前列腺癌生物力学性质的改变与其细胞恶性表型相关性的实验研究
发布时间：2018-05-06 05:21
本文选题：前列腺癌 + 力学性质 ； 参考：《大连医科大学》2014年博士论文
【摘要】：前列腺癌是美国及欧洲国家最常见的恶性肿瘤，而许多前列腺癌是通过直肠指诊时触及的硬结发现的。传统观点认为肿瘤组织相较于正常组织要硬，但在临床工作中，当剖开某些肿瘤后，肿瘤组织整体与局部的质地却不尽相同。肿瘤组织进展和转移发生时伴随着肿瘤组织生物力学性质的变化，而在前列腺癌中，组织的力学性质与肿瘤恶性生物学行为之间的关系还不甚明了。 胶原蛋白collagen是细胞外基质中含量最丰富的蛋白，而且它为组织的力学性质提供支架结构，并且胶原蛋白collagen的变化能够导致细胞外基质刚度发生变化。纤维连接蛋白fibronectin分子结构上同时含有与细胞外基质中的胶原蛋白及细胞膜表面整合蛋白integrin家族的结合位点，能够作为桥梁将肿瘤细胞与细胞外基质相连接。而纤维连接蛋白fibronectin和整合蛋白家族integrin又是力学信号转导通路上的关键因子。在之前的研究中，原子力显微镜已经被用于检测生物组织微观层面的力学性质，，而实时弹性超声成像也已经在生物组织宏观层面的力学性质检测上被广泛使用。但是关于前列腺癌组织微观和宏观层面的力学性质的研究较为少见。 目的 1.检测前列腺癌组织宏观及微观力学性质变化，用以探讨前列腺癌力学性质变化与前列腺癌患者的病理分级及肿瘤转移之间的联系; 2.探讨前列腺癌力学性质变化的原因，并在组织层面探索力学信号转导通路中的重要分子; 3.探讨collagen I对前列腺癌细胞粘附、杨氏模量、侵袭、凋亡的影响。 方法 1.收集43例患者的临床资料（前列腺增生20例，前列腺癌23例），按照Gleason评分系统进行病理分级，按照肿瘤分化程度分成1-5分，分化良好为1分，分化极差为5分，将两区的Gleason分值相加即可以得到Gleason评分。根据Gleason评分将23例Pca患者分成两组：低-中级别组（Gleason2-7分）共11例，高级别组（Gleason8-10分）12例。另外，根据肿瘤是否转移，将23例Pca患者分成转移组7例，非转移组16例。所有患者术前均未行任何手术及放、化疗。所有患者在术前均行TRTE检查。应用于AFM检测的BPH标本取自经尿道前列腺电切术（transurethral resection of the prostate, TURP）, Pca组织标本取自经尿道前列腺癌切除术及耻骨上前列腺癌切除术。所有组织术前及术后均行病理学检查确认。 2.我们首先对前列腺癌组织标本进行免疫组织化学实验,根据Gleason评分将23例Pca患者分成两组;另外，根据肿瘤是否转移，将23例Pca患者分成转移组和非转移组。根据免疫组化染色阳性细胞的数量及切片的染色程度来进行免疫组化评分。 3.制作不同浓度的collagen I进行前列腺癌细胞系三维培养，紧接着使用细胞粘附实验、AFM实验、Transwell实验、TUNEL实验等检测不同浓度collagen I上前列腺癌细胞的粘附、杨氏模量、凋亡、侵袭的变化。 结果 1.前列腺癌宏观及微观力学性质的变化及其临床意义 宏观层面列腺组织的力学性质变化，共43例患者穿刺及手术前行TRTE检测，其中BPH患者20例，Pca患者23例。所有患者行TRTE检测后，均行前列腺穿刺活检，并在术后结合病理诊断确认。前列腺癌病灶SI明显高于BPH组织(P0.05)；根据术后病理诊断及患者转移情况，整理TRTE结果资料发现，高级别Pca组织（Gleason8-10）SI明显高于低-中级别Pca组织（Gleason2-7）SI (P0.05)；而转移组与非转移组Pca组织间SI无明显统计学差异(P=0.075)。 微观层面列腺组织的力学性质变化，我们采用AFM检测43例患者组织切片的杨氏模量。结果显示，Pca组织杨氏模量明显低于BPH组织（P0.05）；其中低-中级别肿瘤（Gleason2-7）杨氏模量明显高于高级别肿瘤（Gleason8-10）组织（P0.05）；并且，未出现转移的Pca组织杨氏模量明显高于转移组（P0.05）。 2.前列腺癌力学性质变化原因及力学信号转导重要分子 我们发现BPH中collagen I, III, IV的表达明显高于Pca组织（P0.05），并且，随着肿瘤Gleason分级的增高及转移的发生，三者表达进一步下降（P0.05）；MMP-2在Pca组织中的表达明显高于BPH组织（P0.05），并且Pca转移组表达比非转移组高(P0.05)；MMP-2在Pca低-中级别组Gleason(2-7)表达高于高级别组Gleason(8-10)（(P0.05）；fibronectin在BPH与Pca组织中fibronectin的表达水平未见明显差异，仅发现表达部位的不同（P=0.189）;在Pca组织中，fibronectin的表达强度在Gleason (2-7)组明显高于Gleason (8-10)组（P0.05），在转移组与非转移组之间未见明显差异（P=0.697）;Integrin α5在Pca组织中的表达高于BPH组织（P0.05）。在Pca组织中，Gleason (2-7)组MMP-2的表达明显高于Gleason (8-10)组（P0.05），但其表达在转移组与非转移组之间无明显差异（P=0.122）。 3.不同浓度collagen I干预前列腺癌细胞粘附、杨氏模量、凋亡及侵袭的实验研究 分别在不同浓度的collagen I上对PC-3及LNCaP细胞系进行三维细胞培养，通过使用说明将collagen I在能形成胶体的范围内以1mg/ml及5mg/ml作为后续实验的最低作用浓度及最高作用浓度。后续实验发现,骨转移灶来源的PC-3细胞在collagen I上的粘附能力高于来源于淋巴结转移灶LNCaP细胞；AFM实验表明，PC-3细胞的杨氏模量在低浓度collagen I (1mg/ml)上最低；Transwell侵袭实验表明，在低浓度collagen I上，PC-3细胞侵袭能力高于高浓度collagen I;TUNEL实验表明，高浓度的collagen I能增加PC-3细胞的凋亡敏感性。 结论 1.随着前列腺癌组织恶性程度增高及转移的发生，其宏观及微观力学性质发生相反的改变，表明前列腺癌生物力学性质的改变与肿瘤的恶性程度及转移相关； 2. MMP-2介导的Collagen I降解及肿瘤细胞杨氏模量的改变可能是导致前列腺癌组织生物力学性质变化的原因，且Collagen I, Fibronectin, MMP-2,integrinα5可能是力学信号传导通路上的重要分子； 3. Collagen I的浓度变化能够改变前列腺癌PC-3细胞的形态、粘附能力、力学性质、侵袭能力及凋亡敏感性，表明Collagen I与前列腺癌细胞的恶性表型及力学性质存在相关性。
[Abstract]:Prostate cancer is the most common malignant tumor in the United States and European countries , while many prostate cancer is found by hard - knot access when rectal fingers are diagnosed . Traditional view suggests that tumor tissue is harder than normal tissue , but in clinical work , tumor tissue is different from local texture when the tumor tissue progresses and metastasis occurs , but in prostate cancer , the relationship between the mechanical properties of tissue and the malignant biological behavior of tumor is not very clear .
Collagen collagen is the most abundant protein in extracellular matrix , and it provides scaffold structure for the mechanical properties of tissue , and the change of collagen collagen can lead to the change of extracellular matrix rigidity .
Purpose
1 . To detect the changes of macroscopic and microscopic mechanical properties of prostate cancer , and to explore the relationship between the change of mechanical properties of prostate cancer and the pathological grade and tumor metastasis in patients with prostate cancer ;
2 . To explore the causes of the change of the mechanical properties of prostate cancer , and to explore the important molecules in the mechanical signal transduction pathway at the tissue level ;
3 . To investigate the effects of collagen I on adhesion , Young ' s modulus , invasion and apoptosis of prostate cancer cells .
method
According to Gleason score , 23 patients with Pca were divided into two groups : low - middle - grade group ( Gleason2 - 7 ) and advanced group ( Gleason8 - 10 ) . All patients underwent TRTE examination before operation . All patients were taken from transurethral resection of prostate and prostatectomy . All patients were confirmed by pathological examination before and after operation .
2 . We first conducted an immunohistochemical study of prostate cancer tissue samples , and 23 patients with Pca were divided into two groups according to Gleason score , and 23 patients with Pca were divided into two groups according to Gleason score .
3 . Three - dimensional culture of prostate cancer cell line was performed with different concentrations of collagen I , followed by cell adhesion test , AFM experiment , Transwell experiment , TUNEL assay and so on . The adhesion , Young ' s modulus , apoptosis and invasion of prostatic cancer cells were detected by TUNEL assay .
Results
1 . Changes of macroscopic and microscopic mechanical properties of prostate cancer and its clinical significance
A total of 43 patients with BPH and 23 patients with Pca were examined by TRTE . All patients underwent prostate biopsy after TRTE test , and confirmed by pathology after operation . The SI of prostate cancer was significantly higher than that of BPH ( P0.05 ) .
According to the postoperative pathological diagnosis and the patient ' s metastasis , the results of TRTE data showed that the high - level Pca tissue ( Gleason8 - 10 ) SI was significantly higher than that of the low - medium level Pca ( Gleason2 - 7 ) SI ( P0.05 ) ;
There was no significant difference in SI between the metastasis group and the non - metastasis group ( P = 0.075 ) .
The results showed that the Young ' s modulus of Pca tissue was significantly lower than that of BPH ( P0.05 ) .
The Young ' s modulus of Gleason2 - 7 was significantly higher than that in high - grade tumor ( Gleason8 - 10 ) ( P0.05 ) .
In addition , the Young ' s modulus of the untreated Pca tissue was significantly higher than that in the metastasis group ( P0.05 ) .
2 . Causes of changes in mechanical properties of prostate cancer and important molecules of mechanical signal transduction
We found that the expression of collagen I , III , IV in BPH was significantly higher than that in Pca ( P0.05 ) .
The expression of MMP - 2 in Pca tissue was significantly higher than that in BPH group ( P0.05 ) , and the expression of MMP - 2 was higher than that in non - metastasis group ( P0.05 ) .
Gleason ( 2 - 7 ) expression of MMP - 2 was higher than Gleason ( 8 - 10 ) in high grade group ( P 0.05 ) .
In Pca tissue , the expression intensity of fibronectin was significantly higher in Gleason ( 2 - 7 ) than in Gleason ( 8 - 10 ) group ( P0.05 ) .
3 . Experimental study on cell adhesion , Young ' s modulus , apoptosis and invasion of prostate cancer cells with different concentrations of collagen I
Three - dimensional cell culture was performed on PC - 3 and LNAs cell lines at different concentrations of collagen I . By using the method , collagen I was used as the lowest effective concentration and the highest concentration in the follow - up experiment .
AFM showed that the Young ' s modulus of PC - 3 cells was lowest in low concentration collagen I ( 1mg / ml ) ;
Transwell渚佃瀹為獙琛ㄦ槑,鍦ㄤ綆娴撳害collagen I涓
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