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医药导报, 2017, 36(4): 379-384
doi: 10.3870/j.issn.1004-0781.2017.04.006
旱生香茶菜素G对S180细胞抗肿瘤活性*
Antitumor Activity of Xerophilusin G on S180 Cells
杨焕芝1,2,, 周轶平2, 翁稚颖2, 罗敏2, 孙汉董3, 李玛琳2,4,

摘要:

目的 研究旱生香茶菜素G对S180细胞体内外抗肿瘤活性及其作用机制。方法 采用改良噻唑蓝(MTT)法测定旱生香茶菜素G对S180细胞株增殖的影响;以荷S180肉瘤小鼠为模型,检测旱生香茶菜素G对荷瘤小鼠肉瘤质量、体质量及其主要脏器的影响;通过流式细胞术检测旱生香茶菜素G对S180细胞株和S180肉瘤组织细胞周期的影响;采用脾脏淋巴细胞转化实验法测定旱生香茶菜素G对荷瘤小鼠脾脏淋巴细胞增殖活性;采用酶联免疫吸附试验双抗夹心法检测旱生香茶菜素G对荷瘤小鼠血清中白细胞介素(IL)-2含量的影响。结果 旱生香茶菜素G对S180细胞株半数抑制浓度(IC50值)为19.80 μg·mL-1。小鼠经腹腔注射旱生香茶菜素G的LD50为121.11 mg·kg-1。在剂量为3,6 mg·kg-1时,对荷S180肉瘤小鼠抑瘤率分别为32.11%,41.60%,差异有统计学意义(P<0.05),小鼠胸腺、肾脏和心脏指数均有一定程度的降低。旱生香茶菜素G使S180细胞株和S180肉瘤组织细胞G0/G1期所占比例增多,对荷瘤小鼠脾脏T 、B淋巴细胞具有一定的增殖促进作用(P<0.05)。小、中剂量组血清中IL-2含量分别较对照组减少约90.9%和77.1%(P<0.05)。结论 旱生香茶菜素G 具有一定的抗肿瘤活性,抑瘤作用可能是通过对肿瘤细胞G0/G1期阻滞,及其促进荷瘤小鼠脾脏T、B淋巴细胞增殖,增强机体免疫功能有关,而与IL-2的表达关系不密切。

关键词: 旱生香茶菜 ; 旱生香茶菜素G ; S180 ; 细胞周期 ; 免疫增强

Abstract:

Objective To study the antitumor activity of Xerophilusin G on S180 cells,and Its mechanism. Methods Modified MTT assay was used to test the effect of Xerophilusin G on the proliferation of S180 tumor cell strain. The influences on tumor growth and immune organs of mice with transplanted sarcoma (S180) were observed. The cell cycle of S180 cell lines and mouse sarcoma (S180) was analyzed by flow cytometry. The lymphocyte proliferation activity of spleen stimulating was tested. The level of IL-2 in serum of mice with transplanted sarcoma (S180) was measured by ELISA. Results The IC50 of Xerophilusin G in S180 cell lines was 19.80 μg·mL-1, the LD50 in mouse for Xerophilusin G was 121.11 mg·kg-1 through intraperitoneal injection. The tumor inhibition rate of Xerophilusin G was 32.11% and 41.60%, respectively at the doses of 3 and 6 mg·kg-1 (P<0.05). Compared with the control, the thymus, kidney and cardiac index were decreased. The cell proportion at G0/G1 phase of mouse sarcoma (S180) was increased. T and B cell proliferation activities in tumor-bearing mice were enhanced (P<0.05). As compared with control group, the serum level of IL-2 was decreased 90.9% and 77.1% in low- and medium-dose groups, respectively (P<0.05). Conclusion Xerophilusin G has remarkable effects in sarcoma (S180) bearing mice. The antitumor mechanism of Xerophilusin G might be related with G0/G1 phase arrest of mouse sarcoma (S180) cells and enhancing the activity of T and B cell but not related with increasing the secreting of IL-2.

Key words: Isodon xerophilus ; G ; S180 ; Cell cycle ; Immunity enhancement

旱生香茶菜是云南特有的香茶菜物种,药用其枝叶,其味苦,性寒,具有清热解毒、活血化瘀、抗菌消炎以及抗肿瘤等功效[1]。该植物中富含有对映-贝壳杉烷型二萜化合物,其中旱生香茶菜素G(Xerophilusin G)含量最多,约占总二萜的2.5%。本课题组前期研究旱生香茶菜总二萜(the total diterpenoids of Isodon Xerophilus,IXD)对人肿瘤细胞株K562、CA、CNE、BGC-823、MKN-28、A549、T-24等体外细胞毒活性,发现它能明显抑制肿瘤细胞增殖,且抗瘤谱较广[2];IXD 对人肝癌细胞株CA体外有较强的细胞毒活性,对荷CA 裸鼠移植瘤生长具有一定的抑制作用[3]。为从旱生香茶菜总二萜中发现新的抗肿瘤先导化合物,笔者以荷S180肉瘤小鼠为模型拟通过对旱生香茶菜素G抗肿瘤活性及其机制进行研究。

1 材料与方法
1.1 瘤株与实验动物

小鼠肉瘤细胞株S180,引自中国科学院上海药物研究所;美国癌症研究所(Institute of Cancer Research,ICR)小鼠,无特定病原体(SPF)级,雌雄各半,体质量18~22 g,由昆明医学院实验动物中心提供,实验动物生产许可证号:SCXK(滇)2005-0008。

1.2 样品

二萜化合物旱生香茶菜素G由中国科学院昆明植物研究所孙汉董研究员提供,从旱生香茶菜中提取并分离纯化得到,为白色粉末,室温下水溶性较差,醇溶性较好。结构式见图1。

图1 旱生香茶菜素G结构式

Fig.1 Molecular formula for Xerophilusin G

1.3 药品与试剂

顺铂(云南个旧生物药业有限公司,规格:每支10 mg,批号:051001);环磷酰胺(江苏恒瑞医股份有限公司,规格:每支0.2 g,批号:H32020857);噻唑蓝(MTT,AMRESCO公司,批号:0174B32);0.9%氯化钠溶液(昆明南疆制药有限公司,批号:200503156);新生牛血清(杭州四季青生物工程材料有限公司,批号:051203);二甲亚砜(DMSO,Sigma公司,批号:DB19372DB);1,2-丙二醇(成都科龙化工试剂厂,批号:20060522);小鼠白细胞介素-2(IL-2)、酶联免疫吸附试验(ELISA) 试剂盒(美国RD公司,编号:EIA05839)。

1.4 仪器

METTLERAE240型电子分析天平(梅特勒-托利多仪器上海有限公司);680型酶标仪(BIO-RAD公司);YJ-1450型医用净化工作台(苏州安泰空气技术有限公司)。

1.5 旱生香茶菜素G对S180细胞株增殖的影响

将旱生香茶菜素G用DMSO分别配成浓度为1 000,100,10,1,0.1 μg·mL-1 5个浓度,1%DMSO为阴性对照。阳性对照为顺铂,用0.9%氯化钠溶液分别配成100,10,1 μg·mL-1 3个浓度。常规培养S180细胞,取处于对数生长期S180细胞,将细胞浓度调整为5×104·mL-1,接种到96孔培养板,每孔90 μL。每孔分别加入10 μL不同浓度的样品。加样组及对照组均设4个复孔,每块板均设有4个空白对照孔。细胞在37 ℃,5%CO2培养箱中分别孵育48 h后,加入MTT(5 mg·mL-1),每孔10 μL。继续培养4 h后,加入三联液,每孔100 μL,放置过夜后,用酶标仪在570,630 nm双波长下测定各孔的A值,计算细胞增殖抑制率及半数抑制浓度(IC50)。

1.6 旱生香茶菜素G急性毒性实验

ICR小鼠,雌雄各半,禁食12 h后随机分为6组,每组10只,设受试样品5个剂量组(50,77.3,119.4,184.4,285.0 mg·kg-1)和阴性对照组。分别按每10 g体质量0.2 mL,一次性腹腔注射,给药后连续观察14 d,记录动物的毒性反应情况、死亡情况及死亡时间。采用Bliss法测定半数致死量(median lethal dose,LD50)。

1.7 旱生香茶菜素G对荷S180肉瘤小鼠肿瘤生长的影响

取ICR小鼠,无菌条件下腹腔接种浓度为1×107·mL-1的S180细胞悬液每只0.3 mL。当接种的初代移植S180肉瘤腹水长出后再传代。取第3代浓度为1×107个活细胞·mL-1的S180肉瘤细胞每只0.2 mL(腹水为乳白色无絮状沉淀物)迅速皮下接种于ICR小鼠(雌性)右前肢腋部皮下。接种后24 h称质量随机分为6组,旱生香茶菜素G小、中、大剂量组分别为3,6,12 mg·kg-1·d-1,环磷酰胺组剂量为30 mg·kg-1·d-1,0.9%氯化钠溶液组为空白对照组,30%丙二醇组为阴性对照组。按0.01 mL·g-1剂量,腹腔注射每天1次,连续给药7 d。停药后观察24 h,处死动物,剥离肿瘤组织、胸腺、脾、肝脏等脏器,并称质量。肿瘤抑制率(%)=(阴性对照组质量-治疗组质量)/阴性对照组质量×100%,脏器指数(mg·g-1)=脏器质量/动物质量。

1.8 旱生香茶菜素G对S180细胞株和肿瘤组织细胞周期的影响

取对数生长期S180细胞,调整密度为2.0×105·mL-1,以每瓶5 mL接种于细胞培养瓶中。0,5,10 μg·mL-1 旱生香茶菜素G分别作用细胞48 h后,收集细胞,预冷洗涤2次,固定细胞。离心收集细胞,弃固定液,洗涤两次后加入1 mL的碘化丙啶溶液室温下避光染色30 min,流式细胞术检测。旱生香茶菜素G小、中、大剂量和阴性对照组荷瘤小鼠(每组4只)停药后观察24 h,取出瘤组织,切取约0.5 g新鲜肿瘤组织,制备密度为106·mL-1的单细胞悬液,70%冰乙醇固定,4 ℃保存,流式细胞术检测。采集细胞的DNA数据,并以Multigraph软件分析结果。

1.9 旱生香茶菜素G对荷瘤小鼠T、B淋巴细胞增殖活性的影响

处死荷S180肉瘤小鼠,无菌取出脾脏,在RPMI1640液中将脾脏制成单个细胞悬液,离心洗涤2次,用含10%小牛血清的培养液制成6×106·mL-1的脾细胞悬液,以每孔90 μL加入96孔培养板中。在脾细胞培养板中分别加入促进T、B淋巴细胞增殖的刺激因子ConA(250 μg·mL-1)和脂多糖(200 μg·mL-1),每孔10 μL,阴性对照为RPM1640,每孔10 μL。培养板于37 ℃,5%CO2培养箱中孵育48 h后,加入MTT(5 mg·mL-1),每孔10 μL。继续培养4 h后,加入三联液,放置过夜后,用酶标仪在570,630 nm双波长下测定各孔的A值。T、B淋巴细胞增殖率(%)=(实验孔A值-对照孔A值)/对照孔A值×100%[4]

1.10 旱生香茶菜素G对荷S180肉瘤小鼠血清IL-2含量的影响

荷瘤小鼠(每组4只)停药后观察24 h,处死动物,摘除荷S180肉瘤小鼠的单侧眼球取静脉血2 mL,用清洁的试管收集血液,室温凝固2 h,离心收集血清。立即分装后-20 ℃冷冻保存。严格按照ELISA试剂盒说明书进行实验操作,运用CurveExpert1.3绘图软件绘制IL-2标准曲线,计算出血清中IL-2含量。

1.11 统计学方法

采用SPSS11.5版统计软件对数据进行处理,计量资料用均数±标准差( ヌ±s) 表示,多组变量采用单因素方差分析,以P<0.05为差异有统计学意义。

2 结果
2.1 旱生香茶菜素G对S180细胞增殖的影响

旱生香茶菜素G在浓度为0.01,0.1 μg·mL-1时,对S180细胞株的增殖没有抑制作用,当浓度增至1.0,10,100 μg·mL-1时,抑制率分别为7.9%,31.5%,86.4%,且呈浓度依赖性,抑制率随浓度增加而增加,IC50为19.80 μg·mL-1,r=0.92。顺铂对S180细胞株的IC50值为0.16 μg·mL-1,r=0.93。

2.2 旱生香茶菜素G急性毒性实验

旱生香茶菜素G的5个剂量组(浓度285,184.4,119.4,77.3,50 mg·kg-1)小鼠死亡率依次分别为100%,90%,60%,0%,0%。小鼠最早死亡时间出现在给药后30 min,最晚出现死亡时间为第3天,死亡高峰出现在给药后1.5 h。对死亡小鼠进行解剖,其皮肤黏膜、心、肺、肝、脾均未见肉眼可见病变,小鼠的五官、粪便未见异常,动物行为未见异常。动物在死亡前出现后肢拉伸、行动不稳、有的小鼠有仰翻抽搐现象、气息微弱等症状,并持续至死亡。阴性对照组中的每只小鼠在观察期都没有任何异常状况,生长良好。经用Bliss法计算旱生香茶菜素G的LD50为121.11 mg·kg-1

2.3 旱生香茶菜素G体内抗肿瘤活性

与阴性对照组比较,旱生香茶菜素G小、中剂量组抑瘤率分别为32.11%,41.60%(P<0.05),而旱生香茶菜素G大剂量组抑瘤作用不显著(P>0.05),旱生香茶菜素G的3个剂量组的抑瘤作用均低于环磷酰胺组(P<0.05),对小鼠体质量均无明显的抑制作用(P>0.05)。环磷酰胺组与空白对照组比较,抑瘤率为68.54%(P<0.05),体质量明显减轻(P<0.05)。见表1。

表1 6组荷S180肉瘤小鼠肿瘤抑制作用比较
Tab.1 Comparison of tumor inhibition among six groups of mice bearing S180 tumor ヌ±s,n=10
组别 剂量/
(mg·kg-1)
初体质量 末体质量
g
肿瘤质量 抑瘤率/
%
空白对照组 18.69±1.28 21.19±1.63 1.24±0.38
阴性对照组 18.77±0.86 20.87±1.32 1.37±0.25
环磷酰胺组 30 18.95±1.59 18.29±2.10*1 0.39±0.17*1 68.54
旱生香茶菜素G
小剂量 3 19.50±1.46 22.27±1.47 0.93±0.44*2*3 32.11
中剂量 6 18.60±1.20 20.11±2.30 0.80±0.29*2*3 41.60
大剂量 12 17.56±2.52 20.58±1.57 1.17±0.33*3 14.59
F 2.232 7.897 10.474
P >0.05 <0.01 <0.01

Compared with blank control group,*1P<0.05;compared with negative control group,*2P<0.05;compared with cyclophosphamide group,*3P< 0.05

与空白对照组比较,*1P<0.05;与阴性对照组比较,*2P<0.05;与环磷酰胺组比较,*3P<0.05

表1 6组荷S180肉瘤小鼠肿瘤抑制作用比较

Tab.1 Comparison of tumor inhibition among six groups of mice bearing S180 tumor ヌ±s,n=10

2.4 旱生香茶菜素G对荷瘤小鼠脏器指数的影响

与空白对照组比较,环磷酰胺组小鼠胸腺指数显著降低,肾脏、心脏指数显著增加(P<0.05),脾脏指数、肝脏指数差异无统计学意义(P>0.05)。与阴性对照组比较,旱生香茶菜素G 3个剂量组小鼠胸腺指数显著降低,小剂量组肾脏、心脏指数均降低(P<0.05),3个剂量组脾脏、肝脏指数差异无统计学意义(P>0.05)。见表2。

表2 6组荷S180肉瘤小鼠脏器指数的比较
Tab.2 Comparison of organ index among six groups of mice bearing S180 tumor ヌ±s,n=10
组别 剂量/
(mg·kg-1)
胸腺指数 脾脏指数 肝脏指数
(mg·g-1)
肾脏指数 心脏指数
空白对照组 2.26±0.45 5.50±1.15 54.63±4.32 10.86±0.83 3.66±0.33
阴性对照组 2.58±0.58 5.87±0.84 54.17±4.51 11.80±0.97 4.64±0.69
环磷酰胺组 30 1.57±0.41*1 4.58±1.88 54.94±4.82 12.84±1.15*1 4.56±0.52*1
旱生香茶菜素G
小剂量 3 2.49±0.37*2*3 5.82±1.23 52.34±4.08 10.63±0.85*2*3 4.22±0.54*2
中剂量 6 2.04±0.62*4 5.64±1.80 51.16±5.53 11.29±1.06*3 4.34±0.44
大剂量 12 2.37±0.38*3*4 6.76±0.79 53.86±5.54 11.04±1.53*3 4.23±0.47
F 6.461 2.554 0.527 7.414 5.915
P <0.01 >0.05 >0.05 <0.01 <0.01

Compared with blank control group,*1P<0.05;compared with negative control group,*2P<0.05,*4P<0.05;compared with cyclophosphamide group,*3P< 0.05

与空白对照组比较,*1P<0.05;与阴性对照组比较,*2P<0.05,*4P<0.01;与环磷酰胺组比较,*3P<0.05

表2 6组荷S180肉瘤小鼠脏器指数的比较

Tab.2 Comparison of organ index among six groups of mice bearing S180 tumor ヌ±s,n=10

2.5 旱生香茶菜素G对S180细胞株及其肿瘤组织细胞周期的影响

流式结果显示,5,10 μg·mL-1 旱生香茶菜素G处理S180细胞48 h,处于G0/G1分别为50.7%,42.0%(空白对照组19.0%),处于G2/M期细胞分别为32.8%,36.5%(空白对照组33.3%);处于S期细胞分别为16.4%,21.5%(空白对照组47.7%)。与空白对照组比较,药物组 G0/G1期细胞所占比例呈相对增多趋势,S期细胞呈相对减少趋势,表明旱生香茶菜素G 可使S180细胞阻滞于G0/G1 期(图2)。

图2 旱生香茶菜素G处理S180细胞48 h细胞周期流式图

Fig.2 Flow cytometry analysis on cell cycle of S180 cells treated with Xerophilusin G

与阴性对照组比较,旱生香茶菜素G 3个剂量组S180小鼠肿瘤组织细胞处于G0/G1,S期细胞比例数值相对增加,但差异无统计学意义(P>0.05);旱生香茶菜素G小、中剂量组肿瘤组织细胞处于G2/M期细胞明显减少,分别减少33%和26%(P<0.05),见表3。

表3 6组荷S180小鼠肿瘤组织细胞周期的比较
Tab.3 Comparison of cell cycle of tumor tissue cells among six groups of mice bearing S180 tumor%,ヌ±s,n=4
组别 剂量/(mg·kg-1) G0/G1 S期 G2/M期
空白对照组 49.25±4.13 22.90±6.82 27.70±8.24
阴性对照组 47.98±9.92 13.48±12.73 38.58±6.42
环磷酰胺组 30 40.85±19.11 26.00±10.32 33.15±14.36
旱生香茶菜素G
小剂量 3 76.82±25.57 17.63±9.63 5.55±3.10*1
中剂量 6 62.30±21.84 25.20±7.34 12.52±8.51*1
大剂量 12 60.92±26.49 17.80±11.32 21.28±7.23
F 1.720 0.676 3.872
P >0.05 >0.05 <0.05

Compared with negative control group,*1P<0.05

与阴性对照组比较,*1P<0.05

表3 6组荷S180小鼠肿瘤组织细胞周期的比较

Tab.3 Comparison of cell cycle of tumor tissue cells among six groups of mice bearing S180 tumor%,ヌ±s,n=4

2.6 旱生香茶菜素G对荷S180肉瘤小鼠脾细胞增殖和小鼠血清中IL-2的影响

与阴性对照组比较,旱生香茶菜素G的3个剂量组均可促进荷瘤小鼠脾脏T,B淋巴细胞增殖(P<0.05),对T淋巴细胞增殖作用以旱生香茶菜素G中剂量组最明显,旱生香茶菜素G大剂量组对B淋巴细胞增殖率是旱生香茶菜素G小剂量组的3.68倍,是旱生香茶菜素G中剂量组的2.57倍。与空白对照组比较,环磷酰胺组血清中IL-2含量明显减少(P<0.05)。与阴性对照组比较,旱生香茶菜素G小、中剂量组血清中IL-2含量分别减少约90.9%和77.1%(P<0.05)。见表4。

表4 6组荷S180肉瘤小鼠脾细胞增殖及血清中IL-2含量的比较
Tab.4 Comparison of splenocyte proliferation and the serum level of IL-2 among six groups of mice bearing S180 tumor ヌ±s,n=4
组别 剂量/
(mg·kg-1)
T淋巴细胞增殖率
%
B淋巴细胞增殖率 IL-2/
(pg·mL-1)
空白对照组 8.81±4.17 7.76±1.64 11.78±0.12
阴性对照组 -17.75±3.85 4.25±2.87 10.31±0.76
环磷酰胺组 30 2.19±1.04 -8.66±3.80 3.07±0.10*1
旱生香茶菜素G
小剂量 3 24.51±8.94*2 35.43±9.42*2 0.94±0.10*2
中剂量 6 34.27±10.25*2 50.66±15.31*2 2.36±0.14*2
大剂量 12 32.56±14.73*2 130.36±16.27*2 8.12±0.16
F 1.870 2.350 4.263
P <0.05 <0.05 <0.05

Compared with blank control group,*1P<0.05;compared with negative control group,*2P<0.05

与空白对照组比较,*1P<0.05;与阴性对照组比较,*2P<0.05

表4 6组荷S180肉瘤小鼠脾细胞增殖及血清中IL-2含量的比较

Tab.4 Comparison of splenocyte proliferation and the serum level of IL-2 among six groups of mice bearing S180 tumor ヌ±s,n=4

3 讨论

前期研究显示,旱生香茶菜素G对K562、HCT和MKN-28细胞株体外无细胞毒活性,对HL-60细胞株IC50为3.93 μg·m L - 1 5 。本研究中对S180细胞株的IC50为19.80 μg·mL-1,显示对S180细胞细胞毒活性不显著。根据我国抗肿瘤药物体内筛选标准操作规程,体内试验抑瘤率>40%,且差异有统计学意义,说明药物有抗肿瘤作用。本研究中旱生香茶菜素G小、中剂量组对荷S180肉瘤小鼠的抑瘤率分别为32.11%,41.6%,说明旱生香茶菜素G在体内有抗肿瘤作用。但旱生香茶菜素G给药剂量与抑瘤作用之间量效关系只在一定范围显现,而超过一定剂量时量效关系不再存在。实验中观察到,各剂量组胸腺指数显著降低,但与环磷酰胺组比较,对小鼠胸腺的毒性小,小剂量组对肾脏、心脏有一定的毒性,对脾脏、肝脏的毒性不明显。

细胞周期调控是当前肿瘤研究的热点,几乎所有癌基因、抑癌基因的生物学效应,最终都归结到细胞周期的机制,在细胞周期调控中,G2/M期是细胞增殖的主要调控点[6]。香茶菜属植物所含化学成分以二萜类化合物为主,该类化合物普遍具有诱导肿瘤细胞凋亡的生物学活性。冬凌草甲素通过将胶质瘤细胞周期阻滞于S期诱导细胞凋亡[7],作用于SGC-7901细胞通过下调Cdk1和cyclinB1蛋白的表达使细胞周期阻滞于G2/M期抑制细胞增殖[8]。毛萼乙素通过激活p53信号通路诱导激活caspase-3和将细胞周期阻滞于G2/M期诱导人胰腺癌细胞细胞凋亡[9]。本研究发现不同浓度旱生香茶菜素G处理S180细胞48 h后,G0/G1期细胞所占比例呈上升趋势,S期细胞呈减少趋势。各剂量组对荷S180小鼠肿瘤组织细胞处于G0/G1期细胞比例呈增多趋势;小、中剂量组肿瘤组织细胞处于G2/M期的细胞比例均下降。表明旱生香茶菜素G 可能通过阻滞细胞周期于G0/G1期而达到抑制细胞增殖作用。IXD中细胞毒活性最强的化合物旱生香茶菜素B,可通过诱导K562细胞凋亡发挥其抗肿瘤作用,并可影响凋亡通路中的多个环节;对K562细胞中致病融合蛋白BCR-ABL的表达具有直接的抑制作用[10]。研究证明旱生香茶菜素G、冬凌草甲素和毛萼乙素对于一些肿瘤细胞具有细胞周期阻滞作用,但旱生香茶菜素G是否具有诱导细胞凋亡作用有待进一步研究。

旱生香茶菜素G在体外的细胞毒活性不明显,体内抗肿瘤活性及毒性显示其大剂量组低于小剂量组,提示体内抗肿瘤活性可能与其细胞毒活性不密切。各剂量组与阴性对照组比较,均可促进荷瘤小鼠脾脏T、B淋巴细胞增殖,对T淋巴细胞增殖作用以中剂量组最明显,大剂量组对B淋巴细胞增殖率最高。小、中剂量组对肿瘤的抑制作用与T、B淋巴细胞增殖率呈线性相关,推测旱生香茶菜素G的抗肿瘤作用可能与其促进小鼠脾脏T、B淋巴细胞增殖,增强机体免疫功能有关。T细胞主要有两类,C D 4 + T辅助细胞和C D 8 + 细胞毒性T细胞。C D 4 + T细胞可以发生克隆性增殖,释放出多种细胞因子,主要为IL-2、干扰素、肿瘤坏死因子[11]。本研究结果发现旱生香茶菜素G小、中剂量组荷瘤小鼠血清IL-2含量较阴性对照组,明显减少,显示旱生香茶菜素G对荷S180小鼠肿瘤的抑制作用不是通过促进IL-2分泌而实现的。本课题组前期研究发现:0.01,0.1,1,10,100 μg·mL-1 旱生香茶菜素G在体外对T淋巴细胞增殖抑制率依次是21.6%,9.4%,-48.9%,52.9%,69.0%,显示旱生香茶菜素G在一定剂量范围内对T淋巴细胞增殖具有双向调节作用[10]。本研究中体内实验各剂量组胸腺指数显著降低,但体外实验可促进荷瘤小鼠脾脏T、B淋巴细胞增殖,推测旱生香茶菜素G对免疫系统可能具有双向调节作用,有待进一步研究。

综上所述,旱生香茶菜素G在体外能抑制S180细胞增殖,体内对荷S180肉瘤小鼠肿瘤生长具有抑制作用,而其抗肿瘤活性的发挥可能与阻滞肿瘤细胞周期有关。今后,将扩大细胞株和瘤株的选择范围,对其抗肿瘤活性进一步研究。

The authors have declared that no competing interests exist.

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关键词(key words)
旱生香茶菜
旱生香茶菜素G
S180
细胞周期
免疫增强

Isodon xerophilus
G
S180
Cell cycle
Immunity enhancement

作者
杨焕芝
周轶平
翁稚颖
罗敏
孙汉董
李玛琳

YANG Huanzhi
ZHOU Yiping
WENG Zhiying
LUO Min
SUN Handong
LI Malin