泪腺腺样囊性癌嗜神经侵袭的机制研究
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摘要
目的
1.检测泪腺腺样囊性癌(ACC)组织中胶质源性神经营养因子(GDNF), GDNF家族受体α1(GFRα-1)、酪氨酸激酶受体(Ret)和神经细胞粘附因子(NCAM)以及凋亡抑制蛋白(Survivin)的表达,并观察其与泪腺ACC组织嗜神经侵袭(PNI)等生物学行为之间的关系,以及彼此之间的相关性。
2.研究GDNF对体外培养人腺样囊性癌细胞(ACC-2)增殖和迁移的影响,为进一步明确GDNF在泪腺腺样囊性癌PNI机制提供实验依据。
3.分别探讨人腺样囊性癌细胞(ACC-2)中GDNF、GFRα-1、NCAM、Survivin蛋白质和]mRNA表达的相关性,进一步了解它们在泪腺ACC PNI微环境中的相互作用与关系,为临床寻找控制早期PNI的有效靶点提供参考依据。
方法
1.免疫组织化学SABC法检测51例泪腺ACC患者手术标本和5例正常泪腺组织中GDNF、GFRα-1、Ret、NCAM以及Survivin的表达状况。
2.通过MTT法、绘制细胞生长曲线,以及流式细胞术检测不同浓度(时间)的GDNF对人腺样囊性癌细胞(ACC-2)增殖和凋亡的影响；Transwell小室法研究不同浓度(时间)的GDNF对人腺样囊性癌细胞(ACC-2)迁移能力的影响。
3.分别通过(?)Vestern Blotting蛋白印迹法和RT-PCR法检测一定浓度的GDNF对人腺样囊性癌细胞(ACC-2)中GDNF、GFRα-1、NCAM以及Survivin蛋白质和]nRNA表达的影响。
结果
1.泪腺ACC组织中GDNF、GFRα-1、Ret、NCAM以及Survivin的表达率依次为62.7%(32/51)、62.7%(32/51)、54.9%(28/51)、60.8%(31/51)、60.8%(31/51),显著高于泪腺正常组织；实体型ACC、复发组ACC的嗜神经侵袭率显著高于腺样-管状型ACC、原发组ACC的嗜神经侵袭率,差别具有统计学意义(P<0.05),有/无疼痛组ACC间嗜神经侵袭率的差异无统计学意义(P>0.05)。
2.实体型ACC组织中NCAM、Survivin的表达率显著高于其在腺样-管状型ACC组织中的表达率,复发组ACC组织中GFRα-1、Ret、NCAM以及Survivin的表达率显著高于其在原发组ACC组织中的表达率,有嗜神经侵袭组ACC中GDNF GFRα-1、Ret、NCAM的表达率显著高于其在无嗜神经侵袭组ACC中的表达率,上述差别均具有统计学意义(P<0.05)。
3.泪腺ACC组织中GDNF的阳性表达与GFRα-1、Ret、NCAM的阳性表达均具有明显相关性,GDNF的阳性表达与Survivin的阳性表达无明显相关性。
4.标准培养48h,GDNF浓度在20～120ng/ml之间,可促进ACC-2细胞系的增殖,与对照组相比差异具有统计学意义(P<0.05),其中,80ng/ml的GDNF促进ACC-2增殖作用最明显。在第72h,80ng/ml GDNF促进ACC-2细胞系的增殖作用最显著(P<0.05)。上述结果与80ng/ml GDNF作用下的ACC-2细胞生长曲线的趋势一致。
5.标准培养48h后,应用流式细胞仪检测,80ng/ml GDNF干预组ACC-2细胞中的G1期ACC-2细胞比例较对照组减少,S期ACC-2细胞比例较对照组增加。
6.标准培养40h, GDNF在60～160ng/ml浓度下促进ACC-2细胞迁移(P<0.05),其中,160ng/ml的GDNF促进ACC-2迁移作用最强。从第24h起,100ng/ml GDNF在各时间点(24h,30h,40h)均可明显促进ACC-2细胞系迁移,与对照组相比差异均有统计学意义(P<0.05)。
7.80ng/ml GDNF能够一致性上调ACC-2细胞的GFRα-1、NCAM、Survivin蛋白质和(?)m RNA的表达,下调了ACC-2细胞中GDNF m RNA和蛋白质的表达。
结论
1.泪腺ACC具有嗜神经侵袭性生物学行为,嗜神经侵袭现象可作为泪腺ACC预后不良的临床观察指标之一。
2. GDNF、GFRα-1、Ret在泪腺ACC嗜神经侵袭中发挥重要作用,三者可以作为判断有无嗜神经侵袭现象的生物学指标,而后两者有望成为预测及评估预后的因素之一。
3. GDNF可以促进体外培养的人ACC-2细胞的增殖和迁移能力,并且分别具有剂量和时间依赖性,但是GDNF对ACC-2细胞的凋亡水平无明显的调控作用。
4. GDNF通过GFRα-1-Ret信号传导途径,或者通过NCAM这一非依赖Ret的信号途径,沿着以周围神经为中心的GDNF的浓度梯度,以及凭借NCAM的异/同嗜性粘附作用,使得泪腺ACC组织与周围神经相互吸引,形成了有利于肿瘤嗜神经侵袭的微环境,增强了泪腺ACC肿瘤细胞的神经侵袭能力。
5. NCAM的高表达与泪腺ACC的病理类型相关,同时成为判断泪腺ACC嗜神经侵袭行为与预后的指标,通过检测NCAM在泪腺ACC肿瘤细胞周围的表达,特别是在肿瘤周围神经中的表达,了解泪腺ACC嗜神经侵袭的情况,可能发现肿瘤细胞的残留或早期的神经侵犯,以期指导临床治疗。
6. Survivin的表达与泪腺ACC的病理类型、预后密切相关,却与泪腺ACC的嗜神经侵袭现象无明显的相关性；Survivin与GDNF可能具有促进ACC-2细胞增殖的协同作用。
Objective
1. To detect the expression and correlation of GDNF, GFRa-1, Ret, NCAM and Survivin in adenoid cystic carcinoma of lacrimal gland as well as their relations with perineural invasion of adenoid cystic carcinoma(ACC) of lacrimal gland.
2. To study the effect of GDNF on ACC-2cells proliferation and migration, and to provide experimental evidence for the mechanisms of perineural invasion in adenoid cystic carcinoma of lacrimal gland.3. To investigate the relations of protein and mRNA of GDNF, GFRa-1, NCAM and Survivin in ACC-2cells respectively, and we would grasp their interaction and association in the microenvironment of ACC to provide references for clinically effective targets.
Methods
1. The expression of GDNF, GFRa-1, Ret, NCAM and Survivin was evaluated in the51operative samples of adenoid cystic carcinoma of lacrimal gland as well as5samples of normal lacrimal gland by SABC immunohistochemical staining.
2. MTT assay, the growth curve, flow cytometry was employed to observe the influence on the proliferation and apoptosis of ACC-2cells with different concentrations (time) courses of GDNF. Meanwhile, Transwell chamber was used to observe the migration effect of GDNF on ACC-2cells.
3. When exposed to determinate concentrations of GDNF, protein and mRNA of GDNF, GFRa-1, NCAM and Survivin in ACC-2cells were detected by Western blotting and RT-PCR respectively.
Results
1. Expression rate of GDNF, GFRa-1, Ret, NCAM and Survivin in the samples of adenoid cystic carcinoma(ACC) of lacrimal gland was as following:62.7%(32/51),62.7%(32/51),54.9%(28/51),60.8%(31/51),60.8%(31/51)each of which was higher than in normal lacrimal gland tissue. The perineural invasion(PNI) rate of solid pattern ACC and recurrent tumor group were higher than in adenoid-tubular pattern ACC and primary tumor group, there were significant differences between two groups (P<0.05), and there were no significant differences between pain group and no-pain group (P>0.05).
2. Expression rate of NCAM and Survivin in solid pattern ACC was higher than in adenoid-tubular pattern ACC, Expression rate of GFRα-1, Ret, NCAM and Survivin in recurrent tumor group was higher than in primary tumor group, Expression rate of GDNF, GFRa-1, Ret and NCAM in PNI tumor group was higher than in no-PNI tumor group, there were significant differences in above-mentioned groups(P<0.05).
3. There was significant correlation between the expression rate of GDNF and GFRa-1, Ret, NCAM in ACC of lacrimal gland, but no significant correlation between the expression rate of GDNF and Survivin.
4. With standard cultivated for48hours, the proliferation of ACC-2cells was stimulated when exposed to incremental concentrations of GDNF(20～120ng/ml), which had statistical significant differences compared with control group. The effect of GDNF on ACC-2cells proliferation reached its maximum at the concentration of80ng/ml. On the72th hour, GDNF(80ng/ml) can promote the proliferation of ACC-2cells significantly which got the coincidental tendency to the growth curve interfered in GDNF(80ng/ml).
5. After standard cultivated for48hours with GDNF(80ng/ml), the ratio of ACC-2cells in G1period was decreased compared with control group, and the ratio of ACC-2cells in S period was increased by flow cytometry.
6. With standard cultivated for40hours, the count of migration cells was increased by GDNF(60-160ng/ml). The effect of GDNF on ACC-2cells migration reached its maximum at the concentration of160ng/ml. From the24th hour, GDNF (100ng/ml) can promote the migration of ACC-2cells in a time-dependent manner(24h,30h,40h), which had statistical significant differences compared with control group(P<0.05).
7. GDNF(80ng/ml) enhanced the mRNA and protein expression of GFRα-1, NCAM, Survivin in ACC-2cells, and weakened the mRNA and protein expression of GDNF in ACC-2cells. Conclusions
1. Aggressive biological behavior of ACC of lacrimal gland includes perineural invasion phenomenon, which can be used as a poor prognosis clinical observation of one of the indicators in the lacrimal gland ACC.
2. GDNF, GFRa-1, Ret play an important role in the perineural invasion in ACC of lacrimal gland, which can become biological indicators to judge whether exists PNI phenomenon, and the latter two are expected to become one of the factors of prediction and assessment for prognosis.
3. GDNF can promote cultured human ACC-2cells proliferation and migration, in a dose and time dependent manner respectively, but no significant role in regulation of GDNF on the level of ACC-2cells apoptosis.
4. GDNF via GFRa-1-Ret signaling pathway, or by non-dependent Ret signaling pathway-NCAM, through the concentration gradient along the peripheral nerve-center of GDNF, as well as by virtue of NCAM different/same tropism adhesion, ACC and peripheral nerve attracted each other, forming a micro-environment conducive to PNI, and enhanced the ability of PNI for ACC cells.
5. The expression of NCAM is associated with the pathological type of ACC of lacrimal gland, which become indicators for PNI and prognosis to ACC of lacrimal gland at the same time. Through the detection of NCAM expression around ACC of lacrimal gland, especially the expression in peripheral nerve nearby, you may find residual tumor cells or early PNI in ACC of lacrimal gland, and guide clinical treatment.
6. The expression of Survivin is closely related to the pathological type and prognosis,of ACC of lacrimal gland but no obvious correlation with PNI of ACC of lacrimal gland, Survivin and GDNF may promote synergistic effect of ACC-2cells proliferation.
1.检测泪腺腺样囊性癌(ACC)组织中胶质源性神经营养因子(GDNF), GDNF家族受体α1(GFRα-1)、酪氨酸激酶受体(Ret)和神经细胞粘附因子(NCAM)以及凋亡抑制蛋白(Survivin)的表达,并观察其与泪腺ACC组织嗜神经侵袭(PNI)等生物学行为之间的关系,以及彼此之间的相关性。
2.研究GDNF对体外培养人腺样囊性癌细胞(ACC-2)增殖和迁移的影响,为进一步明确GDNF在泪腺腺样囊性癌PNI机制提供实验依据。
3.分别探讨人腺样囊性癌细胞(ACC-2)中GDNF、GFRα-1、NCAM、Survivin蛋白质和]mRNA表达的相关性,进一步了解它们在泪腺ACC PNI微环境中的相互作用与关系,为临床寻找控制早期PNI的有效靶点提供参考依据。
方法
1.免疫组织化学SABC法检测51例泪腺ACC患者手术标本和5例正常泪腺组织中GDNF、GFRα-1、Ret、NCAM以及Survivin的表达状况。
2.通过MTT法、绘制细胞生长曲线,以及流式细胞术检测不同浓度(时间)的GDNF对人腺样囊性癌细胞(ACC-2)增殖和凋亡的影响；Transwell小室法研究不同浓度(时间)的GDNF对人腺样囊性癌细胞(ACC-2)迁移能力的影响。
3.分别通过(?)Vestern Blotting蛋白印迹法和RT-PCR法检测一定浓度的GDNF对人腺样囊性癌细胞(ACC-2)中GDNF、GFRα-1、NCAM以及Survivin蛋白质和]nRNA表达的影响。
结果
1.泪腺ACC组织中GDNF、GFRα-1、Ret、NCAM以及Survivin的表达率依次为62.7%(32/51)、62.7%(32/51)、54.9%(28/51)、60.8%(31/51)、60.8%(31/51),显著高于泪腺正常组织；实体型ACC、复发组ACC的嗜神经侵袭率显著高于腺样-管状型ACC、原发组ACC的嗜神经侵袭率,差别具有统计学意义(P<0.05),有/无疼痛组ACC间嗜神经侵袭率的差异无统计学意义(P>0.05)。
2.实体型ACC组织中NCAM、Survivin的表达率显著高于其在腺样-管状型ACC组织中的表达率,复发组ACC组织中GFRα-1、Ret、NCAM以及Survivin的表达率显著高于其在原发组ACC组织中的表达率,有嗜神经侵袭组ACC中GDNF GFRα-1、Ret、NCAM的表达率显著高于其在无嗜神经侵袭组ACC中的表达率,上述差别均具有统计学意义(P<0.05)。
3.泪腺ACC组织中GDNF的阳性表达与GFRα-1、Ret、NCAM的阳性表达均具有明显相关性,GDNF的阳性表达与Survivin的阳性表达无明显相关性。
4.标准培养48h,GDNF浓度在20～120ng/ml之间,可促进ACC-2细胞系的增殖,与对照组相比差异具有统计学意义(P<0.05),其中,80ng/ml的GDNF促进ACC-2增殖作用最明显。在第72h,80ng/ml GDNF促进ACC-2细胞系的增殖作用最显著(P<0.05)。上述结果与80ng/ml GDNF作用下的ACC-2细胞生长曲线的趋势一致。
5.标准培养48h后,应用流式细胞仪检测,80ng/ml GDNF干预组ACC-2细胞中的G1期ACC-2细胞比例较对照组减少,S期ACC-2细胞比例较对照组增加。
6.标准培养40h, GDNF在60～160ng/ml浓度下促进ACC-2细胞迁移(P<0.05),其中,160ng/ml的GDNF促进ACC-2迁移作用最强。从第24h起,100ng/ml GDNF在各时间点(24h,30h,40h)均可明显促进ACC-2细胞系迁移,与对照组相比差异均有统计学意义(P<0.05)。
7.80ng/ml GDNF能够一致性上调ACC-2细胞的GFRα-1、NCAM、Survivin蛋白质和(?)m RNA的表达,下调了ACC-2细胞中GDNF m RNA和蛋白质的表达。
结论
1.泪腺ACC具有嗜神经侵袭性生物学行为,嗜神经侵袭现象可作为泪腺ACC预后不良的临床观察指标之一。
2. GDNF、GFRα-1、Ret在泪腺ACC嗜神经侵袭中发挥重要作用,三者可以作为判断有无嗜神经侵袭现象的生物学指标,而后两者有望成为预测及评估预后的因素之一。
3. GDNF可以促进体外培养的人ACC-2细胞的增殖和迁移能力,并且分别具有剂量和时间依赖性,但是GDNF对ACC-2细胞的凋亡水平无明显的调控作用。
4. GDNF通过GFRα-1-Ret信号传导途径,或者通过NCAM这一非依赖Ret的信号途径,沿着以周围神经为中心的GDNF的浓度梯度,以及凭借NCAM的异/同嗜性粘附作用,使得泪腺ACC组织与周围神经相互吸引,形成了有利于肿瘤嗜神经侵袭的微环境,增强了泪腺ACC肿瘤细胞的神经侵袭能力。
5. NCAM的高表达与泪腺ACC的病理类型相关,同时成为判断泪腺ACC嗜神经侵袭行为与预后的指标,通过检测NCAM在泪腺ACC肿瘤细胞周围的表达,特别是在肿瘤周围神经中的表达,了解泪腺ACC嗜神经侵袭的情况,可能发现肿瘤细胞的残留或早期的神经侵犯,以期指导临床治疗。
6. Survivin的表达与泪腺ACC的病理类型、预后密切相关,却与泪腺ACC的嗜神经侵袭现象无明显的相关性；Survivin与GDNF可能具有促进ACC-2细胞增殖的协同作用。
Objective
1. To detect the expression and correlation of GDNF, GFRa-1, Ret, NCAM and Survivin in adenoid cystic carcinoma of lacrimal gland as well as their relations with perineural invasion of adenoid cystic carcinoma(ACC) of lacrimal gland.
2. To study the effect of GDNF on ACC-2cells proliferation and migration, and to provide experimental evidence for the mechanisms of perineural invasion in adenoid cystic carcinoma of lacrimal gland.3. To investigate the relations of protein and mRNA of GDNF, GFRa-1, NCAM and Survivin in ACC-2cells respectively, and we would grasp their interaction and association in the microenvironment of ACC to provide references for clinically effective targets.
Methods
1. The expression of GDNF, GFRa-1, Ret, NCAM and Survivin was evaluated in the51operative samples of adenoid cystic carcinoma of lacrimal gland as well as5samples of normal lacrimal gland by SABC immunohistochemical staining.
2. MTT assay, the growth curve, flow cytometry was employed to observe the influence on the proliferation and apoptosis of ACC-2cells with different concentrations (time) courses of GDNF. Meanwhile, Transwell chamber was used to observe the migration effect of GDNF on ACC-2cells.
3. When exposed to determinate concentrations of GDNF, protein and mRNA of GDNF, GFRa-1, NCAM and Survivin in ACC-2cells were detected by Western blotting and RT-PCR respectively.
Results
1. Expression rate of GDNF, GFRa-1, Ret, NCAM and Survivin in the samples of adenoid cystic carcinoma(ACC) of lacrimal gland was as following:62.7%(32/51),62.7%(32/51),54.9%(28/51),60.8%(31/51),60.8%(31/51)each of which was higher than in normal lacrimal gland tissue. The perineural invasion(PNI) rate of solid pattern ACC and recurrent tumor group were higher than in adenoid-tubular pattern ACC and primary tumor group, there were significant differences between two groups (P<0.05), and there were no significant differences between pain group and no-pain group (P>0.05).
2. Expression rate of NCAM and Survivin in solid pattern ACC was higher than in adenoid-tubular pattern ACC, Expression rate of GFRα-1, Ret, NCAM and Survivin in recurrent tumor group was higher than in primary tumor group, Expression rate of GDNF, GFRa-1, Ret and NCAM in PNI tumor group was higher than in no-PNI tumor group, there were significant differences in above-mentioned groups(P<0.05).
3. There was significant correlation between the expression rate of GDNF and GFRa-1, Ret, NCAM in ACC of lacrimal gland, but no significant correlation between the expression rate of GDNF and Survivin.
4. With standard cultivated for48hours, the proliferation of ACC-2cells was stimulated when exposed to incremental concentrations of GDNF(20～120ng/ml), which had statistical significant differences compared with control group. The effect of GDNF on ACC-2cells proliferation reached its maximum at the concentration of80ng/ml. On the72th hour, GDNF(80ng/ml) can promote the proliferation of ACC-2cells significantly which got the coincidental tendency to the growth curve interfered in GDNF(80ng/ml).
5. After standard cultivated for48hours with GDNF(80ng/ml), the ratio of ACC-2cells in G1period was decreased compared with control group, and the ratio of ACC-2cells in S period was increased by flow cytometry.
6. With standard cultivated for40hours, the count of migration cells was increased by GDNF(60-160ng/ml). The effect of GDNF on ACC-2cells migration reached its maximum at the concentration of160ng/ml. From the24th hour, GDNF (100ng/ml) can promote the migration of ACC-2cells in a time-dependent manner(24h,30h,40h), which had statistical significant differences compared with control group(P<0.05).
7. GDNF(80ng/ml) enhanced the mRNA and protein expression of GFRα-1, NCAM, Survivin in ACC-2cells, and weakened the mRNA and protein expression of GDNF in ACC-2cells. Conclusions
1. Aggressive biological behavior of ACC of lacrimal gland includes perineural invasion phenomenon, which can be used as a poor prognosis clinical observation of one of the indicators in the lacrimal gland ACC.
2. GDNF, GFRa-1, Ret play an important role in the perineural invasion in ACC of lacrimal gland, which can become biological indicators to judge whether exists PNI phenomenon, and the latter two are expected to become one of the factors of prediction and assessment for prognosis.
3. GDNF can promote cultured human ACC-2cells proliferation and migration, in a dose and time dependent manner respectively, but no significant role in regulation of GDNF on the level of ACC-2cells apoptosis.
4. GDNF via GFRa-1-Ret signaling pathway, or by non-dependent Ret signaling pathway-NCAM, through the concentration gradient along the peripheral nerve-center of GDNF, as well as by virtue of NCAM different/same tropism adhesion, ACC and peripheral nerve attracted each other, forming a micro-environment conducive to PNI, and enhanced the ability of PNI for ACC cells.
5. The expression of NCAM is associated with the pathological type of ACC of lacrimal gland, which become indicators for PNI and prognosis to ACC of lacrimal gland at the same time. Through the detection of NCAM expression around ACC of lacrimal gland, especially the expression in peripheral nerve nearby, you may find residual tumor cells or early PNI in ACC of lacrimal gland, and guide clinical treatment.
6. The expression of Survivin is closely related to the pathological type and prognosis,of ACC of lacrimal gland but no obvious correlation with PNI of ACC of lacrimal gland, Survivin and GDNF may promote synergistic effect of ACC-2cells proliferation.
引文
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