幾種SS和MSTN基因質(zhì)粒對Hela細(xì)胞生長和增殖及凋亡的影響
發(fā)布時(shí)間：2018-12-31 08:48
【摘要】：本研究應(yīng)用細(xì)胞培養(yǎng)、質(zhì)粒提取、脂質(zhì)體轉(zhuǎn)染、逆轉(zhuǎn)錄PCR、共聚焦顯微定位、流式細(xì)胞儀分類、MTT、實(shí)時(shí)熒光定量PCR等技術(shù),通過比較幾種SS和MSTN基因質(zhì)粒轉(zhuǎn)染對宮頸癌Hela細(xì)胞生長和增殖的作用,旨在：(1)篩選促進(jìn)Hela細(xì)胞凋亡效果最佳的質(zhì)粒,為開發(fā)腫瘤基因治療新技術(shù)奠定基礎(chǔ);(2)探討SS和MSTN抗腫瘤的作用通路,為進(jìn)一步提高腫瘤基因治療效果奠定基礎(chǔ)；(3)從轉(zhuǎn)錄和表達(dá)角度比較各種質(zhì)粒在真核細(xì)胞中的表達(dá)量,為探討SS基因疫苗作用機(jī)制,開發(fā)SS和MSTN基因疫苗奠定基礎(chǔ)。主要研究內(nèi)容和結(jié)果如下： 1.應(yīng)用LipofectamineTM2000脂質(zhì)體轉(zhuǎn)染方法將SS基因質(zhì)粒pEGS/2SS、pEGS2SS-V、pGS/2SS-M4GFP-asd、pGS/2SS-IL6-asd和MSTN基因質(zhì)粒pEGMS及SS和MSTN混合基因質(zhì)粒pEGS/2SS+pEGMS、pEGS2SS-V+pEGMS轉(zhuǎn)染到Hela細(xì)胞中,轉(zhuǎn)染后48h,提取總RNA,反轉(zhuǎn)錄后電泳檢測到SS和MSTN,說明SS和MSTN基因質(zhì)粒在Hela細(xì)胞中能夠正常轉(zhuǎn)錄； 2.上述基因質(zhì)粒轉(zhuǎn)染Hela細(xì)胞后48h,用DAPI避光染色、應(yīng)用共聚焦顯微鏡亞細(xì)胞定位,發(fā)現(xiàn)SS融合蛋白在細(xì)胞核中表達(dá),MSTN融合蛋白則在細(xì)胞核和細(xì)胞質(zhì)中都有表達(dá)； 3.上述基因質(zhì)粒轉(zhuǎn)染Hela細(xì)胞后24h、48h、72h和96h,用MTT法檢測細(xì)胞增殖的情況,并與對照組比較,發(fā)現(xiàn)試驗(yàn)組明顯抑制細(xì)胞生長(P0.05),尤其是用pEGMS質(zhì)粒轉(zhuǎn)染后72h的細(xì)胞生長抑制作用最明顯。 4.應(yīng)用Annexin V-APC/7-AAD雙染色法檢測SS和MSTN基因質(zhì)粒轉(zhuǎn)染Hela細(xì)胞后48h的凋亡情況,并與對照組比較,發(fā)現(xiàn)試驗(yàn)組的R5區(qū)細(xì)胞(即早期凋亡細(xì)胞)明顯增加(P0.05),其中,pEGMS試驗(yàn)組的凋亡率(27.474±2.86%)最高,比對照組高8.15%。表明SS和MSTN基因質(zhì)�？纱龠M(jìn)Hela細(xì)胞的凋亡 5.上述基因質(zhì)粒轉(zhuǎn)染Hela細(xì)胞后48h,利用流式細(xì)胞儀檢測細(xì)胞周期,發(fā)現(xiàn)質(zhì)粒轉(zhuǎn)染組細(xì)胞周期分布變化明顯：與對照組相比,轉(zhuǎn)染質(zhì)粒組G0-G1期細(xì)胞比例顯著降低,S期和G2-M期細(xì)胞比例顯著升高(P0.05)。說明SS和MSTN基因質(zhì)粒主要將Hela細(xì)胞阻滯在S期。 6.從轉(zhuǎn)染SS和MSTN基因質(zhì)粒后48h的Hela細(xì)胞中提取總RNA,逆轉(zhuǎn)錄PCR擴(kuò)增,檢測到SSTR-1, SSTR-2, SSTR-3, SSTR-4和SSTR-5五種受體均能在Hela細(xì)胞中表達(dá),說明這5種受體參與SS表達(dá)的調(diào)控。 7.應(yīng)用實(shí)時(shí)熒光定量PCR檢測細(xì)胞凋亡相關(guān)基因(Bax, Bcl-2和p53)的相對表達(dá)量,并與對照組相比,發(fā)現(xiàn)轉(zhuǎn)染組能顯著增加Bax和p53基因mRNA的表達(dá)量,降低Bcl-2基因mRNA的表達(dá)量(P0.05),說明SS和MSTN可能通過調(diào)控細(xì)胞凋亡相關(guān)基因的表達(dá)而起作用。 上述結(jié)果表明,SS和MSTN能夠抑制Hela細(xì)胞的增殖,其中SS組中pEGS2SS-V和pGS/2SS-IL6-asd(?)的抑制率較高,MSTN組(pEGMS)細(xì)胞抑制效果最好,而pEGS/2SS+pEGMS和pEGS2SS-V+pEGMS組的抑制率并沒有高于SS和MSTN組。SS和MSTN基因質(zhì)粒抑制Hela細(xì)胞的增殖主要通過誘導(dǎo)細(xì)胞周期停滯和細(xì)胞凋亡實(shí)現(xiàn)的。其中,主要將細(xì)胞阻滯在S期,并且還能顯著上調(diào)Bax和p53基因mRNA的表達(dá)量,下調(diào)Bcl-2基因mRNA的表達(dá)量來誘導(dǎo)細(xì)胞凋亡。從轉(zhuǎn)錄和表達(dá)角度發(fā)現(xiàn)pEGS2SS-V和pGS/2SS-IL6-asd在真核細(xì)胞中的表達(dá)較好,間接表明pEGS2SS-V和pGS/2SS-IL6-asd基因疫苗效果較好,為開發(fā)SS基因疫苗奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363
本文編號：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363
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