本文選題：Tet-on + advanced　； 參考：《暨南大學(xué)》2011年碩士論文

【摘要】：目的利用可調(diào)控的Tet-on advanced系統(tǒng)和SV40T基因構(gòu)建永生化小鼠胰島p細(xì)胞系,觀察回復(fù)后胰島p細(xì)胞生物學(xué)特性,評(píng)估可逆性永生化小鼠胰島β細(xì)胞系回復(fù)后的安全性和功能性,為解決胰島移植的細(xì)胞來源提供實(shí)驗(yàn)依據(jù)。 方法采用Gateway基因重組技術(shù),構(gòu)建Tet-on advanced系統(tǒng)調(diào)控表達(dá)的慢病毒載體pLV.EX2d.P/puro-EF1 ArtTA(M2)和pLV.EX3d.P/neo-TRESV40TIRES/eGFP。脂質(zhì)體lipotamin-2000轉(zhuǎn)染法,將重組的兩種慢病毒載體分別轉(zhuǎn)染入包裝細(xì)胞293FT,獲得高滴度的病毒顆粒；再將病毒顆粒共同轉(zhuǎn)染純化培養(yǎng)的原代小鼠胰島細(xì)胞,轉(zhuǎn)染24小時(shí)后加入強(qiáng)力霉素誘導(dǎo)SV40T表達(dá),48小時(shí)后加入嘌呤霉素和G418進(jìn)行抗生素篩選獲得體外擴(kuò)增能力的永生化小鼠胰島細(xì)胞,對(duì)多個(gè)永生化細(xì)胞克隆進(jìn)行ELISA檢測(cè)基礎(chǔ)胰島素分泌量,RT-PCR檢測(cè)胰島p細(xì)胞相關(guān)因子,篩選出有生理功能的可逆性永生化小鼠胰島p細(xì)胞系。選取Tet-on調(diào)控下的RIB細(xì)胞株第10代細(xì)胞,去掉DOX誘導(dǎo)使永生化基因SV40T沉默而獲得回復(fù)后的RIB細(xì)胞。通過RT-PCR及細(xì)胞免疫熒光技術(shù)檢測(cè)回復(fù)前后SV40T及胰島p細(xì)胞相關(guān)基因在mRNA和蛋白水平的表達(dá)；染色體核型分析、軟瓊脂克隆形成實(shí)驗(yàn)、細(xì)胞增殖試驗(yàn)和葡萄糖刺激胰島素釋放實(shí)驗(yàn)監(jiān)測(cè)可逆性永生化胰島β細(xì)胞在DOX調(diào)控前后的安全性和功能性變化。 結(jié)果重組慢病毒載體pLV.EX2d.P/puro-EF 1 ArtTA(M2)和pLV.EX3d.P/neo-TRESV40T IRES/eGFP經(jīng)PCR電泳檢測(cè)顯示構(gòu)建成功,并驗(yàn)證了重組載體的正確性；分別轉(zhuǎn)染包裝細(xì)胞293FT,24小時(shí)后可見到轉(zhuǎn)染pLV.EX3d.P/neo-TRESV40TIRES/eGFP載體的293FT有綠色熒光表達(dá),轉(zhuǎn)染效率高達(dá)90%以上,48后小時(shí)收集兩種病毒上清進(jìn)行濃縮,經(jīng)龍膽紫染色法檢測(cè)病毒滴度分別能達(dá)到107和108；將獲得的病毒顆粒同時(shí)轉(zhuǎn)染小鼠原代胰島細(xì)胞,轉(zhuǎn)染后24小時(shí)加入DOX誘導(dǎo),可見到處于終末分化狀態(tài)的胰島細(xì)胞有增殖現(xiàn)象,轉(zhuǎn)染后48小時(shí)進(jìn)行G418和嘌呤霉素的篩選,穩(wěn)定轉(zhuǎn)染的細(xì)胞繼續(xù)增殖,余下細(xì)胞死亡；細(xì)胞克隆中經(jīng)胰島素ELISA檢測(cè)陽性的有20株,再進(jìn)行小鼠胰島β細(xì)胞的相關(guān)基因RT-PCR檢測(cè),篩選到一株細(xì)胞克隆能表達(dá)Pdx、Nkx6.1、Pax6、Insulin胰島β細(xì)胞相關(guān)基因,成功建立了可逆性永生化小鼠胰島p細(xì)胞系(RIB系)。RIB系在有DOX誘導(dǎo)下增殖能力旺盛,去除DOX進(jìn)行回復(fù)后的RIB細(xì)胞增殖速度減慢,5天后完全停止生長(zhǎng)；RT-PCR及細(xì)胞免疫熒光檢測(cè)RIB系在DOX誘導(dǎo)下SV40T基因和蛋白水平均有表達(dá),去除DOX即在短期內(nèi)停止增殖不表達(dá)SV40T,胰島β細(xì)胞相關(guān)標(biāo)志基因和蛋白在回復(fù)前后均有表達(dá)；回復(fù)前后的RIB細(xì)胞核型分析均為正常二倍體,平板克隆實(shí)驗(yàn)未見其致瘤性；RIB系葡萄糖刺激胰島素釋放實(shí)驗(yàn)胰島素分泌能力較弱,回復(fù)后胰島素分泌能力增強(qiáng),但仍與原代胰島細(xì)胞有差距,約為正常胰島分泌量的1/3-1/2。 結(jié)論利用Tet-on advanced系統(tǒng)調(diào)控的重組慢病毒載體pLV.EX2d.P/puro-EF1 ArtTA(M2)和PLV.EX3d.P/neo-TRESV40TIRES/eGFP成功建立的RIB系,其生存期延長(zhǎng),在體外增殖能力較強(qiáng),且核型檢測(cè)正常,胰島β細(xì)胞相關(guān)基因和蛋白仍繼續(xù)表達(dá),回復(fù)后RIB細(xì)胞系停止增殖,不表達(dá)永生化基因SV40T,而胰島素分泌能力增強(qiáng),但分泌量仍與正常胰島有差距。
[Abstract]:Objective To investigate the biological characteristics of islet 尾 - cell line after recovery and evaluate the safety and functionality of 尾 - cell line after recovery of pancreatic islet 尾 - cell line , and to provide experimental basis for solving the source of pancreatic islet transplantation .

Methods The recombinant plasmids pLV . EX22d . P / puro - EF1 and pLV . EX3d . P / neo - TRESV40TIRES / eGFP were constructed by Gateway gene recombination . The recombinant plasmids were transfected into the packed cells 293FT by lipotamin - 2000 transfection .
After 24 hours of transfection , the cultured primary mouse pancreatic islet cells were co - transfected and purified . After 24 hours of transfection , it was induced to express SV40T . After 48 hours , Puromycin and G418 were added to screen the islet cells .
Chromosome karyotype analysis , soft agar colony formation assay , cell proliferation assay , and glucose - stimulated insulin release assay were used to monitor the safety and functional changes of 尾 - cells in 尾 - cells before and after control .

Results The recombinant lentivirus vectors pLV . EX22d . P / puro - EF 1 and pLV . EX3d . P / neo - TRESV40T were constructed successfully by PCR electrophoresis , and the correctness of the recombinant vector was verified .
The transfected pLV . EX3d . P / neo - TRESV40TIRES / eGFP vector was transfected into 293 FT . The transfection efficiency was over 90 % . After 48 hours , two kinds of supernatant were collected and concentrated .
The virus particles were transfected into the mouse primary islet cells at the same time . After 24 hours after transfection , the cells were induced to proliferate . After transfection , the cells were screened by G418 and puromycin 48 hours after transfection , and the cells stably transfected continue to proliferate and the remaining cells die ;
In the cell clones , 20 strains were detected by ELISA and RT - PCR was performed on the 尾 - cells of mouse pancreatic islets .
RT - PCR and immunofluorescence were used to detect the expression of SV40T gene and protein in vitro .
The results showed that there was no tumor genicity in the normal diploid and plate clone experiments .
The results showed that the insulin secretion was weak and the insulin secretion increased after recovery , but it was still different from that of the primary islet cells , which was about 1 / 3 - 1 / 2 of the normal islet secretion .

Conclusion The recombinant lentivirus vector pLV . EX22d . P / puro - EF1 , TA ( M2 ) and PLV.EX3d . P / neo - TRESV40TIRES / eGFP have been successfully established . The survival time is prolonged , the ability to proliferate in vitro is strong , and the related genes and proteins of 尾 - cells in the pancreatic islets are still expressed .
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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