發(fā)布時間：2020-12-14 07:35

　　第一部分經(jīng)BMP9誘導(dǎo)后C3H10T1/2細(xì)胞JNK和ERK5的激活情況目的：檢測經(jīng)高滴度攜帶GFP和BMP9基因的重組腺病毒轉(zhuǎn)染C3H10T1/2細(xì)胞后JNK和ERK5的激活情況。方法：培養(yǎng)HEK293細(xì)胞，并用其對AdBMP9和AdGFP進(jìn)行擴(kuò)增，提高AdBMP9和AdGFP的滴度。高滴度AdBMP9和AdGFP轉(zhuǎn)染C3H10T1/2細(xì)胞24h后，熒光顯微鏡觀察GFP的表達(dá)情況并用流式細(xì)胞儀檢測轉(zhuǎn)染效率，免疫熒光技術(shù)定位p-JNK和p-ERK5在細(xì)胞內(nèi)的表達(dá)部位，利用CCK-8檢測特異性抑制劑對細(xì)胞生長情況的影響，Western blot檢測加入JNK或ERK5信號通路特異性抑制劑SP600125或BIX02189并轉(zhuǎn)染后JNK和ERK5的激活情況。結(jié)果：1.流式細(xì)胞儀檢測高滴度AdBMP9和AdGFP轉(zhuǎn)染C3H10T1/2細(xì)胞24h后的轉(zhuǎn)染效率均可達(dá)50%左右。2.倒置顯微鏡下觀察AdBMP9轉(zhuǎn)染后細(xì)胞的形態(tài)隨培養(yǎng)時間的延長由多邊形逐漸向長梭形轉(zhuǎn)變，且細(xì)胞間鏈接更加緊密。3.免疫熒光技術(shù)結(jié)果顯示各組細(xì)胞的胞質(zhì)、胞核均可見p-JNK及p-ERK5的表達(dá)，但經(jīng)AdBMP9誘導(dǎo)后p-J... 

【文章來源】：重慶醫(yī)科大學(xué)重慶市

【文章頁數(shù)】：54 頁

【學(xué)位級別】：碩士

【部分圖文】：

不同濃度藥物作用下C3H10T1/2細(xì)胞的生存率曲線

A:HEK293 cells cultured 24h; B:HEK293 cells cultured 48h; C:C3H10T1/2 cells cultured 24h; D:C3H10T1/2 cells cultured 48h圖 1.1 倒置顯微鏡下的 HEK293 細(xì)胞及 C3H10T1/2 細(xì)胞(×100)Fig 1.1 HEK293 cells and C3H10T1/2 cells under inverted microscope(×100)A1:AdGFP transfection after 24h under Inverted microscope; A2:AdGFP transfection after 24h under Fluorescence microscopeB1:AdBMP9 transfection after 24h under Inverted microscope; B2:AdBMP9 transfection after 24h under Fluorescence microscopeC1: AdGFP transfection after 72h under Inverted microscope; C2: AdGFP transfection after 72h under Fluorescence microscopeD1:AdBMP9 transfection after 72h under Inverted microscope; D2 :AdBMP9 transfection after 72h under Fluorescence microscope圖 1.2 倒置顯微鏡及熒光顯微鏡下的 HEK293 細(xì)胞(×100)

流式細(xì)胞術(shù)檢測轉(zhuǎn)染效率


本文編號：2916076