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目的探讨蝎丝氨酸蛋白酶抑制剂(Sj7170)多肽在神经胶质瘤U87细胞体外增殖中的影响。方法选取人胶质瘤细胞系U87细胞,使用重组Sj7170(r Sj7170)对其进行处理,处理浓度设置:0μM、2μM、4μM、6μM、8μM、10μM。r Sj7170(10μM)处理时间设置:0、1、2、3 d,并用血球计数板分别进行计数;收集未处理(对照组)和r Sj7170处理过的(加肽组)U87细胞,流式细胞仪检测细胞周期;取对数生长期的对照组和加肽组的U87细胞,采用软琼脂克隆形成实验观察U87细胞克隆形成情况;采用Western Blot检测周期相关蛋白的相对表达水平。结果U87细胞增殖率均随r Sj7170浓度递增而递增,且加肽组各浓度的细胞增殖率均显著高于对照组(P均<0.05);10μM r Sj7170处理过的U87细胞增殖率均随时间推移(1d→2d→3d)而递增(P均<0.05),且加肽组2、3 d时的细胞增殖率均显著高于对照组(P均<0.05)。r Sj7170在不同浓度和不同时间作用下均可促进U87细胞的增殖。对照组和加肽组U87细胞均在软琼脂培养基中形成了克隆,加肽组中克隆形成的数目比明显大于对照组[(284.67±26.53)%vs(100.00±0.00)%,P<0.01]。在10μM r Sj7170处理过的U87细胞中,其细胞周期蛋白(Cyclins)D1、CyclinD3、E2F转录因子1、磷酸化视网膜神经胶质瘤蛋白家族(pRb)2/p130的表达量明显高于未处理的对照细胞,而视网膜神经胶质瘤蛋白家族(Rb)2/p130和E2F5的表达量则明显低于对照细胞,其他几个周期相关蛋白CyclinD2、CyclinE1、CyclinE2、CDK2、CDK4、CDK6、P21、P27表达量两组相当。结论蝎丝氨酸蛋白酶抑制剂Sj7170能促神经胶质瘤细胞U87的增殖,该作用可能与细胞周期蛋白有关。
Objective To investigate the effect of scorpion serine protease inhibitor (Sj7170) on the proliferation of glioma U87 cells in vitro. Methods Human glioma cell line U87 cells were selected and treated with recombinant Sj7170 (r Sj7170). The treatment concentrations were set at 0μM, 2μM, 4μM, 6μM, 8μM and 10μM. r Sj7170 (10μM) treatment time set: 0, 1, 2, 3 d, and were counted with a hemocytometer; untreated (control group) and r Sj7170-treated (peptide group) U87 cells were collected, flow cytometry The cell cycle of U87 cells was detected by logarithmic growth phase assay. U87 cells were added with U87 cells in the logarithmic growth phase. The colony formation of U87 cells was observed by soft agar colony formation assay. The relative expression level of the protein was determined by Western Blot. Results The proliferation rate of U87 cells increased with the increase of rSj7170 concentration, and the cell proliferation rate of each concentration of peptide group was significantly higher than that of the control group (all P <0.05). The proliferation rate of U87 cells treated with 10μM r Sj7170 all increased with time (P <0.05), and the cell proliferation rates of the two groups were significantly higher than that of the control group (P <0.05). r Sj7170 can promote the proliferation of U87 cells under different concentrations and different time. The number of clones in the control group and the added group of U87 cells were all formed in soft agar medium, and the number of colonies in the group of adding peptide was significantly larger than that in the control group [(284.67 ± 26.53)% vs (100.00 ± 0.00)%, P <0.01 ]. The expression of Cyclins D1, CyclinD3, E2F transcription factor 1, phosphorylated retinoblastoma protein family (pRb) 2 / p130 in U87 cells treated with 10μM r Sj7170 was significantly higher than that of untreated (Rb) 2 / p130 and E2F5 were significantly lower than those in control cells. The expression of several other cyclinD2, CyclinE1, CyclinE2, CDK2, CDK4, CDK6, P21, The expression of P27 was comparable between the two groups. Conclusion Sj7170, a serine protease inhibitor, can promote the proliferation of glioma U87, which may be related to cyclin.