.05,1,2μmol/L)作用于SK-N-SH细胞12～72h,细胞抑制率出现逐渐升高的趋势,与单独YM155组、单独顺铂组、对照组比较,差异具有显著性(P0.05)。不同浓度(0.05,1,2μmol/L)顺铂联合YM155作用72h,各组SK-N-SH细胞的抑制率比较,差异无统计学意义(P0.05),YM155可以增加SK-N-SH细胞对顺铂的敏感性,细胞生长的抑制作用加强,即使减少顺铂的使用剂量也可以达到明显的抗肿瘤作用。顺铂联合YM155作用于SK-N-SH细胞12-72h,细胞凋亡率均明显增高,明显高于单独YM155组、单独顺铂组、对照组,差异具有显著性(P0.05)。作用72h时,不同浓度顺铂(0.05,1,2u mol/L)联合YM155作用SK-N-SH细胞的凋亡率比较,差异无显著性(P0.05),说明YM155对顺铂的化疗增敏作用明显,可以增加SK-N-SH细胞对顺铂的敏感性,有效诱导细胞凋亡,减少化疗中顺铂的使用剂量。 YM155联合顺铂(0.05,1,2μmol/L)组和单独YM155组的Survivin蛋白表达均被完全抑制,而单独顺铂组和对照组Survivin蛋白表达均无明显变化。顺铂单独作用SK-N-SH细胞,对Survivin蛋白表达无抑制作用。结论 YM155对顺铂的增敏作用机制可能与YM155特异性地抑制凋亡抑制蛋白Survivin的表达密切相关。第四部分YM155对神经母细胞瘤裸鼠移植瘤模型顺铂增敏作用研究目的建立神经母细胞瘤荷瘤鼠模型,进一步评价体内YM155对顺铂化疗敏感性的影响。方法建立神经母细胞瘤荷瘤鼠模型,在肿瘤及肿瘤周围分别注射顺铂、YM155及YM155联合顺铂,观察用药3周时间内移植瘤生长情况,计算肿瘤体积；处死老鼠后,剥离肿瘤称重,计算抑瘤率(IR),进行肿瘤组织常规病理检查,采用TUNEL法观察肿瘤组织中细胞凋亡的情况,应用RT-PCR法和免疫组织化学法检测肿瘤组织中survivin mRNA和蛋白质表达水平。结果各实验组给药3周后,处死裸鼠,剥取肿瘤并称重,计算抑瘤率。结果表明,顺铂+YM155组的肿瘤重量明显轻于其余两组,而抑瘤率明显高于顺铂组及YM155组(P0.01),用药治疗的3周期间,顺铂(cispatin)+YM155组的肿瘤体积增长明显放缓,其曲线最低,与YM155组、顺铂组和对照组比较,均有显著性差异(P0.05)。顺铂和YM155对NB裸鼠移植瘤生长均具有一定抑制作用,两者联合应用能更有效抑制NB裸鼠移植瘤生长,明显优于单独使用顺铂或YM155。用药结束后检测四组肿瘤组织中细胞凋亡的情况,发现顺铂+YM155组的细胞凋亡指数AI最高,次之是YM155组,其次是顺铂组。顺铂+YM155组的AI明显高于YM155组、顺铂组和对照组(P0.05),而YM155组的AI也明显高于顺铂组。这说明顺铂和YM155均能诱导NB的肿瘤细胞发生凋亡,但YM155的作用更显著,且两者联合应用时,能更有效地诱导肿瘤细胞凋亡,明显优于单独使用顺铂或YM155。 YM155组和顺铂+YM155的裸鼠移植瘤中survivin mRNA表达显著下降,明显低于对照组和顺铂组(P0.05),而对照组和顺铂组比较,差异无显著性(P0.05)。免疫组织化学检测结果显示,各组裸鼠移植瘤组织中均发现不同程度的survivin蛋白阳性表达,其阳性表达主要位于细胞浆或细胞膜。对照组和顺铂组中有大量强阳性的survivin蛋白表达,YM155组和顺铂+YM155的survivin阳性表达率明显降低,survivin阳性表达的强度和区域面积均较对照组和顺铂组明显降低。结论 YM155联合顺铂应用可能是治疗NB的一个有效的新方法。
[Abstract]:Background of Study :

Neuroblastomas ( NB ) is one of the most common malignant extracranial solid tumors in children and infants . It is difficult for clinical treatment . Especially for children with advanced stage NB , the survival rate is still maintained between 20 % and 30 % , the recurrence rate is high . Therefore , it is important to search for new tumor gene therapy methods , improve the curative rate of NB children and improve the long - term prognosis of children .

Survivin protein belongs to the family of apoptosis - inhibiting proteins , and is highly expressed in many malignant tumors . It suggests that it may play an important role in the pathogenesis of NB . Survivin may play an important role in the development and evolution of NB tumor cells .

Objective : To investigate the effect of survivin ASONDN on the migration and invasion of neuroblastomas .

Survivin antisense oligonucleotides were transfected into human glioma cell line SK - N - SH at different concentrations , and the effects of Survivin on proliferation , apoptosis , migration and invasion of SK - N - SH were observed .

method

Survivin antisense oligonucleotides ( ASODN ) were designed and synthesized . The expression of Survivin mRNA and protein in SK - N - SH cells was determined by RT - PCR and Western blot .
MTT assay was used to detect the inhibitory effect of survivin ASODN on cell growth , and the apoptosis rate was detected by flow cytometry .
The effects of survivin ASODN on the in vitro migration and invasion of SK - N - SH cells were analyzed by Transwell experiment .

Results

The expression of Survivin mRNA was down - regulated after 48 h of SK - N - SH cells with different concentrations of Survivin ASODN . Compared with control group , liposome - transfected control group and sense oligonucleotide SODN group , the expression of Survivin decreased significantly ( P0.05 ) .

After transfected into SK - N - SH cells with different concentrations of Survivin ASODN , the growth and proliferation of the tumor cells were inhibited to some extent , and the inhibitory rates of ASODN - transfected group were significantly higher than those in the control group ( P0.05 ) .

Survivin ASODN transfected SK - N - SH cells with the same concentration ( 800 nmol / L ) .

After 24 , 48 and 72 h transfection , the number of migration and invasion - through membrane cells of survivin ASODN transfected group were significantly decreased compared with control group , liposome - transfected control group and SODN control group .

Survivin ASODN can specifically inhibit the expression of Survivin mRN and protein in SK - N - SH cells , induce apoptosis of SK - N - SH cells , inhibit tumor proliferation , and reduce the invasion and migration ability of SK - N - SH cells .

The effect of the second part YM155 on the migration and invasion of glioma cells

Purpose

Objective To investigate the effects of survivin inhibitor YM155 on proliferation , apoptosis , migration and invasion of human glioma cell line SK - N - SH and its mechanism of action .

method

SK - N - SH was treated with different concentrations of YM155 . The expression of Survivin mRNA and protein in SK - N - SH cells was analyzed by RT - PCR and Western blot .
The effect of YM155 on the in vitro migration and invasion of SK - N - SH cells was investigated by MTT assay .

Results

The expression of Survivin mRNA and protein in SK - N - SH cells decreased significantly ( P0.05 ) , and the expression of Survivin in SK - N - SH cells decreased significantly ( P0.05 ) .

The inhibition rate of SK - N - SH cells was significantly higher than that in the control group ( P0.05 ) , and the inhibition rate of SK - N - SH cells was significantly higher than that in the blank control group ( P0.05 ) .

The apoptosis rate of SK - N - SH cells increased with the increase of YM155 ( 10 - 500 nmol / L ) . The apoptosis rate of SK - N - SH cells increased gradually with the concentration of YM155 ( 10 - 500 nmol / L ) .

In the experiment of cell migration and invasion , 10 - 500 nmol / L YM155 acts on SK - N - SH cells at 12 , 24 , 48 and 72 h , and the number of cell - penetrating cells and the number of invasive and penetrating cells in tumor cells decreased significantly . Compared with the blank control group , there was a significant difference ( P0.05 ) , and the dose and time - dependence were also presented .

YM155 , as a specific Survivin inhibitor , may inhibit the expression of Survivin in NB tumor cells , inhibit the proliferation of tumor cells , and induce tumor cell apoptosis and reduce its migration and invasion ability .

The effect of the third part YM155 on the cisplatin - chemotherapy sensitization of neuroblastoid cells

Purpose

To investigate the effect of YM155 and cisplatin on the proliferation and apoptosis of human glioma cell line SK - N - SH , and to investigate the effect of YM155 and cisplatin on the sensitivity of cisplatin chemotherapy and its mechanism .

method

SK - N - SH was treated with cisplatin , YM155 , YM155 and cisplatin respectively . The growth inhibition of treated cells was determined by MTT assay . The expression of Survivin in SK - N - SH cells was analyzed by Western blot analysis .

YM155 combined cisplatin ( 0.05 , 1 , 2 渭mol / L ) on SK - N - SH cells for 12 - 72h , and the inhibition rate of the cells increased gradually . Compared with control group alone , the inhibition rate of SK - N - SH cells increased significantly ( P0.05 ) , and YM155 could increase the sensitivity of SK - N - SH cells to cisplatin and enhance the inhibition of cell growth .

The apoptosis rate of SK - N - SH cells was significantly higher than that of YM155 alone ( P0.05 ) .

Survivin protein expression was completely inhibited by YM155 combined with cisplatin ( 0.05 , 1 , 2渭mol / L ) and single YM155 alone .

The mechanism of sensitization of YM155 to cisplatin may be closely related to the inhibition of the expression of Survivin in YM155 .

Objective : To study the effect of the fourth part YM155 on the cisplatin sensitization in the nude mice transplanted tumor

The effect of YM155 in vivo on the sensitivity of cisplatin chemotherapy was further evaluated .

The tumor and tumor were injected with cisplatin , YM155 and YM155 respectively to observe the growth of transplanted tumor within 3 weeks , and the tumor volume was calculated .
After the mice were killed , the tumor was weighed , the tumor inhibition rate ( IR ) was calculated , the tumor tissue was examined by routine pathology , and the expression level of survivin mRNA and protein in tumor tissues was detected by TUNEL method .

The results showed that the tumor weight of cisplatin + YM155 group was significantly lower than that of cisplatin group and YM155 group ( P0.01 ) . The results showed that the tumor volume of cisplatin + YM155 group was significantly slower than that of cisplatin group and YM155 group ( P0.01 ) .

It was found that the AI of the cisplatin + YM155 group was higher than that in the group YM155 , the cisplatin group and the control group ( P0.05 ) , but the AI of the group YM155 was significantly higher than that in the cisplatin group .

Survivin mRNA expression was significantly lower in YM155 and cisplatin + YM155 nude mice than that in control group and cisplatin group ( P0.05 ) , but there was no significant difference between control group and cisplatin group ( P0.05 ) .

The positive expression of survivin was found to be significantly lower in the control group and the cisplatin group than in the control group and the cisplatin group . Conclusion The positive expression of survivin in the control group and the cisplatin group was significantly lower than that in the control group and the cisplatin group .

The combination of YM155 and cisplatin may be an effective new method for the treatment of NB .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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