本文選題：TPO + IL-6　； 參考：《第三軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：目的： 通過(guò)構(gòu)建TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三個(gè)真核表達(dá)載體，并用脂質(zhì)體轉(zhuǎn)染法共轉(zhuǎn)染人骨髓基質(zhì)細(xì)胞，觀察三個(gè)真核表達(dá)載體在人骨髓基質(zhì)細(xì)胞中的表達(dá)情況。 方法： 1、分別對(duì)TPO、IL-6、IL-11三個(gè)基因片段克隆，再用PCR技術(shù)將克隆出來(lái)的TPO、IL-6、IL-11基因片段和pEGFP-N1、pcDNA3.1(+)、pcDNA3.1(-)這三個(gè)質(zhì)粒分別連接，構(gòu)建成TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三個(gè)真核表達(dá)載體，并進(jìn)行酶切鑒定及測(cè)序分析。 2、用貼壁篩選法對(duì)人骨髓基質(zhì)細(xì)胞進(jìn)行分離，選用分裂能力強(qiáng)的第三代細(xì)胞做為轉(zhuǎn)基因的靶細(xì)胞，，用脂質(zhì)體轉(zhuǎn)染法將TPO-pEGFP-N1、 IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三個(gè)真核表達(dá)載體共轉(zhuǎn)染人骨髓基質(zhì)細(xì)胞，并采用細(xì)胞免疫組化及Western blot法等方法檢測(cè)多基因轉(zhuǎn)染人骨髓基質(zhì)細(xì)胞后在mRNA和蛋白水平的表達(dá)。 結(jié)果： 1、酶切及測(cè)序結(jié)果證實(shí)，本實(shí)驗(yàn)成功構(gòu)建TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三個(gè)真核表達(dá)載體。 2、人骨髓基質(zhì)細(xì)胞的體外培養(yǎng)形態(tài)呈異質(zhì)性，原代細(xì)胞呈集落生長(zhǎng)，傳代后則均勻生長(zhǎng)。 3、TPO-pEGFP-N1、IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三個(gè)真核表達(dá)載體轉(zhuǎn)染人骨髓基質(zhì)細(xì)胞后，熒光顯微鏡下可觀察到TPO-pEGFP-N1的表達(dá)，25%細(xì)胞發(fā)出綠色熒光；免疫細(xì)胞化學(xué)染色檢測(cè)出IL-6及IL-11在細(xì)胞內(nèi)表達(dá)；經(jīng)Western blot檢測(cè)：TPO蛋白表達(dá)(TPO/GAPDH IOD比值)在正常對(duì)照組為(0.17±0.01)，空載體轉(zhuǎn)染組為(0.18±0.01)，多基因轉(zhuǎn)染組為(0.62±0.01)；IL-6蛋白表達(dá)(IL-6/GAPDH IOD比值)在正常對(duì)照組為(0.14±0.01)，空載體轉(zhuǎn)染組為(0.18±0.04)，多基因轉(zhuǎn)染組為(0.34±0.06)；IL-11蛋白表達(dá)(IL-11/GAPDH IOD比值)在正常對(duì)照組為的(0.8±0.23)，空載體轉(zhuǎn)染組為(0.19±0.14)，多基因轉(zhuǎn)染組為(1.62±0.23)。多基因轉(zhuǎn)染組細(xì)胞的蛋白水平明顯高于對(duì)照組(P均0.01)，表明轉(zhuǎn)染成功。 結(jié)論： 1、通過(guò)分子生物學(xué)方法可成功構(gòu)建TPO-pEGFP-N1、 IL-6-pcDNA3.1(+)、IL-11-pcDNA3.1(-)三個(gè)真核表達(dá)載體。 2、TPO、IL-6、IL-11三個(gè)基因可共轉(zhuǎn)染人骨髓基質(zhì)細(xì)胞，并在細(xì)胞內(nèi)有效表達(dá)，將轉(zhuǎn)染后的人骨髓基質(zhì)細(xì)胞作為血小板體外培養(yǎng)的滋養(yǎng)細(xì)胞可能對(duì)血小板生成起促進(jìn)作用。
[Abstract]:Objective: Three eukaryotic expression vectors of TPO-pEGFP-N1mIL-6-pcDNA3.1were constructed and co-transfected into human bone marrow stromal cells by liposome transfection. The expression of three eukaryotic expression vectors in human bone marrow stromal cells was observed. Methods: 1. TPO-pEGFP-N1IL-6-pcDNA3.1 (pEGFP-N1) plasmid was cloned into three eukaryotic expression vectors of TPO-pEGFP-N1IL-6-pcDNA3.1by PCR technique, and identified by restriction endonuclease digestion, and sequenced and analyzed by restriction endonuclease digestion and sequencing. The three eukaryotic expression vectors of TPO-pEGFP-N1IL-6-pcDNA3.1 (TPO-pEGFP-N1IL-6-pcDNA3.1m-1) were constructed. 2. Human bone marrow stromal cells were isolated by adherent screening, and the third generation cells with strong mitotic ability were selected as target cells. Three eukaryotic expression vectors, TPO-pEGFP-N1, IL-6-pcDNA3.1 (TPO-pEGFP-N1, IL-6-pcDNA3.1) were co-transfected into human bone marrow stromal cells by liposome transfection, and the three eukaryotic expression vectors (TPO-pEGFP-N1, IL-6-pcDNA3.1) were co-transfected into human bone marrow stromal cells. The expression of mRNA and protein in human bone marrow stromal cells was detected by immunohistochemistry and Western blot. Results: 1. The results of restriction endonuclease digestion and sequencing confirmed that three eukaryotic expression vectors, TPO-pEGFP-N1, IL-6-pcDNA3.1 (TPO-pEGFP-N1), were successfully constructed. 2. The morphology of human bone marrow stromal cells was heterogeneity in vitro, and the primary cells grew in colony, and then grew homogeneously after passage. (3) after transfection of three eukaryotic expression vectors of TPO-pEGFP-N1 and IL-6-pcDNA3.1into human bone marrow stromal cells, 25% of the cells expressed TPO-pEGFP-N1 were observed to emit green fluorescence under fluorescence microscope, and IL-6 and IL-11 were detected by immunocytochemical staining. The ratio of TPO / GAPDH IOD in normal control group, empty vector transfection group and polygene transfection group was 0.17 鹵0.01, 0.62 鹵0.01 and 0.62 鹵0.01 respectively. The ratio of TPO / GAPDH IOD was 0.14 鹵0.01 in normal control group, 0.18 鹵0.04 in empty vector transfection group and 0.18 鹵0.04 in polygene transfection group. The ratio of IL-11 / GAPDH IOD was 0.34 鹵0.06 in the control group, 0.19 鹵0.14 in the empty vector transfection group and 1.62 鹵0.23 in the multigene transfection group. The protein level of the cells in the polygene transfection group was significantly higher than that in the control group (P < 0.01), indicating that the transfection was successful. Conclusion: 1.Three eukaryotic expression vectors of TPO-pEGFP-N1, IL-6-pcDNA3.1 were successfully constructed by molecular biological methods. 2TPO-IL-6 / IL-11 gene can be co-transfected into human bone marrow stromal cells and expressed effectively in the cells. The transfection of human bone marrow stromal cells as trophoblastic cells in vitro may promote platelet-forming.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 張可瑩;劉江;賈延軍;單小燕;張志欣;;體外培養(yǎng)血小板研究進(jìn)展[J];中國(guó)輸血雜志;2009年08期

2 陸華;趙樹(shù)銘;;體外生產(chǎn)血小板的研究進(jìn)展[J];重慶醫(yī)學(xué);2011年36期

3 李香琴;劉天慶;朱琳;宋克東;馬學(xué)虎;崔占峰;;氣升式環(huán)流中空纖維膜生物反應(yīng)器內(nèi)骨髓間充質(zhì)干細(xì)胞的培養(yǎng)與擴(kuò)增[J];高�；瘜W(xué)工程學(xué)報(bào);2008年06期

4 賁志云;季玉紅;傅晉翔;汪曉鶯;孫曉雷;;多種細(xì)胞因子對(duì)人骨髓基質(zhì)細(xì)胞生長(zhǎng)的影響[J];交通醫(yī)學(xué);2006年05期

5 黃艷紅,王綺如;三類骨髓基質(zhì)細(xì)胞條件培養(yǎng)液體外擴(kuò)增巨核系細(xì)胞[J];生理學(xué)報(bào);2005年02期

6 史繼靜;劉朝奇;;白介素6與腫瘤相關(guān)性的研究進(jìn)展[J];生命的化學(xué);2008年01期

7 馬曉偉;馬芹穎;王彥永;顧平;王銘維;;骨髓間充質(zhì)干細(xì)胞分泌的蛋白及其功能[J];中國(guó)組織工程研究與臨床康復(fù);2009年14期

8 倪明;謝慧琪;陳俊偉;楊志明;秦嶺;李剛;;利用旋轉(zhuǎn)式生物反應(yīng)器快速擴(kuò)增骨髓間充質(zhì)干細(xì)胞[J];醫(yī)用生物力學(xué);2009年01期

本文編號(hào)：1991529