本文選題：臍帶間充質(zhì)干細(xì)胞 + 膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子�。� 參考：《鄭州大學(xué)》2012年碩士論文

【摘要】：目的 構(gòu)建GDNF基因修飾的臍帶間充質(zhì)干細(xì)胞(UCMSCs),為組織工程化神經(jīng)的構(gòu)建打下基礎(chǔ)。 方法 1.用CaCl2法制備感受態(tài)大腸桿菌,重組逆轉(zhuǎn)錄病毒載體質(zhì)粒(第三軍醫(yī)大學(xué)阮懷珍教授惠贈(zèng))轉(zhuǎn)化入感受態(tài)細(xì)菌并進(jìn)行抗性篩選,抽提純化質(zhì)粒。酶切、測(cè)序檢測(cè)目的基因的正確性。 2.復(fù)蘇包裝細(xì)胞PA317,用貼壁培養(yǎng)法培養(yǎng)、傳代擴(kuò)增,待細(xì)胞生長(zhǎng)至80%融合時(shí),脂質(zhì)體轉(zhuǎn)染法將重組逆轉(zhuǎn)錄病毒PLXSN-GDNF轉(zhuǎn)染進(jìn)入PA317包裝細(xì)胞,G418篩選產(chǎn)毒克隆細(xì)胞,收集病毒上清液并測(cè)定病毒滴度。 3.取健康剖腹產(chǎn)產(chǎn)婦捐獻(xiàn)的臍帶,用組織塊法培養(yǎng)獲得原代細(xì)胞、胰酶消化傳代,取第2代臍帶間充質(zhì)干細(xì)胞(UCMSCs),免疫組織化學(xué)法檢測(cè)細(xì)胞表面抗原CD44、CD105、CD106、CD34、CD45的表達(dá)情況。 4.病毒上清感染臍帶間充質(zhì)干細(xì)胞后篩選轉(zhuǎn)基因細(xì)胞,即GDNF-UCMSCs,收集細(xì)胞并用反轉(zhuǎn)錄聚合酶鏈反應(yīng)法(RT-PCR)檢測(cè)GDNF在mRNA水平的表達(dá)。 結(jié)果 1.Xhol Ⅰ單酶切重組質(zhì)粒,出現(xiàn)0.66kb和5.93kb兩個(gè)片段,與預(yù)期結(jié)果相符。測(cè)序檢測(cè)結(jié)果顯示重組逆轉(zhuǎn)錄病毒PLXSN-GDNF中大鼠GDNF的編碼序列完整。 2.用小鼠成纖維細(xì)胞NIH3T3檢測(cè)逆轉(zhuǎn)錄病毒滴度,挑選四個(gè)克隆檢測(cè),最高病毒滴度為6.56×104cfu/ml,選用該病毒上清液感染細(xì)胞。 3.原代培養(yǎng)1周時(shí)組織塊之間出現(xiàn)貼壁的成纖維樣細(xì)胞,14天左右集落細(xì)胞達(dá)到80%-90%融合,成螺旋狀或放射狀。免疫組化檢測(cè)細(xì)胞表面抗原示：CD44、CD105強(qiáng)陽(yáng)性,CD106弱陽(yáng)性,CD34、CD45陰性,與臍帶間充質(zhì)干細(xì)胞(UCMSCs)的生物學(xué)特性相符。 4. RT-PCR法檢測(cè)顯示,GDNF-UCMSCs組呈陽(yáng)性,而UCMSCs組呈陰性,轉(zhuǎn)基因組GDNF mRNA水平表達(dá)明顯強(qiáng)于對(duì)照組,P0.01,差異有統(tǒng)計(jì)學(xué)意義,可知GDNF基因在mRNA水平得以表達(dá)。 結(jié)論 1.獲贈(zèng)的逆轉(zhuǎn)錄病毒載體PLXSN-GDNF中大鼠GDNF基因的編碼序列完整。 2.成功培養(yǎng)出人臍帶間充質(zhì)干細(xì)胞,免疫組織化學(xué)染色鑒定結(jié)果與臍帶間充質(zhì)干細(xì)胞的生物學(xué)特征相符合。 3.成功構(gòu)建了GDNF基因修飾的臍帶間充質(zhì)干細(xì)胞,并在mRNA水平得以表達(dá),為組織化工程神經(jīng)的構(gòu)建打下了基礎(chǔ)。
[Abstract]:Purpose The construction of GDNF gene modified umbilical cord mesenchymal stem cells (UCM SCS) lays the foundation for the construction of tissue engineered nerve. Method 1. The competent E. coli was prepared by CaCl2 method. The recombinant retroviral vector plasmid (Professor Huai Chin Nguyen Huai-chen, third military Medical University) was transformed into the receptive bacteria and screened for resistance, and the plasmid was extracted and purified. The correctness of the target gene was detected by enzyme digestion and sequencing. 2. The resuscitation packaging cells PA317 were cultured with adherent culture method and amplified by passage. When the cells grew to 80% fusion, the recombinant retrovirus PLXSN-GDNF was transfected into the PA317 packaging cells by liposome transfection, and the toxic clones were screened by G418. Collect the supernatant of the virus and determine the titer of the virus. 3. The umbilical cord donated by healthy caesarean women was cultured with tissue block method. The primary cells were cultured and subcultured by trypsin digestion. The UCM SCS were collected from the second generation of umbilical cord mesenchymal stem cells. The expression of CD44 + CD105 + CD106 + CD34 + CD34 + CD45 was detected by immunohistochemical method. 4. After the virus supernatant was infected with umbilical cord mesenchymal stem cells, GDNF-UCMSCswere selected, and the expression of GDNF at mRNA level was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Result The recombinant plasmid of 1.Xhol 鈪，

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