【摘要】： 背景與目的 在多種生長因子中，轉(zhuǎn)化生長因子β(TGFβ)在組織纖維化中扮演重要角色。在哺乳動物細(xì)胞內(nèi)已經(jīng)發(fā)現(xiàn)三種亞型：TGFβ1、β2和β3，這三種亞型基因在不同種屬間高度保守。TGFβ可以被涉及創(chuàng)面愈合的大部分細(xì)胞分泌，包括：巨噬細(xì)胞、成纖維細(xì)胞等。它們在創(chuàng)面愈合中扮演不同角色，TGFβ1和β2能夠誘導(dǎo)瘢痕形成，而TGFβ3卻能抑制瘢痕形成。本實驗將pcDNA3.1(-)／TGFβ3穩(wěn)定轉(zhuǎn)染到原代培養(yǎng)的Wistar大鼠皮膚成纖維細(xì)胞內(nèi)，篩選轉(zhuǎn)基因細(xì)胞。采用流式細(xì)胞術(shù)檢測轉(zhuǎn)染前后細(xì)胞內(nèi)TGFβ含量的變化，對基因轉(zhuǎn)染成纖維細(xì)胞TGFβ分泌等生物學(xué)行為進行初步研究，為臨床應(yīng)用提供實驗依據(jù)。 方法 1采用植塊培養(yǎng)法和分離細(xì)胞培養(yǎng)法，，原代培養(yǎng)Wistar大鼠皮膚真皮層中成纖維細(xì)胞。 2通過脂質(zhì)體介導(dǎo)的轉(zhuǎn)染技術(shù)和G418篩選法，將已經(jīng)構(gòu)建好的pcDNA3.1(-)／TGFβ3穩(wěn)定轉(zhuǎn)染成纖維細(xì)胞；得到單克隆細(xì)胞，并擴增得到轉(zhuǎn)基因細(xì)胞。 3流式細(xì)胞儀檢測細(xì)胞內(nèi)TGFβ1、β3含量。 結(jié)果 1成功獲得穩(wěn)定表達細(xì)胞克隆。 2流式細(xì)胞檢測結(jié)果：正常細(xì)胞內(nèi)TGFβ總量為60.86，TGFβ1／TGFβ3=1.38；而轉(zhuǎn)染后TGFβ總量為225.23，TGFβ1／TGFβ3=0.09。上述結(jié)果表明，轉(zhuǎn)基因細(xì)胞內(nèi)TGFβ3表達明顯比正常細(xì)胞高，而TGFβ1表達比正常細(xì)胞低。 結(jié)論 通過脂質(zhì)體介導(dǎo)的轉(zhuǎn)染技術(shù)和G418篩選法，將pcDNA3.1(-)／TGFβ3穩(wěn)定轉(zhuǎn)染成纖維細(xì)胞，得到的轉(zhuǎn)基因細(xì)胞能夠表達TGFβ3蛋白，并且能夠抑制TGFβ1蛋白的表達。從而為其應(yīng)用于創(chuàng)面移植提供了理論基礎(chǔ)。
[Abstract]:Background & objective Transforming growth factor 尾 (TGF 尾) plays an important role in tissue fibrosis. Three subtypes: TGF- 尾 _ 1, 尾 _ 2 and 尾 _ 3 have been found in mammalian cells. These three subtypes are highly conserved among different species. TGF 尾 can be secreted by most of the cells involved in wound healing, including macrophages, fibroblasts and so on. They play different roles in wound healing. TGF- 尾 1 and 尾 2 can induce scar formation, while TGF 尾 3 can inhibit scar formation. In this experiment, pcDNA3.1 (-) / TGF- 尾 3 was stably transfected into the primary cultured fibroblasts of Wistar rat skin to screen the transgenic cells. The changes of TGF 尾 content in cells before and after transfection were detected by flow cytometry, and the biological behaviors such as TGF 尾 secretion of gene transfected fibroblasts were preliminarily studied, which provided experimental basis for clinical application. Methods 1. Graft culture and cell culture were used. Fibroblasts from the dermis of Wistar rats were cultured in primary culture. 2 pcDNA3.1 (-) / TGF- 尾 3 was stably transfected into fibroblasts by liposome-mediated transfection and G418 screening. Monoclonal cells were obtained, and transgenic cells were amplified. 3 the contents of TGF 尾 1 and 尾 3 in the cells were detected by flow cytometry. Results 1 stable expression cell clones were successfully obtained. 2 flow cytometry analysis showed that the total amount of TGF 尾 in normal cells was 60.86TGF- 尾 1/TGF 尾 -1.38; The total amount of TGF 尾 after transfection was 225.23 TGF- 尾 1/TGF 尾 30.09. The results showed that the expression of TGF 尾 3 in transgenic cells was significantly higher than that in normal cells, while the expression of TGF 尾 1 in transgenic cells was lower than that in normal cells. Conclusion pcDNA3.1 (-) / TGF- 尾 3 was stably transfected into fibroblasts by liposome-mediated transfection and G418 screening, and the transgenic cells could express TGF 尾 3 protein. And it can inhibit the expression of TGF 尾 1 protein. It provides a theoretical basis for its application in wound transplantation.
【學(xué)位授予單位】：汕頭大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R329

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