西黄丸含药血清经Wnt信号转导通路细胞周期蛋白D1调控人肺癌干细胞增殖的机制

Containing Xihuang Pill Drug Serum Regulates Growth of Lung Cancer Stem Cells by Controlling Cyclin D1 of Wnt Signaling Pathway

目的:研究西黄丸含药血清经Wnt信号转导通路关键蛋白细胞周期蛋白D1(Cyclin D1)调控人肺癌干细胞增殖的机制。方法:10只日本大耳白兔随机分为5组,西黄丸浸出液高、中、低剂量组按32,16,8 g·kg-1·d-1ig,空白组给以等量生理盐水ig;环磷酰胺(CTX)组按7.5 mg·kg-1·d-1ip,各组连续给药3 d后腹主动脉采血,离心,获得含药血清。体外培养人肺腺癌细胞系(LAC),应用流式细胞荧光激活(FACS)技术,筛选鉴定具干细胞特性的SP细胞(side population cells)。50只C57BL/6小鼠分西黄丸高、中、低剂量组,CTX组,模型组5组,SP细胞以培养基稀释调整瘤细胞密度1×107/mL,于每只小鼠右腋皮下接种0.2 mL瘤细胞悬液,每组分别给予西黄丸药液32,16,8 g·kg-1·d-1ig,等量生理盐水ig和CTX7.5 mg·kg-1·d-1,ip,连续14 d,处死小鼠,剥取瘤块称瘤重,并计算抑瘤率。SP细胞密度调至2×106/mL,分别种入培养瓶,设西黄丸高、中、低剂量组,CTX组,空白组5组,分别加入如前所述制备的各组血清(血清、培养液体积比30%),培养24 h后上流式细胞仪检测各组细胞周期。按同样方法制备5组SP细胞,加入各组含药血清培养24 h后用逆转录-聚合酶链反应(RT-PCR)法和蛋白印迹(Western blot)法分别检测Cyclin D1 mRNA及蛋白表达。结果:与模型组相比,西黄丸高、中剂量组和CTX组瘤重明显降低(P<0.01);与空白组30%血清比较,西黄丸高、中剂量30%含药血清组和CTX 30%含药血清组G1期细胞数量比例明显增高,S期、G2期细胞数量比例明显减少(P<0.05);与空白组30%血清比较,西黄丸高、中剂量30%含药血清组及CTX 30%含药血清组Cyclin D1 mRNA及蛋白表达均明显低于空白血清组(P<0.05,P<0.01)。结论:西黄丸可通过抑制Wnt信号通路中关键蛋白Cyclin D1表达,阻止肺癌干细胞从G1向S期转化,从而达到抑制肺癌细胞增殖的作用。

Objective：To study the mechanism of regulating the growth of the lung cancer stem cells containing by Xihuang pill drug serum.Method：Ten rabbits were divided 5 groups,the therapeutic dose of 32,16,8 g ·kg-1·d-1 ig of Xihuang pill liquid and 7.5 mg ·kg-1 ·d-1 ip of cyclophosphamide （CTX） were given.Drug serum of rabbits were prepared.Side population cells （SP cells） of the human lung adenocarcinoma cells （LAC） were sorted by Fluorescence-activated cell sorting assay （FACS）.Fifty C57BL/6 mice were divided model group,Xihuang pill low,middle and high-dose group,and the CTX group.SP cells were regulated to 1 × 107/mLand sc,Xihuang pill liquid （32,16,8 g ·kg-1·d-1,ig） and CTX （7.5 mg ·kg-1·d-1,ip） were given lasted 2 weeks.Then the tumors were weighted for calculating the tumor growth inhibition rate.SP cells were regulated to 2 × 106/mL and put into 5 culture bottles,divided into control group,Xihuang pill low,middle and high-dose groups,and the CTX group.Drug serum of groups were put into corresponding SP cells （drug serum occupies 70％ of cell medium）.The cell cycle was analyzed by flow cytometry （FCM）.In the same way,SP cells were affected by drug serum of groups and the expression of Cyclin D1 mRNA and protein was detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot.Result：The Xihuang pill middle,high-dose group and CTX group significantly inhibited tumors of all groups,compared with the control group （P ＜ 0.01）.Cells in G1 phase of the Xihuang pill middle,high-dose group and CTX group increased,cells in G2 phase and S phase of the Xihuang pill middle,high-dose group and CTX group decreased （P ＜ 0.05）.The Xihuang pill middle and high-dose group could regulate down the expression of Cyclin D1 mRNA and protein compared with the model group （P ＜0.05,P ＜0.01）.Conclusion：Xihuang pill could down-regulate the expression of Cyclin D1of lung cancer stem cells.It could prevent their G1 period turning into S period,so as to block the growth of lung cancer cells.