本文關(guān)鍵詞： 惡性黑色素瘤 促血管生成2 RNA干擾 微血管密度　出處：《福建醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：構(gòu)建人惡性黑色素瘤裸鼠移植瘤模型，利用Ang2-siRNA慢病毒載體介導(dǎo)的RNA干擾作用，沉默Ang2基因在裸鼠移植瘤中的表達(dá)，研究在體內(nèi)Ang2-siRNA慢病毒對(duì)移植瘤中Ang2基因的影響，并檢測(cè)移植瘤微血管密度，腫瘤的增殖情況和體積，評(píng)價(jià)及初步明確Ang2表達(dá)水平、微血管密度與腫瘤增殖之間的關(guān)系，為腫瘤的基因治療提供一定的基礎(chǔ)實(shí)驗(yàn)研究依據(jù)。 方法：1、將15雄性裸鼠隨機(jī)分成空白組（PBScontrolgroup）、空載組（Vectorcontrolgroup）、實(shí)驗(yàn)組（RNAigroup）3組，每組5只。 2、正常培養(yǎng)、傳代A375細(xì)胞，制成單細(xì)胞懸液，調(diào)整細(xì)胞密度至約為5×107個(gè)/ml。 3、在裸鼠右腋皮下，用100μl微量進(jìn)樣器接種制備好的A375單細(xì)胞懸液，，100μl/只，建立人惡性黑色素瘤裸鼠移植瘤模型。 4、空白組、空載組、實(shí)驗(yàn)組3組裸鼠皮下移植瘤分別多點(diǎn)注射PBS、pNL-EGFP-Vector和pNL-EGFP-Ang2-siRNA慢病毒液，200μl/只，并于腹腔內(nèi)加強(qiáng)注射同種溶液500μl/只，進(jìn)行干擾試驗(yàn)研究。 5、觀察并記錄裸鼠移植瘤增殖情況和體積，統(tǒng)計(jì)、分析各組之間體積差異，并繪制腫瘤生長(zhǎng)曲線。 6、用實(shí)時(shí)熒光定量RT-PCR法檢測(cè)移植瘤中Ang2基因的相對(duì)表達(dá)量，統(tǒng)計(jì)分析各組之間Ang2基因表達(dá)水平的差異和腫瘤增殖程度的關(guān)系。 7、用免疫組織化學(xué)法檢測(cè)移植瘤的微血管密度，統(tǒng)計(jì)分析各組之間差異及其與腫瘤增殖程度的關(guān)系。 結(jié)果：1、成功建立了人惡性黑色素瘤裸鼠移植瘤模型。 2、實(shí)驗(yàn)組移植瘤體積顯著小于空白組和空載組，有統(tǒng)計(jì)學(xué)意義（P＜0.01），空白組和空載組移植瘤體積之間差異無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。 3、實(shí)驗(yàn)組移植瘤Ang2基因相對(duì)表達(dá)水平顯著低于空白組和空載組，有統(tǒng)計(jì)學(xué)意義（P＜0.01），空白組和空載組Ang2基因相對(duì)表達(dá)水平之間差異無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。 4、實(shí)驗(yàn)組微血管密度顯著低于空白組和空載組，有統(tǒng)計(jì)學(xué)意義（P＜0.01），空白組和空載組之間差異無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。 結(jié)論：利用Ang2-siRNA慢病毒載體介導(dǎo)的RNA干擾作用，能夠成功降低人惡性黑色素瘤裸鼠移植瘤中Ang2基因的表達(dá)水平，減少移植瘤中微血管的生成，從而抑制腫瘤的生長(zhǎng)、增殖。
[Abstract]:Objective: to construct a human malignant melanoma xenograft model in nude mice, and to silence the expression of Ang2 gene in nude mice xenografts by RNA interference mediated by Ang2-siRNA lentivirus vector. To study the effect of Ang2-siRNA lentivirus on the Ang2 gene in transplanted tumor, and to detect the microvessel density, tumor proliferation and volume. The expression level of Ang2, the relationship between microvessel density and tumor proliferation were evaluated and preliminarily determined, which provided a basis for the basic experimental study of tumor gene therapy. Methods 15 male nude mice were randomly divided into control group and control group. The experimental group was treated with RNAi group (n = 5). 2. A375 cells were cultured and subcultured to form a single cell suspension, and the cell density was adjusted to about 5 脳 107 / ml. 3Subcutaneously in the right axilla of nude mice, 100 渭 l / mouse A375 single cell suspension was inoculated with 100 渭 l microsampler to establish the transplanted tumor model of human malignant melanoma in nude mice. (4) PBS was injected into nude mice subcutaneously in blank group, no-load group and experimental group respectively. PNL-EGFP-Vector and pNL-EGFP-Ang2-siRNA lentivirus were injected intraperitoneally with 500 渭 l / rat lentivirus. The interference test was carried out. 5. The proliferation and volume of xenografts in nude mice were observed and recorded. The volume difference was analyzed and the tumor growth curve was drawn. 6. The relative expression of Ang2 gene in transplanted tumor was detected by real-time fluorescence quantitative RT-PCR, and the relationship between the expression of Ang2 gene and the degree of tumor proliferation was statistically analyzed. 7. The microvessel density of transplanted tumor was detected by immunohistochemical method, and the relationship between the microvessel density and the degree of tumor proliferation was analyzed statistically. Results: 1, the transplanted tumor model of human malignant melanoma was successfully established in nude mice. 2. The volume of transplanted tumor in the experimental group was significantly smaller than that in the blank group and the no-load group (P < 0.01), but there was no significant difference between the blank group and the no-load group (P > 0.05). 3The relative expression level of Ang2 gene in experimental group was significantly lower than that in blank group and no-load group (P < 0.01). There was no significant difference in relative expression of Ang2 gene between blank group and no-load group (P > 0.05). 4. The microvessel density in the experimental group was significantly lower than that in the blank group and the no-load group (P < 0.01), but there was no significant difference between the blank group and the no-load group (P > 0.05). Conclusion: RNA interference mediated by Ang2-siRNA lentivirus vector can successfully reduce the expression of Ang2 gene in human malignant melanoma xenografts in nude mice. Reduce the formation of microvessels in the tumor, thereby inhibiting the growth and proliferation of the tumor.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.5

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