<rp id="vgn1y"></rp>
<rp id="vgn1y"><object id="vgn1y"></object></rp>

  • <rp id="vgn1y"><ruby id="vgn1y"><input id="vgn1y"></input></ruby></rp>
  • <th id="vgn1y"></th>
      當前位置:首頁 > 智慧健康列表 > 科學儀器 > 詳細信息
      HSP70-2基因慢病毒載體的構建及其對鼻咽癌6-10B細胞增殖的影響
      來源: | 作者: | 發布時間:2013-11-11 10:08:29

      HSP70-2基因慢病毒載體的構建及其對鼻咽癌6-10B細胞增殖的影響
      孫偉偉1,唐發清2,李名薇1,鄒飛雁1
      基金項目:國家自然科學基金(81071718) 作者簡介:孫偉偉(1988年),女,發育調控與腫瘤發生 通信聯系人:鄒飛雁(1963年),女,副教授,發育調控與腫瘤發生.

      (1. 暨南大學生命科學技術學院發育與再生生物學系,廣東廣州 510632;2. 暨南大學附屬珠海醫院檢驗科和實驗中心,廣東珠海 519000)
      摘要:背景與目的 HSP70-2在鼻咽癌6-10B細胞中過表達及功能研究國內外未見報道。本研究構建HSP70-2基因慢病毒載體及其轉染6-10B細胞,觀察HSP70-2基因對6-10B細胞生長增殖的影響。方法 利用慢病毒技術,使目的基因HSP70-2與pLenti/V5-DEST載體連接,經轉化篩選鑒定后得到pLV-HSP70-2-IRES/eGFP-Neo慢病毒載體,包裝慢病毒并測定病毒滴度,感染6-10B細胞,經G418篩選后得到高表達HSP70-2的6-10B穩轉細胞株,即6-10B-HSP70-2/eGFP細胞。同時設兩對照組,空載對照組為6-10B空載慢病毒穩轉細胞株,即6-10B-blank/eGFP細胞;空白對照組為6-10B未感染細胞株,即6-10B細胞。應用Western blotting檢測HSP70-2的蛋白表達水平;CCK8檢測細胞增殖情況;流式細胞儀分析細胞增殖指數(PI)、S期細胞比例(SPF)和凋亡百分比。結果 HSP70-2蛋白表達,培養24h,6-10B-HSP70-2/eGFP細胞與6-10B-blank/eGFP細胞或6-10B細胞比較,升高4.15±0.68%(P<0.01)或4.12±0.65%(P<0.01)。細胞增殖率,培養24h、48h和72h,6-10B-HSP70-2/eGFP細胞與6-10B-blank/eGFP細胞比較,分別升高29.18±0.014%(P<0.01)、81.72±0.124%(P<0.001)和41.84±0.004%(P<0.001);與6-10B細胞比較,分別升高25.69±0.031%20 (P<0.01)、84.53±0.060%(P<0.001)和45.28±0.044%(P<0.001)。PI和SPF,培養24h,6-10B-HSP70-2/eGFP細胞與6-10B-blank/eGFP細胞比較,分別增加73.31±5.882%(P<0.001)和71.09±7.495%(P<0.01);與6-10B細胞比較,分別增加72.60±1.300%(P<0.001)和69.29±13.42%(P<0.01)。細胞凋亡百分比,各組間比較無明顯變化(P>0.05)。結論 成功構建pLV-HSP70-2-IRES/eGFP-Neo慢病毒載體以及6-10B-HSP70-2/eGFP細胞和25 6-10B-blank/eGFP細胞,初步觀察到HSP70-2基因可能有促進鼻咽癌6-10B細胞生長與增殖作用。
      關鍵詞:慢病毒;HSP70-2;6-10B細胞
      中圖分類號:R739.62

      Construction of HSP70-2 gene lentiviral vector and Its Effects on 6-10B Cells’ Growth
      SUN weiwei1, TANG faqing2, LI mingwei1, ZOU feiyan1
      (1. Department of Developmental and Regenerative Biology,College of Life Science and Technology, Jinan University,Guangdong Guangzhou 510632; 35 2. Clinical Laboratory and Medical Research Center,the Affiliated Zhuhai Hospital,Jinan University,Guangzhou Zhuhai 519000)
      Abstract: Background and Objective Over expression and function study of HSP70-2 in nasopharyngeal carcinoma cell lines 6-10B has not been reported at home and abroad. In this study, HSP70-2 gene lentiviral vector and high expression of HSP70-2 in the stably transfected  cell lines 6-10B were constructed. The effect of HSP70-2 gene on 6-10B cell growth and proliferation was observed. Methods The target gene of HSP70-2 was connected to the pLenti/V5-DEST vector by lentiviral technology. After the screening and identification, the product was transformed into pLV-HSP70-2-IRES/eGFP-Neo lentiviral vector. The lentivirus was packed and measured virus titer, and then the lentivirus infected the 6-10B cells. It could get high expression of HSP70-2 in the stably transfected cell lines 6-10B by G418 screening, i.e.6-10-HSP70-2/eGFP cells. While the experiment set two groups: the blank control group was the stably transfected 6-10B cell lines with the empty vector infected, i.e.6-10B-blank/eGFP cells and the no-treatment control group was the uninfected 6-10B cell lines, i.e.6-10B cells. HSP70-2 protein expression of the three groups was analyzed by western blotting; cell proliferation of the three groups was  detected by CCK8; cell proliferation index (PI), S-phase fraction (SPF) and cell apoptosis percentage of the three groups were analyzed by flow cytometry. Results Western blotting assay showed that 6-10B-HSP70-2/eGFP cells compared with 6-10B-blank/eGFP cells or 6-10B cells, HSP70-2 protein expression was respectively increased by 4.15 ± 0.65% (P<0.01) or 4.12 ± 0.68% (P <0.01) after 24h of culture. 6-10B-HSP70-2/eGFP cells compared with 6-10B-blank/eGFP cells,  the proliferation rates were respectively increased by 29.18±0.014% (P<0.01), 81.72±0.124% (P <0.001) and 41.84 ± 0.004% (P <0.001) after 24h, 48h or 72h of culture; or compared with 6-10B cells, the proliferation rates were respectively increased by 25.69 ± 0.031% (P <0.01), 84.53 ± 0.060% (P <0.001) and 45.28 ± 0.044% (P <0.001) after 24h, 48h or 72h of culture. 6-10B- HSP70-2/eGFP cells compared with 6-10B-blank/eGFP cells, PI and SPF were respectively  increased by 73.31 ± 5.882% (P <0.001) and 71.09 ± 7.495% (P <0.01) after 24h of culture; or compared with 6-10B cells, PI and SPF were respectively increased by 72.60 ± 1.300% (P <0.001) and 69.29 ± 13.42% (P <0.01) after 24h of culture. Cell apoptosis percentage was no significantly changed among the three groups (P> 0.05). Conclusion pLV-HSP70-2-IRES/eGFP-Neo lentiviral vector, 6-10B-HSP70-2/eGFP cells and 6-10B-blank/eGFP cells were successfully constructed.  There might be initially observed that HSP70-2 gene could promote the growth and proliferation of nasopharyngeal carcinoma cell lines 6-10B.
      Key words: HSP70-2; Lentivirus; 6-10B cells

      奇米色8888