几种SS和MSTN基因质粒对Hela细胞生长和增殖及凋亡的影响
发布时间：2018-12-31 08:48
【摘要】：本研究应用细胞培养、质粒提取、脂质体转染、逆转录PCR、共聚焦显微定位、流式细胞仪分类、MTT、实时荧光定量PCR等技术,通过比较几种SS和MSTN基因质粒转染对宫颈癌Hela细胞生长和增殖的作用,旨在：(1)筛选促进Hela细胞凋亡效果最佳的质粒,为开发肿瘤基因治疗新技术奠定基础;(2)探讨SS和MSTN抗肿瘤的作用通路,为进一步提高肿瘤基因治疗效果奠定基础；(3)从转录和表达角度比较各种质粒在真核细胞中的表达量,为探讨SS基因疫苗作用机制,开发SS和MSTN基因疫苗奠定基础。主要研究内容和结果如下： 1.应用LipofectamineTM2000脂质体转染方法将SS基因质粒pEGS/2SS、pEGS2SS-V、pGS/2SS-M4GFP-asd、pGS/2SS-IL6-asd和MSTN基因质粒pEGMS及SS和MSTN混合基因质粒pEGS/2SS+pEGMS、pEGS2SS-V+pEGMS转染到Hela细胞中,转染后48h,提取总RNA,反转录后电泳检测到SS和MSTN,说明SS和MSTN基因质粒在Hela细胞中能够正常转录； 2.上述基因质粒转染Hela细胞后48h,用DAPI避光染色、应用共聚焦显微镜亚细胞定位,发现SS融合蛋白在细胞核中表达,MSTN融合蛋白则在细胞核和细胞质中都有表达； 3.上述基因质粒转染Hela细胞后24h、48h、72h和96h,用MTT法检测细胞增殖的情况,并与对照组比较,发现试验组明显抑制细胞生长(P0.05),尤其是用pEGMS质粒转染后72h的细胞生长抑制作用最明显。 4.应用Annexin V-APC/7-AAD双染色法检测SS和MSTN基因质粒转染Hela细胞后48h的凋亡情况,并与对照组比较,发现试验组的R5区细胞(即早期凋亡细胞)明显增加(P0.05),其中,pEGMS试验组的凋亡率(27.474±2.86%)最高,比对照组高8.15%。表明SS和MSTN基因质粒可促进Hela细胞的凋亡 5.上述基因质粒转染Hela细胞后48h,利用流式细胞仪检测细胞周期,发现质粒转染组细胞周期分布变化明显：与对照组相比,转染质粒组G0-G1期细胞比例显著降低,S期和G2-M期细胞比例显著升高(P0.05)。说明SS和MSTN基因质粒主要将Hela细胞阻滞在S期。 6.从转染SS和MSTN基因质粒后48h的Hela细胞中提取总RNA,逆转录PCR扩增,检测到SSTR-1, SSTR-2, SSTR-3, SSTR-4和SSTR-5五种受体均能在Hela细胞中表达,说明这5种受体参与SS表达的调控。 7.应用实时荧光定量PCR检测细胞凋亡相关基因(Bax, Bcl-2和p53)的相对表达量,并与对照组相比,发现转染组能显著增加Bax和p53基因mRNA的表达量,降低Bcl-2基因mRNA的表达量(P0.05),说明SS和MSTN可能通过调控细胞凋亡相关基因的表达而起作用。 上述结果表明,SS和MSTN能够抑制Hela细胞的增殖,其中SS组中pEGS2SS-V和pGS/2SS-IL6-asd(?)的抑制率较高,MSTN组(pEGMS)细胞抑制效果最好,而pEGS/2SS+pEGMS和pEGS2SS-V+pEGMS组的抑制率并没有高于SS和MSTN组。SS和MSTN基因质粒抑制Hela细胞的增殖主要通过诱导细胞周期停滞和细胞凋亡实现的。其中,主要将细胞阻滞在S期,并且还能显著上调Bax和p53基因mRNA的表达量,下调Bcl-2基因mRNA的表达量来诱导细胞凋亡。从转录和表达角度发现pEGS2SS-V和pGS/2SS-IL6-asd在真核细胞中的表达较好,间接表明pEGS2SS-V和pGS/2SS-IL6-asd基因疫苗效果较好,为开发SS基因疫苗奠定基础。
[Abstract]:The effects of several SS and MSTN gene plasmid transfection on the growth and proliferation of cervical cancer Hela cells were studied by cell culture, plasmid extraction, liposome transfection, reverse transcription PCR, confocal microscopy, flow cytometry, MTT and real-time fluorescence quantitative PCR. (1) screening the best plasmid to promote the apoptosis of Hela cells, laying the foundation for developing new technology for tumor gene therapy, (2) exploring the role of the SS and the MSTN anti-tumor, and laying a foundation for further improving the gene therapy effect of the tumor; and (3) comparing the expression of various plasmids in the eukaryotic cells from the angle of transcription and expression, and laying the foundation for exploring the mechanism of the action of the SS gene vaccine, developing the SS and the MSTN gene vaccine. The main contents and results are as follows: 1. The plasmid pEGS/ 2SS, pEGS2SS-V, pGS/ 2SS-M4GFP-asd, pGS/ 2SS-IL6-asd, and MSTN gene plasmid pEGMS and SS and MSTN hybrid gene plasmid pEGS/ 2SS + pEGMS and pEGS2SS-V + pEGMS were transfected into Hela cells by lipofectamine TM2000 liposome transfection method, and then the total RNA was extracted after transfection, and the SS and MST were detected by reverse transcription after reverse transcription. N, indicating that the SS and MSTN gene plasmids can be normally transferred in Hela cells recording; 2. the above-mentioned gene plasmid was transfected with Hela cells for 48h, and stained with DAPI for protection from light, and the confocal microscope subcell positioning was applied, and the SS fusion protein was found to be in the nucleus. The MSTN fusion protein is expressed in both the nucleus and the cytoplasm. Expression; 3. The above-mentioned gene plasmid was transfected with Hela cells for 24h, 48h, 72h and 96h, and the cell proliferation was detected by MTT method, and the cell growth was significantly inhibited in the test group compared with the control group (P 0. 05), especially cell growth inhibition after transfection with pEGMS plasmid The results showed that the apoptosis of the cells (i.e., the early apoptotic cells) in the test group was significantly increased (P <0.05) after the transfection of Hela cells with the Annexin V-APC/ 7-AAD double staining method, and the apoptotic rate of the pEGMS test group (27. 474-2) was found.. 86%) The highest, specific to the control The results showed that the plasmid of SS and MSTN could promote H. The cell cycle was detected by flow cytometry. The cell cycle distribution of the transfected Hela cells was detected by flow cytometry, and the cell cycle distribution in the transfected group was found to be obvious: the transfection plasmid group G0-G1 was transfected with the control group. The proportion of the phase cells decreased significantly, and the proportion of the cells in the S phase and the G2-M phase was significant. The expression of SS and MSTN gene was mainly described as He (P. 05). The total RNA and RT-PCR were extracted from Hela cells transfected with SS and MSTN gene, and the five receptors of SSTR-1, SSTR-2, SSTR-3, SSTR-4 and SSTR-5 could be expressed in Hela cells. The expression of Bax, Bcl-2 and p53 was detected by real-time fluorescence quantitative PCR, and the expression of Bax and p53 gene mRNA was significantly increased in the transfected group compared with the control group, and the expression of Bcl-2 gene mR was decreased. The expression of NA (P0.05), indicating that SS and MSTN may be regulated by regulatory cells The results showed that SS and MSTN could inhibit the proliferation of Hela cells, of which pEGS2SS-V and p in the SS group G The inhibition rate of S/ 2SS-IL6-asd (?) is higher, and the inhibition effect of pEGS/ 2SS + pEGMS and pEGS2SS-V + pEGMS is the best. The rate of production was not higher than that of SS and MSTN groups. SS and MSTN gene plasmids inhibited the proliferation of Hela cells. the expression of the Bax and p53 gene mRNA can be significantly increased, the Bcl-2 gene is down-regulated, The expression of pEGS2SS-V and pGS/ 2SS-IL6-asd in eukaryotic cells was found to be better than that of pEGS2SS-V and pGS/ 2SS-IL6-asd.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
本文编号：2396347
[Abstract]:The effects of several SS and MSTN gene plasmid transfection on the growth and proliferation of cervical cancer Hela cells were studied by cell culture, plasmid extraction, liposome transfection, reverse transcription PCR, confocal microscopy, flow cytometry, MTT and real-time fluorescence quantitative PCR. (1) screening the best plasmid to promote the apoptosis of Hela cells, laying the foundation for developing new technology for tumor gene therapy, (2) exploring the role of the SS and the MSTN anti-tumor, and laying a foundation for further improving the gene therapy effect of the tumor; and (3) comparing the expression of various plasmids in the eukaryotic cells from the angle of transcription and expression, and laying the foundation for exploring the mechanism of the action of the SS gene vaccine, developing the SS and the MSTN gene vaccine. The main contents and results are as follows: 1. The plasmid pEGS/ 2SS, pEGS2SS-V, pGS/ 2SS-M4GFP-asd, pGS/ 2SS-IL6-asd, and MSTN gene plasmid pEGMS and SS and MSTN hybrid gene plasmid pEGS/ 2SS + pEGMS and pEGS2SS-V + pEGMS were transfected into Hela cells by lipofectamine TM2000 liposome transfection method, and then the total RNA was extracted after transfection, and the SS and MST were detected by reverse transcription after reverse transcription. N, indicating that the SS and MSTN gene plasmids can be normally transferred in Hela cells recording; 2. the above-mentioned gene plasmid was transfected with Hela cells for 48h, and stained with DAPI for protection from light, and the confocal microscope subcell positioning was applied, and the SS fusion protein was found to be in the nucleus. The MSTN fusion protein is expressed in both the nucleus and the cytoplasm. Expression; 3. The above-mentioned gene plasmid was transfected with Hela cells for 24h, 48h, 72h and 96h, and the cell proliferation was detected by MTT method, and the cell growth was significantly inhibited in the test group compared with the control group (P 0. 05), especially cell growth inhibition after transfection with pEGMS plasmid The results showed that the apoptosis of the cells (i.e., the early apoptotic cells) in the test group was significantly increased (P <0.05) after the transfection of Hela cells with the Annexin V-APC/ 7-AAD double staining method, and the apoptotic rate of the pEGMS test group (27. 474-2) was found.. 86%) The highest, specific to the control The results showed that the plasmid of SS and MSTN could promote H. The cell cycle was detected by flow cytometry. The cell cycle distribution of the transfected Hela cells was detected by flow cytometry, and the cell cycle distribution in the transfected group was found to be obvious: the transfection plasmid group G0-G1 was transfected with the control group. The proportion of the phase cells decreased significantly, and the proportion of the cells in the S phase and the G2-M phase was significant. The expression of SS and MSTN gene was mainly described as He (P. 05). The total RNA and RT-PCR were extracted from Hela cells transfected with SS and MSTN gene, and the five receptors of SSTR-1, SSTR-2, SSTR-3, SSTR-4 and SSTR-5 could be expressed in Hela cells. The expression of Bax, Bcl-2 and p53 was detected by real-time fluorescence quantitative PCR, and the expression of Bax and p53 gene mRNA was significantly increased in the transfected group compared with the control group, and the expression of Bcl-2 gene mR was decreased. The expression of NA (P0.05), indicating that SS and MSTN may be regulated by regulatory cells The results showed that SS and MSTN could inhibit the proliferation of Hela cells, of which pEGS2SS-V and p in the SS group G The inhibition rate of S/ 2SS-IL6-asd (?) is higher, and the inhibition effect of pEGS/ 2SS + pEGMS and pEGS2SS-V + pEGMS is the best. The rate of production was not higher than that of SS and MSTN groups. SS and MSTN gene plasmids inhibited the proliferation of Hela cells. the expression of the Bax and p53 gene mRNA can be significantly increased, the Bcl-2 gene is down-regulated, The expression of pEGS2SS-V and pGS/ 2SS-IL6-asd in eukaryotic cells was found to be better than that of pEGS2SS-V and pGS/ 2SS-IL6-asd.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363
【参考文献】
相关期刊论文 前10条
1 刘宇斌;邝素娟;黄小穗;简志祥;刘子贤;林叶;;生长抑素类似物抑制人肝癌细胞生长和诱导其凋亡[J];南方医科大学学报;2008年12期
2 谢礼波;卢一平;;肾癌基因治疗研究进展[J];华西医学;2011年03期
3 曹少先;何晓红;孙延鸣;刘铁铮;杨利国;;生长抑素基因疫苗pES/2SS的构建与表达[J];扬州大学学报(农业与生命科学版);2007年02期
4 曹少先,张文伟,茆达干,管峰,杨利国;生长抑素基因疫苗质粒pcS/2SS的构建、表达及免疫[J];农业生物技术学报;2005年04期
5 于洪波;黄宗海;孔恒;李在宝;俞金龙;厉周;李强;;奥曲肽联合5-FU对人胃癌细胞株SGC-7901生长的影响[J];山东医药;2008年43期
6 孙卫民,张大鹏;淋巴细胞活化的抑制性受体及其作用[J];生命的化学;1999年01期
7 刘稀逢;胡维新;;肿瘤HSV-tk/GCV自杀基因治疗系统的增效策略[J];现代肿瘤医学;2010年02期
8 吴丹;胡兰;;MSTN RNAi双元表达载体对成肌细胞增殖及分化的影响[J];沈阳农业大学学报;2009年01期
9 刘晓华;税青林;;乳腺癌的基因治疗研究[J];现代诊断与治疗;2011年01期
10 舒邓群;茆达干;吴志敏;程宝;杨利国;;以减毒沙门氏菌为载体的GM-CSF与生长抑素融合表达质粒对小鼠淋巴细胞增殖和GH及IGF-Ⅰ分泌的影响[J];畜牧兽医学报;2006年08期
相关硕士学位论文 前4条
1 赵兴华;IL-6与GM-CSF基因融合表达生长抑素基因疫苗对小鼠生长的影响[D];华中农业大学;2010年
2 边睿;Myostatin诱导肿瘤细胞凋亡的分子机制[D];中国协和医科大学;2006年
3 金定恩;小梅山猪MSTN三种重组蛋白的制备及其免疫对小鼠生长的影响[D];华中农业大学;2008年
4 冯细钢;生长抑素DNA疫苗免疫小鼠的基因表达与免疫应答规律研究[D];华中农业大学;2009年
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