本文選題：前列腺腫瘤 + Gli/Hedgehog信號(hào)通路�。� 參考：《西北農(nóng)林科技大學(xué)》2014年博士論文

【摘要】：前列腺癌是男性泌尿生殖系統(tǒng)的惡性腫瘤之一。在西方國(guó)家，它僅次于肺癌，是導(dǎo)致男性因癌癥死亡的第二大疾病。治療前列腺腫瘤的主要方法為激素療法和化學(xué)療法，但是這兩種治療方法在治愈后經(jīng)常復(fù)發(fā)高致病力腫瘤。因此，新的治療方法，如通過(guò)調(diào)節(jié)前列腺腫瘤中異常表達(dá)的信號(hào)通路來(lái)達(dá)到抑制前列腺腫瘤的生長(zhǎng)具有重要的臨床應(yīng)用前景。 Gli/Hedgehog(Hh)信號(hào)通路是癌細(xì)胞增殖和腫瘤生長(zhǎng)所必須的，同時(shí)它在調(diào)節(jié)前列腺上皮生長(zhǎng)方面也發(fā)揮著重要的作用。此信號(hào)通路靶向基因的表達(dá)量升高會(huì)驅(qū)使前列腺前體細(xì)胞向癌細(xì)胞轉(zhuǎn)化，甚至導(dǎo)致癌細(xì)胞擴(kuò)散。已有研究表明利用Gli/Hh信號(hào)通路的抑制劑環(huán)巴胺可以在體內(nèi)或體外顯著地抑制前列腺癌細(xì)胞的生長(zhǎng)。但是，由于環(huán)巴胺本身存在多種缺點(diǎn)，如：半衰期短，非特異性毒性和它在高劑量時(shí)的非有效靶向作用都導(dǎo)致其不是最佳的抑制劑。因此，篩選Gli/Hh信號(hào)通路的高效抑制劑可能是預(yù)防或者治療前列腺腫瘤的一種有效方法。 Sutherlandia Frutescens（S.Frutescens）是一種傳統(tǒng)藥用植物，它常用來(lái)治療發(fā)熱、咳嗽、感染、胃病、糖尿病和癌癥等多種人類疾病。近來(lái)，它又在改善艾滋病患者的身體機(jī)能方面發(fā)揮了重要的作用。已有研究證明，S.Frutescens提取物可以抑制人前列腺癌細(xì)胞的增殖，但是它的作用機(jī)理還不是十分清楚。一些來(lái)源于植物的活性成分可以通過(guò)對(duì)Gli/Hh信號(hào)通路的調(diào)控來(lái)抑制前列腺癌細(xì)胞的增殖。因此，本研究在體外檢測(cè)了S. Frutescens甲醇提取物（SLE）對(duì)前列腺癌細(xì)胞中Gli/Hh信號(hào)通路的影響；利用穩(wěn)定轉(zhuǎn)染Gli報(bào)告基因的小鼠胚胎成纖維細(xì)胞Shh Light II篩選SLE中抑制Gli/Hh信號(hào)通路的主要化學(xué)活性成分。在體內(nèi)檢測(cè)了S. Frutescens對(duì)TRAMP小鼠腫瘤的影響。得到了以下研究結(jié)果： 1.利用不同濃度的SLE處理人前列腺癌細(xì)胞PC3、LNCaP和小鼠前列腺癌細(xì)胞TRAMP-C224h、48h和72h后，發(fā)現(xiàn)SLE對(duì)三種癌細(xì)胞的生長(zhǎng)抑制與劑量和時(shí)間呈正相關(guān)，即濃度越高或作用時(shí)間越長(zhǎng)，SLE對(duì)細(xì)胞的抑制力也越強(qiáng)。而且在72h時(shí)，它對(duì)三種癌細(xì)胞的增殖抑制達(dá)到一半。它對(duì)三種前列腺腫瘤細(xì)胞的半數(shù)抑制濃度分別為：PC3167μg/ml、LNCaP200μg/ml和TRAMP-C2100μg/ml。但是，SLE對(duì)人正常前列腺上皮細(xì)胞RWPE-1無(wú)影響。 2.利用穩(wěn)定轉(zhuǎn)染Gli報(bào)告基因的小鼠胚胎成纖維細(xì)胞Shh Light II和小鼠前列腺癌細(xì)胞TRAMP-C2QGli檢測(cè)SLE對(duì)Gli/Hh信號(hào)通路的影響。熒光素酶實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn)，無(wú)論是否用Gli/Hh信號(hào)通路的激活劑CM或者SAG激活，SLE均抑制Gli報(bào)告基因的表達(dá)，并隨著劑量增加對(duì)Gli報(bào)告基因的抑制作用增強(qiáng)。但是，環(huán)巴胺（5μM）對(duì)Gli報(bào)告基因在未被激活的Shh Light II細(xì)胞中的表達(dá)沒(méi)有影響。以上實(shí)驗(yàn)證明S. frutescens提取物不僅可以抑制處于活化狀態(tài)的Gli/Hh信號(hào)通路，而且對(duì)非活化狀態(tài)細(xì)胞Gli基礎(chǔ)水平的表達(dá)也有抑制作用。 3.利用實(shí)時(shí)定量PCR檢測(cè)SLE對(duì)人和小鼠前列腺癌細(xì)胞中Gli/Hh信號(hào)通路的影響。結(jié)果顯示，當(dāng)小鼠前列腺癌細(xì)胞株TRAMP-C2用含有Gli/Hh信號(hào)通路配體（ShhN+）的CM激活時(shí)，SLE和環(huán)巴胺都顯著地抑制Gli1和Ptch1的表達(dá)；反之，，若沒(méi)有CM激活，SLE和環(huán)巴胺的抑制作用消失。在人前列腺癌細(xì)胞PC3上，即使用CM激活PC細(xì)胞，環(huán)巴胺對(duì)Gli1和Ptch1的表達(dá)沒(méi)有明顯影響，SLE卻仍然能顯著抑制Gli1和Ptch1的表達(dá)。利用免疫印跡試驗(yàn)得出SLE抑制蛋白GLI1在人骨髓橫紋肌肉癌細(xì)胞RMS13細(xì)胞中的表達(dá)。 4.利用高效液相色譜-蒸發(fā)光散射檢測(cè)法（HPLC-ELSD）從SLE中分離鑒定環(huán)木菠蘿烷Sutherlandiosides（A-D）和黃酮類Sutherlandins（A-D）的化合物或者同系化合物的混合物。熒光素酶實(shí)驗(yàn)篩選比較發(fā)現(xiàn)Sutherlandioside D對(duì)Gli報(bào)告基因的抑制力最強(qiáng)，Sutherlandioside D對(duì)Gli報(bào)告基因的半數(shù)抑制濃度（IC50）為1.8μg/ml，而SLE的IC50則是30μg/ml，分析發(fā)現(xiàn)Sutherlandioside D在SLE中的含量約為0.3%，這些數(shù)據(jù)提示Sutherlandioside D很可能是SLE中調(diào)節(jié)Gli/Hh信號(hào)通路的主要化學(xué)活性成分。 5.用含有三種濃度的S. frutescens（0.05%、0.25%和1%）日糧飼喂轉(zhuǎn)基因前列腺癌小鼠（TRAMP：Transgenic adenocarcinoma of the mouse prostate），與對(duì)照組相比，0.05%的S. frutescens能明顯降低TRAMP小鼠前列腺低度分化腫瘤（gross-PDC）的發(fā)生率，而且，0.05%的S. frutescens實(shí)驗(yàn)組的小鼠具有最小的前列腺重量。但是，在TRAMP小鼠皮下注射環(huán)巴胺既不能明顯降低前列腺腫瘤的發(fā)生率，也不能推遲前列腺腫瘤的發(fā)生，甚至對(duì)前列腺腫瘤的大小也沒(méi)有明顯的作用。 6.免疫組化檢測(cè)GLI1蛋白在0.05%S. frutescens實(shí)驗(yàn)組TRAMP小鼠不同分化類型腫瘤中的表達(dá)，結(jié)果表明GLI1蛋白的表達(dá)不會(huì)隨著TRAMP小鼠前列腺腫瘤的癌性損傷而改變。 研究表明，S. frutescens提取物在體外可以通過(guò)對(duì)Gli/Hh信號(hào)通路主要元件Gli1和Ptch1的調(diào)控來(lái)抑制人和小鼠前列腺癌細(xì)胞的生長(zhǎng)；在體內(nèi)，用含有0.05%S. frutescens日糧飼喂TRAMP小鼠可以顯著地抑制小鼠前列腺低度分化腫瘤的發(fā)生率。
[Abstract]:Prostate cancer is one of the malignant tumors in the male genitourinary system. In western countries, it is second only to lung cancer, which is the second major disease that causes death for men. The main method for treating prostate cancer is hormone therapy and chemotherapy, but these two treatments often relapse high pathogenic tumor after cure. Therefore, new treatment is a new treatment. Therapies such as regulating abnormal signaling pathways in prostate tumors to inhibit the growth of prostate cancer have important clinical application prospects.
Gli/Hedgehog (Hh) signal pathway is necessary for cancer cell proliferation and tumor growth, and it also plays an important role in regulating the growth of prostate epithelium. The increase of the target gene expression in this signal pathway will drive the prostatic cells to transform to the cancer cells and even lead to the proliferation of cancer cells. Research has shown that the use of Gli/Hh The signal pathway inhibitor, cyclic amine, can significantly inhibit the growth of prostate cancer cells in vivo or in vitro. However, due to the existence of a variety of shortcomings, such as short half-life, non specific toxicity, and its non effective targeting at high doses, it is not the best inhibitor. Therefore, the Gli/Hh signal pathway is screened. Highly effective inhibitors may be an effective way to prevent or treat prostate cancer.
Sutherlandia Frutescens (S.Frutescens) is a traditional medicinal plant. It is often used to treat a variety of human diseases such as fever, cough, infection, stomach disease, diabetes and cancer. Recently, it has played an important role in improving the physical function of AIDS patients. Research has shown that the S.Frutescens extract can inhibit the human prostate. Cancer cell proliferation, but its mechanism is not very clear. Some of the active components of the plant can inhibit the proliferation of prostate cancer cells by regulating the Gli/Hh signaling pathway. Therefore, the effect of S. Frutescens methanol extract (SLE) on the Gli/Hh signaling pathway in prostate cancer cells is detected in this study. The main chemical active components of the Gli/Hh signaling pathway in SLE were screened using Shh Light II, a mouse embryonic fibroblast stable transfected with Gli reporter gene. The effects of S. Frutescens on the TRAMP mouse tumor were detected in the body. The following results were obtained:
1. using different concentrations of SLE to treat human prostate cancer cells PC3, LNCaP and mouse prostate cancer cells TRAMP-C224h, 48h and 72h, it was found that the growth inhibition of SLE on three kinds of cancer cells was positively correlated with the dose and time, that is, the higher the concentration or the longer the action time, the stronger the inhibition of SLE to the cells. And at 72h, it is to three kinds of cancer cells. The inhibitory concentration of proliferation to three kinds of prostate tumor cells was PC3167 mu g/ml, LNCaP200 mu g/ml and TRAMP-C2100 micron g/ml., but SLE had no effect on human normal prostate epithelial cells RWPE-1.
2. the effect of SLE on Gli/Hh signaling pathway was detected by Shh Light II in mouse embryonic fibroblasts and TRAMP-C2QGli of mouse prostate cancer cells with stable transfection of Gli reporter gene. The luciferase test found that SLE inhibited the expression of the Gli reporter gene, whether or not it was activated by the activator of Gli/Hh signaling pathway, and with the agent. The inhibition of the Gli reporter gene was enhanced. However, the expression of the Gli reporter gene was not affected by the expression of the Gli reporter gene in the non activated Shh Light II cells. The above experiments showed that the S. frutescens extract could not only inhibit the Gli/Hh signaling pathway in the activated state, but also the table of the non activated state cell Gli base level. It also has an inhibitory effect.
3. the effect of SLE on the Gli/Hh signaling pathway in human and mouse prostate cancer cells was detected by real-time quantitative PCR. The results showed that both SLE and cyclic amine significantly inhibited the expression of Gli1 and Ptch1 when the mouse prostate cancer cell line TRAMP-C2 was activated by CM containing the Gli/Hh signaling ligand (ShhN+); conversely, if no CM activation was performed, SLE and cyclic amines were not activated. The inhibitory effect disappeared. On human prostate cancer cell PC3, the use of CM to activate PC cells, the expression of Gli1 and Ptch1 was not significantly affected by cyclic amine, but SLE still significantly inhibited the expression of Gli1 and Ptch1. The expression of SLE suppressor protein GLI1 in the RMS13 cells of human bone marrow rhabdomystriated muscle cancer cells was obtained by immunoblot test.
4. high performance liquid chromatography - evaporative light scattering detection (HPLC-ELSD) was used to separate and identify compounds or homologous compounds of pineapple pineapple Sutherlandiosides (A-D) and flavonoids Sutherlandins (A-D) from SLE. The luciferase test showed that Sutherlandioside D had the strongest inhibitory effect on the Gli reporter gene, Sutherlandio. The median inhibitory concentration (IC50) of side D to the Gli reporter gene was 1.8 mu g/ml, while the IC50 of SLE was 30 mu g/ml. The analysis found that Sutherlandioside D was about 0.3% in SLE.
5. the transgenic prostate cancer mice (TRAMP:Transgenic adenocarcinoma of the mouse prostate) were fed with three concentrations of S. frutescens (0.05%, 0.25% and 1%). Compared with the control group, 0.05% S. frutescens could significantly reduce the incidence of low-grade prostate cancer (gross-PDC) in TRAMP mice, and 0.05% The mice in the experimental group had the smallest weight of the prostate. However, the subcutaneous injection of cyclic amine in TRAMP mice could not significantly reduce the incidence of prostate tumors, nor delayed the occurrence of prostate tumors, and even had no significant effect on the size of the prostate tumor.
6. immunohistochemistry was used to detect the expression of GLI1 protein in 0.05%S. frutescens experimental group TRAMP mice with different differentiated types of tumor. The results showed that the expression of GLI1 protein did not change with the carcinomatous damage of the prostate tumor of TRAMP mice.
The study shows that S. frutescens extract can inhibit the growth of human and mouse prostate cancer cells by regulating the main components of Gli/Hh signaling pathway Gli1 and Ptch1 in vitro. In vivo, the rate of low differentiation tumor of mouse anterior gland can be inhibited significantly by feeding TRAMP mice with 0.05%S. frutescens diet.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25

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本文編號(hào)：1975818