KISS-1基因表达物对骨肉瘤细胞凋亡和自噬调控机制的研究

Study on the Effect of KISS-1 Expression on the Osteosarcoma Cell Apoptosis and Autophagy Regulation Mechanism

目的该研究就KISS-1基因表达物在骨肉瘤细胞中的凋亡和自噬作用进行比较分析,探讨KISS-1基因表达对骨肉瘤细胞凋亡和自噬的调控机制。方法于2016年4月—2017年1月在该院细胞实验室分别向K7M2骨肉瘤细胞培养中分别加入KISS1蛋白Kp(Kisspeptin)或抗Kp蛋白,将其对应分为Kp组和抗Kp蛋白组,并分别与普通培养的K7M2细胞形成的控制组进行比较,待细胞培养24,48、72、96 h后,每组各时间点选取6孔对数生长期的细胞纳入实验观察,MTT观察细胞增殖情况,并采用流式细胞仪检测细胞凋亡率,应用RT-PCR检测细胞培养72 h后凋亡标记物(P53、Cyt C)及自噬标记物(P62、Beclin1)的m RNA表达水平。结果细胞培养96 h后,对照组细胞增殖率均数百分比由(0.660±0.075)%变为(1.330±0.214)%,Anti-Kp组细胞增殖率均数百分比由(0.677±0.093)%变为(1.465±0.194)%,而Kp组细胞增殖率均数百分比由(0.530±0.083)%变为(0.328±0.044)%,凋亡率均数百分比由(24.53±3.65)%变为(39.75±8.32)%。Anti-Kp组增殖率显著高于对照组(P<0.05)。细胞接种培养72 h后,对照组的P53和Cyt C的m RNA表达水平均数为(0.213±0.045)和(0.220±0.046),Kp组P53和Cyt C的m RNA表达量均数为(0.393±0.107)和(0.509±0.145)。同时,P62和Beclin1在控制组的m RNA表达量均数为(0.169±0.055)和(0.120±0.043),而在Kp组P62和Beclin1m RNA表达量均数为(0.426±0.120)和(0.357±0.103)。P62和Beclin1的m RNA表达水平在Kp组最高,Anti-Kp组最低,差异有统计学意义(P<0.05)。结论 KISS-1基因表达物具有促进细胞凋亡和自噬来控制骨肉瘤细胞增殖的作用。

Objective To compare and analyze the effect of KISS-1 expression on the Osteosarcoma cell apoptosis and Autophagy regulation mechanism. Methods Kp（Kisspeptin）or anti-Kp protein were respectively increased into the K7 M2 osteosarcoma cell culture in the cell laboratory in our hospital from April 2016 to January 2017, and the corresponding groups were the Kp group and the anti-Kp protein group, and compared with the control group of K7 M2 cell formed by the common culture, after the culture at 24 h, 48 h,72 h, 96 h, the cells in the logarithmic growth period of the 6 hole at various time points in each group were for experimental observation, and the cell proliferation was observed by the MTT, and the cell apoptosis rate was tested by the flow cytometer, and the m RNA expression levels of P53,CytC and P62,Beclin1 after72 h culture was tested by the RT-PCR. Results The percentage of cell proliferation in the control group changed from（0.660 ±0.075）% to（1.330 ±0.214）% within 96 hours of cell culture and in anti-Kp group the indicator changed from（0.677±0.093）% to（1.465±0.194）%. The average value of Cell proliferation rate in the Kp group changed from（0.530±0.083）% to（0.328±0.044）%, and the percentage of average value of apoptosis rate changed from（24.53±3.65）% to（39.75±8.32）%, and the proliferation rate of the anti-Kp group was significantly higher than that of the control group（P〈0.05）.The m RNA expression average levels of p53 and CytC in the control group were（0.213±0.045） and（0.220±0.046）and in Kp group were（0.393±0.107） and（0.509±0.145）. The average m RNA expression amounts of P62, Beclin1 in the control group and in the Kp group were respectively（0.169±0.055）,（0.120±0.043） and（0.426±0.120）,（0.357±0.103）, and the m RNA expression levels of P62, Beclin1 were the highest in the Kp group and the lowest in the anti-Kp group, and the differences were statistically significant（P〈0.05）. Conclusion KISS-1 gene expression can promote the apoptosis and autophagy to control the osteosarcoma cell proliferation.