化痰祛瘀方对人喉癌原代细胞STAT3信号通路影响

Effects of Huatan Quyu Formula on STAT3 signaling pathway in human laryngeal carcinoma primary cells

目的:观察化痰祛瘀方(HTQYF)对喉癌原代细胞STAT3信号通路的影响。方法:取病理检查诊断为喉鳞状细胞癌患者喉癌组织,组织贴壁法分离喉癌细胞,以喉癌细胞Tu212作为阳性对照,运用细胞免疫荧光技术鉴定细胞,通过CCK8实验探讨HTQYF对喉癌原代细胞增殖的影响,并计算细胞抑制率,Western Blot、PCR法检测不同组别STAT3、Cyclin D1、P27蛋白和mRNA表达情况。结果:HTQYF对喉癌原代细胞具有明显的抑制作用,随着药物浓度的升高,抑制率不断上升,浓度为3.2、6.4mg/mL时,平均抑制率分别为47.3%、89.9%;与空白组比较,各剂量组能显著抑制喉癌原代细胞STAT3蛋白的表达(P<0.05,P<0.01),同时高剂量组上调P27蛋白(P<0.01),而各组对Cyclin D1蛋白表达无影响;高剂量组可显著抑制喉癌原代细胞STAT3、Cyclin D1mRNA表达(P<0.01),但是对P27mRNA表达无影响。结论:通过组织贴壁法成功培养出人喉癌原代细胞,HTQYF能抑制原代喉癌细胞增殖,且能抑制STAT3的表达,有可能是调控下游Cyclin D1和P27信号因子起作用。

Objective:To observe the effects of Huatan Quyu Formula(HTQYF)on STAT3 signaling pathway in primary laryngeal cancer cells which cultured by obtaining tissue from patients with laryngeal cancer.Methods:Tissue dissected from laryngeal squamous cell carcinoma diagnosed by clinic opathological examination was cultured primary cells by tissueadherent method.Laryngeal cancer cells Tu212 were purified repeatedly using different time adhesion technique.Cells were identified by immunofluorescence technique.CCK8 experiment was carried out to calculate the cell inhibition rate.The protein and mRNA expression of STAT3,Cyclin D1 and P27 in different groups were detected by Western Blot and PCR.Results:HTQYF had obvious inhibitory effect on the proliferation of primary laryngeal cancer cells.Accompanied by the increase of drug concentration,the inhibition rate was increased.When the drug concentration was 3.2 and 6.4 mg/mL,the average inhibition rate was 47.3%and 89.9%,respectively.Compared with the blank group,the dosage groups could inhibited the expression of STAT3 protein in primary laryngeal cancer cells(P<0.05,P<0.01),and the high dose group upregulated P27 protein(P<0.01).There was no effect on the expression of Cyclin D1 protein.The mRNA expression of STAT3 and Cyclin D1 in primary laryngeal cancer cells were inhibited by high dose group of HTQYF(P<0.01),but it had no effect on the mRNA expression of P27.Conclusion:Human primary laryngeal cancer cells are successfully cultured by tissue-adherent method.HTQYF inhibites the proliferation of primary laryngeal cancer cells.It inhibites the expression of STAT3 through regulation of downstream Cyclin D1 and P27 signaling factors.