本文選題：穩(wěn)定轉(zhuǎn)染　切入點(diǎn)：脂質(zhì)體　出處：《南華大學(xué)》2006年碩士論文

【摘要】：[目的]將人canstatin cDNA分泌型真核表達(dá)載體pSecTag2B／canstatin穩(wěn)定轉(zhuǎn)染中國(guó)倉鼠卵巢(CHO-K1)細(xì)胞并獲得穩(wěn)定表達(dá)canstatin蛋白產(chǎn)物的細(xì)胞株。再用含canstatin的上清液作用人臍靜脈內(nèi)皮細(xì)胞(HUVEC-12)以進(jìn)一步研究canstatin的生物學(xué)作用。 [方法]將人canstatin cDNA的重組質(zhì)粒pSecTag2B／canstatin穩(wěn)定轉(zhuǎn)染中國(guó)倉鼠卵巢(CHO-K1)細(xì)胞并獲得穩(wěn)定表達(dá)canstatin蛋白產(chǎn)物的細(xì)胞株，應(yīng)用RT-PCR檢測(cè)細(xì)胞中canstatin基因的表達(dá)。收集細(xì)胞培養(yǎng)上清液中的蛋白，采用western-blotting法鑒定表達(dá)產(chǎn)物，用雞胚絨毛尿囊膜(CAM)實(shí)驗(yàn)初步鑒定其生物學(xué)活性。通過繪制細(xì)胞生長(zhǎng)曲線和流式細(xì)胞學(xué)技術(shù)進(jìn)一步研究canstatin對(duì)血管內(nèi)皮細(xì)胞的生物學(xué)作用。 [結(jié)果]成功的將人canstatin cDNA的重組質(zhì)粒pSecTag2B／canstatin穩(wěn)定轉(zhuǎn)染CHO-K1細(xì)胞，RT-PCR法證實(shí)質(zhì)粒pSecTag2B／canstatin已成功轉(zhuǎn)染進(jìn)入CHO-K1細(xì)胞。離心收集細(xì)胞培養(yǎng)上清液，western-blotting法鑒定上清液中有目的蛋白的表達(dá)。該細(xì)胞培養(yǎng)上清液在體內(nèi)能抑制雞胚絨毛尿囊膜中微血管的生成，可見CHO-K1，，pSecTag2B／canstatin組給藥區(qū)及其周圍血管明顯減少甚至未見血管紋理，小血管分支少，而其余四組雞胚尿囊膜血管生成沒有明顯影響。通過雞胚處理前后血管生成記數(shù)，CHO-K1，pSecTag2B／canstatin組與其余四組之間經(jīng)統(tǒng)計(jì)學(xué)處理差異有顯著性(P＜0.05)。通過繪制細(xì)胞生長(zhǎng)曲線顯示
[Abstract]:[objective] to stably transfect human canstatin cDNA secretory eukaryotic expression vector pSecTag2B/canstatin into Chinese hamster ovary cell line CHO-K1 and to obtain a cell line with stable expression of canstatin protein product.Human umbilical vein endothelial cells (HUVEC-12) were treated with supernatant containing canstatin to further study the biological effects of canstatin.[methods] the recombinant plasmid pSecTag2B/canstatin of human canstatin cDNA was stably transfected into Chinese hamster ovary (CHO-K1) cells and the cell lines expressing canstatin protein products stably were obtained. RT-PCR was used to detect the expression of canstatin gene in the cells.The protein in the supernatant of cell culture was collected and the expression product was identified by western-blotting method. The biological activity of the protein was preliminarily identified by chorioallantoic membrane assay.The biological effects of canstatin on vascular endothelial cells were further studied by cell growth curve and flow cytometry.[results] Recombinant plasmid pSecTag2B/canstatin of human canstatin cDNA was successfully transfected into CHO-K1 cells by RT-PCR and confirmed that the plasmid pSecTag2B/canstatin had been successfully transfected into CHO-K1 cells.The expression of the target protein in the supernatant was identified by Western-blotting method.The supernatant of cell culture could inhibit the formation of microvessels in chorioallantoic membrane of chicken embryo in vivo.The other four groups had no significant effect on angiogenesis of allantoic membrane.There was a significant difference between CHO-K1PSecTag2B / canstatin group and the other four groups in the number of angiogenesis before and after embryo treatment (P < 0.05).By drawing the cell growth curve.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 李光雷;siRNA靶向干擾APE1基因?qū)δX膠質(zhì)瘤細(xì)胞增殖與凋亡的影響[D];遼寧醫(yī)學(xué)院;2012年

本文編號(hào)：1713991