腹水来源的外泌体对卵巢上皮性癌干细胞样细胞的干性特征及侵袭能力的影响

Effects of ascites derived exosomes on the stemness and invasion ability of ovarian cancer stem-like cell

目的探讨卵巢上皮性癌(卵巢癌)患者腹水来源的外泌体对卵巢癌干细胞样细胞(OCS-LC)干性特征及侵袭能力的影响。方法(1)采用无血清悬浮培养法诱导卵巢癌细胞系A2780细胞生成OCS-LC,并采用成球实验、分化功能实验以及流式细胞仪检测等方法鉴定OCS-LC的成球克隆生长、定向分化潜能、干性标志物CD_(133)表达等干性特征。(2)采用超速离心法提取卵巢癌来源外泌体(OCDE),包括卵巢癌患者腹水来源及A2780细胞来源的外泌体[即腹水来源外泌体(ADE)及细胞来源外泌体(CDE)],采用透射电镜观察外泌体形态、纳米颗粒跟踪分析(NTA)法测量外泌体粒径、蛋白印迹(western blot)法检测特异性蛋白分子热休克蛋白70(HSP-70)、CD_(63)、CD_(9)的表达等方法对外泌体ADE和CDE进行鉴定。(3)将ADE和CDE分别与OCS-LC共培养,即ADE+OCS-LC组、CDE+OCS-LC组,以磷酸盐缓冲液(PBS)与OCS-LC共培养作为空白对照组。通过观察3组OCS-LC的成球周期、最大球直径及成球率,评估各组OCS-LC的成球能力;采用免疫荧光染色法检测3组OCS-LC的干性标志物CD_(133)的表达;实时荧光定量PCR技术检测3组OCS-LC中干细胞转录因子八聚体结合转录因子4(Oct-4)和Nanog mRNA的表达;穿膜小室(transwell小室)实验检测3组OCS-LC的侵袭能力。结果(1)OCS-LC的鉴定:成球实验显示,OCS-LC单细胞悬液在无血清培养基中可二次成球生长;分化功能实验显示,OCS-LC细胞球在含血清培养基中改变生长方式,分化成呈贴壁生长的A2780细胞;流式细胞仪检测显示,OCS-LC中CD_(133)阳性(CD_(133)^(+))细胞的比例为(18.9±0.9)%,显著高于其对照(即A2780细胞)的(0.6±0.5)%(t=38.570,P<0.01)。(2)OCDE的鉴定:透射电镜下观察,外泌体ADE和CDE均可见清晰的脂质双层膜结构,一侧呈凹陷的半球形或杯托样;NTA法测量显示,外泌体粒径主要在30~100 nm范围,平均粒径67.2 nm;western blot法检测显示,特异性蛋白分子HSP-70、CD_(63)、CD_(9)均呈阳性表达。(3)共培养后,成球实验显示,空白对照组、ADE+OCS-LC组、CDE+OCS-LC组OCS-LC均可继续成球生长,其中ADE+OCS-LC组细胞呈现两种生长方式,大部分细胞继续维持干性成球生长,小部分细胞出现分化,呈贴壁生长;3组OCS-LC的成球周期分别为(15.3±1.5)、(10.3±0.6)、(6.7±0.6)d,细胞球最大直径分别为(100.3±3.2)、(145.2±5.1)和(170.0±2.1)μm,成球率分别为(1.05±0.20)%、(4.15±0.10)%和(10.45±0.25)%,3组分别比较,差异均有统计学意义(P均<0.05),且CDE+OCS-LC组分别与ADE+OCS-LC组比较,差异也均有统计学意义(P均<0.05)。免疫荧光染色法检测显示,空白对照组、ADE+OCS-LC组,CDE+OCS-LC组OCS-LC细胞球中呈绿色荧光的CD_(133)^(+)细胞数依次增多,3组OCS-LC细胞球中CD_(133)^(+)细胞比例[分别为(26.6±1.5)%、(46.2±2.1)%和(58.4±2.2)%]比较,差异有统计学意义(F=187.588,P<0.05),且CDE+OCS-LC组较ADE+OCS-LC组更高(t=6.753,P<0.05)。实时荧光定量PCR技术检测显示,空白对照组、ADE+OCS-LC组、CDE+OCS-LC组OCS-LC中Oct-4 mRNA的表达水平分别为1.04±0.12、3.46±0.24、4.03±0.31,Nanog mRNA表达水平分别为1.00±0.07、1.57±0.32、2.66±0.15,3组分别比较,差异均有统计学意义(P均<0.05),且CDE+OCS-LC组均显著高于ADE+OCS-LC组(P均<0.05)。transwell小室实验显示,空白对照组、ADE+OCS-LC组、CDE+OCS-LC组的侵袭细胞数依次增多,分别为(30±5)、(102±4)、(210±7)个,3组比较,差异有统计学意义(F=820.800,P<0.05),且CDE+OCS-LC组显著高于ADE+OCS-LC组(t=23.202,P<0.05)。结论卵巢癌ADE能够增强和维持OCS-LC的干性特征,促进肿瘤细胞的侵袭转移,但CDE优于ADE。

Objective To investigate the effects of ovarian cancer ascites-derived exosomes on the stem cell properties and invasion ability of ovarian cancer stem-like cell(OCS-LC).Methods(1)A2780 cells were induced into OCS-LC in serum-free medium,and authenticating their stem-like properties by sphere-forming test,differentiation test and CD_(133) marker detection.(2)Exosomes from ascites and A2780 cell were extracted by ultracentrifugation,then authenticating them.The morphology of exosomes was observed by the transmission electron microscope,exosome particle size was measured by nanoparticle tracking analysis(NTA).The expressions of heat shock protein 70(HSP-70),CD_(63) and CD_(9) were detected by western blot.(3)The exosomes from ovarian cancer ascites and tumor cell supernate were co-cultured with OCS-LC.The groups were divided into control group,ascites-derived exosomes(ADE)group(ADE+OCS-LC group),and cells-derived exosomes(CDE)group(CDE+OCS-LC group).The sphere-forming ability was evaluated by sphere-forming cycle,maximum sphere diameter and sphere-forming rate of each group;the expression of CD_(133) was detected by immunofluorescence staining under microscope;quantitative real-time(qRT)-PCR was used to detected the expression levels of octamer-4(Oct-4),Nanog mRNA of the signature genes in the stem cells of each group;the metastasis ability of each group was measured by transwell assay.Results(1)Identification of OCS-LC:sphere-forming experiment showed that the suspension of OCSC single cells was grown in serum-free medium in secondary sphere-forming.Differentiation function experiment showed that OCS-LC were differentiated into adherent A2780 cells by changing the growth mode in serum containing medium.Flow cytometry showed that the proportion of CD_(133) positive(CD_(133)^(+))cells in OCS-LC group was(18.9±0.9)%,significantly higher than that of control group(0.6±0.5)%(t=38.570,P<0.01).(2)Under transmission electron microscope,clear lipid bilayer structure was observed in ADE and CDE,and one side presented a concave hemispheric or cup like structure.NTA showed that the diameter of exosomes mainly ranged from 30 to 100 nm,with an average diameter of 67.2 nm.Western blot analysis showed that the specific protein molecules HSP-70,CD_(63) and CD_(9) were positive.(3)Three groups′OCS-LC could continue to grow into spheres,and the group of ADE+OCS-LC showed two growth modes,most of the cells continued to grow into spheres,while a small part of cells grew in adherent differentiation.The sphere-forming rate of OCS-LC in the control group,ADE+OCS-LC group,and CDE+OCS-LC group were(1.05±0.20)%,(4.15±0.10)%,and(10.45±0.25)%,the sphere-forming cycle of OCS-LC in the three groups were(15.3±1.5),(10.3±0.6),and(6.7±0.6)days,and the maximum diameters of OCS-LC were(100.3±3.2),(145.2±5.1)and(170.0±2.1)μm,respectively.And the differences were statistically significant(all P<0.05).After co-culture of exosomes with OCS-LC,the sphere-forming ability of cells in the group of CDE+OCS-LC was prior to ADE+OCS-LC group(all P<0.05).Immunofluorescence staining showed that the number of CD_(133) green fluorescent chromophore cells in OCS-LC groups[(46.2±2.1)%,(58.4±2.2)%]was significantly higher than that in the control group[(26.6±1.5)%]after the addition of exosomes in co-culture,the positive rate of CD_(133) was higher than that in the control group(F=187.588,P<0.05).The qRT-PCR results showed that the expression levels of Oct-4 mRNA in ADE+OCS-LC and CDE+OCS-LC groups were 3.46±0.24,4.03±0.31,compared with that in control group(1.04±0.12),the differences were statistically significant(F=134.932,P<0.05).The mRNA expression levels of Nanog were 1.57±0.32,2.66±0.15,which were significantly higher than that in the control group(1.00±0.07),and the differences were statistically significant(F=49.329,P<0.05).And the expression of both in CDE+OCS-LC group increased more significantly than ADE+OCS-LC group(all P<0.05).The number of invasive cells in the three groups of OCS-LC were:control group 30±5,ADE+OCS-LC group 102±4,CDE+OCS-LC group 210±7,and there were statistically significant differences among three groups(F=820.800,P<0.05).Compared with the control group,the number of invaded cells in the co-culture group were significantly increased(P<0.05),and the CDE+OCS-LC group had the higher cell invasion ability then the ADE+OCS-LC group(t=23.202,P<0.05).Conclusions Exosomes derived from ovarian cancer ascites could enhance and maintain the stemness of OSC-LC,and promote the invasion of tumor cells.Moreover,CDE is superior to ADE.