【摘要】： 第一部分MSCs的培養(yǎng)及HIF-1αsiRNA的篩選 目的:貼壁法培養(yǎng)骨髓間充質(zhì)干細(xì)胞(MSCs,mesenchymal stem cells), RT-PCR法篩選抑制效果最強(qiáng)的RNA干擾序列。 方法:貼壁法培養(yǎng)MSCs,取第3-5代細(xì)胞用于實驗,倒置顯微鏡下觀察細(xì)胞形態(tài),并用流式細(xì)胞儀檢測其表面標(biāo)志物CD11b/c、CD34、CD44、CD90。將MSCs分為五組,單純?nèi)毖踅M(未經(jīng)任何干預(yù))、脂質(zhì)體組(轉(zhuǎn)染空載的脂質(zhì)體)、siRNA609組(轉(zhuǎn)染siRNA609)、siRNA658組(轉(zhuǎn)染siRNA658)、siRNA2070組(轉(zhuǎn)染siRNA2070)。將以上五組細(xì)胞在缺氧條件下培養(yǎng)24h,RT-PCR檢測其HIF-1α的基因表達(dá)。 結(jié)果:1.流式細(xì)胞儀檢測CD11b/c陰性、CD34陰性、CD44陽性、CD90陽性細(xì)胞分別為84.2%±0.2%、97.91%±0.7%、99.8%±0.9%、97.7%±0.4%,CD44+/CD34-細(xì)胞數(shù)為99.4%±0.4%。2.轉(zhuǎn)染siRNA細(xì)胞內(nèi)均能檢測到綠色熒光信號,lipsome組和空白對照組未見到綠色熒光信號。3.RT-PCR結(jié)果顯示,與單純?nèi)毖踅M相比siRNA609、siRNA658、siRNA2070組HIF-1α的基因表達(dá)明顯降低(P 0.05),其中siRNA658組抑制效果最強(qiáng)(P 0.05)。 結(jié)論:1.培養(yǎng)的細(xì)胞表達(dá)MSCs相應(yīng)的表面標(biāo)記物。2.MSCs可以轉(zhuǎn)染HIF-1αsiRNA序列。3. siRNA658干擾序列抑制效果最強(qiáng)。 第二部分HIF-1α小干擾RNA對MSCs的HIF-1α、SDF-1α和VEGF基因表達(dá)的影響 目的:HIF-1α小干擾RNA對MSCs HIF-1α、基質(zhì)細(xì)胞衍生因子-1α(SDF-1α,stromal derived factor 1α)和血管內(nèi)皮生長因子(VEGF,Vascular endothelial growth factor)基因表達(dá)的影響。 方法:將MSCs四組,常氧對照組(無干預(yù)因素)、缺氧組(缺氧24h)、脂質(zhì)體對照組(轉(zhuǎn)染空載脂質(zhì)體后缺氧24h)、RNA干擾組(轉(zhuǎn)染脂質(zhì)體介導(dǎo)的RNA干擾序列后缺氧24h)。RT-PCR法檢測MSCs的HIF-1α、SDF-1α和VEGF mRNA表達(dá)水平, ELISA檢測MSCs培養(yǎng)上清HIF-1α、SDF-1α和VEGF蛋白表達(dá)水平,CCK8檢測MSCs培養(yǎng)上清對大鼠平滑肌細(xì)胞增殖的影響。 結(jié)果:1.RT-PCR顯示,同常氧對照組相比缺氧組HIF-1α、SDF-1α、VEGF基因表達(dá)增高(P 0.05),同脂質(zhì)體對照組相比RNA干擾組HIF-1α、SDF-1α、VEGF基因表達(dá)降低。2.ELISA檢測發(fā)現(xiàn),同常氧對照組相比缺氧組條件培養(yǎng)液中的HIF-1α、SDF-1α、VEGF含量增加(P 0.05),同脂質(zhì)體對照組相比RNA干擾組HIF-1α、SDF-1α、VEGF含量降低(p0.05)。3.CCK8檢測發(fā)現(xiàn),同常氧對照組的相比缺氧組條件培養(yǎng)上清可以刺激平滑肌細(xì)胞增殖(P 0.05),同脂質(zhì)體對照組相比RNA干擾組缺氧培養(yǎng)上清促進(jìn)增殖的能力減弱(P 0.05)。 結(jié)論:1.抑制HIF-1α表達(dá)可以使MSCs的SDF-1α和VEGF基因表達(dá)降低。2.缺氧可以使HIF-1α,SDF-1α和VEGF基因表達(dá)增高。3.HIF-1α是MSC調(diào)控SDF-1α和VEGF基因表達(dá)的主要因素之一。4.RNA干擾可以降低缺氧培養(yǎng)上清對大鼠平滑肌細(xì)胞的促增殖作用。
[Abstract]:Part one: culture of MSCs and screening of HIF-1 偽 siRNA objective: to screen the most effective RNA interference sequence of bone marrow mesenchymal stem cells (MSCs / mesenchymal stem cells), RT-PCR) cultured by adherent method. Methods: MSCs were cultured by adherent method. The cells of the 3-5 passage were used in the experiment. The morphology of the cells was observed under inverted microscope, and the surface marker CD11b / cpCD34, CD444 and CD90 were detected by flow cytometry. MSCs was divided into five groups: hypoxia group (without any intervention), liposome group (transfection of empty liposome) siRNA609 group (transfected siRNA609), siRNA658 group (transfected siRNA658) and siRNA2070 group (transfected siRNA2070). The gene expression of HIF-1 偽 was detected by RT-PCR in the above five groups cultured in anoxic condition for 24 h. The result is 1: 1. Flow cytometry showed that the positive cells of CD44 and CD44 were 84.2% 鹵0.2% and 97.91% 鹵0.710% respectively by flow cytometry. The number of CD44 / CD34- cells was 99.4% 鹵0.40.41% 鹵0.97% 鹵0.97%. The results of RT-PCR showed that the gene expression of HIF-1 偽 in siRNA609 siRNA658siRNA2070 group was significantly lower than that in hypoxic group (P 0.05), and the inhibitory effect of siRNA658 group was the highest (P 0.05). Conclusion 1. The cultured cells expressed the corresponding surface marker of MSCs. 2. MSCs could transfect HIF-1 偽 siRNA sequence. 3. SiRNA658 interference sequence had the strongest inhibitory effect. The second part is the effect of HIF-1 偽 small interfering RNA on the expression of HIF-1 偽 -SDF-1 偽 and VEGF gene in MSCs. Objective to investigate the effects of small interfering RNA on the expression of MSCs HIF-1 偽, SDF-1 偽 -stromal derived factor 1 偽 and vascular endothelial growth factor) in MSCs. Methods: four groups of MSCs, Normoxic control group (no intervention factor), hypoxia group (hypoxia 24 h), liposome control group (hypoxia 24 h after transfection of no-loaded liposome) RNA interference group (hypoxia 24 h after transfection of liposome mediated RNA interference sequence) .RT-PCR method to detect HIF-1 偽 -SDF-1 偽 and VEGF mRNA expression water in MSCs The expression levels of HIF-1 偽 -SDF-1 偽 and VEGF protein in the supernatant of MSCs culture were detected by ELISA and the effect of MSCs supernatant on the proliferation of rat smooth muscle cells was detected by CCK8. Results 1. RT-PCR showed that the expression of HIF-1 偽 -SDF-1 偽 mRNA in hypoxia group was higher than that in normoxic control group (P0. 05), and that in RNA interference group was lower than that in liposome control group. Compared with the normoxic control group, the content of HIF-1 偽 -SDF-1 偽 -VEGF in conditioned medium of hypoxia group was increased (P 0.05), and the content of HIF-1 偽 -SDF-1 偽 -VEGF in RNA interference group was lower than that in liposome control group (p0.05). Compared with the normoxic control group, the conditioned supernatant of hypoxia group could stimulate the proliferation of smooth muscle cells (P0. 05), and the supernatant of hypoxia culture supernatant of RNA interference group was weaker than that of liposome control group (P 0. 05). Conclusion 1. Inhibiting the expression of HIF-1 偽 could decrease the expression of SDF-1 偽 and VEGF genes in MSCs. Hypoxia can increase the expression of HIF-1 偽 -SDF-1 偽 and VEGF genes. 3.HIF-1 偽 is one of the main factors of MSC regulating the expression of SDF-1 偽 and VEGF genes. 4. RNA interference can reduce the proliferation of rat smooth muscle cells induced by hypoxia culture supernatant.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李剛,李湘平,彭英,劉雄,李曉華;應(yīng)用RNA干擾抑制EB病毒潛伏膜蛋白-1表達(dá)對鼻咽癌細(xì)胞生長的影響(英文)[J];第一軍醫(yī)大學(xué)學(xué)報;2004年03期

2 梁云,周韌;RNA干擾——治療HIV感染的新方法[J];國外醫(yī)學(xué).病毒學(xué)分冊;2005年01期

3 陳道榮,王丕龍,閆歌;RNA干擾技術(shù)的研究進(jìn)展[J];胃腸病學(xué)和肝病學(xué)雜志;2005年01期

4 安立峰,董震;RNA干擾——腫瘤研究的新工具[J];中華腫瘤雜志;2005年07期

5 陳雪梅,杜顯剛,譚志巍,王亞琴,官鵬;基因沉默的研究進(jìn)展[J];法律與醫(yī)學(xué)雜志;2005年03期

6 帖君,房殿春,寧曉燕;載體介導(dǎo)的siRNA對SGC-7901胃癌細(xì)胞中hPOT1表達(dá)的抑制作用[J];解放軍醫(yī)學(xué)雜志;2005年09期

7 祝蓉;張新;白春學(xué);;RNA干擾在哺乳動物中的應(yīng)用與腫瘤基因治療[J];國際呼吸雜志;2006年02期

8 陳剛;耿江;張元芳;;NKX3.1短發(fā)卡RNA表達(dá)載體對NKX3.1基因表達(dá)的抑制作用[J];中華實驗外科雜志;2006年02期

9 周穎;凌斌;高婷;陳敏;馮定慶;程志祥;朱園園;趙衛(wèi)東;石永云;王梅梅;祝懷平;;siRNA靶向Her-2逆轉(zhuǎn)錄病毒載體構(gòu)建及其在SKOV3中的表達(dá)[J];腫瘤防治研究;2007年04期

10 徐云智;梁彩霞;趙春禮;鄭德宇;段德義;;人類白細(xì)胞抗原A表達(dá)沉默對大鼠腦內(nèi)人細(xì)胞移植排斥反應(yīng)的調(diào)節(jié)[J];中國組織工程研究與臨床康復(fù);2008年40期

相關(guān)會議論文 前10條

1 張輝;于力;朱龍英;萬延慧;朱為民;;Hair-pin RNA介導(dǎo)的番茄抗TYLCV效果初步研究[A];慶祝中國園藝學(xué)會創(chuàng)建80周年暨第11次全國會員代表大會論文摘要集[C];2009年

2 李艷;嚴(yán)明;;RNA干擾[A];第一屆全國化學(xué)工程與生物化工年會論文摘要集(下)[C];2004年

3 閆虎斌;陸智明;;基于RNA干擾的植物抗病毒育種原理及研究進(jìn)展[A];中國柑橘科技創(chuàng)新與產(chǎn)業(yè)發(fā)展戰(zhàn)略論壇暨中國柑橘學(xué)會2007年年會論文集[C];2007年

4 趙午莉;邵榮光;;MR-1基因在白血病K562細(xì)胞中的功能研究[A];2009醫(yī)學(xué)前沿論壇暨第十一屆全國腫瘤藥理與化療學(xué)術(shù)會議論文集[C];2009年

5 陳始明;陶澤璋;肖伯奎;;RNA干擾hTERT基因抑制Hep-2細(xì)胞生長增殖的實驗研究[A];湖北省抗癌協(xié)會頭頸腫瘤專業(yè)委員會第四次學(xué)術(shù)會議資料匯編[C];2005年

6 潘q，

本文編號：2193090