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目的探讨Src蛋白对体外三维培养的胃癌多细胞团簇(multicellular aggregates,MCAs)增殖及凋亡的影响。方法体外三维培养人胃腺癌细胞BGC823和MKN45成MCAs,Western blot检测MCAs和单层贴壁培养的胃癌细胞中Src蛋白表达情况。siRNA法干扰Src表达,转染胃癌细胞后检测其对胃癌细胞BGC823和MKN45 MCAs形态学的影响。分别采用CCK-8和流式细胞术检测干扰Src表达对胃癌BGC823和MKN45 MCAs细胞活力和细胞凋亡率的影响。结果利用BGC823和MKN5成功建立胃癌MCAs体外三维培养模型,Western blot检测结果表明,与对照组比较,MCAs中Src的表达水平明显高于单层贴壁培养的胃癌细胞[BGC823:(0.615±0.068)vs(0.219±0.017),P<0.01;MKN45:(0.657±0.087)vs(0.420±0.015),P<0.05]。干扰胃癌细胞BGC823和MKN45 Src表达,可以显著降低MCAs形成能力,并明显影响MCAs活力。流式细胞术检测结果表明,干扰Src表达可以显著诱导MCAs发生凋亡[BGC823:(9.456±0.209)vs(3.026±0.201),P<0.01;MKN45:(9.356±0.416)vs(2.541±0.251),P<0.05]。结论 Src是胃癌MCAs抗失巢凋亡的关键分子,抑制Src表达可以显著抑制胃癌BGC823 MCAs和MKN45 MCAs形成,影响胃癌MCAs增殖,并诱导胃癌MCAs凋亡。
Objective To investigate the effects of Src protein on the proliferation and apoptosis of multicellular aggregates (MCs) cultured in vitro. Methods Human gastric adenocarcinoma BGC823 and MKN45 cells were cultured in vitro three-dimensionally as MCAs. Western blot was used to detect the expression of Src protein in MCAs and monolayer adherent cultured gastric cancer cells. The expression of Src was knocked down by siRNA, and the effect of Src on the morphology of gastric cancer cells BGC823 and MKN45 MCAs was detected after transfected into gastric cancer cells. The effects of interfering Src expression on cell viability and apoptosis rate of BGC823 and MKN45 MCAs were detected by CCK-8 and flow cytometry respectively. Results The three-dimensional culture model of gastric cancer MCAs was successfully established by using BGC823 and MKN5. The Western blot results showed that the expression of Src in MCAs was significantly higher than that in monolayer adherent cultured gastric cancer cells [(BGC823: 0.615 ± 0.068) vs (0.219 ± 0.017), P <0.01; MKN45: (0.657 ± 0.087) vs (0.420 ± 0.015), P <0.05]. Interference of gastric cancer cells BGC823 and MKN45 Src expression, can significantly reduce the formation of MCA, and significantly affect the activity of MCAs. The result of flow cytometry showed that the interference of Src expression could significantly induce the apoptosis of MCAs [BGC823: (9.456 ± 0.209) vs (3.026 ± 0.201, P <0.01; MKN45: (9.356 ± 0.416) vs (2.541 ± 0.251) , P <0.05]. Conclusion Src is a key molecule of anti-anoikis for gastric cancer MCAs. Inhibiting Src expression can significantly inhibit the formation of gastric cancer BGC823 MCAs and MKN45 MCAs, affect the proliferation of gastric cancer MCAs and induce the apoptosis of gastric cancer MCAs.