本文選題：Tet-on + advanced��； 參考：《暨南大學》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細胞系,觀察回復后胰島p細胞生物學特性,評估可逆性永生化小鼠胰島β細胞系回復后的安全性和功能性,為解決胰島移植的細胞來源提供實驗依據(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)的原代小鼠胰島細胞,轉(zhuǎn)染24小時后加入強力霉素誘導SV40T表達,48小時后加入嘌呤霉素和G418進行抗生素篩選獲得體外擴增能力的永生化小鼠胰島細胞,對多個永生化細胞克隆進行ELISA檢測基礎(chǔ)胰島素分泌量,RT-PCR檢測胰島p細胞相關(guān)因子,篩選出有生理功能的可逆性永生化小鼠胰島p細胞系。選取Tet-on調(diào)控下的RIB細胞株第10代細胞,去掉DOX誘導使永生化基因SV40T沉默而獲得回復后的RIB細胞。通過RT-PCR及細胞免疫熒光技術(shù)檢測回復前后SV40T及胰島p細胞相關(guān)基因在mRNA和蛋白水平的表達；染色體核型分析、軟瓊脂克隆形成實驗、細胞增殖試驗和葡萄糖刺激胰島素釋放實驗監(jiān)測可逆性永生化胰島β細胞在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電泳檢測顯示構(gòu)建成功,并驗證了重組載體的正確性；分別轉(zhuǎn)染包裝細胞293FT,24小時后可見到轉(zhuǎn)染pLV.EX3d.P/neo-TRESV40TIRES/eGFP載體的293FT有綠色熒光表達,轉(zhuǎn)染效率高達90%以上,48后小時收集兩種病毒上清進行濃縮,經(jīng)龍膽紫染色法檢測病毒滴度分別能達到107和108；將獲得的病毒顆粒同時轉(zhuǎn)染小鼠原代胰島細胞,轉(zhuǎn)染后24小時加入DOX誘導,可見到處于終末分化狀態(tài)的胰島細胞有增殖現(xiàn)象,轉(zhuǎn)染后48小時進行G418和嘌呤霉素的篩選,穩(wěn)定轉(zhuǎn)染的細胞繼續(xù)增殖,余下細胞死亡；細胞克隆中經(jīng)胰島素ELISA檢測陽性的有20株,再進行小鼠胰島β細胞的相關(guān)基因RT-PCR檢測,篩選到一株細胞克隆能表達Pdx、Nkx6.1、Pax6、Insulin胰島β細胞相關(guān)基因,成功建立了可逆性永生化小鼠胰島p細胞系(RIB系)。RIB系在有DOX誘導下增殖能力旺盛,去除DOX進行回復后的RIB細胞增殖速度減慢,5天后完全停止生長；RT-PCR及細胞免疫熒光檢測RIB系在DOX誘導下SV40T基因和蛋白水平均有表達,去除DOX即在短期內(nèi)停止增殖不表達SV40T,胰島β細胞相關(guān)標志基因和蛋白在回復前后均有表達；回復前后的RIB細胞核型分析均為正常二倍體,平板克隆實驗未見其致瘤性；RIB系葡萄糖刺激胰島素釋放實驗胰島素分泌能力較弱,回復后胰島素分泌能力增強,但仍與原代胰島細胞有差距,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,在體外增殖能力較強,且核型檢測正常,胰島β細胞相關(guān)基因和蛋白仍繼續(xù)表達,回復后RIB細胞系停止增殖,不表達永生化基因SV40T,而胰島素分泌能力增強,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 .
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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