Platelet-rich Plasma Promotes the Proliferation of Human Endometrial Mesenchymal Stem Cells (EnMSCs) through the PI3K/AKT/mTOR Signaling Pathway
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摘要:
目的 探讨PRP促进EnMSCs增殖的机制,为EnMSCs的扩增及临床应用提供理论基础。 方法 将EnMSCs随机分为对照组、2%PRP组、2%PRP + LY294002组,CCK-8法检测细胞增殖情况;流式细胞仪监测细胞周期及Western Blot和ELISA检测细胞中p-PI3K、AKT、p-AKT、mTOR及p-mTOR的蛋白表达。 结果 (1) CCK-8结果显示:与对照组相比较,2%PRP组EnMSCs的增殖水平显著较高(P < 0.05);与2%PRP组相比,2%PRP+LY294002组EnMSCs的增殖水平显著较低( P < 0.05);(2)流式细胞仪分析细胞周期结果显示:2%PRP组G2/M期细胞比例显著高于对照组和2%PRP+ LY294002组,G0/G1细胞比例明显低于对照组和2%PRP+ LY294002组,差异均有统计学意义( P < 0.05);(3) 2%PRP组EnMSCs中p-PI3K、AKT、p-AKT、mTOR、p-mTOR的蛋白表达明显高于对照组及2%PRP+LY294002组,差异均有统计学意义( P < 0.05)。 结论 EnMSCs的增殖由PI3K/AKT/ mTOR信号通路调控,PRP通过促进AKT、mTOR的蛋白表达及其磷酸化以激活该通路,促进EnMSCs由G1期向G2期转变,从而促进EnMSCs的增殖。 -
关键词:
- 富血小板血浆 /
- 子宫内膜间充质干细胞 /
- PI3K/AKT/mTOR /
- 细胞增殖 /
- 细胞周期
Abstract:Objective To explore the mechanism of PRP promoting EnMSCs proliferation, and to provide a theoretical basis for the expansion and clinical application of EnMSCs. Methods EnMSCs were randomly divided into the control group, 2%PRP group and 2%PRP + LY294002 group. Cell proliferation was detected by CCK-8 method. Cell cycle was monitored by flow cytometry and protein expressions of p-PI3K, AKT, p-AKT, mTOR and p-mTOR were detected by Western Blot. Results (1) CCK-8 results: Compared with the control group, the proliferation level of EnMSCs in 2%PRP group was significantly higher (P < 0.05). Compared with the 2%PRP group, the proliferation level of EnMSCs in the 2%PRP+LY294002 group was significantly lower ( P < 0.05). (2) The results of cell cycle analysis by flow cytometry showed that the proportion of cells in G2/M phase in the 2% PRP group was significantly higher than that in the control group and 2% PRP+LY294002 group, and the proportion of cells in G0/G1 phase was significantly lower than that in the control group and 2% PRP+ LY294002 group. All of these had statistically significant differences ( P < 0.05). (3) The protein expressions of p-PI3K, AKT, p-AKT, mTOR and p-mTOR in EnMSCs in the 2%PRP group were significantly higher than those in the control group and the 2%PRP+LY294002 group, with statistical significance ( P < 0.05). Conclusion The proliferation of EnMSCs is regulated by the PI3K/Akt/mTOR signaling pathway. PRP activates this pathway by promoting the protein expression and phosphorylation of AKT and mTOR, thereby promoting the proliferation of EnMSCs. -
茯苓为多孔菌科真菌茯苓Poria cocos (Schw.) Wolf的干燥菌核,寄生于松科植物的根茎上,埋于土壤下繁衍而成。茯苓又名松苓、茯菟、茯灵,其野生资源稀少,栽培品种主要产于安徽、湖北、湖南、云南、贵州等地。茯苓味甘、淡,性平,归心、肺、脾、肾经;具有利水渗湿,健脾,宁心功效;用于水肿尿少,痰饮眩悸,脾虚食少,便溏泄泻,心神不安,惊悸失眠。茯苓始载于《神农本草经》,应用广泛,不仅是多种方剂和中成药的原料,有“十药九茯苓”之说,也添加在各种食品中,是沿用千年的药食两用的中药材[1]。羊毛甾烷四环三萜类化合物是茯苓主要成分之一,其结构类型主要分为四种,分别为羊毛甾-8-烯型三萜、羊毛甾-7,9(11)-二烯型三萜、3,4-开环-羊毛甾-8-烯型三萜、3,4-开环-羊毛甾-7,9(11)-二烯型三萜[2]。现代药理学研究结果显示,茯苓三萜类化合物是茯苓传统药效和拓展功效的物质基础[3-9]。本文采用超高效液相色谱串联离子阱飞行时间质谱(UPLC-IT-TOF/MS) 技术定性分析云南大理产茯苓95%乙醇提取物的三萜类化学成分,并对其裂解规律进行总结,为该产地茯苓的药效物质基础解析和质量控制提供参考依据。
1. 材料与方法
1.1 仪器
LC-MS分析所用仪器为岛津公司(Shimaduz,Kyoto,Japan)生产的LCMS-IT-TOF仪。液相色谱仪控制器型号为CBM-20A,泵型号为LC-30AD,自动进样器型号为SIL-30AC,脱气装置型号为DGU-20A5,二极管阵列检测器为SPD-M20A,柱温箱型号为CTO-20AC。
1.2 试药和试剂
茯苓购自昆明市螺蛳湾中药材市场(产自云南大理),经昆明医科大学唐丽萍教授鉴定为多孔菌科真菌茯苓Poria cocos (Schw.) Wolf的干燥菌核;色谱纯乙腈购自德国默克公司,色谱纯甲酸购自上海阿拉丁试剂公司,超纯水由默克MingCheTM-D24UV设备制备。
1.3 供试品溶液制备
干燥药材茯苓10 g,粉碎,用95%乙醇250 mL浸泡1 h,加热至微沸,加热1 h,过滤浓缩得浸膏1.8 g。取5 mg 浸膏,加入2 mL甲醇超声溶解,离心(10000 r/min×10 min)取上清液进样。
1.4 液相色谱条件
色谱柱为UHPLC XB-C18色谱柱 (2.1×100 mm,1.8 µm),柱温30℃,紫外检测波长范围为190~400 nm。流动相A:甲酸/水(0.05/100,v/v),B:乙腈;流速为0.20 mL/min;梯度洗脱条件:起始梯度为5% B,0~50 min,5%~90 % B;50~55 min,90% B;55~56 min,90%~5% B;56~60 min,5% B。进样量为:2 µL。
1.5 质谱条件
质谱条件:离子源为ESI源,三氟乙酸钠校正,电喷雾正负离子同时检测。分析条件如下:喷雾电压为4.50 kV或− 3.50 kV;检测电压为1.65 kV;旋转式真空泵压力为71.0 Pa;干燥气压力为110.0 kPa;喷雾气为氮气,流速为1.5 L/min;曲形裂解器(CDL)温度为200 ℃;加热模块温度为200 ℃;离子捕获时间为10 ms;母离子选择范围为m/z ±3.0 Da;裂解能量(CID)为50%;裂解气为氩气,扫描范围为m/z 100~1000。
1.6 茯苓的三萜类化学成分分析方法
Shimadzu Composition Formula Predictor软件用于预测分子式,并通过多级质谱数据分析,结合氮规则和Scifinder数据库检索推断化合物的基本结构信息。
2. 结果
茯苓的95%乙醇提取物在实验色谱、质谱条件下响应良好,经过分析发现其主要化合物为羊毛甾烷型四环三萜,保留时间集中在15~50 min之间,最大离子流图(BPC)如图1所示。共分析出24个羊毛甾烷型四环三萜类成分,鉴定了22个,化学结构如图2所示。
色谱峰的鉴定结果如表1所示,主要展示色谱峰保留时间、分子量、分子式、二级质谱碎片以及化合物名称等。根据化合物的结构不同,将识别到的色谱峰分为三类,包括羊毛甾-8-烯型三萜、羊毛甾-7,9(11)-二烯型三萜和其他羊毛甾三萜类型,通过不同类型化合物的结构特征来说明其鉴别过程。
表 1 茯苓中色谱峰的LC/ESI–MSn鉴定结果(1)Table 1. Characterization of the peaks in LC/ESI–MSn chromatogram of Poria cocos (1)色谱峰 保留时间 分子式 分子量 ESI+ (误差,mDa) ESI− (误差,mDa) 化合物名称 1 17.51 C30H48O5 488 [M+Na]+ 511.3328 (−6.6)
MSn: 511-493.3316 (C30H46O4, +2.8),
453.3319 (C30H44O3, −4.4)[M−H]−487.3402 (−2.7) Daedaleanic acid B[10] 2 19.15 C30H46O6 502 [M+Na]+ 525.3301 (+11.4) [M−H]−501.3164 (−5.8) Pinicolic acid F [9] 3 19.90 C31H48O5 500 MSn: 483.3468 (C31H46O4, −0.1), 465.3319 (C31H44O3, −4.4) [M−H]−499.3427 (−0.2) 29α-羟基去氢土莫酸
29α-Hydroxydehy-dropachymic acid[14]4 19.90 C30H48O6 504 [M+Na]+ 527.3331 (−1.2)
MSn: 527- 495.3077 (C29H44O5, +6.3), 437.2886 (C27H42O3, −14.0)[M−H]−503.3373 (−0.5) - 5 21.69 C31H48O6 516 [M+Na]+539.3047 (+6.8)
MSn: 539- 521.2809 (C31H46O5, −6.5), 507.2633 (C30H44O5, −4.6)[M−H]−515.3056 (+4.2) 5,8α-Dioxy-3β,16α-dihydroxylanosta-7,
24- dien-21-oic acid [18]6 21.97 C31H46O6 514 [M+H]+515.3331 (−3.6)
MSn: 515- 497.3360 (C31H44O5, +9.8), 443.2839
(C31H38O2, −10.6), 433.3267 (C30H40O2, +16.6), 425.2996
(C28H40O3, −5.4), 415.2933 (C30H38O, −6.2), 385.2397
(C24H32O4, +2.4), 377.2199 (C25H28O3, +8.8), 335.2455
(C24H30O, +8.6), 253.1655 (C18H20O, +6.8), 247.1670
(C16H22O2, −2.3), 229.1583 (C16H20O, −0.4), 201.1704 (C15H20, +6.6)[M−H]−513.3151 (−7.1)
MSn: 513- 483.3167 (C31H46O4, −5.6)5α,8α-过氧化去氢土莫酸
5α,8α-Peroxydehydrotumulosic acid[19]7 22.54 C31H46O5 498 [M+H]+ 499.3287 (−13.1)
MSn: 481.3308 ( (C31H44O4, −0.4), 463.3147 (C31H42O3, −6.0)[M−H]−497.3193 (−7.9)
MSn: 497- 403.2418 (C24H26O5, −7.2), 389.2435 (C27H34O2, −5.1),
371.2336(C27H32O, −4.4), 355.2131 (C26H28O, +6.4)6α-羟基猪苓酸C
6α-Hydroxypolyporenic acid C[11]8 23.69 C33H48O8 572 [M+H]+573.3312 (−11.1) [M−H]−571.3213 (−6.3) 3-(2-羟基乙酰氧基)- 5α,8α-过氧化去氢土莫酸
3-(2-hydroxyacetoxy)- 5α,
8α-peroxydehydrotumulosic acid [20]9 25.78 C31H46O5 498 [M+H]+ 499.3433 (+1.5)
MSn: 481.3408 ( (C31H44O4, +9.6)- 451.3147 (C30H42O3, −1.4),
435.2955 (C29H38O3, +6.2), 325.2132 (C22H28O2, −3.0),
299.2112 (C20H26O2, +10.6)[M−H]−497.3236 (−3.6) - 表 1 茯苓中色谱峰的LC/ESI–MSn鉴定结果(2)Table 1. Characterization of the peaks in LC/ESI–MSn chromatogram of Poria cocos (2)色谱峰 保留时间 分子式 分子量 ESI+ (误差,mDa) ESI- (误差,mDa) 化合物名称 10 28.42 C32H50O6 530 [M+Na]+553.3443 (−5.7)
MSn: 553- 535.3422 (C32H48O5, +2.8), 495.3437
(C30H48O4, −0.8), 477.3274 (C30H46O3, −6.5), 435.3364
(C28H44O2,+13.0), 353.2548 (C22H34O2, +9.7)[M−H]−529.3461 (−7.4) 3-乙酰氧基-16α,26-二羟基-羊毛甾-8,24-二烯-21-酸
3- Acetoxy-16α,26-dihydroxy-lanosta-8,24-dien-21-oic acid[10]11 29.01 C33H50O6 542 MSn: 525.3513 (C33H48O5, −6.2)- 507.3459
(C33H46O4, −1.0), 465.3365 (C31H44O3, +0.2),
447.3544 (C32H46O, −7.7)[M−H]−541.3460 (−7.5) 6α-羟基去氢茯苓酸
6α-Hydroxy-dehydropachymic acid[13]12 31.15 C31H48O4 484 [M+Na]+485.3592 (−3.3)
MSn: 485- 467.3492 (C31H46O3, −2.9), 449.3408 (C31H44O2, −0.6),
311.2388 (C22H30O, +1.9), 293.2259 (C22H28, −0.5)[M−H]−483.3413 (−6.7)
MSn: 483- 437.3393 (C30H46O2, −3.2), 421.2978 (C29H42O2, −13.4)3β,15α-二羟基-羊毛甾-7,9(11),24-三烯-21-酸
3β,15α-dihydroxylanosta-7,9(11),24-trien-21-oic acid[15]13 30.27 C31H50O4 486 [M+H]+ 487.3722 (−6.0)
MSn: 487- 469.3719 (C31H48O3, +4.3),
451.3553 (C31H46O2, −1.8), 343.2701
(C23H34O2, +6.9), 313.2464 (C22H32O, −6.2),
295.2405 (C22H30, −1.5)[M−H]−485.3570 (−6.6)
MSn: 483- 423.3371 (C29H44O2,+10.2)土莫酸
Tumulosic acid[10-11]14 31.74 C31H46O5 498 [M+H]+ 499.3368 (−5.0)
MSn: 499- 481.3292 (C31H44O4, −2.0), 463.3291 (C31H42O3, +8.4),
421.3024 (C29H40O2, −7.7), 325.2198 (C22H28O2, +3.8), 307.2102 (C22H26O, +4.6)[M−H]−497.3199 (−7.3)
MSn: 497- 425.2942 (C31H38O, +9.2)29-羟基猪苓酸 C
29-Hydroxypolyporenic acid C[16]15 33.26 C33H52O6 544 [M+H]+ 545.3733 (−10.4)
MSn: 545- 527.3696 (C33H50O5, −3.5), 451.3097 (C31H46O2, +2.6), 433.3267 (C31H44O, −19.8), 295.2433 (C22H30,+2.3)[M−H]−543.3623 (−6.8)
MSn: 543- 467.3227 (C31H48O3, +9.6)25-羟基茯苓酸
25-Hydroxypachimic acid[11]16 33.57 C31H46O4 482 [M+H]+ 483.3420 (−4.9)
MSn: 483- 465.3327 (C31H44O3, −3.6), 447.3246 (C31H42O2, −1.2), 309.2214 (C22H28O, +0.1)[M−H]−481.3266 (−5.7)
MSn: 481- 311.1817 (C21H28O2, −14.0)猪苓酸 C
Polyporenic acid C[11]17 34.28 C31H48O4 484 [M+H]+ 485.33637 (+1.2)
MSn: 483- 467.3486 (C31H46O3, −3.4), 449.3532 (C31H44O2, +11.8), 311.2343 (C22H30O, −2.6), 293.2330 (C22H28,+6.6)[M−H]−483.3378 (−10.2)
MSn:3-表去氢土莫酸
3-epi-Dehydrotumulosic acid acid[11]表 1 茯苓中色谱峰的LC/ESI–MSn鉴定结果(3)Table 1. Characterization of the peaks in LC/ESI–MSn chromatogram of Poria cocos (3)色谱峰 保留时间 分子式 分子量 ESI+ (误差,mDa) ESI− (误差,mDa) 化合物名称 18 34.89 C31H48O4 484 [M+H]+ 485.3589 (−3.6)
MSn: 483- 467.3526 (C31H46O3, +0.6), 449.3377 (C31H44O2, −3.7), 311.2408 (C22H30O, +3.9), 293.2265 (C22H28, 0.1)[M−H]−483.3406 (−7.4)
MSn: 481- 337.2465 (C24H34O, −7.2)去氢土莫酸
Dehydrotumulosic acid[17]19 38.63 C33H50O5 526 [M+H]+ 527.3671(−6.0)
MSn: 527- 509.3564 (C33H48O4, −6.1), 449.3398 (C31H44O2, −1.6), 293.2240 (C22H28, −2.4)[M−H]−525.3493 (−9.2)
MSn:3-表去氢茯苓酸
3-epi-Dehydropachymic acid[14]20 39.64 C32H50O5 514 [M+H]+ 515.3708(−2.3)
MSn: 515- 497.3555 (C32H46O4, −7.0), 437.3398 (C30H44O2, −1.6), 295.2422 (C22H30,+0.2)[M−H]−513.3532 (−5.3) 3-O-乙酰基-16α-羟基松苓酸
3-O-Acetyl-16α-hydroxytrametenolic acid[12]21 40.64 C33H50O5 526 [M+H]+ 527.3697 (−3.4)
MSn: 527- 509.3594 (C33H48O4, −3.1), 449.3446 (C31H44O2, +3.2), 353.2501 (C24H32O2, +2.6),
293.2254 (C22H28, −1.0)[M−H]−525.3511 (−7.4)
MSn: 525- 479.3471 (C32H48O3, −6.0), 465.3351 (C31H46O3, −2.3), 355.2295 (C23H32O3, +1.6),去氢茯苓酸dehydropachymic acid[11] 22 41.87 C33H52O5 528 [M+H]+ 529.3820 (−6.8)
MSn: 529- 511.3735 (C33H50O4, −4.7), 451.3599 (C31H46O2, −3.0), 295.2419 (C22H30, −0.1)[M−H]−527.3666 (−7.6)
MSn: 527- 465.3330 (C31H46O3, −4.4)茯苓酸
Pachymic acid[10-11]23 45.90 C30H46O3 454 [M+H]+ 455.3532 (+1.2)
MSn: 455- 437.3457 (C30H44O2, +4.3), 311.2370 (C22H30O, +0.1), 295.2381 (C22H30, −3.9)[M−H]−453.3342 (−3.2) 3-羟基-羊毛甾-7,9(11),24-三烯-21酸
Dehydrotrametenolic acid[12]24 47.30 C30H48O3 456 [M+H]+ 457.3663 (−1.3)
MSn: 457- 439.3502 (C30H46O2, −6.9), 313.2445 (C22H32O, −8.1), 295.2463 (C22H30, +4.3)[M−H]−455.3471 (−6.0) 3-氢化松苓酸
trametenolic acid[13]A型羊毛甾-8-烯型三萜(1,10,13,15,20,22,24)主要结构特征核心结构四元环中的C-8和C-9位之间为双键,C-3位均有羟基或乙酰氧基取代;除了化合物24,C-16位均有α位羟基取代;化合物1中C-17侧链的C-24是羰基取代,其他化合物C-24位均为双键。对于A型化合物而言,较容易失去C-17位支链,且同时失去一个H2O,形成含[M+H-C9H16O2-H2O]+的碎片离子。以色谱峰13为例,在正负离子模式下容易检测到其[M+H]+和 [M-H]-离子,分别为m/z 487.3722和485.3570,预测其分子式为C31H50O4,以m/z 487.3722为母离子进行轰击,检测到5个碎片,分别为m/z 469.3719 (C31H48O3,+4.3),451.3553 (C31H46O2,−1.8),343.2701 (C23H34O2,+6.9),313.2464 (C22H32O,−6.2),295.2405 (C22H30,−1.5),推测其分别对应于[M+H-H2O]+,[M+H-2H2O]+,[M+H-CO2-2H2O-4CH4]+,[M+H-C9H16O2-H2O]+,[M+H-C9H16O2-2H2O]+;通过查阅文献比对,茯苓中化合物土莫酸(Tumulosic acid)满足该裂解条件,因此将色谱峰13鉴定为土莫酸(Tumulosic acid)。色谱峰22的[M+H]+和[M-H]-离子分别为m/z 529.3820和527.3666,预测其分子式为C33H52O5,检测到丢失H2O离子碎片m/z 511.3735 (C33H50O4,−4.7),进一步裂解还形成了451.3599 (C31H46O2,−3.0),295.2419 (C22H30,−0.1)等碎片离子,分别对应[M+H-CH3COOH-H2O]+,[M+H-CH3COOH-H2O-C9H16O2]+,与茯苓酸(Pachymic acid)的裂解方式相符,因此将色谱峰22鉴定为茯苓酸(Pachymic acid)。图3展示了主要色谱峰的质谱图。
B型羊毛甾-7,9(11)-二烯型三萜(2,3,7,11~12,14,16~19,21,23)主要结构特征为核心结构四元环中的C-7、C-8位和C-9、C-11位之间形成异环双键,C-3位均有羟基、乙酰氧基或氧代取代,与A型相似,较容易失去C-17位支链,且同时失去一分子H2O,形成含[M+H-C9H16O2-H2O]+的碎片离子。色谱峰14检测到其[M+H]+峰为m/z 499.3368,因此预测其分子式为C31H46O5,m/z 481.3292 (C31H44O4,−2.0),463.3291 (C31H42O3,+8.4)是连续失去二个H2O产生的碎片,m/z 421.3024 (C29H40O2,−7.7)对应的是[M+H-H2O-CO2-CH4]+碎片离子,m/z 325.2198 (C22H28O2,+3.8)为失去C-17侧链和一个H2O生成的[M+H-C9H16O2-H2O]+的碎片离子,m/z 307.2102 (C22H26O,+4.6)为失去C-17侧链和二个H2O生成的[M+H-C9H16O2-2H2O]+的碎片离子,这些裂解行为与29-羟基猪苓酸 C (29-Hydroxypolyporenic acid C)相符,因此色谱峰14被鉴定为29-羟基猪苓酸 C (29-Hydroxypolyporenic acid C)。图3展示了主要色谱峰的质谱图。
C型其他羊毛甾三萜类型(5,6,8)的主要结构特征为C-5位和C-8位通过过氧键链接,因此该类型化合物的主要质谱裂解特征行为是C-5位和C-8位过氧基的丢失,色谱峰5和6分别检测到失去过氧基的碎片离子m/z 507.2633 (C30H44O5,−4.6)和385.2397 (C24H32O4,+2.4)、229.1583 (C16H20O,−0.4);以及侧链羧基先裂解,再进一步裂解C-17侧链,形成丰富的离子碎片,并结合数据库搜索,可将其结构初步鉴定。
3. 讨论
我国茯苓人工栽培历史悠久,且有广泛的茯苓资源,但是茯苓的不同品种和不同地域是导致其有效成分差异的主要原因。UPLC-IT-TOF/MS结合了液相的快速分离能力和飞行时间质谱的准确结构鉴定功能的优点,具有高效分离能力、高分辨和高灵敏度的特性,故本文利用UPLC-IT-TOF/MS技术分析鉴定云南大理的茯苓95%乙醇提取物的化学成分,发现其主要化合物为羊毛甾烷四环三萜,结构类型包括A型羊毛甾-8-烯型三萜(1,10,13,15,20,22,24)[9-13]、B型羊毛甾-7,9(11)-二烯型三萜(2,3,7,11~12,14,16~19,21,23)[11-17]和C型其他羊毛甾三萜类型。该类化合物在ESI源正离子模式响应较好,较易检测到[M+H]+和[M+Na]+离子,其二级质谱碎片较为丰富。通过以[M+H]+或[M+Na]+离子作为母离子进行轰击,其主要裂解途径为失去核心结构四元环的羟基或者乙酰基,形成碎片同时C-17位侧链也容易失去,C-17位侧链含有羧基的化合物较容易脱羧再裂解C-17位侧链,形成含有22个碳原子的离子碎片,并通过将化合物的结构进行简单分离,总结其质谱裂解规律。
三萜类化合物是茯苓的主要化学成分之一,其已被报道具有免疫调节、抗炎及抗肿瘤等多种生物功效。研究结果显示,产自云南大理的茯苓95%乙醇提取物的主要成分是羊毛甾烷四环三萜类化合物,化合物结构特征是其他羊毛甾三萜类型(5,6,8)的C-5位和C-8位均通过过氧键链接,为进一步研究该种茯苓的化学成分和生物活性提供了科学依据。
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表 1 3组EnMSCs细胞周期比例(%)比较(n = 3)
Table 1. The EnMSCs cell cycle proportion comparison in the three groups (n = 3)
组别 G1/G0(%) S(%) G2/M(%) 对照组 70.04 ± 2.67 25.49 ± 2.68 4.47 ± 1.04 2%PRP组 54.63 ± 4.23**▲ 30.66 ± 2.85*▲ 14.71 ± 2.72**▲ 2%PRP+ LY294002组 72.16 ± 4.26 17.45 ± 3.49** 10.39 ± 2.84** 与对照组比较,*P < 0.05, **P < 0.01;与2%PRP+ LY294002组比较, ▲P < 0.01。 -
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