【摘要】：目的構(gòu)建β-神經(jīng)生長因子(β-NGF)慢病毒表達(dá)載體p LVX-TRE3G-IRES-β-NGF,應(yīng)用Tet-on 3G四環(huán)素誘導(dǎo)表達(dá)系統(tǒng),探討β-NGF在人胚腎(HEK293FT)細(xì)胞中的過表達(dá)情況,及其對大鼠腎上腺嗜鉻細(xì)胞瘤(PC12)細(xì)胞分化的影響。方法從SD大鼠腦組織提取總RNA,反轉(zhuǎn)錄成c DNA,利用分子生物學(xué)技術(shù),克隆鼠β-NGF基因完整編碼區(qū),將β-NGF基因定向插入載體p LVX-TRE3G-IRES中。將重組載體p LVX-TRE3G-IRES-β-NGF和載體p LVX-Tet3G分別轉(zhuǎn)染空白HEK293FT細(xì)胞,制備收獲慢病毒,共轉(zhuǎn)染HEK293FT細(xì)胞,同時用不同劑量強(qiáng)力霉素(Dox)誘導(dǎo)NGF表達(dá)(分為未轉(zhuǎn)染組、0、100、500和1000μg/L Dox誘導(dǎo)表達(dá)組)。48h后收集細(xì)胞,免疫印跡法(Western blotting)檢測各組細(xì)胞內(nèi)β-NGF表達(dá)情況。同時收集培養(yǎng)上清,1.ELISA檢測各組上清內(nèi)β-NGF的分泌量;2.制作條件培養(yǎng)基,加入PC12細(xì)胞培養(yǎng)基中進(jìn)行誘導(dǎo)培養(yǎng),觀察PC12細(xì)胞誘導(dǎo)分化情況。結(jié)果雙酶切和測序鑒定重組質(zhì)粒p LVX-TRE3G-IRES-β-NGF序列和方向正確;β-NGF在轉(zhuǎn)染細(xì)胞內(nèi)被誘導(dǎo)表達(dá),隨著Dox劑量增加,表達(dá)量增強(qiáng);β-NGF在培養(yǎng)基的上清內(nèi)表達(dá),表達(dá)量隨Dox劑量增加而增多。誘導(dǎo)表達(dá)的條件培養(yǎng)基可誘導(dǎo)PC12細(xì)胞形態(tài)改變,表達(dá)神經(jīng)元特異性烯醇化酶(NSE)蛋白。結(jié)論成功重組慢病毒載體p LVX-TRE3G-IRES-β-NGF,轉(zhuǎn)染后β-NGF基因能夠在HEK293FT細(xì)胞中表達(dá)和分泌,且該蛋白具有誘導(dǎo)PC12細(xì)胞向神經(jīng)元樣細(xì)胞分化的能力;運用Tet-On 3G四環(huán)素誘導(dǎo)表達(dá)系統(tǒng),可成功誘導(dǎo)表達(dá)獲得不同劑量β-NGF蛋白。
[Abstract]:Objective to construct 尾-nerve growth factor (尾-NGF) lentivirus expression vector p LVX-TRE3G-IRES- 尾-NGF, and to investigate the overexpression of 尾-NGF in human embryonic kidney (HEK293FT) cells by using Tet-on 3G tetracycline induced expression system. And its effect on the differentiation of rat adrenal pheochromocytoma (PC12) cells. Methods Total RNA, was extracted from the brain tissue of SD rats and transcribed into c DNA,. The complete coding region of mouse 尾-NGF gene was cloned by molecular biology technique, and the 尾-NGF gene was inserted into vector p LVX-TRE3G-IRES. The recombinant vector p LVX-TRE3G-IRES- 尾-NGF and vector p LVX-Tet3G were transfected into blank HEK293FT cells respectively. Lentivirus was prepared and co-transfected into HEK293FT cells. NGF expression was induced by different doses of doxycycline (Dox) (divided into untransfected group). 0100500 渭 g / L and 1000 渭 g / L Dox induced expression groups). 48 hours later, the cells were collected and the expression of 尾-NGF in each group was detected by immunoblotting (Western blotting). At the same time, the culture supernatant was collected and the secretion of 尾-NGF in each group was detected by 1.ELISA. The conditioned medium was prepared and cultured in PC12 cell medium to observe the differentiation of PC12 cells. Results the sequence and direction of the recombinant plasmid p LVX-TRE3G-IRES- 尾-NGF were identified by double enzyme digestion and sequencing, and the expression of 尾-NGF was induced in the transfected cells, and the expression increased with the increase of Dox dose. 尾-NGF was expressed in the culture medium, and the expression increased with the increase of Dox dose. The conditioned medium could induce the morphological changes of PC12 cells and express neuron-specific enolase (NSE) protein. Conclusion the recombinant lentivirus vector p LVX-TRE3G-IRES- 尾-NGF, can express and secrete 尾-NGF gene in HEK293FT cells, and the protein can induce PC12 cells to differentiate into neuron-like cells. Different doses of 尾-NGF protein could be successfully induced by Tet-On 3G tetracycline induced expression system.
【作者單位】： 貴州醫(yī)科大學(xué)組織學(xué)胚胎學(xué)教研室;貴州醫(yī)科大學(xué)貴州省細(xì)胞工程生物醫(yī)藥技術(shù)國家地方聯(lián)合工程實驗室貴州省再生醫(yī)學(xué)重點實驗室;
【基金】：貴州省優(yōu)秀科技教育人才省長專項資金[黔省專合字(2012)41號] 貴州省科技廳-貴陽醫(yī)學(xué)院聯(lián)合基金[黔科合LG字(2012)026號]
【分類號】：R772.2

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