用血清药理学方法研究灵芝浸膏GLE的抗肿瘤作用机制
作者:张群豪 於东晖 林志彬
单位:北京大学基础医学院药理学系,北京 100083
关键词:血清药理学;灵芝浸膏;细胞凋亡;肿瘤坏死因子α;干扰素γ
北京医科大学学报000305 [摘 要] 目的:探讨灵芝浸膏(Ganoderma lucidum Extract, GLE)的抗肿瘤作用及其机制。方法:采用血清药理学与体内外抑瘤实验相结合方法。MTT法测肿瘤细胞增殖程度,流式细胞仪测细胞凋亡,同时采用生物法测TNF-α,ELISA测定IFN-γ,并用RT-PCR方法检测mRNA表达。结果:(1)GLE(5、10、20 g.kg-1)灌胃显著抑制小鼠移植性肉瘤S180的生长;(2)GLE直接加入S180细胞体外培养不能抑制其生长,亦无诱导其凋亡的作用;(3)GLE血清显著抑制S180细胞生长并诱导其凋亡,GLE血清中TNF-α、INF-γ水平显著升高,而且GLE显著促进其mRNA的表达。结论:GLE无直接的抗肿瘤作用,其抗肿瘤作用是通过促进TNF-α和INF-γ mRNA表达及其生成而实现的。
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[中图分类号] R282.7105 [文献标识码] A
[文章编号] 1000-1530(2000)03-0210-04
Study on the antitumor mechanism of Ganoderma lucidum Extract(GLE)
by serologic pharmacological method
ZHANG Qun-Hao, YU Dong-Hui, LIN Zhi-Bin
(Department of Pharmacology, School of Basical Medical Sciences,Peking University, Beijing 100083, China)
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ABSTRACT Objective: To study the antitumor activity and the mechanism of Ganoderma lucidum Extract (GLE) Methods: Serologic pharmacological method, and both in vivo and in vitro antitumor experiments were used in the studies. Proliferation of tumor cells was detected by MTT, TNF-α was detected by biological assay, and INF-γ by ELISA. The level of mRNA was detected with the method of RT-PCR. Results: (1) GLE (5, 10, 20 g.kg-1, i.g.) inhibited the growth of implanted sarcoma 180 in vivo significantly and dose-dependently. (2) GLE directly adding to their cultured medium neither induced sarcoma 180 apoptosis nor restrained its proliferation in vitro. (3)GLE-treated serum significantly induced sarcoma 180 apoptosis and inhibited its proliferation. The TNF-α and INF-γ raised in the cultured medium treated with GLE-treated serum, GLE (5, 10, 20 g.kg-1, i.g.) significantly increased TNF-α and INF-γ mRNA expression in mice. Conclusion: The anti-tumor activity of GLE was derived from promoting mRNA expression of TNF-α and INF-γ, resulting in the production of TNF-α and INF-γ.
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KEY WORDS Serologic pharmacology; Ganoderma lucidum Extract; Apoptosis; TNF-α; INF-γ▲
灵芝浸膏(Ganoderma lucidum Extract,GLE)系从灵芝[赤芝,Ganoderma lucidum (Leyss.ex Fr.) Karst.]子实体中提取的水溶部分。一系列研究证明灵芝及其所含多糖具有抗肿瘤作用,并推测其抗肿瘤作用与其免疫增强作用有关[1],但关于灵芝的抗肿瘤机制至今尚未完全阐明。本文采用血清药理学方法[2],从整体、细胞、分子水平,体内、外实验相结合,观察了GLE的抗肿瘤作用及其机制。
1 材料与方法
1.1 药品与材料
GLE:由天安制药(厦门)有限公司提供,系灵芝子实体热水提取物,每10 g生药提取1 g GLE。
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依托泊苷(Etoposide, VP-16):连云港制药厂。环磷酰胺(Cyclophosphamide, CY):上海华联制药公司。
L929细胞:购自北京大学基础医学院免疫学系。HL-60、S180细胞:购自北京肿瘤研究所。小鼠ELISA INF-γ试剂盒:美国Endogen公司产品。TRIZOL试剂(RNA提取剂):Gibco BRL公司产品。RT-PCR试剂盒:美国Promega公司产品。
1.2 实验动物
BALB/c近交系小鼠,体重18~22 g,合格证号01-3046,购自我校实验动物部。
1.3 试验方法
肿瘤细胞体外增殖试验:采用MTT方法[3],小鼠S180肉瘤的抑瘤试验采用常规方法[4],肿瘤细胞凋亡采用流式细胞仪测定[5]。
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GLE血清制备:GLE(20、10、5 g.kg-1,按其生药含量折算)分别溶于0.4 ml蒸馏水中,正常对照组用生理盐水0.4 ml,以上过程每天1次,连续灌胃10 d,于最后一次灌胃后1 h,摘眼球取血,无菌分离血清,用0.45 μm微孔滤膜过滤除菌,-20℃保存备用。
细胞因子检测:收集GLE血清,用结晶紫染料摄入法[6]测定培养上清液中TNF-α的含量,用小鼠INF-γ ELISA试剂盒测定INF-γ含量。
RT-PCR方法:正常健康BALB/c小鼠颈椎脱臼处死,常规制备BALB/c小鼠腹腔巨噬细胞(peritoneal macrophages, PM)和脾淋巴细胞,分别于含PM和脾淋巴细胞的细胞培养瓶中加入不同浓度的药物和LPS(5 mg.L-1)和ConA(2.5 mg.L-1),培养48 h后用TRIZOL试剂提取细胞总RNA,紫外可见光分光光度计对RNA样品定量。RT-PCR检测TNF-α、INF-γ及β-actin mRNA的表达,扩增反应条件参照试剂盒操作手册,反应体系为50 μl,反转录与PCR一步完成。引物根据文献资料设计[7,8],由中科院微生物所合成。序列为:TNF-α产物422 bp; INF-γ产物220 bp;β-actin产物478 bp。
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取RT-PCR产物10 μl,加入2 μl上样缓冲液混合,加样于15 g.L-1的琼脂糖凝胶,分别以385、485、585 bp和154、220、344 bp的DNA片段为DNA marker,0.5×TBE为电泳缓冲液,5 V/cm恒压电泳2 h检测RT-PCR产物,紫外透射仪观察并照像。RT-PCR结果用图像分析仪对胶片上每一泳带进行光密度扫描定量,结果用各组的光密度值与β-actin的光密度之比表示。RT-PCR结果以3次以上实验测定值与相应对照的比值的±s表示。
1.4 统计学方法
各组间的显著性检验均采用Duncan's test, ANOVA,SAS 6.04。
2 结果
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2.1 灌胃GLE对BALB/c小鼠移植性S180肉瘤的抑制作用
给小鼠皮下接种S180后,连续灌胃GLE (5、10、20 g.kg-1) 10 d可以显著抑制S180的生长,并呈剂量依赖关系,其抑瘤率分别为22.77%、41.58%和60.89%(表1),与生理盐水组比较,差异有显著性。
表1 灌胃GLE对BALB/c小鼠移植性S180肉瘤生长的影响(n=10,±s)
Table 1 Effect of GLE on growth of implanted Sarcoma 180
in BALB/c mice (n=10, ±s) Groups
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Dose
(i.g.×10 d)
m(body)/g
m(tumor)
/mg
Inhibitory rate
(%)
before
difference
NS
-
23.61±1.40
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10.32±2.60
2.02±0.16
GLE(g.kg-1)
5
22.86±1.78
6.24±1.50*
1.56±0.50
22.77
10
23.16±1.53
7.01±1.43**
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1.18±0.41*
41.58
20
23.67±1.24
6.52±1.36**
0.79±0.45**
60.89
CY(mg.kg-1)
20
23.84±1.43
5.84±1.68**
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0.44±0.54**
78.02
m, mass; NS, normal saline; GLE, Ganoderma lucidum Extract; CY, cyclophosphamide; *P<0.05, **P<0.01 vs NS.
2.2 GLE、GLE血清对S180细胞体外增殖的影响
表2显示,直接加GLE至体外培养的S180细胞培养基中,GLE对S180细胞的生长并无抑制作用。而给小鼠灌服GLE(5、10、20 g.kg-1)后取其血清加至体外培养的S180细胞中,则对S180细胞的生长有显著的抑制作用,并呈剂量依赖关系,与RPMI1640对照组及生理盐水血清对照组相比,差异有显著性。
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表2 GLE、GLE血清对S180细胞体外增殖(n=8)和
凋亡(n=3)的影响(±s)
Table 2 Effect of GLE、GLE-treated serum on proliferation(n=8)
and apoptosis (n=3) of Sarcoma 180 cells in vitro(±s) Groups
Dose
D(570 nm)
Apoptotic cell/%
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RPMI1640
-
0.439±0.037
0.57±0.22
NS-treated serum
-
0.425±0.029
0.81±0.24
GLE-treated
5
0.354±0.030*#
29.20±4.21**##
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serum(g.kg-1)
10
0.326±0.028*#
31.43±5.17**##
20
0.279±0.041**##
32.55±5.12**##
ρ(GLE)/g.L-1
50
0.424±0.037
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0.64±0.30
100
0.455±0.071
0.75±0.41
200
0.476±0.144
0.70±0.36
ρ(VP-16)/mg.L-1
80
0.242±0.092**##
69.61±8.04**
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D, optical density; ρ, mass concentration; GLE, Ganoderma lucidum Extract; VP-16, Etoposide; *P<0.05, **P<0.01 vs RPMI1640; #P<0.05, ##P<0.01 vs NS-treated serum.
2.3 GLE、GLE血清对S180细胞凋亡的影响
表2所示,GLE(50、100、200 mg.L-1)直接加药与S180细胞共同培养71 h,与RPMI1640对照组相似,未见有诱导S180细胞凋亡作用。而灌胃GLE(5、10、20 g.kg-1)小鼠血清则可显著诱导S180细胞发生凋亡,与RPMI1640对照组及生理盐水灌胃后的小鼠血清对照组比较差异均有显著性。
2.4 GLE血清中TNF-α、INF-γ的检测
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表3结果显示,GLE血清中TNF-α活性的INF-γ含量,随灌胃GLE的剂量提高而增强,与灌胃生理盐水血清对照组比较差异均有显著性。
表3 GLE血清中TNF-α(n=8)活性及INF-γ(n=3)的检测(±s)
Table 3 Defect of TNF-α (n=8) activity and content
of INF-γ (n=3) in GLE-treated serum(±s) Groups
Dose
D(570 nm)
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ρ(INF-γ)/ng.L-1
Control
-
0.452±0.145
42.58± 5.16
GLE-treated
5
0.375±0.117*
177.15±16.20*
serum(g.kg-1)
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10
0.302±0.219**
256.30±14.34**
20
0.271±0.052**
345.28±18.24**
ρ(TNF-α)/mg.L-1
25
0.246±0.105**
D, optical density; ρ, mass concentration; *P<0.05 vs control (NS-treated serum); **P<0.01 vs control.
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2.5 灌胃GLE对小鼠PM TNF-α和脾细胞INF-γ mRNA表达的影响
图1、2和表4显示,灌服GLE(5、10、20 g.kg-1)的小鼠腹腔巨噬细胞TNF-α mRNA和脾细胞INF-γ mRNA表达均明显高于对照组,其辉度扫描的表达量经统计学检验,与灌胃生理盐水血清对照组比较差异有显著性。
Lane A, DNA marker; Lane B, D, F, H, TNF-α expression with different drug treated cell RNA; Lane C, E, G, I, β-actin expression with different drug treated cell RNA; B, C, 1640; D, E, GLE 5 g.kg-1; F, G, GLE 10 g.kg-1; H, I, GLE 20 g.kg-1.
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图1 GLE整体给药对小鼠PM TNF-α mRNA的影响
Figure 1 Effect of various dose of GLE on expression of murine PM TNF-α mRNA. Mice were treated with GLE (5, 10, 20 g.kg-1) once a day for 10 days. Mice were killed and PM was collected. Total cellular RNA was isolated and RT-PCR product was separated by 1.5% agarose gel electrophoresis.
Lane A, DNA marker; Lane B, D, F, H, INF-γ expression with different drug treated cell RNA; Lane C, E, G, I, β-actin expression with different drug treated cell RNA; B, C, 1640; D, E, GLE 5 g.kg-1; F, G, GLE 10 g.kg-1; H, I, GLE 20 g.kg-1.
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图2 GLE整体给药对小鼠脾细胞INF-γ mRNA的影响
Figure 2 Effect of various dose of GLE on expression of murine spleen cell INF-γ mRNA. Mice were treated with GLE (5, 10, 20 g.kg-1) once a day for 10 days. Mice were killed and spleen was removed. Total cellular RNA was isolated and RT-PCR product was separated by 1.5% agarose gel electrophoresis.
3 讨论
为了深入探讨灵芝的抗肿瘤作用机制,我们给移植S180肉瘤的BALB/c小鼠灌胃GLE,结果与文献报道一致,GLE 5、10、20 g.kg-1均有显著抑制小鼠移植性S180肉瘤生长的作用,抑制率分别达到22.77%、41.58%、60.89%。基于以上的实验结果,我们把GLE(50~200 g.L-1)直接加到体外培养的S180细胞中,结果GLE对S180细胞无抑制作用。流式细胞仪分析亦未发现有诱导S180肿瘤细胞凋亡的作用。然而,当我们按整体实验的剂量给小鼠灌服GLE 5、10、20 g.kg-1连续10 d后,取其血清加至S180细胞体外培养,结果却发现3个剂量组均有显著抑制S180细胞增殖的作用。流式细胞仪检测也发现有显著诱导S180细胞凋亡的作用。而灌胃生理盐水的小鼠血清则未抑制肿瘤细胞增殖,亦不能促其凋亡。
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表4 GLE整体给药对TNF-α和INF-γ mRNA
表达的影响(n=3, ±s)
Table 4 Effect of GLE on TNF-α and INF-γ mRNA expression
in mice (n=3, ±s) Groups
Dose
(g.kg-1, i.g.)
mRNA
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(TNF-α/β-actin)
(INF-γ/β-actin)
Control
-
0.124±0.023
0.100±0.030
GLE
5
0.599±0.116*
0.443±0.113*
10
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0.858±0.075**
0.653±0.122**
20
1.191±0.240**
0.787±0.098**
*P<0.05, **P<0.01 vs control (NS-treated serum).
为什么GLE血清与GLE不同,前者在体外可抑制肿瘤细胞并促其凋亡?为此,我们分别采用生物学活性检测法和ELISA法检测了GLE血清中TNF-α的活性和INF-γ的含量。结果显示,GLE血清中TNF-α的活性和INF-γ的含量均呈剂量依赖性升高。进一步采用RT-PCR法检测此二细胞因子mRNA表达的结果也与上述生物法和ELISA法的结果一致。已知TNF-α和INF-γ在抗肿瘤及诱导肿瘤细胞凋亡中具重要作用,TNF-α对肿瘤细胞有细胞毒和生长抑制作用,可诱导许多不同来源肿瘤细胞凋亡[9~12]。INF-γ有抗肿瘤细胞增生作用,并且可与TNF-α协同增强诱导肿瘤细胞凋亡[13~15]。故以上实验结果表明:GLE本身无直接抑瘤及诱导肿瘤细胞凋亡的作用,GLE通过口服吸收后,促进 PMTNF-α mRNA、脾细胞INF-γ mRNA的表达。刺激诱导机体产生TNF-α和INF-γ,进而达到诱导肿瘤细胞凋亡,抑制肿瘤细胞增殖,从而达到整体的抗肿瘤效果。
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由于受条件限制,本研究中未对含GLE血清进行进一步化学分析,故含GLE血清诱生TNF-α和INF-γ的作用究竟是GLE本身或是其在体内代谢产物的作用尚不清,值得深入研究。■
基金项目:中国博士后基金资助课题;
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收稿日期:2000-03-22, 百拇医药
单位:北京大学基础医学院药理学系,北京 100083
关键词:血清药理学;灵芝浸膏;细胞凋亡;肿瘤坏死因子α;干扰素γ
北京医科大学学报000305 [摘 要] 目的:探讨灵芝浸膏(Ganoderma lucidum Extract, GLE)的抗肿瘤作用及其机制。方法:采用血清药理学与体内外抑瘤实验相结合方法。MTT法测肿瘤细胞增殖程度,流式细胞仪测细胞凋亡,同时采用生物法测TNF-α,ELISA测定IFN-γ,并用RT-PCR方法检测mRNA表达。结果:(1)GLE(5、10、20 g.kg-1)灌胃显著抑制小鼠移植性肉瘤S180的生长;(2)GLE直接加入S180细胞体外培养不能抑制其生长,亦无诱导其凋亡的作用;(3)GLE血清显著抑制S180细胞生长并诱导其凋亡,GLE血清中TNF-α、INF-γ水平显著升高,而且GLE显著促进其mRNA的表达。结论:GLE无直接的抗肿瘤作用,其抗肿瘤作用是通过促进TNF-α和INF-γ mRNA表达及其生成而实现的。
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[中图分类号] R282.7105 [文献标识码] A
[文章编号] 1000-1530(2000)03-0210-04
Study on the antitumor mechanism of Ganoderma lucidum Extract(GLE)
by serologic pharmacological method
ZHANG Qun-Hao, YU Dong-Hui, LIN Zhi-Bin
(Department of Pharmacology, School of Basical Medical Sciences,Peking University, Beijing 100083, China)
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ABSTRACT Objective: To study the antitumor activity and the mechanism of Ganoderma lucidum Extract (GLE) Methods: Serologic pharmacological method, and both in vivo and in vitro antitumor experiments were used in the studies. Proliferation of tumor cells was detected by MTT, TNF-α was detected by biological assay, and INF-γ by ELISA. The level of mRNA was detected with the method of RT-PCR. Results: (1) GLE (5, 10, 20 g.kg-1, i.g.) inhibited the growth of implanted sarcoma 180 in vivo significantly and dose-dependently. (2) GLE directly adding to their cultured medium neither induced sarcoma 180 apoptosis nor restrained its proliferation in vitro. (3)GLE-treated serum significantly induced sarcoma 180 apoptosis and inhibited its proliferation. The TNF-α and INF-γ raised in the cultured medium treated with GLE-treated serum, GLE (5, 10, 20 g.kg-1, i.g.) significantly increased TNF-α and INF-γ mRNA expression in mice. Conclusion: The anti-tumor activity of GLE was derived from promoting mRNA expression of TNF-α and INF-γ, resulting in the production of TNF-α and INF-γ.
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KEY WORDS Serologic pharmacology; Ganoderma lucidum Extract; Apoptosis; TNF-α; INF-γ▲
灵芝浸膏(Ganoderma lucidum Extract,GLE)系从灵芝[赤芝,Ganoderma lucidum (Leyss.ex Fr.) Karst.]子实体中提取的水溶部分。一系列研究证明灵芝及其所含多糖具有抗肿瘤作用,并推测其抗肿瘤作用与其免疫增强作用有关[1],但关于灵芝的抗肿瘤机制至今尚未完全阐明。本文采用血清药理学方法[2],从整体、细胞、分子水平,体内、外实验相结合,观察了GLE的抗肿瘤作用及其机制。
1 材料与方法
1.1 药品与材料
GLE:由天安制药(厦门)有限公司提供,系灵芝子实体热水提取物,每10 g生药提取1 g GLE。
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依托泊苷(Etoposide, VP-16):连云港制药厂。环磷酰胺(Cyclophosphamide, CY):上海华联制药公司。
L929细胞:购自北京大学基础医学院免疫学系。HL-60、S180细胞:购自北京肿瘤研究所。小鼠ELISA INF-γ试剂盒:美国Endogen公司产品。TRIZOL试剂(RNA提取剂):Gibco BRL公司产品。RT-PCR试剂盒:美国Promega公司产品。
1.2 实验动物
BALB/c近交系小鼠,体重18~22 g,合格证号01-3046,购自我校实验动物部。
1.3 试验方法
肿瘤细胞体外增殖试验:采用MTT方法[3],小鼠S180肉瘤的抑瘤试验采用常规方法[4],肿瘤细胞凋亡采用流式细胞仪测定[5]。
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GLE血清制备:GLE(20、10、5 g.kg-1,按其生药含量折算)分别溶于0.4 ml蒸馏水中,正常对照组用生理盐水0.4 ml,以上过程每天1次,连续灌胃10 d,于最后一次灌胃后1 h,摘眼球取血,无菌分离血清,用0.45 μm微孔滤膜过滤除菌,-20℃保存备用。
细胞因子检测:收集GLE血清,用结晶紫染料摄入法[6]测定培养上清液中TNF-α的含量,用小鼠INF-γ ELISA试剂盒测定INF-γ含量。
RT-PCR方法:正常健康BALB/c小鼠颈椎脱臼处死,常规制备BALB/c小鼠腹腔巨噬细胞(peritoneal macrophages, PM)和脾淋巴细胞,分别于含PM和脾淋巴细胞的细胞培养瓶中加入不同浓度的药物和LPS(5 mg.L-1)和ConA(2.5 mg.L-1),培养48 h后用TRIZOL试剂提取细胞总RNA,紫外可见光分光光度计对RNA样品定量。RT-PCR检测TNF-α、INF-γ及β-actin mRNA的表达,扩增反应条件参照试剂盒操作手册,反应体系为50 μl,反转录与PCR一步完成。引物根据文献资料设计[7,8],由中科院微生物所合成。序列为:TNF-α产物422 bp; INF-γ产物220 bp;β-actin产物478 bp。
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取RT-PCR产物10 μl,加入2 μl上样缓冲液混合,加样于15 g.L-1的琼脂糖凝胶,分别以385、485、585 bp和154、220、344 bp的DNA片段为DNA marker,0.5×TBE为电泳缓冲液,5 V/cm恒压电泳2 h检测RT-PCR产物,紫外透射仪观察并照像。RT-PCR结果用图像分析仪对胶片上每一泳带进行光密度扫描定量,结果用各组的光密度值与β-actin的光密度之比表示。RT-PCR结果以3次以上实验测定值与相应对照的比值的±s表示。
1.4 统计学方法
各组间的显著性检验均采用Duncan's test, ANOVA,SAS 6.04。
2 结果
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2.1 灌胃GLE对BALB/c小鼠移植性S180肉瘤的抑制作用
给小鼠皮下接种S180后,连续灌胃GLE (5、10、20 g.kg-1) 10 d可以显著抑制S180的生长,并呈剂量依赖关系,其抑瘤率分别为22.77%、41.58%和60.89%(表1),与生理盐水组比较,差异有显著性。
表1 灌胃GLE对BALB/c小鼠移植性S180肉瘤生长的影响(n=10,±s)
Table 1 Effect of GLE on growth of implanted Sarcoma 180
in BALB/c mice (n=10, ±s) Groups
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Dose
(i.g.×10 d)
m(body)/g
m(tumor)
/mg
Inhibitory rate
(%)
before
difference
NS
-
23.61±1.40
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10.32±2.60
2.02±0.16
GLE(g.kg-1)
5
22.86±1.78
6.24±1.50*
1.56±0.50
22.77
10
23.16±1.53
7.01±1.43**
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1.18±0.41*
41.58
20
23.67±1.24
6.52±1.36**
0.79±0.45**
60.89
CY(mg.kg-1)
20
23.84±1.43
5.84±1.68**
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0.44±0.54**
78.02
m, mass; NS, normal saline; GLE, Ganoderma lucidum Extract; CY, cyclophosphamide; *P<0.05, **P<0.01 vs NS.
2.2 GLE、GLE血清对S180细胞体外增殖的影响
表2显示,直接加GLE至体外培养的S180细胞培养基中,GLE对S180细胞的生长并无抑制作用。而给小鼠灌服GLE(5、10、20 g.kg-1)后取其血清加至体外培养的S180细胞中,则对S180细胞的生长有显著的抑制作用,并呈剂量依赖关系,与RPMI1640对照组及生理盐水血清对照组相比,差异有显著性。
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表2 GLE、GLE血清对S180细胞体外增殖(n=8)和
凋亡(n=3)的影响(±s)
Table 2 Effect of GLE、GLE-treated serum on proliferation(n=8)
and apoptosis (n=3) of Sarcoma 180 cells in vitro(±s) Groups
Dose
D(570 nm)
Apoptotic cell/%
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RPMI1640
-
0.439±0.037
0.57±0.22
NS-treated serum
-
0.425±0.029
0.81±0.24
GLE-treated
5
0.354±0.030*#
29.20±4.21**##
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serum(g.kg-1)
10
0.326±0.028*#
31.43±5.17**##
20
0.279±0.041**##
32.55±5.12**##
ρ(GLE)/g.L-1
50
0.424±0.037
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0.64±0.30
100
0.455±0.071
0.75±0.41
200
0.476±0.144
0.70±0.36
ρ(VP-16)/mg.L-1
80
0.242±0.092**##
69.61±8.04**
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D, optical density; ρ, mass concentration; GLE, Ganoderma lucidum Extract; VP-16, Etoposide; *P<0.05, **P<0.01 vs RPMI1640; #P<0.05, ##P<0.01 vs NS-treated serum.
2.3 GLE、GLE血清对S180细胞凋亡的影响
表2所示,GLE(50、100、200 mg.L-1)直接加药与S180细胞共同培养71 h,与RPMI1640对照组相似,未见有诱导S180细胞凋亡作用。而灌胃GLE(5、10、20 g.kg-1)小鼠血清则可显著诱导S180细胞发生凋亡,与RPMI1640对照组及生理盐水灌胃后的小鼠血清对照组比较差异均有显著性。
2.4 GLE血清中TNF-α、INF-γ的检测
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表3结果显示,GLE血清中TNF-α活性的INF-γ含量,随灌胃GLE的剂量提高而增强,与灌胃生理盐水血清对照组比较差异均有显著性。
表3 GLE血清中TNF-α(n=8)活性及INF-γ(n=3)的检测(±s)
Table 3 Defect of TNF-α (n=8) activity and content
of INF-γ (n=3) in GLE-treated serum(±s) Groups
Dose
D(570 nm)
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ρ(INF-γ)/ng.L-1
Control
-
0.452±0.145
42.58± 5.16
GLE-treated
5
0.375±0.117*
177.15±16.20*
serum(g.kg-1)
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10
0.302±0.219**
256.30±14.34**
20
0.271±0.052**
345.28±18.24**
ρ(TNF-α)/mg.L-1
25
0.246±0.105**
D, optical density; ρ, mass concentration; *P<0.05 vs control (NS-treated serum); **P<0.01 vs control.
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2.5 灌胃GLE对小鼠PM TNF-α和脾细胞INF-γ mRNA表达的影响
图1、2和表4显示,灌服GLE(5、10、20 g.kg-1)的小鼠腹腔巨噬细胞TNF-α mRNA和脾细胞INF-γ mRNA表达均明显高于对照组,其辉度扫描的表达量经统计学检验,与灌胃生理盐水血清对照组比较差异有显著性。
Lane A, DNA marker; Lane B, D, F, H, TNF-α expression with different drug treated cell RNA; Lane C, E, G, I, β-actin expression with different drug treated cell RNA; B, C, 1640; D, E, GLE 5 g.kg-1; F, G, GLE 10 g.kg-1; H, I, GLE 20 g.kg-1.
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图1 GLE整体给药对小鼠PM TNF-α mRNA的影响
Figure 1 Effect of various dose of GLE on expression of murine PM TNF-α mRNA. Mice were treated with GLE (5, 10, 20 g.kg-1) once a day for 10 days. Mice were killed and PM was collected. Total cellular RNA was isolated and RT-PCR product was separated by 1.5% agarose gel electrophoresis.
Lane A, DNA marker; Lane B, D, F, H, INF-γ expression with different drug treated cell RNA; Lane C, E, G, I, β-actin expression with different drug treated cell RNA; B, C, 1640; D, E, GLE 5 g.kg-1; F, G, GLE 10 g.kg-1; H, I, GLE 20 g.kg-1.
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图2 GLE整体给药对小鼠脾细胞INF-γ mRNA的影响
Figure 2 Effect of various dose of GLE on expression of murine spleen cell INF-γ mRNA. Mice were treated with GLE (5, 10, 20 g.kg-1) once a day for 10 days. Mice were killed and spleen was removed. Total cellular RNA was isolated and RT-PCR product was separated by 1.5% agarose gel electrophoresis.
3 讨论
为了深入探讨灵芝的抗肿瘤作用机制,我们给移植S180肉瘤的BALB/c小鼠灌胃GLE,结果与文献报道一致,GLE 5、10、20 g.kg-1均有显著抑制小鼠移植性S180肉瘤生长的作用,抑制率分别达到22.77%、41.58%、60.89%。基于以上的实验结果,我们把GLE(50~200 g.L-1)直接加到体外培养的S180细胞中,结果GLE对S180细胞无抑制作用。流式细胞仪分析亦未发现有诱导S180肿瘤细胞凋亡的作用。然而,当我们按整体实验的剂量给小鼠灌服GLE 5、10、20 g.kg-1连续10 d后,取其血清加至S180细胞体外培养,结果却发现3个剂量组均有显著抑制S180细胞增殖的作用。流式细胞仪检测也发现有显著诱导S180细胞凋亡的作用。而灌胃生理盐水的小鼠血清则未抑制肿瘤细胞增殖,亦不能促其凋亡。
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表4 GLE整体给药对TNF-α和INF-γ mRNA
表达的影响(n=3, ±s)
Table 4 Effect of GLE on TNF-α and INF-γ mRNA expression
in mice (n=3, ±s) Groups
Dose
(g.kg-1, i.g.)
mRNA
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(TNF-α/β-actin)
(INF-γ/β-actin)
Control
-
0.124±0.023
0.100±0.030
GLE
5
0.599±0.116*
0.443±0.113*
10
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0.858±0.075**
0.653±0.122**
20
1.191±0.240**
0.787±0.098**
*P<0.05, **P<0.01 vs control (NS-treated serum).
为什么GLE血清与GLE不同,前者在体外可抑制肿瘤细胞并促其凋亡?为此,我们分别采用生物学活性检测法和ELISA法检测了GLE血清中TNF-α的活性和INF-γ的含量。结果显示,GLE血清中TNF-α的活性和INF-γ的含量均呈剂量依赖性升高。进一步采用RT-PCR法检测此二细胞因子mRNA表达的结果也与上述生物法和ELISA法的结果一致。已知TNF-α和INF-γ在抗肿瘤及诱导肿瘤细胞凋亡中具重要作用,TNF-α对肿瘤细胞有细胞毒和生长抑制作用,可诱导许多不同来源肿瘤细胞凋亡[9~12]。INF-γ有抗肿瘤细胞增生作用,并且可与TNF-α协同增强诱导肿瘤细胞凋亡[13~15]。故以上实验结果表明:GLE本身无直接抑瘤及诱导肿瘤细胞凋亡的作用,GLE通过口服吸收后,促进 PMTNF-α mRNA、脾细胞INF-γ mRNA的表达。刺激诱导机体产生TNF-α和INF-γ,进而达到诱导肿瘤细胞凋亡,抑制肿瘤细胞增殖,从而达到整体的抗肿瘤效果。
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由于受条件限制,本研究中未对含GLE血清进行进一步化学分析,故含GLE血清诱生TNF-α和INF-γ的作用究竟是GLE本身或是其在体内代谢产物的作用尚不清,值得深入研究。■
基金项目:中国博士后基金资助课题;
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收稿日期:2000-03-22, 百拇医药