TGFβ3基因对Wistar大鼠皮肤成纤维细胞分泌TGFβ的影响

发布时间：2018-08-19 15:08

【摘要】： 背景与目的在多种生长因子中，转化生长因子β(TGFβ)在组织纤维化中扮演重要角色。在哺乳动物细胞内已经发现三种亚型：TGFβ1、β2和β3，这三种亚型基因在不同种属间高度保守。TGFβ可以被涉及创面愈合的大部分细胞分泌，包括：巨噬细胞、成纤维细胞等。它们在创面愈合中扮演不同角色，TGFβ1和β2能够诱导瘢痕形成，而TGFβ3却能抑制瘢痕形成。本实验将pcDNA3.1(-)／TGFβ3稳定转染到原代培养的Wistar大鼠皮肤成纤维细胞内，筛选转基因细胞。采用流式细胞术检测转染前后细胞内TGFβ含量的变化，对基因转染成纤维细胞TGFβ分泌等生物学行为进行初步研究，为临床应用提供实验依据。方法 1采用植块培养法和分离细胞培养法，，原代培养Wistar大鼠皮肤真皮层中成纤维细胞。 2通过脂质体介导的转染技术和G418筛选法，将已经构建好的pcDNA3.1(-)／TGFβ3稳定转染成纤维细胞；得到单克隆细胞，并扩增得到转基因细胞。 3流式细胞仪检测细胞内TGFβ1、β3含量。结果 1成功获得稳定表达细胞克隆。 2流式细胞检测结果：正常细胞内TGFβ总量为60.86，TGFβ1／TGFβ3=1.38；而转染后TGFβ总量为225.23，TGFβ1／TGFβ3=0.09。上述结果表明，转基因细胞内TGFβ3表达明显比正常细胞高，而TGFβ1表达比正常细胞低。结论通过脂质体介导的转染技术和G418筛选法，将pcDNA3.1(-)／TGFβ3稳定转染成纤维细胞，得到的转基因细胞能够表达TGFβ3蛋白，并且能够抑制TGFβ1蛋白的表达。从而为其应用于创面移植提供了理论基础。
[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.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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