Oct4、Sox2、c-Myc、Klf4-慢病毒載體構(gòu)建與包裝及感染人皮膚成纖維細(xì)胞的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-09-13 06:28
【摘要】： 目的本研究構(gòu)建并包裝含有Oct4、Sox2、c-Myc、Klf4四基因的慢病毒載體(lentivirus vector),并進(jìn)行目的基因過(guò)表達(dá)慢病毒顆粒感染原代培養(yǎng)的人皮膚成纖維細(xì)胞(human foreskin fibroblast HFF),旨在初步探討誘導(dǎo)性多能干細(xì)胞((induced pluripotent stem cell ,iPSC)成功誘導(dǎo)的關(guān)鍵技術(shù)。 方法(1)從含有Oct4、Sox2、c-Myc、Klf4的質(zhì)粒中獲取目的基因。將目的基因與酶切線性化的慢病毒載體進(jìn)行定向連接,其產(chǎn)物轉(zhuǎn)化細(xì)菌感受態(tài)細(xì)胞,對(duì)長(zhǎng)出的陽(yáng)性克隆進(jìn)行測(cè)序和比對(duì)分析,比對(duì)正確的即為構(gòu)建成功的目的質(zhì)粒.。然后將構(gòu)建好的目的質(zhì)粒進(jìn)行超純?nèi)?nèi)毒素抽提。采用Western Blot法檢測(cè)目的基因質(zhì)粒表達(dá)。通過(guò)三質(zhì)粒系統(tǒng)共轉(zhuǎn)染293T細(xì)胞對(duì)慢病毒進(jìn)行包裝并進(jìn)行滴度測(cè)定;(2)采用Ⅰ型膠原酶消化結(jié)合組織塊培養(yǎng)法分離培養(yǎng)HFF,以胰蛋白酶消化法分離培養(yǎng)小鼠胚胎成纖維細(xì)胞(Mouse embryonic fibroblast MEF),分別觀察兩種細(xì)胞的生長(zhǎng)情況,并以免疫細(xì)胞化學(xué)染色SABC法對(duì)兩種細(xì)胞進(jìn)行鑒定;絲裂霉素C處理MEF用于建立干細(xì)胞培養(yǎng)所需要的飼養(yǎng)層;(3)用包裝有綠色熒光蛋白的空病毒感染HFF,摸索出慢病毒感染HFF的最佳MOI值;(4)將生長(zhǎng)狀態(tài)良好的第3代HFF隨機(jī)分為3組:目的基因感染組、空病毒感染組、正常對(duì)照組,采用含四種目的基因的慢病毒混合感染HFF并連續(xù)感染兩次的方法,在二次感染后4天給予HFF細(xì)胞干細(xì)胞培養(yǎng)條件,在倒置顯微鏡下觀察其形態(tài)變化,篩選最佳實(shí)驗(yàn)室條件。 結(jié)果(1)成功構(gòu)建并包裝出Oct4、Sox2、c-Myc、Klf4 慢病毒載體,基因測(cè)序結(jié)果與目標(biāo)序列完全一致,滴度為1×109 TU/ml;(2) 0.02%Ⅰ型膠原酶消化結(jié)合組織塊貼壁培養(yǎng)法可以在體外建立穩(wěn)定的HFF系,培養(yǎng)4d時(shí)即有細(xì)胞游走出,細(xì)胞呈長(zhǎng)梭形、有分支,6d時(shí)有較多的細(xì)胞游離出,匯集成束,10d左右細(xì)胞鋪滿瓶底,0.25%胰蛋白酶消化傳代。采用0.0625%胰蛋白酶分次消化法可以獲得高活力高產(chǎn)量的MEF,細(xì)胞呈長(zhǎng)梭形、多角形,24h內(nèi)細(xì)胞完全貼壁。免疫細(xì)胞化學(xué)鑒定兩種細(xì)胞均呈陽(yáng)性結(jié)果;(3)用含有15ug/ml的絲裂霉素C的培養(yǎng)液1ml處理長(zhǎng)滿25CM2培養(yǎng)瓶的MEF1.5h,可以獲得良好功能的干細(xì)胞培養(yǎng)所需的飼養(yǎng)層;(4)摸索出慢病毒感染HFF的最佳MOI值為30;與正常組相比目的基因慢病毒感染HFF組雖未見(jiàn)到明顯的多能干細(xì)胞,但出現(xiàn)了明顯的細(xì)胞形態(tài)變化,由長(zhǎng)梭型變成橢圓形或半橢圓形,相同條件下的空病毒感染組HFF形態(tài)未發(fā)生明顯的變化,仍為典型的長(zhǎng)梭型。 結(jié)論本研究成功地進(jìn)行了HFF和MEF的原代培養(yǎng),構(gòu)建并包裝出高滴度的Oct4、Sox2、c-Myc、Klf4 慢病毒載體,并感染人皮膚成纖維細(xì)胞,通過(guò)目的基因轉(zhuǎn)導(dǎo)可誘導(dǎo)HFF發(fā)生形態(tài)變化,初步探討了iPSC研究的關(guān)鍵技術(shù)。
[Abstract]:Objective to construct and package lentivirus vector (lentivirus vector), containing four genes of Oct4,Sox2,c-Myc,Klf4 and overexpression lentivirus particles in primary cultured human skin fibroblasts (human foreskin fibroblast HFF), in order to explore the inducible pluripotency. The key technology of the successful induction of cell (induced pluripotent stem cell / iPSC. Methods (1) the target gene was obtained from the plasmid containing Oct4,Sox2,c-Myc,Klf4. The target gene was directly ligated with the lentivirus vector which was digested by enzyme digestion. The product was transformed into bacterial receptive cells and the positive clones were sequenced and compared. Then the constructed target plasmid was extracted by super pure endotoxin removal. The expression of target gene plasmid was detected by Western Blot method. Packaging and titer determination of lentivirus by three plasmid system co-transfection 293T cells. (2) isolation and culture of HFF, by type I collagenase digestion combined with tissue mass culture and isolation and culture of mouse embryonic fibroblasts by trypsin digestion Cell (Mouse embryonic fibroblast MEF), was used to observe the growth of two kinds of cells. Two kinds of cells were identified by immunocytochemical staining (SABC). Mitomycin C treatment of MEF was used to establish the feeder layer needed for stem cell culture; (3) the best MOI value of lentivirus infected HFF was found by HFF, infected with empty virus wrapped with green fluorescent protein; (4) the third generation HFF with good growth was followed. The machine was divided into three groups: target gene infection group, In the empty virus infection group and the normal control group, HFF cell stem cells were cultured 4 days after the second infection with lentivirus containing four target genes. The morphologic changes were observed under inverted microscope and the optimum laboratory conditions were screened. Results (1) the Oct4,Sox2,c-Myc,Klf4 lentivirus vector was successfully constructed and packaged. The result of gene sequencing was identical with the target sequence. The titer of 1 脳 109 TU/ml; (2) 0.02% collagenase digestion combined with tissue mass adherence culture could establish stable HFF lines in vitro. After 4 days of culture, some cells swam out, and the cells were fusiform. After 6 days of branching, more cells were free out, and the cells were collected into bundles about 10 days after digestion and passage of 0.25% trypsin. MEF, cells with high activity and high yield could be obtained by 0.0625% trypsin fractionation method, and the cells with polygonal shape could be completely adhered to within 24 hours. (3) the culture medium 1ml containing mitomycin C of 15ug/ml can obtain the feeder layer of stem cell culture with good function by treating MEF1.5h, filled with 25CM2 culture flask; (4) to find out the necessary feeder layer for stem cell culture with good function. (4) to find out the necessary feeder layer for the culture of stem cells with good function. (3) to use 1ml as a culture medium containing mitomycin C of 15ug/ml. The optimal MOI value of infected HFF was 30. Compared with the control group, there were no significant pluripotent stem cells in the HFF group infected with the target gene lentivirus. However, there were obvious changes in cell morphology, from long spindle type to elliptic or semi elliptic shape. The HFF morphology of empty virus infected group did not change obviously under the same condition, and it was still a typical long spindle type. Conclusion the primary culture of HFF and MEF was successfully carried out in this study, and the high titer Oct4,Sox2,c-Myc,Klf4 lentivirus vector was constructed and packaged, and infected with human skin fibroblasts. The morphological changes of HFF could be induced by target gene transduction. The key technologies of iPSC research are discussed.
【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】：2010
【分類號(hào)】：R751
本文編號(hào)：2240351
[Abstract]:Objective to construct and package lentivirus vector (lentivirus vector), containing four genes of Oct4,Sox2,c-Myc,Klf4 and overexpression lentivirus particles in primary cultured human skin fibroblasts (human foreskin fibroblast HFF), in order to explore the inducible pluripotency. The key technology of the successful induction of cell (induced pluripotent stem cell / iPSC. Methods (1) the target gene was obtained from the plasmid containing Oct4,Sox2,c-Myc,Klf4. The target gene was directly ligated with the lentivirus vector which was digested by enzyme digestion. The product was transformed into bacterial receptive cells and the positive clones were sequenced and compared. Then the constructed target plasmid was extracted by super pure endotoxin removal. The expression of target gene plasmid was detected by Western Blot method. Packaging and titer determination of lentivirus by three plasmid system co-transfection 293T cells. (2) isolation and culture of HFF, by type I collagenase digestion combined with tissue mass culture and isolation and culture of mouse embryonic fibroblasts by trypsin digestion Cell (Mouse embryonic fibroblast MEF), was used to observe the growth of two kinds of cells. Two kinds of cells were identified by immunocytochemical staining (SABC). Mitomycin C treatment of MEF was used to establish the feeder layer needed for stem cell culture; (3) the best MOI value of lentivirus infected HFF was found by HFF, infected with empty virus wrapped with green fluorescent protein; (4) the third generation HFF with good growth was followed. The machine was divided into three groups: target gene infection group, In the empty virus infection group and the normal control group, HFF cell stem cells were cultured 4 days after the second infection with lentivirus containing four target genes. The morphologic changes were observed under inverted microscope and the optimum laboratory conditions were screened. Results (1) the Oct4,Sox2,c-Myc,Klf4 lentivirus vector was successfully constructed and packaged. The result of gene sequencing was identical with the target sequence. The titer of 1 脳 109 TU/ml; (2) 0.02% collagenase digestion combined with tissue mass adherence culture could establish stable HFF lines in vitro. After 4 days of culture, some cells swam out, and the cells were fusiform. After 6 days of branching, more cells were free out, and the cells were collected into bundles about 10 days after digestion and passage of 0.25% trypsin. MEF, cells with high activity and high yield could be obtained by 0.0625% trypsin fractionation method, and the cells with polygonal shape could be completely adhered to within 24 hours. (3) the culture medium 1ml containing mitomycin C of 15ug/ml can obtain the feeder layer of stem cell culture with good function by treating MEF1.5h, filled with 25CM2 culture flask; (4) to find out the necessary feeder layer for stem cell culture with good function. (4) to find out the necessary feeder layer for the culture of stem cells with good function. (3) to use 1ml as a culture medium containing mitomycin C of 15ug/ml. The optimal MOI value of infected HFF was 30. Compared with the control group, there were no significant pluripotent stem cells in the HFF group infected with the target gene lentivirus. However, there were obvious changes in cell morphology, from long spindle type to elliptic or semi elliptic shape. The HFF morphology of empty virus infected group did not change obviously under the same condition, and it was still a typical long spindle type. Conclusion the primary culture of HFF and MEF was successfully carried out in this study, and the high titer Oct4,Sox2,c-Myc,Klf4 lentivirus vector was constructed and packaged, and infected with human skin fibroblasts. The morphological changes of HFF could be induced by target gene transduction. The key technologies of iPSC research are discussed.
【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】：2010
【分類號(hào)】：R751
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 李振宇;慢病毒載體構(gòu)建及結(jié)構(gòu)優(yōu)化[J];國(guó)外醫(yī)學(xué)(分子生物學(xué)分冊(cè));2002年05期
2 張怡,趙連三,汪成孝,雷秉鈞;小鼠胚胎成纖維細(xì)胞的分離與培養(yǎng)[J];四川大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2003年02期
3 米薔;何志旭;段茂利;王志華;楊燕;;人成纖維細(xì)胞飼養(yǎng)層的制備及其對(duì)胚胎干細(xì)胞生長(zhǎng)支持作用[J];復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版);2008年05期
4 徐曉明;楊慧;王曉萍;;慢病毒載體的構(gòu)建及優(yōu)化[J];中國(guó)臨床康復(fù);2006年09期
5 劉民,李柏青;小鼠胚胎成纖維細(xì)胞的分離、擴(kuò)增和抑制[J];中國(guó)實(shí)驗(yàn)動(dòng)物學(xué)雜志;2002年04期
6 安立龍,楊奇,馮秀亮,楊春榮,雷安民,高志民,竇忠英,邱懷;牛類胚胎干細(xì)胞的分離與克隆[J];中國(guó)農(nóng)業(yè)科學(xué);2001年05期
，本文編號(hào)：2240351
本文鏈接：http://sikaile.net/yixuelunwen/pifb/2240351.html
最近更新
教材專著
