發(fā)布時(shí)間：2019-04-17 17:56

【摘要】：目的構(gòu)建人鼻咽癌細(xì)胞CNE-1與人臍靜脈血管內(nèi)皮細(xì)胞(human umbilical vein endothelial cells,HUVEC)的交互培養(yǎng)體系,初步觀察HUVEC微環(huán)境對(duì)鼻咽癌細(xì)胞生物學(xué)行為以及化療藥物對(duì)細(xì)胞生長(zhǎng)抑制的影響。方法將鼻咽癌細(xì)胞分為A、B和C組,A組加入100%CNE-1上清液,B組加入50%HUVEC上清液和50%CNE-1上清液,C組加入100%HUVEC上清液。采用噻唑藍(lán)(MTT)法檢測(cè)細(xì)胞貼壁率、生長(zhǎng)曲線和不同濃度順鉑(DDP)、5-氟尿嘧啶(5-FU)對(duì)鼻咽癌細(xì)胞的生長(zhǎng)抑制作用,激光掃描共聚焦顯微技術(shù)(laser scanning confocal microscopy,LSCM)檢測(cè)各組活細(xì)胞線粒體膜電位和鈣離子濃度。結(jié)果成功構(gòu)建了CNE-1細(xì)胞與HUVEC細(xì)胞的交互培養(yǎng)體系。不同時(shí)間段CNE-1細(xì)胞貼壁率B組和C組均大于A組,2h時(shí)B組和C組與A組的貼壁率差異最為顯著,F值分別為37.303和20.299,P值分別為0.004和0.001。A組細(xì)胞倍增時(shí)間為(42.26±0.21)h,B組為(41.24±0.30)h,C組為(45.90±0.53)h,交互培養(yǎng)B組細(xì)胞增殖速率顯著快于A組,F=33.506,P=0.004;而C組慢于A組,F=109.353,P0.001。5-FU作用3組細(xì)胞后,A組細(xì)胞的半數(shù)抑制濃度(IC50)為(81.65±0.07)μg/mL,B組為(118.38±0.11)μg/mL,C組為(82.95±0.06)μg/mL,與A組相比,B組增加,差異有統(tǒng)計(jì)學(xué)意義,F=2.381×105,P0.001,C組差異無統(tǒng)計(jì)學(xué)意義。DDP作用3組細(xì)胞后,A組的IC50值為(12.44±0.04)μg/mL,B組為(16.70±0.01)μg/mL,C組為(8.13±0.01)μg/mL,B組大于A組,F=3.203×103,P0.001;C組小于A組,F=3.278×103,P0.001。提示交互培養(yǎng)B組細(xì)胞對(duì)化療藥物的敏感性降低。與A組細(xì)胞膜電位比較,B組細(xì)胞線粒體跨膜電位顯著升高,F=25.511,P=0.007;C組顯著降低,F=54.251,P=0.002。與A組鈣離子濃度比較,B組鈣離子濃度顯著降低,F=260.972,P0.001;C組也降低,F=562.385,P0.001。結(jié)論改變腫瘤細(xì)胞的微環(huán)境,將改變細(xì)胞的生物學(xué)特性,血管內(nèi)皮細(xì)胞有利于維持鼻咽癌細(xì)胞的正常功能,提高細(xì)胞的活力,并導(dǎo)致交互培養(yǎng)體系中鼻咽癌細(xì)胞對(duì)化療藥物敏感性降低。
[Abstract]:Aim to construct a co-culture system between human nasopharyngeal carcinoma cell line CNE-1 and human umbilical vein endothelial cell (human umbilical vein endothelial cells,HUVEC), and to observe the effect of HUVEC microenvironment on the biological behavior of nasopharyngeal carcinoma cells and the inhibitory effect of chemotherapeutic drugs on the growth of nasopharyngeal carcinoma cells. Methods Nasopharyngeal carcinoma cells were divided into A, B and C groups. 100%CNE-1 supernatant was added to group A, 50%HUVEC supernatant and 50%CNE-1 supernatant were added to group B, and 100%HUVEC supernatant was added to group C. Cell adhesion rate, growth curve and inhibitory effect of cisplatin (DDP), 5-fluorouracil (5-FU) on nasopharyngeal carcinoma (NPC) cells were detected by thiazolyl blue (MTT) assay. Laser scanning confocal microscopy (laser scanning confocal microscopy,) was used to detect the inhibitory effect of cisplatin on the growth of nasopharyngeal carcinoma (NPC) cells. LSCM) was used to detect the mitochondrial membrane potential and calcium ion concentration of living cells in each group. Results the co-culture system of CNE-1 cells and HUVEC cells was successfully constructed. The adherent rate of CNE-1 cells in group B and C was higher than that in group A at different time points. At 2 h, the adhesion rate of cells in group B and C was the most significantly higher than that in group A (F = 37.303 and 20.299, respectively). The cell doubling time was (42.26 鹵0.21) h in group A, (41.24 鹵0.30) h in group B and (45.90 鹵0.53) h in group C, respectively. The cell proliferation rate in group B was significantly faster than that in group A (P < 0.05). F = 33. 506, P = 0. 004; In group C, the inhibition concentration (IC50) was (81.65 鹵0.07) 渭 g / mL and (118.38 鹵0.11) 渭 g / L, respectively, compared with that in group A (109.353) and P0.001.5 渭 g / U (P < 0.05), and that in group B was (118.38 鹵0.11) 渭 g / L, while that in group C was (81.65 鹵0.07) 渭 g / mL. Group C was (82.95 鹵0.06) 渭 g / L, compared with group A, group B increased significantly (F = 2.381 脳 105, P0.001), and there was no significant difference between group C (P < 0.05) and group C (P < 0.01). After treated with cisplatin, the number of cells in group B was significantly higher than that in group A (P < 0.01). The IC 50 was (12.44 鹵0.04) 渭 g / L in group A, (16.70 鹵0.01) 渭 g / L in group B, (8.13 鹵0.01) 渭 g / L in group C, higher than that in group A (F = 3.203 脳 103, P0.001). Group C smaller than Group A, F = 3.278 脳 103, P0.001. The results suggest that the sensitivity of group B cells to chemotherapeutic drugs is lower than that of group B cells. Compared with group A, the transmembrane potential of mitochondria in group B was significantly higher than that in group A (P < 0.05). The mitochondrial transmembrane potential in group B was significantly higher than that in group A (P < 0.05), while that in group B was significantly higher than that in group B (P < 0.05). Compared with group A, the concentration of calcium in group B was significantly lower than that in group A, the concentration of calcium in group B was significantly lower than that in group A, and the concentration of calcium in group B was significantly lower than that in group A (P < 0.01). Conclusion changing the microenvironment of tumor cells will change the biological characteristics of the cells. Vascular endothelial cells are beneficial to maintain the normal function of nasopharyngeal carcinoma cells and enhance the viability of the cells. Furthermore, the chemosensitivity of nasopharyngeal carcinoma cells to chemotherapeutic drugs was decreased in the interactive culture system.
【作者單位】： 廣西醫(yī)科大學(xué)藥學(xué)院;
【基金】：國(guó)家自然科學(xué)基金(81460561) 廣西自然基金(2014GXNSFAA118225)
【分類號(hào)】：R739.63

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