比較E.coli與293T cell表現之人類resistin在結構上及功能上的差異

Structural and functional comparison of human resistin expressed by E.coli and 293T cell

10.6342/NTU.2006.02707

廖唯宇

蛋白純化 ； 膠體過濾 ； resistin ； nickel column ； hexamer ； dimer

臺灣大學生物化學暨分子生物學研究所學位論文

2006年

碩士

呂紹俊

繁體中文

Resistin在動物實驗中被認為是肥胖時由脂肪細胞所分泌而造成第二型糖尿病的重要因子。但是多數的臨床研究顯示，human resistin似乎和發炎反應較具有相關性，而與肥胖或糖尿病沒有明顯的相關性。因此resistin也被認為是屬於和發炎有關的細胞激素之ㄧ。近幾年的研究中多利用大腸桿菌表現的recombinant human resistin (rhResistin)來探討其功能，並觀察到rhResistin會促進肝臟的糖質新生，也會造成心血管內皮細胞的細胞附著分子表現量上升以及促進心血管平滑肌細胞的細胞增生。Resisitn有十一個cysteines，這些cysteines當中的十個cysteines會形成五對的分子內雙硫鍵，剩下的一個cysteine會形成一對的分子間雙硫鍵而造成dimer的形式，這些dimer則可以再聚合成hexamer 。我們懷疑由大腸桿菌所表現之rhResistin即使再refolding之後是否真能準確的摺疊成哺乳類細胞所表現出來的hexameric resistin相同。首先我們將於大腸桿菌和293T細胞中大量表現的rhResistin以Ni-NTA純化出並以sliver stain的方式分析其純度。也以LC/MS/MC確定其為rhResistin。再將自大腸桿菌、293T細胞純化後的rhResistin以及英國PeproTech生技公司所提供之E.coli expressed rhResistin分別以superose 6和superose 12 管柱分析，發現只有自293T細胞所表現之rhResistin才會形成hexamer的形式，而由大腸桿菌所表現的rhResistin則是不會形成hexamer的形式。以non-denaturing or denaturing的 SDS-PAGE分析發現，由大腸桿菌所表現的rhResistin較容易形成aggregates，由293T所表現的rhResistin則沒有這種現象。因此，爲了解由大腸桿菌以及293T細胞所表現rhResistin在發炎以及細胞增生有關的生物活性上是否相同，我們比較由293T細胞所表現的rhResistin之conditional medium和英國PeproTech生技公司所提供的大腸桿菌所表現之rhResistin對Raw264.7細胞株之與發炎有關基因的表現以及心血管內皮細胞增生情況的影響。實驗結果發現，以生技公司所提供之rhResistin處理Raw 264.7細胞會使得TNF-α以及IL-6之mRNA表現上升，也會提高人類內皮細胞之細胞增生。若以293T表現之rhResistin的conditional medium來細胞處理，會發現到單只是pcDNA3 myc-His vector only conditional medium的情況下欲觀察之基因其表現情況皆會大量上升，導致我們無法有效地比較在conditional medium作為背景值的情況之下，293T細胞以及大腸桿菌表現之rhResistin在生物活性上影響為何。因此接下來我們利用純化後的293T細胞所表現rhResistin去處理細胞並比較與生技公司所提供之rhResistin在活性上的差異，我們觀察到293T表現rhResistin似乎是比生技公司所提供之rhResistin更能促進Raw264.7細胞TNF-α、resistin及iNOS等基因之mRNA表現，但兩者對於造成HCAECs的增生並沒有差異。因此未來我們將以純化的293T表現rhResistin來處理細胞，再進一步觀察並釐清由293T所表現之rhResistin與E.coli表現之rhResistin在生物活性上有何異同。

Resistin has been identified to be synthesized and secreted in adipocytes, and was thought to be a link of obesity and diabetes in mouse. However, clinical studies did not support that resistin link obesity and type II diabetes in human. Several lines of evidences suggest that resistin may play a role in inflammation. However, the physiological function of rhResistin is still unclear. Recently, functional studies using E.coli rhResistin suggested that the resistin may increase gluconeogenesis in the liver, increase production of adhesion molecules by aortic endothelial cells and increase proliferation of aortic smooth muscle cells. Resistin, which has 11 cysteines, contains five intramolecular disulfide bonds through ten cysteines and forms homo-dimmer through an intermolecular disulfide bond. And three dimers form a hexamer. We speculate that E.coli expressed rhResistin may not fold correctly and form hexamer as that produced by mammalian cells. Therefore, biological function tested using a E.coli expressed rhResistin may be different from that using rhResistin expressed by mammalian cells. The aims of this study are to investigate the structure and function of rhResistin protein produced by 293T cells or E.coli. We purified rhResistin from E.coli and 293T cells using Ni-NTA and analysed the degree of purity by sliver stain. The purified rhResistin were confirmed by LC/MS/MS. Gel filtration analysis of purified rhResistin from E.coli, 293T cells or purchased from PeproTech(E.coli expressed) using superose 6 or superose 12 column. The results show that 293T expressed rhResistin were eluted at ~75KD, however, E.coli expressed rhResistin was eluted at ~40kDa. The results suggest that 293T expressed rhResistin are a hexamer, but the E.coli expressed rhResistin do not form hexamer structure. In non-denaturing or denaturing SDS-PAGE, 293T expressed rhResistin show dimmer and monomer but E.coli expressed rhResistin tend to aggregate. Furthermore, we compared the biological activity of rhResistin from PeproTech (E.coli expressed) or 293T expressed rhResistin in promotion of inflammation in Raw264.7 cells and cell proliferation in aortic endothelial cells. We treated cells using conditional medium of rhResistin transfected 293T cells and E.coli expressed rhResistin from PeproTech. Our preliminary data show that E.coli expressed rhResistin induced TNF-α and IL-6 mRNA production in mouse macrophages and induce human endothelial cell proliferation. However, we were unable to clarify the effects of rhResistin expressed by 293T cells. Because the levels of TNF-α, IL-6, resistin and iNOS mRNA were increased when Raw264.7 cells were treated with conditional medium from cells transfected with pcDNA3 myc-His vector. Then we treated Raw 264.7 and HCAEC with the purified 293T expressed rhResistin and found that purified 293T expressed rhResistin induced more TNF-α, resistin and iNOS mRNA expression in Raw264.7 cell than E.coli expressed rhResistin from PeproTech. In future, we will compare the biological function of purified rhResistin from 293T cell and E.coli.