摘要
第一部分强力霉素调控表达的人肝癌HepG2Tet-on细胞系的建立
目的构建可以用强力霉素调控表达的人肝癌HepG2Tet-on细胞系
方法脂质体转染法将pWHE146质粒转染到人肝癌HepG2细胞中,G418筛选出稳定表达细胞克隆;单克隆分别扩增后,瞬时转染pTRE-hyg-luc质粒,强力霉素诱导表达后,检测荧光素酶表达活性,挑选出受强力霉素调控的低背景、高表达的HepG2Tet-on细胞株。
结果成功构建了一株受强力霉素调控的高表达低背景的HepG2Tet-on细胞株。
结论HepG2Tet-on细胞株可用于外源基因的真核调控高表达,为研究真核基因功能提供了一种有力的实验手段。
第二部分强力霉素调控HIF-1α表达的人肝癌HepG2Tet-on-HIF-1α双稳细胞系构建
目的建立强力霉素调控HIF-1α表达的人肝癌HepG2Tet-on-HIF-1α双稳细胞系。
方法脂质体转染法将重组质粒pTRE-HIF-1α转染入HepG2Tet-on细胞株,潮霉素(hyg)筛选出稳定表达细胞克隆HepG2Tet-on-HIF-1α;用强力霉素(1μg/ml)诱导后,用RT-PCR和Western-blot检测HIF-1α表达。
结果强力霉素(1μg/ml)可诱导HepG2Tet-on-HIF-1α细胞HIF-1αmRNA(5.899±2.176倍)和蛋白表达增加(2.179±0.742倍)。
结论成功建立强力霉素调控HIF-1α表达的人肝癌HepG2Tet-on-HIF-1α双稳细胞系,为深入研究HIF-1α在肝癌细胞中的作用提供了有力的实验手段。
第三部分调控HIF-1α表达对人肝癌HepG2细胞增殖及侵袭能力的影响
目的探讨体外常氧下,诱导表达HIF-1α对HepG2细胞增殖及侵袭能力的影响。
方法常氧下,四甲基偶氮唑盐法检测缺氧诱导因子1α对细胞增殖、黏附能力的影响,Transwell法检测其对侵袭能力的影响。
结果增殖实验中,Dox(+)组与Dox(-)组各时段A490nm值无差异(P>0.05);黏附实验中,Dox(+)组的A490nm值明显高于Dox(-)组(P=0.008);Dox(+)组侵袭细胞数(37.611±8.424)明显高于Dox(-)组(25.333±8.117)(P<0.001)。
结论常氧下,缺氧诱导因子1α不影响HepG2细胞的增殖,但明显增加其黏附和侵袭能力。
第四部分强力霉素调控HIF-1α表达的裸鼠肝癌皮下移植瘤模型的建立
目的建立可以由强力霉素(Dox)诱导HIF-1α表达的裸鼠肝癌皮下移植瘤模型。
方法注射法建立裸鼠肝癌HepG2Tet-on-HIF-1α细胞皮下移植瘤模型;口服强力霉素7周,RT-PCR和Western-blot检测皮下移植瘤中HIF-1α表达。
结果与Dox(-)组相比,Dox(+)组裸鼠HepG2Tet-on-HIF-1α细胞皮下移植瘤中HIF-1αmRNA(4.303±1.004倍)和蛋白(1.666±0.079倍)表达增加。
结论口服强力霉素可以有效地诱导裸鼠HepG2Tet-on-HIF-1α细胞皮下移植瘤中HIF-1αmRNA和蛋白表达增加,为体内研究HIF-1α对肝癌的作用提供了有效的实验手段。
第五部分强力霉素调控HIF-1α表达对裸鼠肝癌皮下移植瘤的作用
目的探讨体内调控表达HIF-1α对裸鼠肝癌皮下移植瘤生长和凋亡的影响。
方法强力霉素诱导裸鼠肝癌HepG2Tet-on-HIF-1α细胞皮下移植瘤缺氧诱导因子1α表达;观察上调缺氧诱导因子1α表达对裸鼠皮下移植瘤生长的影响;Tunel法检测两组皮下移植瘤原位凋亡的情况;免疫组化和western blot检测两组皮下移植瘤中血管生成情况。
结果Dox(+)组在肿瘤体积、重量、生长速度上都明显超过Dox(-)组,肿瘤内坏死面积明显小于Dox(-)组;同Dox(-)组相比,Dox(+)组裸鼠体重下降更为明显,两组均无肝、肺转移发生;Dox(+)组皮下移植瘤的细胞凋亡较Dox(-)组少,而血管则较多。
结论体内实验中,缺氧诱导因子1α可促进肿瘤的生长;其机制可能是促进肿瘤内部血管生成,及减少肿瘤细胞凋亡。
PartⅠ: Establishment of HepG2Tet-on cell line controlled by the Tet-On regulatory system
Objective To establish tetracycline-controlled inducible system ( Tet-On) in HepG2 cell.
Methods The HepG2 cells were transfected with pWHE146 vector by using liposome transfection reagent . The transfected cells were selected in medium containing G418 and G418-resistant clones were isolated. All individual G418-resistant clones were screened by transient transfection with plasmid pTRE-hyg-luc for clones with low background and high induction of luciferase in response to Dox.
Results One HepG2 cell line, which exhibited high levels of induction and high gene expression levels, was obtained.
Conclusion The HepG2 cell line can be used to highly express eukaryotic gene and this Tet-On system is available for use in eukaryotic gene function studies.
PartⅡ: Establishment of Tet-On system in HepG2 cells to regulate HIF-1αexpression
Objective To develop the HepG2Tet-on-HIF-1αcell line which can regulate the HIF-1αexpression by doxycycline.
Methods The HepG2Tet-on cells were transfected with pTRE-HIF-1αvector by using liposome transfection reagent. The transfected cells were selected in medium containing hygromycin(hyg) and the double-stable cell lines(G418- and hyg-resistant) HepG2Tet-on-HIF-1αwere isolated. Induced by Dox(1μg/ml), RT-PCR and Western-blot to test the expression of HIF-1α.
Results the HIF-1αmRNA and protein of HepG2Tet-on-HIF-1αcells could be induced up to 5.899±2.176 and 2.179±0.742 folds by doxycycline(1μg/ml).
Conclusion The double-stable cell line HepG2Tet-on-HIF-1αwas successfully established, which could be induced HIF-1αexpression by doxycycline and provided an ideal experimental platform for further functional study of HIF-1α.
PartⅢ: Inducible Expression of Hypoxia-Inducible factor 1αon the Proliferation and Invasion Property of HepG2 cells under Normoxia in Vitro
Objective To study the inducible expression of HIF-1αon the proliferation and invasion property of HepG2 cells under normoxia in vitro.
Methods Under normoxia in vitro, MTT assay was used to observe the proliferative and adhesive activity of cells, and the invasive activity was determined by transwell cell culture chamber method.
Results There were no difference of A490nm between the Dox(+)and Dox(-)group in experiment detecting the proliferation activity(P>0.05); But in adhesive experiment, the A490nm of Dox ( + ) group was 0.662±0.058, higher than the Dox ( - ) group 0.526±0.808(P=0.008); The invasived cell number of Dox(+)group was 37.611±8.424, but in the Dox(-)group, the number was 25.333±8.117(P<0.001).
Conclusion HIF-1αhas no influence with the proliferation activity, but it could enhance the adhesive and invasive properties of HepG2 cells.
PartⅣ:Construction of Subcutaneous Implanted Hepatoma Model Inducible Expression of HIF-1αin Nude Mice
Objective To constructe the subcutaneous implanted hepatoma model in nude mice with HepG2Tet-on-HIF-1αcell line which could be induced the expression of HIF-1αby doxycycline.
Methods Constructed the subcutaneous implanted hepatoma model in nude mice with HepG2Tet-on-HIF-1αcell line; The HIF-1αmRNA and protein level of subcutaneous implanted tumors were detected with RT-PCR and Western-blot after seven weeks taking doxycycline orally.
Results The HIF-1αmRNA and protein of subcutaneous implanted tumor in nude mice could be induced up to 4.303±1.004 and 1.666±0.079 folds by taking doxycycline orally(1mg/ml).
Conclusion The HIF-1αmRNA and protein of subcutaneous implanted tumor with HepG2Tet-on-HIF-1αcell line in nude mice could be induced by taking doxycycline orally. It provided an ideal experimental platform for further study of the effect of HIF-1αon hepatoma in vivo.
PartⅤ:Impact of Induced Hypoxia-Inducible Factor-1αon the Subcutaneous Implanted Hepatoma in Nude Mice in Vivo
Objective To study the impact of induced HIF-1αon the growth and apoptosis of subcutaneous implanted hepatoma in nude mice in vivo.
Methods The HIF-1αof the subcutaneous implanted hepatoma in nude mice were induced by doxycycline; Observed the impact of HIF-1αinduced by doxycycline on the growth of subcutaneous implanted hepatoma in nude mice and its impact on nude mice. Tunel method was used to analyze the apoptosis index of subcutaneous implanted hepatoma. Immunohistochemistry and western blot methods were used to analyze the vascularization in subcutaneous implanted hepatoma of two groups.
Results Compared with Dox(-)group, the volume, weight and growth velocity of subcutaneous implanted hepatoma were higher in Dox(+)group, but the necrosis period of the tumor were less. The weight loss of nude mice was obvious in Dox(+)group. There was no liver or lung metastasis in either group. In Dox(-)group, the AI was higher than that of Dox(+)group. And there were much more blood vessels in subcutaneous implanted hepatoma of Dox(+) groups.
Conclusion HIF-1αpromote the growth of subcutaneous implanted hepatoma in nude mice in vivo. The mechanism may be that HIF-1αdecrease the cell apoptosis and promot the vascularization of subcutaneous implanted hepatoma.
引文
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