本文選題：CD59　切入點(diǎn)：pSUPER載體　出處：《青島大學(xué)》2008年碩士論文

【摘要】： 目的:構(gòu)建針對(duì)CD59基因的pSUPER retro RNAi逆轉(zhuǎn)錄病毒載體,為下一步研究腫瘤細(xì)胞內(nèi)CD59 RNAi奠定基礎(chǔ)。利用本課題組設(shè)計(jì)并合成的CD59短肽封條,檢測(cè)其對(duì)HeLa細(xì)胞凋亡的作用,為腫瘤的治療提供一條新的思路。 方法:根據(jù)GenBank提供的CD59基因mRNA的序列及siRNA的設(shè)計(jì)原則設(shè)計(jì)RNA干擾序列及一條對(duì)照序列,以CD59基因mRNA序列的220-238nt的19個(gè)bp片段作為靶序列,設(shè)計(jì)并合成兩端含有酶切位點(diǎn)的60個(gè)堿基的寡核苷酸鏈。寡核苷酸鏈退火后用T_4連接酶連接至線性化的質(zhì)粒pSUPER.retro.neo+GFP中,轉(zhuǎn)化大腸桿菌JM109,提取質(zhì)粒。然后通過(guò)酶切,PCR,測(cè)序鑒定重組質(zhì)粒。50mg/L CD59短肽封條作用于HeLa細(xì)胞24h后,用透射電鏡檢測(cè)HeLa細(xì)胞的凋亡情況;用MTT的方法來(lái)檢測(cè)HeLa細(xì)胞增殖抑制率;利用免疫組織化學(xué)方法檢測(cè)survivin,caspase-3,bax的表達(dá)水平。 結(jié)果:經(jīng)酶切鑒定,PCR鑒定及測(cè)序結(jié)果表明成功地構(gòu)建了CD59基因的pSUPER retroRNAi逆轉(zhuǎn)錄病毒載體。透射電鏡結(jié)果表明,CD59短肽封條能使HeLa細(xì)胞的胞核發(fā)生碎裂。MTT實(shí)驗(yàn)表明轉(zhuǎn)CD59基因的HeLa細(xì)胞+短肽的細(xì)胞組增殖抑制率大于HeLa細(xì)胞+短肽組(P＜0.01)。免疫組化實(shí)驗(yàn)顯示,轉(zhuǎn)CD59基因的HeLa細(xì)胞+短肽組和正常的HeLa細(xì)胞+短肽組相比較,survivin表達(dá)水平降低,差別有統(tǒng)計(jì)學(xué)意義(P＜0.05);而caspase-3表達(dá)水平增高,差別有統(tǒng)計(jì)學(xué)意義(P＜0.05);bax的表達(dá)量各組比較(P＞0.05),差別無(wú)統(tǒng)計(jì)學(xué)意義。 結(jié)論:酶切,PCR,測(cè)序鑒定成功地構(gòu)建了CD59基因的pSUPER retro RNAi逆轉(zhuǎn)錄病毒載體。電鏡,MTT實(shí)驗(yàn)及免疫組化實(shí)驗(yàn)顯示,CD59特異位點(diǎn)的短肽封條具有下調(diào)survivin的表達(dá),活化caspase-3,從而促進(jìn)HeLa細(xì)胞的凋亡。我們的研究為CD59對(duì)腫瘤細(xì)胞凋亡信號(hào)轉(zhuǎn)導(dǎo)的進(jìn)一步研究奠定基礎(chǔ),為腫瘤的生物治療提供一條新的思路。
[Abstract]:Objective: to construct pSUPER retro RNAi retroviral vector for CD59 gene, to lay the foundation for further research in tumor cells. CD59 RNAi uses this group of design and synthesis of the CD59 peptide seal, to detect the HeLa cell apoptosis, and provide a new way for cancer therapy.
Methods: according to the design principles for RNA interference sequence and siRNA gene of CD59 mRNA provided by GenBank and a control sequence with 19 bp fragment sequence of CD59 gene of mRNA 220-238nt as the target sequence, the oligonucleotide chain 60 at both ends was designed and synthesized with restriction sites of the plasmid pSUPER.retro.neo+GFP with the base. T_4 ligase to linear oligonucleotides were annealed and transformed into Escherichia coli JM109, plasmid was extracted. Then by enzyme digestion, PCR and sequencing of recombinant plasmid.50mg/L CD59 peptide seal in HeLa cells after 24h, apoptosis of HeLa cells by transmission electron microscopy detection; using MTT method to detect the inhibition rate of HeLa cells proliferation; using immunohistochemical method to detect survivin, Caspase-3, the expression level of Bax.
Results: after enzyme digestion, PCR and sequencing results showed that the successful construction of the pSUPER retroRNAi retroviral vector of CD59 gene. The TEM results showed that the CD59 peptide seal can make the HeLa cell nuclear.MTT fragmentation experiments show that cell proliferation of HeLa cells and short peptide CD59 gene inhibition rate more than HeLa cell + short peptide group (P < 0.01). Immunohistochemical experiments showed that HeLa + short peptide group of transfected CD59 and HeLa cells + normal peptide group, the expression level of survivin decreased, the difference was statistically significant (P < 0.05); and the expression of Caspase-3 was increased, the difference was statistically significant (P < 0.05); expression of Bax in each group (P > 0.05), the difference was not statistically significant.
Conclusion: enzyme digestion, PCR sequencing, pSUPER has been successfully constructed retro RNAi retroviral vector of CD59 gene. Electron microscope, MTT assay and immunohistochemical assay showed that the peptide seals specific sites with CD59 expression, down-regulation of Survivin and activation of Caspase-3, thereby promoting the apoptosis of HeLa cells. Our study laid the foundation for the further study of CD59 on apoptosis of tumor cell signal transduction, provide a new way for tumor biological therapy.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392.12

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 崔林林;高美華;趙鵬;;CHO細(xì)胞表面與人CD59結(jié)合的活性短肽序列的篩選[J];青島大學(xué)醫(yī)學(xué)院學(xué)報(bào);2007年03期

2 王海燕,徐如祥,姜曉丹,羅深秋;dsRNA阻斷大鼠骨髓源性神經(jīng)干細(xì)胞Hes5表達(dá)的實(shí)驗(yàn)研究[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2004年01期

3 張艷麗,高美華;人CD59基因的突變和表達(dá)及其活性研究[J];細(xì)胞與分子免疫學(xué)雜志;2005年02期

4 程穎;高美華;;利用噬菌體肽庫(kù)篩選與人CD59特異性結(jié)合的短肽[J];細(xì)胞與分子免疫學(xué)雜志;2006年02期

，

本文編號(hào)：1671586