前列腺外周帶基質(zhì)細(xì)胞過表達(dá)LMO2基因?qū)η傲邢偕掀ぜ?xì)胞的生物學(xué)影響
發(fā)布時(shí)間：2018-09-12 10:48
【摘要】：目的：根據(jù)前列腺帶性結(jié)構(gòu)差異學(xué)說，研究前列腺外周帶基質(zhì)細(xì)胞（Peripheral Zones Stromal Cells，PZs)與移行帶基質(zhì)細(xì)胞(Transitional ZonesStromal Cells，TZs)編碼基因及非編碼基因表達(dá)差異，研究差異基因LMO2在PZs中過表達(dá)的意義，進(jìn)一步探討LMO2基因在基質(zhì)微環(huán)境中對前列腺細(xì)胞增殖、遷移的生物學(xué)影響，并解釋產(chǎn)生上述現(xiàn)象可能的機(jī)制。 方法：1.原代培養(yǎng)正常前列腺PZs、TZs，并通過免疫組織細(xì)胞化學(xué)鑒定，應(yīng)用Affymetrix基因表達(dá)譜芯片及miRNA芯片篩選前列腺不同帶基質(zhì)細(xì)胞之間差異的編碼及非編碼基因。RT-PCR、Western-blotting、免疫熒光、免疫組織化學(xué)驗(yàn)證差異基因LMO2在PZs、TZs中的表達(dá)。2.構(gòu)建LMO2的過表達(dá)慢病毒載體，感染前列腺基質(zhì)細(xì)胞WPMY-1后，獲得穩(wěn)定表達(dá)LMO2的前列腺基質(zhì)細(xì)胞株（WPMY-1-LMO2）；構(gòu)建LMO2特異性shRNA質(zhì)粒，轉(zhuǎn)染PZs，獲得shRNA-LMO2前列腺基質(zhì)細(xì)胞（PZsshRNA-LMO2）。熒光顯微鏡、RT-PCR、Western-blot檢測LMO2的表達(dá)及沉默效率。3.利用Transwell系統(tǒng)構(gòu)建上皮-基質(zhì)共培養(yǎng)模型，觀察前列腺上皮細(xì)胞增殖及遷移能力的特性變化。蛋白因子芯片及RT-PCR檢測細(xì)胞因子FGF-9、IL-11的表達(dá)。CCK-8、EdU實(shí)驗(yàn)及細(xì)胞劃痕實(shí)驗(yàn)觀察FGF-9及IL-11對BPH-1、PC3細(xì)胞增殖及細(xì)胞遷移的影響。Western-blot檢測上皮-基質(zhì)共培養(yǎng)模型中BPH-1及PC3細(xì)胞相關(guān)蛋白分子的變化。4.裸鼠前列腺原位移植瘤模型及皮下移植瘤模型，觀察成瘤時(shí)間、腫瘤體積等，驗(yàn)證前列腺基質(zhì)細(xì)胞WPMY-1-LMO2對PC3細(xì)胞增殖及侵襲能力的影響，動物活體成像技術(shù)觀察腫瘤的體內(nèi)轉(zhuǎn)移，免疫組化方法檢測淋巴結(jié)轉(zhuǎn)移及移植瘤增殖能力。 結(jié)果：1.成功原代培養(yǎng)PZs、TZs，基因芯片篩選512種基因在前列腺不同帶基質(zhì)細(xì)胞有差異表達(dá)。miRNA芯片篩選發(fā)現(xiàn)在PZs、TZs中存在27種差異的miRNA。RT-PCR、Western-blot、免疫組化、免疫熒光技術(shù)證實(shí)LMO2在PZs中過表達(dá)。2.成功構(gòu)建了穩(wěn)定表達(dá)LMO2基因的慢病毒細(xì)胞株WPMY-1-LMO2；構(gòu)建的shRNA-LMO2質(zhì)粒能有效干擾外周帶基質(zhì)細(xì)胞中LMO2基因，獲得PZsshRNA-LMO2細(xì)胞株。3.體外建立上皮-基質(zhì)共培養(yǎng)模型，，前列腺上皮細(xì)胞BPH-1及PC3與WPMY-1-LMO2共培養(yǎng)及用WPMY-1-LMO2的條件培養(yǎng)基孵育后，BPH-1及PC3增殖及遷移能力與對照組比較明顯增強(qiáng)（P0.05），STAT3及AKT磷酸化水平升高，CCND1表達(dá)增加。沉默PZs中的LMO2基因，PZsshRNA-LMO2對前列腺細(xì)胞增殖、遷移能力減弱。蛋白因子芯片發(fā)現(xiàn)FGF-9及IL-11在WPMY-1-LMO2上清液中表達(dá)升高， RT-PCR結(jié)果發(fā)現(xiàn)FGF-9mRNA在WPMY-1-LMO2較對照組細(xì)胞平均上調(diào)4.1倍，IL-11mRNA在WPMY-1-LMO2較對照組細(xì)胞平均上調(diào)5.5倍（P0.05）。4. WPMY-1-LMO2基質(zhì)細(xì)胞可以促進(jìn)腫瘤生長、轉(zhuǎn)移較對照組效果顯著（P0.05）。 結(jié)論：前列腺外周帶和移行帶基質(zhì)細(xì)胞中存在差異基因表達(dá)，篩選LMO2基因在前列腺外周帶基質(zhì)細(xì)胞中過表達(dá)；前列腺基質(zhì)細(xì)胞LMO2蛋白過表達(dá)，明顯增加前列腺上皮細(xì)胞增殖和遷移等能力，并增加腫瘤的生長、侵襲和遠(yuǎn)處轉(zhuǎn)移；前列腺基質(zhì)細(xì)胞表達(dá)LMO2后，誘導(dǎo)FGF-9、IL-11等細(xì)胞因子高表達(dá)，并通過旁分泌效應(yīng)刺激前列腺上皮細(xì)胞生長、遷移；進(jìn)一步證明前列腺基質(zhì)-上皮相互作用在前列腺癌發(fā)生進(jìn)展中的重要性，特別是基質(zhì)的關(guān)鍵作用。以前列腺基質(zhì)為研究切入點(diǎn)，為研究前列腺癌發(fā)生具有帶性差異的分子機(jī)制、前列腺基質(zhì)作為靶向治療和預(yù)后判斷的研究奠定堅(jiān)實(shí)的實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Objective: According to the theory of zonal structural difference of prostate, to study the expression of coding and non-coding genes in peripheral zone stromal cells (PZs) and transitional zone stromal cells (TZs) of prostate, and to study the significance of overexpression of LMO2 in PZs. The biological effects of genes on the proliferation and migration of prostate cells in the matrix microenvironment and the possible mechanisms underlying these phenomena are explained.
Methods: 1. Primary culture of normal prostate PZs, TZs, and through immunohistochemical identification, Affymetrix gene expression profiling chip and microRNA chip screening prostate stromal cells with different coding and non-coding genes. RT-PCR, Western-blotting, immunofluorescence, immunohistochemical verification of the differential gene LMO2 in PZs, TZ. LMO2-specific shRNA plasmid was constructed and transfected into PZs to obtain shRNA-LMO2 prostate stromal cells (PZs shRNA-LMO2). Fluorescence microscopy, RT-PCR and Western-blot were used to detect LMO2 expression. Expression and Silencing Efficiency. 3. Establishment of an epithelial-matrix co-culture model using Transwell system to observe the proliferation and migration of prostatic epithelial cells. Expression of cytokines FGF-9 and IL-11 was detected by protein chip and RT-PCR. Proliferation and cell proliferation of BPH-1 and PC3 cells were observed by CCK-8, EdU and cell scratch assays. Western-blot was used to detect the changes of BPH-1 and PC3 cell-related protein molecules in the co-culture model of epithelium and matrix. 4. Orthotopic prostate transplantation tumor model and subcutaneous prostate transplantation tumor model in nude mice were used to observe the tumor formation time, tumor volume, etc. To verify the effect of prostatic stromal cells WPMY-1-LMO2 on the proliferation and invasion of PC3 cells, and animal survival. The tumor metastasis in vivo was observed by body imaging, and the lymph node metastasis and the proliferation of transplanted tumor were detected by immunohistochemistry.
Results: 1. Primary culture of PZs, TZs, gene chip screening 512 genes differentially expressed in prostate stromal cells with different zones. Microarray screening found 27 different microRNAs. RT-PCR, Western-blot, immunohistochemistry, immunofluorescence technology confirmed that LMO2 was overexpressed in PZs. 2. The stable expression of LMO2 gene was successfully constructed. A lentiviral cell line WPMY-1-LMO2 was constructed, and the plasmid shRNA-LMO2 could effectively interfere with the LMO2 gene in the peripheral zone stromal cells. 3. Establishment of an epithelial-matrix co-culture model in vitro, co-culture of BPH-1 and PC3 with WPMY-1-LMO2 and incubation with WPMY-1-LMO2, BPH-1 and PC3 proliferated. Compared with the control group, the migration ability and STAT3 and AKT phosphorylation level increased, CCND1 expression increased. LMO2 gene in PZs was silenced, and the proliferation and migration ability of prostate cells were weakened by PZsshRNA-LMO2. The expression of FGF-9 and IL-11 in the supernatant of WPMY-1-LMO2 were increased by protein chip, and the expression of FGF-9 mRNA in WPMY-1-LMO2 was detected by RT-PCR. The expression of IL-11 mRNA in WPMY-1-LMO2 was 5.5 times higher than that in control group (P 0.05). 4. WPMY-1-LMO2 stromal cells could promote tumor growth and metastasis significantly (P 0.05).
Conclusion: Differential gene expression exists in the stromal cells of the peripheral zone and transitional zone of prostate, LMO2 gene overexpression is screened in the stromal cells of the peripheral zone of prostate, LMO2 protein overexpression in prostatic stromal cells significantly increases the proliferation and migration of prostatic epithelial cells, and increases tumor growth, invasion and distant metastasis. Glandular stromal cells expressing LMO2 induce the overexpression of cytokines such as fibroblast growth factor-9 and interleukin-11, and stimulate the growth and migration of prostatic epithelial cells by paracrine effect. This further proves the importance of prostatic stromal-epithelial interaction in the development of prostate cancer, especially the key role of stroma. In order to study the molecular mechanism of banding differences in prostate cancer, prostatic stroma as a targeted therapy and prognostic evaluation lays a solid experimental foundation.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.25
本文編號：2238763
[Abstract]:Objective: According to the theory of zonal structural difference of prostate, to study the expression of coding and non-coding genes in peripheral zone stromal cells (PZs) and transitional zone stromal cells (TZs) of prostate, and to study the significance of overexpression of LMO2 in PZs. The biological effects of genes on the proliferation and migration of prostate cells in the matrix microenvironment and the possible mechanisms underlying these phenomena are explained.
Methods: 1. Primary culture of normal prostate PZs, TZs, and through immunohistochemical identification, Affymetrix gene expression profiling chip and microRNA chip screening prostate stromal cells with different coding and non-coding genes. RT-PCR, Western-blotting, immunofluorescence, immunohistochemical verification of the differential gene LMO2 in PZs, TZ. LMO2-specific shRNA plasmid was constructed and transfected into PZs to obtain shRNA-LMO2 prostate stromal cells (PZs shRNA-LMO2). Fluorescence microscopy, RT-PCR and Western-blot were used to detect LMO2 expression. Expression and Silencing Efficiency. 3. Establishment of an epithelial-matrix co-culture model using Transwell system to observe the proliferation and migration of prostatic epithelial cells. Expression of cytokines FGF-9 and IL-11 was detected by protein chip and RT-PCR. Proliferation and cell proliferation of BPH-1 and PC3 cells were observed by CCK-8, EdU and cell scratch assays. Western-blot was used to detect the changes of BPH-1 and PC3 cell-related protein molecules in the co-culture model of epithelium and matrix. 4. Orthotopic prostate transplantation tumor model and subcutaneous prostate transplantation tumor model in nude mice were used to observe the tumor formation time, tumor volume, etc. To verify the effect of prostatic stromal cells WPMY-1-LMO2 on the proliferation and invasion of PC3 cells, and animal survival. The tumor metastasis in vivo was observed by body imaging, and the lymph node metastasis and the proliferation of transplanted tumor were detected by immunohistochemistry.
Results: 1. Primary culture of PZs, TZs, gene chip screening 512 genes differentially expressed in prostate stromal cells with different zones. Microarray screening found 27 different microRNAs. RT-PCR, Western-blot, immunohistochemistry, immunofluorescence technology confirmed that LMO2 was overexpressed in PZs. 2. The stable expression of LMO2 gene was successfully constructed. A lentiviral cell line WPMY-1-LMO2 was constructed, and the plasmid shRNA-LMO2 could effectively interfere with the LMO2 gene in the peripheral zone stromal cells. 3. Establishment of an epithelial-matrix co-culture model in vitro, co-culture of BPH-1 and PC3 with WPMY-1-LMO2 and incubation with WPMY-1-LMO2, BPH-1 and PC3 proliferated. Compared with the control group, the migration ability and STAT3 and AKT phosphorylation level increased, CCND1 expression increased. LMO2 gene in PZs was silenced, and the proliferation and migration ability of prostate cells were weakened by PZsshRNA-LMO2. The expression of FGF-9 and IL-11 in the supernatant of WPMY-1-LMO2 were increased by protein chip, and the expression of FGF-9 mRNA in WPMY-1-LMO2 was detected by RT-PCR. The expression of IL-11 mRNA in WPMY-1-LMO2 was 5.5 times higher than that in control group (P 0.05). 4. WPMY-1-LMO2 stromal cells could promote tumor growth and metastasis significantly (P 0.05).
Conclusion: Differential gene expression exists in the stromal cells of the peripheral zone and transitional zone of prostate, LMO2 gene overexpression is screened in the stromal cells of the peripheral zone of prostate, LMO2 protein overexpression in prostatic stromal cells significantly increases the proliferation and migration of prostatic epithelial cells, and increases tumor growth, invasion and distant metastasis. Glandular stromal cells expressing LMO2 induce the overexpression of cytokines such as fibroblast growth factor-9 and interleukin-11, and stimulate the growth and migration of prostatic epithelial cells by paracrine effect. This further proves the importance of prostatic stromal-epithelial interaction in the development of prostate cancer, especially the key role of stroma. In order to study the molecular mechanism of banding differences in prostate cancer, prostatic stroma as a targeted therapy and prognostic evaluation lays a solid experimental foundation.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R737.25
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 韓蘇軍;張思維;陳萬青;李長嶺;;中國前列腺癌發(fā)病現(xiàn)狀和流行趨勢分析[J];臨床腫瘤學(xué)雜志;2013年04期
2 ;Characteristic pattern of human prostatic growth with age[J];Asian Journal of Andrology;2002年04期
3 ;STAT-3 correlates with lymph node metastasis and cell survival in gastric cancer[J];World Journal of Gastroenterology;2010年42期
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