本文選題：前列腺癌 + 力學(xué)性質(zhì)　； 參考：《大連醫(yī)科大學(xué)》2014年博士論文

【摘要】：前列腺癌是美國及歐洲國家最常見的惡性腫瘤，而許多前列腺癌是通過直腸指診時觸及的硬結(jié)發(fā)現(xiàn)的。傳統(tǒng)觀點(diǎn)認(rèn)為腫瘤組織相較于正常組織要硬，但在臨床工作中，當(dāng)剖開某些腫瘤后，腫瘤組織整體與局部的質(zhì)地卻不盡相同。腫瘤組織進(jìn)展和轉(zhuǎn)移發(fā)生時伴隨著腫瘤組織生物力學(xué)性質(zhì)的變化，而在前列腺癌中，組織的力學(xué)性質(zhì)與腫瘤惡性生物學(xué)行為之間的關(guān)系還不甚明了。 膠原蛋白collagen是細(xì)胞外基質(zhì)中含量最豐富的蛋白，而且它為組織的力學(xué)性質(zhì)提供支架結(jié)構(gòu)，并且膠原蛋白collagen的變化能夠?qū)е录?xì)胞外基質(zhì)剛度發(fā)生變化。纖維連接蛋白fibronectin分子結(jié)構(gòu)上同時含有與細(xì)胞外基質(zhì)中的膠原蛋白及細(xì)胞膜表面整合蛋白integrin家族的結(jié)合位點(diǎn)，能夠作為橋梁將腫瘤細(xì)胞與細(xì)胞外基質(zhì)相連接。而纖維連接蛋白fibronectin和整合蛋白家族integrin又是力學(xué)信號轉(zhuǎn)導(dǎo)通路上的關(guān)鍵因子。在之前的研究中，原子力顯微鏡已經(jīng)被用于檢測生物組織微觀層面的力學(xué)性質(zhì)，，而實(shí)時彈性超聲成像也已經(jīng)在生物組織宏觀層面的力學(xué)性質(zhì)檢測上被廣泛使用。但是關(guān)于前列腺癌組織微觀和宏觀層面的力學(xué)性質(zhì)的研究較為少見。 目的 1.檢測前列腺癌組織宏觀及微觀力學(xué)性質(zhì)變化，用以探討前列腺癌力學(xué)性質(zhì)變化與前列腺癌患者的病理分級及腫瘤轉(zhuǎn)移之間的聯(lián)系; 2.探討前列腺癌力學(xué)性質(zhì)變化的原因，并在組織層面探索力學(xué)信號轉(zhuǎn)導(dǎo)通路中的重要分子; 3.探討collagen I對前列腺癌細(xì)胞粘附、楊氏模量、侵襲、凋亡的影響。 方法 1.收集43例患者的臨床資料（前列腺增生20例，前列腺癌23例），按照Gleason評分系統(tǒng)進(jìn)行病理分級，按照腫瘤分化程度分成1-5分，分化良好為1分，分化極差為5分，將兩區(qū)的Gleason分值相加即可以得到Gleason評分。根據(jù)Gleason評分將23例Pca患者分成兩組：低-中級別組（Gleason2-7分）共11例，高級別組（Gleason8-10分）12例。另外，根據(jù)腫瘤是否轉(zhuǎn)移，將23例Pca患者分成轉(zhuǎn)移組7例，非轉(zhuǎn)移組16例。所有患者術(shù)前均未行任何手術(shù)及放、化療。所有患者在術(shù)前均行TRTE檢查。應(yīng)用于AFM檢測的BPH標(biāo)本取自經(jīng)尿道前列腺電切術(shù)（transurethral resection of the prostate, TURP）, Pca組織標(biāo)本取自經(jīng)尿道前列腺癌切除術(shù)及恥骨上前列腺癌切除術(shù)。所有組織術(shù)前及術(shù)后均行病理學(xué)檢查確認(rèn)。 2.我們首先對前列腺癌組織標(biāo)本進(jìn)行免疫組織化學(xué)實(shí)驗(yàn),根據(jù)Gleason評分將23例Pca患者分成兩組;另外，根據(jù)腫瘤是否轉(zhuǎn)移，將23例Pca患者分成轉(zhuǎn)移組和非轉(zhuǎn)移組。根據(jù)免疫組化染色陽性細(xì)胞的數(shù)量及切片的染色程度來進(jìn)行免疫組化評分。 3.制作不同濃度的collagen I進(jìn)行前列腺癌細(xì)胞系三維培養(yǎng)，緊接著使用細(xì)胞粘附實(shí)驗(yàn)、AFM實(shí)驗(yàn)、Transwell實(shí)驗(yàn)、TUNEL實(shí)驗(yàn)等檢測不同濃度collagen I上前列腺癌細(xì)胞的粘附、楊氏模量、凋亡、侵襲的變化。 結(jié)果 1.前列腺癌宏觀及微觀力學(xué)性質(zhì)的變化及其臨床意義 宏觀層面列腺組織的力學(xué)性質(zhì)變化，共43例患者穿刺及手術(shù)前行TRTE檢測，其中BPH患者20例，Pca患者23例。所有患者行TRTE檢測后，均行前列腺穿刺活檢，并在術(shù)后結(jié)合病理診斷確認(rèn)。前列腺癌病灶SI明顯高于BPH組織(P0.05)；根據(jù)術(shù)后病理診斷及患者轉(zhuǎn)移情況，整理TRTE結(jié)果資料發(fā)現(xiàn)，高級別Pca組織（Gleason8-10）SI明顯高于低-中級別Pca組織（Gleason2-7）SI (P0.05)；而轉(zhuǎn)移組與非轉(zhuǎn)移組Pca組織間SI無明顯統(tǒng)計學(xué)差異(P=0.075)。 微觀層面列腺組織的力學(xué)性質(zhì)變化，我們采用AFM檢測43例患者組織切片的楊氏模量。結(jié)果顯示，Pca組織楊氏模量明顯低于BPH組織（P0.05）；其中低-中級別腫瘤（Gleason2-7）楊氏模量明顯高于高級別腫瘤（Gleason8-10）組織（P0.05）；并且，未出現(xiàn)轉(zhuǎn)移的Pca組織楊氏模量明顯高于轉(zhuǎn)移組（P0.05）。 2.前列腺癌力學(xué)性質(zhì)變化原因及力學(xué)信號轉(zhuǎn)導(dǎo)重要分子 我們發(fā)現(xiàn)BPH中collagen I, III, IV的表達(dá)明顯高于Pca組織（P0.05），并且，隨著腫瘤Gleason分級的增高及轉(zhuǎn)移的發(fā)生，三者表達(dá)進(jìn)一步下降（P0.05）；MMP-2在Pca組織中的表達(dá)明顯高于BPH組織（P0.05），并且Pca轉(zhuǎn)移組表達(dá)比非轉(zhuǎn)移組高(P0.05)；MMP-2在Pca低-中級別組Gleason(2-7)表達(dá)高于高級別組Gleason(8-10)（(P0.05）；fibronectin在BPH與Pca組織中fibronectin的表達(dá)水平未見明顯差異，僅發(fā)現(xiàn)表達(dá)部位的不同（P=0.189）;在Pca組織中，fibronectin的表達(dá)強(qiáng)度在Gleason (2-7)組明顯高于Gleason (8-10)組（P0.05），在轉(zhuǎn)移組與非轉(zhuǎn)移組之間未見明顯差異（P=0.697）;Integrin α5在Pca組織中的表達(dá)高于BPH組織（P0.05）。在Pca組織中，Gleason (2-7)組MMP-2的表達(dá)明顯高于Gleason (8-10)組（P0.05），但其表達(dá)在轉(zhuǎn)移組與非轉(zhuǎn)移組之間無明顯差異（P=0.122）。 3.不同濃度collagen I干預(yù)前列腺癌細(xì)胞粘附、楊氏模量、凋亡及侵襲的實(shí)驗(yàn)研究 分別在不同濃度的collagen I上對PC-3及LNCaP細(xì)胞系進(jìn)行三維細(xì)胞培養(yǎng)，通過使用說明將collagen I在能形成膠體的范圍內(nèi)以1mg/ml及5mg/ml作為后續(xù)實(shí)驗(yàn)的最低作用濃度及最高作用濃度。后續(xù)實(shí)驗(yàn)發(fā)現(xiàn),骨轉(zhuǎn)移灶來源的PC-3細(xì)胞在collagen I上的粘附能力高于來源于淋巴結(jié)轉(zhuǎn)移灶LNCaP細(xì)胞；AFM實(shí)驗(yàn)表明，PC-3細(xì)胞的楊氏模量在低濃度collagen I (1mg/ml)上最低；Transwell侵襲實(shí)驗(yàn)表明，在低濃度collagen I上，PC-3細(xì)胞侵襲能力高于高濃度collagen I;TUNEL實(shí)驗(yàn)表明，高濃度的collagen I能增加PC-3細(xì)胞的凋亡敏感性。 結(jié)論 1.隨著前列腺癌組織惡性程度增高及轉(zhuǎn)移的發(fā)生，其宏觀及微觀力學(xué)性質(zhì)發(fā)生相反的改變，表明前列腺癌生物力學(xué)性質(zhì)的改變與腫瘤的惡性程度及轉(zhuǎn)移相關(guān)； 2. MMP-2介導(dǎo)的Collagen I降解及腫瘤細(xì)胞楊氏模量的改變可能是導(dǎo)致前列腺癌組織生物力學(xué)性質(zhì)變化的原因，且Collagen I, Fibronectin, MMP-2,integrinα5可能是力學(xué)信號傳導(dǎo)通路上的重要分子； 3. Collagen I的濃度變化能夠改變前列腺癌PC-3細(xì)胞的形態(tài)、粘附能力、力學(xué)性質(zhì)、侵襲能力及凋亡敏感性，表明Collagen I與前列腺癌細(xì)胞的惡性表型及力學(xué)性質(zhì)存在相關(guān)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綆嫻撳害collagen I涓

本文編號：1850969