松针不同提取部位体外抗肿瘤作用的实验研究.docx

1、松针不同提取部位体外抗肿瘤作用的实验研究松针不同提取部位体外抗肿瘤作用的实验研究周微a，郑晓珂b，王小兰a，冯卫生a （河南中医学院，a药学院；b基础医学院，郑州 450008）摘 要：目的 探讨松针不同提取部位的体外抗肿瘤作用。方法 采用系统溶剂萃取法将松针醇提物进行部位分离，得到石油醚、乙酸乙酯、正丁醇及水提取部位，通过四甲基偶氮唑盐比色法（MTT法）观察各提取部位对人肝癌细胞株SMMC-7721、HepG2，人乳腺癌细胞株MCF-7，人前列腺癌细胞株PC-3细胞增殖的抑制作用。采用流式细胞术分析细胞周期分布，初探松针抗肿瘤作用机理。结果 石油醚部位对肿瘤细胞的抑制作用较强，在一定剂量范围

2、内呈现出时间和剂量依赖性；乙酸乙酯部位的作用次之；正丁醇和水部位的抑制作用不明显。SMMC-7721细胞经0.6 mgml-1、0.8 mgml-1石油醚提取部位处理48h后的凋亡百分率分别为18.7%、21.4%，它对该细胞增殖的抑制是将细胞周期阻滞在G2-M期。结论 松针提取物在体外具有抗肿瘤作用，石油醚部位呈现出较强的抑制作用，其体外抑制SMMC-7721细胞增殖的机制可能与松针提取物的细胞毒作用、诱导细胞凋亡和抑制细胞分裂有关。关键词：松针；肿瘤细胞株；MTT法；流式细胞术Experimental Study on Antitumor Effect of Different Fract

3、ions of Pine Needles in vitroZHOU Weia,ZHENG Xiao-keb,WANG Xiao-lana,FENG Wei-shenga(Henan University of Traditional Chinese Medicine,a.School of Pharmacy;b.Basic Medical College,Zhengzhou 450008)Abstract: Objective To study the antitumor effect of different fractions of pine needles in vitro. Metho

4、ds Pine needles were extracted and separated by the technique of solvent-refining.MTT assay was used to observe the inhibition of extracts on the proliferation of human hepatocellular carcinoma SMMC-7721, HepG2, breast carcinoma MCF-7 and prostatic carcinoma PC-3.Apoptosis rates and cell cycle distr

5、ibution were detected by flow cytometry analysis.Results Petroleum ether extract from pine needles possesses the role of antitumor effect in cell culture. The inhibitory effect on the proliferation of tumor cells has remarkable time-dependence and dose-dependence. Petroleum ether extract induces SMM

6、C-7721 apoptosis and arrests in G2-M phase . Apoptotic percentage of Petroleum ether extract 0.6mgml-1 and 0.8mgml-1 on SMMC-7721 is 18.7% and 21.4% in 48 hours. Conclusion Pine needles fractions have the antitumor effect in vitro and the petroleum ether extract possesses the role of antitumor effec

7、t.Anticancer mechanism of petroleum ether extract on SMMC-7721 may be direct cytotoxic effect,inducing cell apoptosis and inhibiting cell segmentation.Key words: Pine needles; carcinoma cell lines; MTT; flow cytometry analysis松针(Pine needles)为松科(Pinaceae)松属(Pinus)植物马尾松(Pinus massoniana Lamb.)的针状叶1，味

8、苦、涩、性温，归肝肾经。国外许多研究已证实松针具有抗氧化、清除自由基、抑制肿瘤细胞转移、调节免疫系统的功能。本实验室在前期对其化学成分进行了系统研究，已从中得到多个木脂素类、黄酮类化合物2-8，并对木脂素类化合物进行了体外抑癌筛选，发现该类成分能抑制多种肿瘤细胞增殖，并呈时间和剂量依赖性。目前有关松针抗肿瘤系统的实验研究报道不多，为此，本文采用MTT法，以体外培养的人肝癌细胞株SMMC-7721、HepG2，人乳腺癌细胞株MCF-7，人前列腺癌细胞株PC-3为模型，考察了松针提取物的体外抗肿瘤作用，并对其作用机理进行初步探讨，为深入研究该植物抗肿瘤活性成分及作用机理奠定实验基础。1 实验材料1

9、.1 肿瘤细胞株：人肝癌细胞株SMMC-7721、HepG2，人乳腺癌细胞株MCF-7，人前列腺癌细胞株PC-3。由本实验室常规培养于含10%小牛血清的RPMI 1640培养基中（含青霉素100 U/ml，链霉素100 U/ml），置37、5%CO2饱和湿度培养箱中培养，2-3d传代1次，取对数生长期细胞用于实验。1.2 药物与试剂：马尾松松针采自河南伏牛山区；RPMI 1640培养基及新生小牛血清(FCS)，Gibco公司；二甲基亚砜（DMSO)及四甲基偶氮唑盐(MTT)，Amresco公司；胰蛋白酶(Trypsin)，Gibco公司；五氟尿嘧啶（5-FU），上海旭东海普药业有限公司，批号0

10、60703；碘化丙啶（PI）及RNase A，Sigma公司；其余试剂均为国产分析纯。1.3 仪器设备：流式细胞仪，COULTER Epics XL；ELIE细胞培养箱，美国REVCO公司；SW-CJ-IFD洁净工作台，苏净集团安泰公司制造；KDC-160HR高速冷冻离心机，科大创新股份有限公司中佳分公司；TS100倒置显微镜，日本Nikon公司；酶标仪，美国BIO-RAD Model 680；超纯水仪，Sartorius 611VF；单道可调微量移液器，Nichipet EX plus；PHS-3C酸度计，上海精密科学仪器有限公司；培养皿、96孔培养板、细胞冻存管，Corning公司。2 实

11、验方法2.1 药物制备：干燥马尾松松针1kg，粉碎后以70%乙醇10倍量浸泡过夜，加热连续回流提取2次，每次2h，过滤合并，减压浓缩至稠膏。将稠膏悬浮于适量水中，依次用石油醚、乙酸乙酯、正丁醇萃取，将萃取液浓缩得各部位，烘干后称重，计算得率。各提取物用适宜溶媒（水或50%乙醇）溶解，调浓度为10 mgml-1 ，0.22m 孔径滤膜过滤除菌, 4冰箱内保存备用。使用时用无血清RPMI 1640培养基稀释到所需剂量。2.2 松针不同提取部位对肿瘤细胞增殖的抑制作用9:收集对数生长期细胞，以含10%FCS的RPMI 1640培养液调整细胞密度至5104个/ml，按每孔180l加入96孔培养板中，置

12、37、5%CO2饱和湿度培养箱中孵育，细胞贴壁后,实验组分别加入不同稀释浓度的松针提取物20l，使其终浓度为0.2、0.4、0.6、0.8、1 mgml-1；阳性对照组加入20l的5-FU，使其终浓度为0.05 mgml-1；阴性对照组加入等体积的无血清RPMI 1640完全培养液,每个浓度设6个复孔(实验重复3次)，连续培养24h、48h和72h。培养结束前4h各孔加入MTT（5mgml-1）10l，继续孵育4h后吸弃上清液，每孔加入DMSO 150l，蓝紫色结晶充分溶解后，在酶标仪上于490nm处测定各孔吸光度（OD）值。按下式计算药物对肿瘤细胞生长的抑制率:CI=（A-B）/A100%式

13、中：CI-抑制率，%；A-对照组平均OD值；B-实验组平均OD值。并计算样品的半数抑制浓度IC50。2.3 石油醚部位诱导SMMC-7721细胞DNA倍体分析及细胞周期分析：收集对照组和经0.6、0.8、1 mgml-1石油醚提取物处理48h的SMMC-7721细胞，制成约1106个/ml单细胞悬液，PBS洗涤后离心沉淀细胞，70%预冷乙醇（0-4）固定，500g离心5min，弃上清。悬浮管底的细胞沉淀，在细胞悬液中加2mlPBS，混匀后300g离心5min，弃上清液。留下约50l的细胞悬液，加100lRNase溶液，37水浴30min。加入800lPBS、50lPI染色液，避光染色30min

14、，进行FCM检测，重复3次。2.4 统计学方法：采用SPSS 13.0统计软件分析，实验数据用平均数标准差（s）表示，实验组与对照组间均数比较采用One-Way ANOVO程序进行单因素方差分析。3 实验结果3.1 药物制备:各提取部位称重分别为12.30g、16.98g、19.30g、46.22g，得率（相对于总提物）分别为 10.6%、 14.6%、 16.6%、 39.7%。3.2 松针不同提取部位对肿瘤细胞增殖的抑制作用3.2.1 对SMMC-7721细胞增殖的抑制作用：松针石油醚部位在1mgml-1时对SMMC-7721细胞作用48h的抑制率达到63.94%，在0.6 mgml-1时

15、作用72h的抑制率达到50%以上。乙酸乙酯部位在测定浓度范围内对SMMC-7721作用72h的抑制率为50%以上。石油醚部位和乙酸乙酯部位在测定范围内随着药物浓度的增加及作用时间的延长，其抑制率逐渐升高，呈现出时间和剂量依赖性。正丁醇部位和水部位对该细胞的抑制作用变化不明显，仅呈现出时间依赖性，但抑制率均未达到50%（见表1）。Table 1 Inhibition of different fractions on the proliferation of human hepatocellular carcinoma SMMC-7721 cell lines(s,n=6)GroupDose/(

16、mgml-1)ODCI /(%)24h48h72h24h48h72hControl00.28400.011480.43470.05170.80470.05760005-FU0.050.20510.01540.18790.00750.19520.031327.795.422156.771.734575.743.8857petroleum ether extract0.20.25440.03940.36150.05470.46530.011010.4313.866316.8412.585842.171.36890.40.24110.03190.32550.02130.42180.025715.10

17、11.226325.124.894847.593.19650.60.24100.00170.30650.03210.32530.026115.150.609829.487.383659.583.24680.80.18970.02510.28810.02220.30440.039233.238.852935.303.897062.174.876810.16260.01410.15680.01120.27440.012342.774.949163.942.584665.901.5235acetic ether extract0.20.20950.02950.34880.02940.39860.02

18、8026.2410.390219.776.767550.473.47890.40.19430.02070.29760.00690.40240.016031.587.302731.541.593149.991.98300.60.20850.01680.29310.01400.40660.021026.605.932032.563.221949.472.61550.80.19820.01640.26400.01560.34300.012730.235.773839.273.580957.371.577910.18600.01510.24990.01200.20330.017634.525.3113

19、42.522.752474.742.1842n-butanol extract0.20.25500.02000.34670.01710.44150.013510.227.058820.243.944045.131.67770.40.24130.01180.31700.02010.44130.036615.044.151727.074.631745.164.54570.60.23200.02060.33200.02990.44530.033018.327.249323.626.886344.674.10090.80.22280.02030.31600.01860.42420.045821.567

20、.139127.304.274347.295.686510.22200.01710.24830.02680.42030.068921.846.009142.876.172547.768.5609water extract0.20.24370.00900.28430.01470.51350.057614.213.175034.593.373836.187.15600.40.23680.02540.29740.02260.51850.006416.638.935731.585.188335.560.79090.60.22970.01500.29430.04220.47480.051519.145.

21、286429.338.865341.006.40340.80.22860.01240.311003830.46860.046019.524.351928.108.341541.765.716210.21270.00540.27820.18790.45930.057625.131.911136.008.862942.927.1592*P0.05,*P0.01 vs control group3.2.2 对HepG2细胞增殖的抑制作用：松针石油醚部位在1 mgml-1时对HepG2细胞作用48h的抑制率达到53.40%，在0.6 mgml-1时作用72h的抑制率达到55.33%，各剂量组与阴性对照

22、组比较差异显著（P0.05），在测定范围内呈现出时间和剂量依赖性。乙酸乙酯部位在0.6 mgml-1时作用72h的抑制率达到59.02%，呈现出剂量依赖性。正丁醇部位和水部位对该细胞的抑制作用变化不明显，且均未达到50%（见表2）。Table 2 Inhibition of different fractions on the proliferation of human hepatocellular carcinoma HepG2 cell lines(s,n=6)GroupDose/(mgml-1)ODCI /(%)24h48h72h24h48h72hControl00.28270.051

23、50.51560.09580.77430.08270005-FU0.050.18860.02390.24240.04200.16030.010533.318.443852.988.153779.301.3581petroleum ether extract0.20.24710.02150.51250.01840.44770.047812.597.58960.613.574342.186.17280.40.21990.02040.46870.06320.38940.031122.247.19779.1012.262449.704.01550.60.22440.03530.38540.03680.

24、34590.046320.6212.492825.257.142355.335.97910.80.20970.02990.32740.03570.29640.025925.8310.586436.506.917661.713.341310.19140.01220.24030.03110.25030.028032.294.316653.406.034667.673.6154acetic ether extract0.20.22400.02140.43160.06780.43730.096520.777.563716.3013.139143.5212.46320.40.17870.02470.44

25、900.07600.40030.151936.798.734712.9214.729848.3019.61170.60.17370.01310.42540.06740.31730.073138.564.646217.4913.068759.029.43970.80.17430.01250.37500.05040.28040.058138.364.430327.279.778063.787.499810.20530.02880.30900.06270.27110.062327.3910.174740.0712.166664.988.0483n-butanol extract0.20.27350.

26、01720.51450.00070.73570.06673.276.07480.220.13714.988.61600.40.27920.00890.51190.04210.73390.04761.253.13770.738.17265.226.14880.60.26760.00770.47970.03660.68390.03455.362.73866.977.089311.684.45940.80.25290.01920.44400.01870.66390.034810.576.801513.893.621314.264.494210.24260.01310.41300.02850.5506

27、0.022214.214.624419.905.535420.312.8648water extract0.20.26340.03620.50010.03130.56840.06106.8312.81573.006.075826.587.87210.40.25810.03170.47910.02310.51570.03698.7011.22077.084.485033.394.76580.60.27990.02540.45460.04040.45230.02101.028.971911.847.828941.592.71610.80.27720.04980.44540.03620.51500.

28、06371.9717.597013.627.022833.498.227510.28000.01000.40200.03050.49200.04250.973.536922.045.920636.465.4923*P0.05,*P0.01 vs control group3.2.3 对MCF-7细胞增殖的抑制作用：松针石油醚部位在0.8 mgml-1时对MCF-7细胞作用72h的抑制率达到58.65%，乙酸乙酯部位在1 mgml-1时作用72h的抑制率达到57.31%，在测定范围内均呈现出时间和剂量依赖性。正丁醇部位和水部位对该细胞的抑制作用变化不明显，且均未达到50%（见表3）。Table 3 Inhibition of different fractions on the proliferation of human breast carcinoma MCF-7 cell lines(s,n=6)GroupDose/(mgml-1)ODCI /(%)24h48h72h24h48h72hControl00.35430.03360.52680.04590.82280.04760005-FU0.050.29920.02050.14840.01