健脾丸抗肝癌作用及其作用机制的实验研究
|
摘要
目的:通过体内外实验,探讨健脾丸的抗肝癌作用,并提示其可能的作用机制,为该方治疗肝癌提供理论依据,并树立中医肝癌“本”是“脾虚”的观点。
方法:1体外实验:运用中药血清药理学原理和方法,制备健脾丸含药血清。采用四氮唑盐(MTT)法检测不同浓度健脾丸含药血清对人肝癌细胞SMMC-7721作用24h、48h、72h增殖抑制作用的影响。应用流式细胞技术(FCM)检测不同浓度健脾丸含药血清对人肝癌细胞SMMC-7721作用72h后细胞凋亡率和细胞周期,及细胞bcl-2和bax基因蛋白表达。2体内实验:取60只昆明种小鼠,无菌条件下将小鼠肝癌细胞H22腹水瘤细胞接种到小鼠右腋皮下,0.2ml/只(2×105个细胞)。动物随机分为5组,每组12只,化疗(5-氟尿嘧啶)组每天腹腔注射0.2ml/只,即0.5mg/只;中药低、中、高剂量组分别给予0.302g/ml,0.604g/ml, 1.208g/ml灌胃,0.4ml/只;阴性对照组每日灌服生理盐水0.4 ml/只。每日灌胃一次,连续给药10天,于次日处死,剥离瘤组织,称重,计算抑瘤率。取脾脏称重,计算脾指数。采用流式细胞技术(FCM)检测各组瘤组织细胞凋亡率和细胞周期,及细胞bcl-2和bax基因蛋白表达。采用免疫组化法观察不同浓度瘤组织细胞形态,及bcl-2和bax基因蛋白表达情况。
结果
1体外实验结果
1.1健脾丸含药血清对肝癌细胞SMMC-7721作用24小时后,低、中、高剂量组细胞抑制率分别为:7.28%、11.44%、26.14%,与阴性对照组相比能显著抑制肝癌细胞SMMC-7721的生长(P<0.05)。作用48小时后,低、中、高剂量细胞凋亡抑制率分别为:15.67%、20.90%、33.47%,与阴性对照组相比能显著抑制肝癌细胞SMMC-7721的生长(P<0.05)。作用72小时后,低、中、高剂量细胞凋亡抑制率分别为:17.55%、25.47%、34.29%,与阴性对照组相比能显著抑制肝癌细胞SMMC-7721的生长(P<0.05)。并且中药对肝癌细胞SMMC-7721的生长抑制作用呈剂量时间依赖性。本实验结果显示,在一定浓度范围内中药健脾丸能抑制肝癌细胞SMMC-7721的生长。
1.2健脾丸含药血清低、中、高剂量组作用于肝癌细胞SMMC-772172h后,G0/G1期细胞比例分别为:58.800%、62.600%、67.667%;阴性对照组G0/G1期细胞比例为:50.700%,各中药组与其相比均有显著差异(P<0.05)。S期细胞比例分别为:34.167%、24.767%、19.633%;阴性对照组S期细胞比例为:35.933%,各中药组与其相比均无显著差异(P>0.05)。错误!链接无效。凋亡率分别为:6.985%、7.615%、8.883%,阴性对照组凋亡率5.688%,各中药组与其相比均有显著差异(P<0.05)。且细胞凋亡率随中药健脾丸剂量增加而升高。
1.3流式细胞仪定量分析,结果显示:健脾丸含药血清低、中、高剂量组细胞bcl-2基因蛋白的荧光指数(FI)分别为1.1059±0.0069、1.0779±0.0053、1.0422±0.0431,阴性对照组为1.1814±0.0035,各实验组与阴性对照组相比均有显著差异(P<0.05),且随健脾丸含药剂量增加蛋白表达呈降低趋势。bax基因蛋白的荧光指数(FI)分别为1.086±0.0338、1.2024±0.0112、1.2561±0.0148,阴性对照组为1.0392±0.0208,各组与阴性对照组相比均有显著差异(P<0.05),且随健脾丸含药剂量增加bax蛋白表达呈增加趋势。
2体内实验结果
2.1健脾丸低、中、高组对H22移植瘤具有明显抑制作用,各组抑瘤率分别为19.62%,39.71%,48.74%,与阴性对照组相比均有显著差异(P<0.01),并呈剂量依赖性。化疗组抑制率为52.76%,健脾丸高剂量组与之相比无显著差异(P>0.01)。
2.2健脾丸低、中、高剂量组荷瘤小鼠的脾指数分别为:8.038±0.9318、9.778±0.8659、11.584±1.4685,化疗组脾指数为:5.278±0.4226,与化疗组相比有明显差异(P<0.01)。
2.3低、中、高剂量组瘤组织细胞经流式分析仪检测后,G0/G1期细胞比例增加,分别为66.667%、76.100%、81.633%,阴性对照组为:58.400%,与对照组相比均有显著性差异(P<0.05)。低、中、高剂量组肿瘤细胞凋亡率分别为:22.16%、28.42%、37.94%,阴性对照组为:9.660%;与阴性对照组相比均有显著性差异(P<0.05)。5-FU组细胞凋亡率为47.71%,健脾丸高剂量组低于阳性对照组(P<0.05)。
2.4健脾丸低、中、高剂量组,瘤组织细胞bcl-2基因蛋白的荧光指数(FI)分别为1.2049±0.0077、1.1423±0.0054、1.0859±0.0173,阴性对照组为1.2454±0.0052,5-FU组为1.0420±0.0682;各实验组与阴性对照组相比均有显著差异(P<0.01),且随健脾丸含药剂量增加蛋白表达而降低。bax基因蛋白的荧光指数(FI)分别为1.1559±0.0121、1.2396±0.0109、1.2989±0.0119,阴性对照组为1.0349±0.0190,5-FU组为1.2611±0.0855;各组与阴性对照组相比均有显著差异(P<0.01),且随健脾丸剂量增加bax蛋白表达而增加。
2.5免疫组化检测中,因样本含量较少不足以进行统计学分析,但经组织形态学分析,中药低、中、高剂量组细胞浆中出现棕黄色颗粒说明bcl-2基因蛋白表达阳性,且可看到随中药剂量增加bcl-2表达逐渐减弱。中药低、中、高剂量组细胞浆中亦出现棕黄色颗粒说明bax基因蛋白表达阳性,且随中药剂量增加bax表达逐渐增强。以上基因蛋白表达趋势与流式定量分析所得结果相符。
结论
1健脾丸可抑制肝癌细胞的生长,与对照组比,各浓度组在24小时、48小时、72小时均能显著抑制肝癌细胞SMMC-7721的生长。并且中药的生长抑制作用呈剂量时间依赖性。本实验结果显示,在一定浓度范围内中药健脾丸能抑制肿瘤细胞的生长。
2本实验显示:健脾丸低、中、高剂量组作用于肿瘤细胞后,G0/G1期细胞比例增加,S期细胞比例减少,提示可将细胞阻滞于G0/G1期,同时肿瘤细胞凋亡率也增加,与对照组相比有显著差异。且中药的凋亡率呈剂量依赖性。
3本实验中,健脾丸低、中、高剂量组,bcl-2基因蛋白的表达随剂量增加而降低,而bax基因蛋白的表达随剂量增加而增加。提示:健脾丸可能通过降低凋亡抑制基因bcl-2表达,增加促凋亡基因bax表达而发挥抗肝癌作用。
4体内实验表明:中药低、中、高剂量组与化疗组相比均能明显提高荷瘤小鼠脾指数,与化疗组相比有显著差异。说明健脾丸可能提高荷瘤小鼠机体免疫力。
5中医健脾丸是治疗脾虚的代表方剂,具有健脾消食,化湿除热的作用,本实验说明:健脾丸对肝癌细胞具有一定抑制作用,一定程度上支持中医肝癌“本”是“脾虚”的观点。
Objective: To study the experimental on Hepatoma with Jianpiwan. The aim was to find its possible mechanism, to produce the scientific evidence for further guiding.And to creat the standpoint that the foundation of Hepatoma was spleen asthenia.
Methods:1 in vitro:Using the method of pharmacology of the serum with Chinese medicine drugs (PSCD), we prepared Jianpiwan-medicated blood serum. TO observed the suppression of different dose of Jianpiwan-medicated blood serum at 24 hours、48 hours、72 hours on Hepatoma cell line SMMC-7721 by using the colorimentric 3-(4,5-dimethy -lthiazol-2-yl)-2,5biphenyl tetrazolium(MTT).To detected the ratio of apoptosis of Hepatoma cell line SMMC-7721 and the cell cycle at different dose of Jianpiwan-medicated blood serum after 72 hours by using the Flow Cytometry (FCM). And observed the expression of bcl-2、bax gene protein. 2 in vivo: Sixty healthy kunming mouse were transplanted by armpit injection of H22 tumor cells (0.2ml per mice) in asepsis conditions. Then these mice were randomly divided into five groups, twelve mice in each group. The mice in five groups were treated with 5-Fluorouracil (0.2ml per mice intraperitoneal injection), Jianpiwan low dose (0.302g/ml intragastric administration), Jianpiwan middle dose (0.604g/ml intragastric administration), Jianpiwan high dose (1.208g/ml intragastric administration), Sodium Chloride (0.4ml per mice) once per day respectively. Ten days later all mice were killed and the tumor were taken and weighed. Then the ratio of tumor suppression could be calculated.At the same time the spleens were taken and weighed.The spleen index could be calculated.To detected the ratio of apoptosis and the cell cycle of the tumor cells, and observed the expression of bcl-2 and bax gene protein by using the Flow Cytometry (FCM) .To observe the morphous of the tumor, and detected the gene protein expression of bcl-2、bax gene protein by using immunohistochemistry(IH).
Results
1 the result in vitro
1.1 The repression rate on Hepatoma cell line SMMC-7721 treated with Jianpiwan low-medicate blood serum、meddle-medicate blood serum and high-medicate blood serum after 24 hours were 7.28%、11.44%、26.14% respectively, comparing to the negative control group there was significant difference(P<0.05). The repression rate on Hepatoma cell line SMMC-7721 treated with Jianpiwan low-medicate blood serum、meddle-medicate blood serum and high-medicate blood serum after 48 hours were 15.67%、20.90%、33.47%respectively, comparing to the negative control group there was significant difference(P<0.05). The repression rate on Hepatoma cell line SMMC-7721 treated with Jianpiwan low-medicate blood serum、meddil-medicate blood serum and high-medicate blood serum after 72 hours were: 17.55%、25.47%、34.29%respectively, comparing to the negative control group there was significant difference(P<0.05).The growth inhibiting depends on the dose and time. The results show that inside the certain density Jianpiwan can inhibit the growth of the tumor.
1.2 The three groups of cells treated with Jianpiwan-medicated blood serum of low dose、middle dose and high dose after 72 hours were analyzed by flow cytometry. The proportion of G0/G1 period were 58.800%、62.600%、67.667% respectively. The proportion of the negative control group was 50.700%.There was significant difference comparing to the negative control group (P<0.05). The proportion of S period were 34.167%、24.767%、19.633%respectivly. The proportion of the negative control group was 35.933%. There was not significant difference comparing to the negative control group(P>0.05).The apoptosis inhibition rate of the three group were 6.985%、7.615%、8.883% respectively. The apoptosis inhibition rate of the negative control group was 5.688%. There was significant difference comparing to the negative control group (P<0.05). And the inhibition rate depends on the dose.
1.3 The flourescence index (FI) of bcl-2 gene protein analyzed by flow cytometry(FCM).The results of the three group treated with Jianpiwan low-medicate blood serum、middle- medicate blood serum、high- medicate blood serum were 1.1059±0.0069、1.0779±0.0053、1.0422±0.0431.The result of the negative control group was 1.1814±0.0035.Comparing to the negative control group there was significant difference (P<0.05).And the expression of bcl-2 gene protein degraded by the dose. The flourescence index (FI) of bax gene protein treated with Jianpiwan low-medicate blood serum、middle- medicate blood serum、high- medicate blood serum were 1.086±0.0338、1.2024±0.0112、1.2561±0.0148,The result of the negative control group was 1.0392±0.0208.Comparing to the negative control group there was significant difference (P<0.05).Besides the expression of bax gene protein upgraded by the dose.
2 the result in vivo
2.1 The inhibition rate of the tumor treated with Jianpiwan low-dose、middle-dose and high-dose were 19.62%,39.71%,48.74% respectively. There was significant difference comparing to the negative control group错误!链接无效。And the inhibition rate depends on the dose. The inhibition rate of the chemo group was 52.76%, but it has not significant difference comparing to the Jianpiwan high-dose (P>0.01).
2.2 The spleen index of the Jianpiwan low -dose、middle-dose and high -dose increased obviously. The results were 8.038±0.9318、9.778±0.8659、11.584±1.4685.The result of the chemo group was 5.278±0.4226. Comparing to the group treated with 5- Fluorouracil there was remarkable difference (P<0.01).
2.3 The three groups of the tumor treated with Jianpiwan high dose、middle dose and low dose were analyzed by flow cytometry. The proportion of G0/G1 period were 66.667%、76.100%、81.633% respectivly. The propotion of the negative control group was 58.400%.There was significant difference comparing to the negative control group (P<0.05). The apoptosis inhibition rate of the three group were 22.16%、28.42%、37.94%respectivly. The apoptosis of the negative control group was 9.660%. There was significant difference comparing to the negative control group (P<0.05). The apoptosis of the chemo group was 47.71%. The apoptosis of the Jianpiwan high-dose was low than the control group (P<0.05).
2.4 The flourescence index (FI) of bcl-2 gene protein analyzed by flow cytometry(FCM).The results of the there group treated with Jianpiwan low-dose、middle- dose、high- dose were 1.2049±0.0077、1.1423±0.0054、1.0859±0.0173.The result of the control group was 1.2454±0.0052. The result of the 5-FU was 1.0420±0.0682.Comparing to the negative control group there was significant difference (P<0.01).And the expression of bcl-2 gene protein degraded by the dose. The flourescence index (FI) of bax gene protein treated with Jianpiwan low-dose、middle- dose、high- dose were 1.1559±0.0121、1.2396±0.0109、1.2989±0.0119,The result of the negative control group was 1.0349±0.0190. The result of the 5-FU was 1.2611±0.0855.Comparing to the negative control group there was significant difference (P<0.05).Besides the expression of bax gene protein upgraded by the dose.
2.5 Bcl-2 and bax gene protein were detected by immunohistochemistry (IH).But the results were not analyzied by statistics.According to histomorphology buffy granules appeared in cytoplasm which were treated with Jianpiwan low dose、middle dose and high dose.It indicated that bcl-2 gene protein expressed.And the expression of bcl-2 gene protein was weakening gradually. Buffy granules also appeared in cytoplasm which were treated with Jianpiwan low dose、middle dose and high dose.It indicated that bax gene protein expressed.And the expression of bax gene protein was strengthening gradually.The tendency agreed to the results analyzed by FCM.
Conclusion
1 Three group treated with Jianpiwan could inhibit the growth of Hepatoma comparing to the negative control group at 24huors、48hours、72hours. The growth inhibiting depends on the dose and time. The results show that inside the certain density Jianpiwan can inhibit the growth of the tumor.
2 The propotion of G0/G1 period and the apoptosis ratio were increased treated with Jianpiwan.Comparing to the negative control group there was significant difference.And the apoptosis depends on the dose.The results show that the Hepatoma cells could be inhibit at G0/G1 period.
3 And the expression of bcl-2 gene protein degraded by the dose. And the expression of bax gene protein upgraded by the dose.They all had significant difference compared to the negative control group.The results show that Jianpiwan could inhibit the growth of Hepatoma cells.The mechanism may be that it could degrade the expression of bcl-2 gene protein and upgrade the expression of bax gene protein.
4 The spleen index treated with Jianpiwan low -dose、middle-dose and high -dose increased obviously. Comparing to the group treated with 5- Fluorouracil there was remarkable difference.It show that Jianpiwan could elevate the immunity of the tumor-bearing mice.
5 Jianpiwan could invigorate the spleen.The results show that Jianpiwan could inhibit the growth of Hepatoma.To some extent the standpoint was that the foundation of Hepatoma was spleen asthenia.
方法:1体外实验:运用中药血清药理学原理和方法,制备健脾丸含药血清。采用四氮唑盐(MTT)法检测不同浓度健脾丸含药血清对人肝癌细胞SMMC-7721作用24h、48h、72h增殖抑制作用的影响。应用流式细胞技术(FCM)检测不同浓度健脾丸含药血清对人肝癌细胞SMMC-7721作用72h后细胞凋亡率和细胞周期,及细胞bcl-2和bax基因蛋白表达。2体内实验:取60只昆明种小鼠,无菌条件下将小鼠肝癌细胞H22腹水瘤细胞接种到小鼠右腋皮下,0.2ml/只(2×105个细胞)。动物随机分为5组,每组12只,化疗(5-氟尿嘧啶)组每天腹腔注射0.2ml/只,即0.5mg/只;中药低、中、高剂量组分别给予0.302g/ml,0.604g/ml, 1.208g/ml灌胃,0.4ml/只;阴性对照组每日灌服生理盐水0.4 ml/只。每日灌胃一次,连续给药10天,于次日处死,剥离瘤组织,称重,计算抑瘤率。取脾脏称重,计算脾指数。采用流式细胞技术(FCM)检测各组瘤组织细胞凋亡率和细胞周期,及细胞bcl-2和bax基因蛋白表达。采用免疫组化法观察不同浓度瘤组织细胞形态,及bcl-2和bax基因蛋白表达情况。
结果
1体外实验结果
1.1健脾丸含药血清对肝癌细胞SMMC-7721作用24小时后,低、中、高剂量组细胞抑制率分别为:7.28%、11.44%、26.14%,与阴性对照组相比能显著抑制肝癌细胞SMMC-7721的生长(P<0.05)。作用48小时后,低、中、高剂量细胞凋亡抑制率分别为:15.67%、20.90%、33.47%,与阴性对照组相比能显著抑制肝癌细胞SMMC-7721的生长(P<0.05)。作用72小时后,低、中、高剂量细胞凋亡抑制率分别为:17.55%、25.47%、34.29%,与阴性对照组相比能显著抑制肝癌细胞SMMC-7721的生长(P<0.05)。并且中药对肝癌细胞SMMC-7721的生长抑制作用呈剂量时间依赖性。本实验结果显示,在一定浓度范围内中药健脾丸能抑制肝癌细胞SMMC-7721的生长。
1.2健脾丸含药血清低、中、高剂量组作用于肝癌细胞SMMC-772172h后,G0/G1期细胞比例分别为:58.800%、62.600%、67.667%;阴性对照组G0/G1期细胞比例为:50.700%,各中药组与其相比均有显著差异(P<0.05)。S期细胞比例分别为:34.167%、24.767%、19.633%;阴性对照组S期细胞比例为:35.933%,各中药组与其相比均无显著差异(P>0.05)。错误!链接无效。凋亡率分别为:6.985%、7.615%、8.883%,阴性对照组凋亡率5.688%,各中药组与其相比均有显著差异(P<0.05)。且细胞凋亡率随中药健脾丸剂量增加而升高。
1.3流式细胞仪定量分析,结果显示:健脾丸含药血清低、中、高剂量组细胞bcl-2基因蛋白的荧光指数(FI)分别为1.1059±0.0069、1.0779±0.0053、1.0422±0.0431,阴性对照组为1.1814±0.0035,各实验组与阴性对照组相比均有显著差异(P<0.05),且随健脾丸含药剂量增加蛋白表达呈降低趋势。bax基因蛋白的荧光指数(FI)分别为1.086±0.0338、1.2024±0.0112、1.2561±0.0148,阴性对照组为1.0392±0.0208,各组与阴性对照组相比均有显著差异(P<0.05),且随健脾丸含药剂量增加bax蛋白表达呈增加趋势。
2体内实验结果
2.1健脾丸低、中、高组对H22移植瘤具有明显抑制作用,各组抑瘤率分别为19.62%,39.71%,48.74%,与阴性对照组相比均有显著差异(P<0.01),并呈剂量依赖性。化疗组抑制率为52.76%,健脾丸高剂量组与之相比无显著差异(P>0.01)。
2.2健脾丸低、中、高剂量组荷瘤小鼠的脾指数分别为:8.038±0.9318、9.778±0.8659、11.584±1.4685,化疗组脾指数为:5.278±0.4226,与化疗组相比有明显差异(P<0.01)。
2.3低、中、高剂量组瘤组织细胞经流式分析仪检测后,G0/G1期细胞比例增加,分别为66.667%、76.100%、81.633%,阴性对照组为:58.400%,与对照组相比均有显著性差异(P<0.05)。低、中、高剂量组肿瘤细胞凋亡率分别为:22.16%、28.42%、37.94%,阴性对照组为:9.660%;与阴性对照组相比均有显著性差异(P<0.05)。5-FU组细胞凋亡率为47.71%,健脾丸高剂量组低于阳性对照组(P<0.05)。
2.4健脾丸低、中、高剂量组,瘤组织细胞bcl-2基因蛋白的荧光指数(FI)分别为1.2049±0.0077、1.1423±0.0054、1.0859±0.0173,阴性对照组为1.2454±0.0052,5-FU组为1.0420±0.0682;各实验组与阴性对照组相比均有显著差异(P<0.01),且随健脾丸含药剂量增加蛋白表达而降低。bax基因蛋白的荧光指数(FI)分别为1.1559±0.0121、1.2396±0.0109、1.2989±0.0119,阴性对照组为1.0349±0.0190,5-FU组为1.2611±0.0855;各组与阴性对照组相比均有显著差异(P<0.01),且随健脾丸剂量增加bax蛋白表达而增加。
2.5免疫组化检测中,因样本含量较少不足以进行统计学分析,但经组织形态学分析,中药低、中、高剂量组细胞浆中出现棕黄色颗粒说明bcl-2基因蛋白表达阳性,且可看到随中药剂量增加bcl-2表达逐渐减弱。中药低、中、高剂量组细胞浆中亦出现棕黄色颗粒说明bax基因蛋白表达阳性,且随中药剂量增加bax表达逐渐增强。以上基因蛋白表达趋势与流式定量分析所得结果相符。
结论
1健脾丸可抑制肝癌细胞的生长,与对照组比,各浓度组在24小时、48小时、72小时均能显著抑制肝癌细胞SMMC-7721的生长。并且中药的生长抑制作用呈剂量时间依赖性。本实验结果显示,在一定浓度范围内中药健脾丸能抑制肿瘤细胞的生长。
2本实验显示:健脾丸低、中、高剂量组作用于肿瘤细胞后,G0/G1期细胞比例增加,S期细胞比例减少,提示可将细胞阻滞于G0/G1期,同时肿瘤细胞凋亡率也增加,与对照组相比有显著差异。且中药的凋亡率呈剂量依赖性。
3本实验中,健脾丸低、中、高剂量组,bcl-2基因蛋白的表达随剂量增加而降低,而bax基因蛋白的表达随剂量增加而增加。提示:健脾丸可能通过降低凋亡抑制基因bcl-2表达,增加促凋亡基因bax表达而发挥抗肝癌作用。
4体内实验表明:中药低、中、高剂量组与化疗组相比均能明显提高荷瘤小鼠脾指数,与化疗组相比有显著差异。说明健脾丸可能提高荷瘤小鼠机体免疫力。
5中医健脾丸是治疗脾虚的代表方剂,具有健脾消食,化湿除热的作用,本实验说明:健脾丸对肝癌细胞具有一定抑制作用,一定程度上支持中医肝癌“本”是“脾虚”的观点。
Objective: To study the experimental on Hepatoma with Jianpiwan. The aim was to find its possible mechanism, to produce the scientific evidence for further guiding.And to creat the standpoint that the foundation of Hepatoma was spleen asthenia.
Methods:1 in vitro:Using the method of pharmacology of the serum with Chinese medicine drugs (PSCD), we prepared Jianpiwan-medicated blood serum. TO observed the suppression of different dose of Jianpiwan-medicated blood serum at 24 hours、48 hours、72 hours on Hepatoma cell line SMMC-7721 by using the colorimentric 3-(4,5-dimethy -lthiazol-2-yl)-2,5biphenyl tetrazolium(MTT).To detected the ratio of apoptosis of Hepatoma cell line SMMC-7721 and the cell cycle at different dose of Jianpiwan-medicated blood serum after 72 hours by using the Flow Cytometry (FCM). And observed the expression of bcl-2、bax gene protein. 2 in vivo: Sixty healthy kunming mouse were transplanted by armpit injection of H22 tumor cells (0.2ml per mice) in asepsis conditions. Then these mice were randomly divided into five groups, twelve mice in each group. The mice in five groups were treated with 5-Fluorouracil (0.2ml per mice intraperitoneal injection), Jianpiwan low dose (0.302g/ml intragastric administration), Jianpiwan middle dose (0.604g/ml intragastric administration), Jianpiwan high dose (1.208g/ml intragastric administration), Sodium Chloride (0.4ml per mice) once per day respectively. Ten days later all mice were killed and the tumor were taken and weighed. Then the ratio of tumor suppression could be calculated.At the same time the spleens were taken and weighed.The spleen index could be calculated.To detected the ratio of apoptosis and the cell cycle of the tumor cells, and observed the expression of bcl-2 and bax gene protein by using the Flow Cytometry (FCM) .To observe the morphous of the tumor, and detected the gene protein expression of bcl-2、bax gene protein by using immunohistochemistry(IH).
Results
1 the result in vitro
1.1 The repression rate on Hepatoma cell line SMMC-7721 treated with Jianpiwan low-medicate blood serum、meddle-medicate blood serum and high-medicate blood serum after 24 hours were 7.28%、11.44%、26.14% respectively, comparing to the negative control group there was significant difference(P<0.05). The repression rate on Hepatoma cell line SMMC-7721 treated with Jianpiwan low-medicate blood serum、meddle-medicate blood serum and high-medicate blood serum after 48 hours were 15.67%、20.90%、33.47%respectively, comparing to the negative control group there was significant difference(P<0.05). The repression rate on Hepatoma cell line SMMC-7721 treated with Jianpiwan low-medicate blood serum、meddil-medicate blood serum and high-medicate blood serum after 72 hours were: 17.55%、25.47%、34.29%respectively, comparing to the negative control group there was significant difference(P<0.05).The growth inhibiting depends on the dose and time. The results show that inside the certain density Jianpiwan can inhibit the growth of the tumor.
1.2 The three groups of cells treated with Jianpiwan-medicated blood serum of low dose、middle dose and high dose after 72 hours were analyzed by flow cytometry. The proportion of G0/G1 period were 58.800%、62.600%、67.667% respectively. The proportion of the negative control group was 50.700%.There was significant difference comparing to the negative control group (P<0.05). The proportion of S period were 34.167%、24.767%、19.633%respectivly. The proportion of the negative control group was 35.933%. There was not significant difference comparing to the negative control group(P>0.05).The apoptosis inhibition rate of the three group were 6.985%、7.615%、8.883% respectively. The apoptosis inhibition rate of the negative control group was 5.688%. There was significant difference comparing to the negative control group (P<0.05). And the inhibition rate depends on the dose.
1.3 The flourescence index (FI) of bcl-2 gene protein analyzed by flow cytometry(FCM).The results of the three group treated with Jianpiwan low-medicate blood serum、middle- medicate blood serum、high- medicate blood serum were 1.1059±0.0069、1.0779±0.0053、1.0422±0.0431.The result of the negative control group was 1.1814±0.0035.Comparing to the negative control group there was significant difference (P<0.05).And the expression of bcl-2 gene protein degraded by the dose. The flourescence index (FI) of bax gene protein treated with Jianpiwan low-medicate blood serum、middle- medicate blood serum、high- medicate blood serum were 1.086±0.0338、1.2024±0.0112、1.2561±0.0148,The result of the negative control group was 1.0392±0.0208.Comparing to the negative control group there was significant difference (P<0.05).Besides the expression of bax gene protein upgraded by the dose.
2 the result in vivo
2.1 The inhibition rate of the tumor treated with Jianpiwan low-dose、middle-dose and high-dose were 19.62%,39.71%,48.74% respectively. There was significant difference comparing to the negative control group错误!链接无效。And the inhibition rate depends on the dose. The inhibition rate of the chemo group was 52.76%, but it has not significant difference comparing to the Jianpiwan high-dose (P>0.01).
2.2 The spleen index of the Jianpiwan low -dose、middle-dose and high -dose increased obviously. The results were 8.038±0.9318、9.778±0.8659、11.584±1.4685.The result of the chemo group was 5.278±0.4226. Comparing to the group treated with 5- Fluorouracil there was remarkable difference (P<0.01).
2.3 The three groups of the tumor treated with Jianpiwan high dose、middle dose and low dose were analyzed by flow cytometry. The proportion of G0/G1 period were 66.667%、76.100%、81.633% respectivly. The propotion of the negative control group was 58.400%.There was significant difference comparing to the negative control group (P<0.05). The apoptosis inhibition rate of the three group were 22.16%、28.42%、37.94%respectivly. The apoptosis of the negative control group was 9.660%. There was significant difference comparing to the negative control group (P<0.05). The apoptosis of the chemo group was 47.71%. The apoptosis of the Jianpiwan high-dose was low than the control group (P<0.05).
2.4 The flourescence index (FI) of bcl-2 gene protein analyzed by flow cytometry(FCM).The results of the there group treated with Jianpiwan low-dose、middle- dose、high- dose were 1.2049±0.0077、1.1423±0.0054、1.0859±0.0173.The result of the control group was 1.2454±0.0052. The result of the 5-FU was 1.0420±0.0682.Comparing to the negative control group there was significant difference (P<0.01).And the expression of bcl-2 gene protein degraded by the dose. The flourescence index (FI) of bax gene protein treated with Jianpiwan low-dose、middle- dose、high- dose were 1.1559±0.0121、1.2396±0.0109、1.2989±0.0119,The result of the negative control group was 1.0349±0.0190. The result of the 5-FU was 1.2611±0.0855.Comparing to the negative control group there was significant difference (P<0.05).Besides the expression of bax gene protein upgraded by the dose.
2.5 Bcl-2 and bax gene protein were detected by immunohistochemistry (IH).But the results were not analyzied by statistics.According to histomorphology buffy granules appeared in cytoplasm which were treated with Jianpiwan low dose、middle dose and high dose.It indicated that bcl-2 gene protein expressed.And the expression of bcl-2 gene protein was weakening gradually. Buffy granules also appeared in cytoplasm which were treated with Jianpiwan low dose、middle dose and high dose.It indicated that bax gene protein expressed.And the expression of bax gene protein was strengthening gradually.The tendency agreed to the results analyzed by FCM.
Conclusion
1 Three group treated with Jianpiwan could inhibit the growth of Hepatoma comparing to the negative control group at 24huors、48hours、72hours. The growth inhibiting depends on the dose and time. The results show that inside the certain density Jianpiwan can inhibit the growth of the tumor.
2 The propotion of G0/G1 period and the apoptosis ratio were increased treated with Jianpiwan.Comparing to the negative control group there was significant difference.And the apoptosis depends on the dose.The results show that the Hepatoma cells could be inhibit at G0/G1 period.
3 And the expression of bcl-2 gene protein degraded by the dose. And the expression of bax gene protein upgraded by the dose.They all had significant difference compared to the negative control group.The results show that Jianpiwan could inhibit the growth of Hepatoma cells.The mechanism may be that it could degrade the expression of bcl-2 gene protein and upgrade the expression of bax gene protein.
4 The spleen index treated with Jianpiwan low -dose、middle-dose and high -dose increased obviously. Comparing to the group treated with 5- Fluorouracil there was remarkable difference.It show that Jianpiwan could elevate the immunity of the tumor-bearing mice.
5 Jianpiwan could invigorate the spleen.The results show that Jianpiwan could inhibit the growth of Hepatoma.To some extent the standpoint was that the foundation of Hepatoma was spleen asthenia.
引文
1 于尔辛. 中医治疗癌肿的思路[J]. 肿瘤, 2001, 21(6):419
2 段富津, 《方剂学》, 上海科技出版社出版, 1995, 6, 第一版
3 刘福英, 吕占军. 实验动物学. 北京: 中国农业科技出版社, 1997:180-182
4 李仪奎. 中药血清药理学实验方法的若干问题. 中药新药与临床药理, 1999, 10(2):95-98
5 蒙一纯, 丁霞, 本长恩, 等. 中药血清药理应用研究进展北京中医药大学学报. 1999, 4(22):15-16
6 刘建文主编. 药理实验方法学: 新技术与新方法. 北京:化学工业出版社, 2002:18-19
7 周明眉, 杨奎, 姜平远, 等. 中药血清药理学的方法研究-含药血清低温保存和血清灭活的影响.中药药理与临床, 1999, 15(2):44
8 黄馨慧, 罗明志, 弃浩, 等龙胆苦苷等6种中草药提取物对 SMMC-7721 人肝癌细胞增殖的影响. 西北药学杂志, 2004, 19(4)
9 Mosman T. Rapid colorimetric assay for cellar growth and survival: application proliferation and cytotoxic by assay [J]. Immune Methods, 1983, 55-65
10 Koike K, Tsutsumi T, Fujie H, et al. Molecular mechanism of hepatocarcinogenesiss. Oncology, 2002, 62:29-7
11 陈洪, 细胞凋亡与肝癌研究进展, 临床肿瘤学杂志, 1999, 4(1):67-70
12 郭强, 刘娜, 张倩. 凋亡基因在恶性肿瘤中的研究进展.山西中医学院学报, 2006, 7(1):25-27
13 Power C. Fanning N. Redmond HP. Cellular apoptosisand organ injury in sepsis: a review.Shock, 2002, 18(3):197-211
14 陈江, 黄文方, 卢贤瑜. 肝癌细胞凋亡基因调控的研究进展. 国外医学临床生物化学与检验学册, 2005, 26(10):715-717
15 邹劲林, 刘富元, 李艳芳, 等. p27和bcl-2与肿瘤[J].中国医学文摘·肿瘤学, 2001, 15(4):347-34
16 熊丽, 陆坚, 聂广, 等. 肝癌的分子发病学及其中医药防治机制探讨. 中西医结合肝病杂志, 2006, 16(1)
17 王筠, 张军平. 中药血清药理学实验方法研究概况. 天津中医药, 2005, 22(1):86-88
18 刘平. 关于中药血清药理学的若干思考. 中国中西医结合志, 1999, 19(5):263-266
19 薛洁, 谢默林, 中药血清药理学的方法学研究近况. 中草药, 2003, 34(6):9-11
20 余黎, 王坚, 朱荃. 中药血清药理学研究中血清处理方法的探讨[J]. 南京中医药大学学报, 2002, 18(7):222
21 肖月升, 耿建芳, 杨瑞合, 等. 初论“中心辨证”. 时珍国医国药2006, 17(6):1062-1063
1 王修杰, 袁淑兰, 王朝俊, 等. 丹参酮抗小鼠肝癌细胞作用和机理的初步研究. 中华肿瘤杂志, 1996, 18(6):412
2 李杰, 孙桂芝, 朴炳奎, 等. 中药肝康冲剂提取诱导人肝癌细胞系 BEL-7402 细胞凋亡的实验研究. 中国肿瘤生物治疗杂志, 1997, 4(3):234
3 张燕军, 夏天, 赵建斌, 等. 苦参碱对 SMMC-7721 细胞系的诱导分化作用. 第四军医大学学报, 1998, 19(3):340
4 黄炜, 黄济群, 张东方, 等. 18β-甘草次酸和甘草酸对人肝癌细胞增殖的抑制和诱导分化作用. 中国中医药科技, 2002, 9(2):92-93
5 徐学军, 周子成, 罗元辉, 等. β-榄香烯诱导人肝癌细胞株 SMMC-7721 凋亡的研究[J]. 第三军医大学学报, 1999, 21(4):268-271
6 徐瑞峰, 赵健雄, 程卫东, 等. 扶正抑瘤颗粒对小鼠肝癌H22 细胞凋亡及 Fas、FasL 表达的影响. 新中医, 2004, 36(12):65-67
7 陈忠伟, 林勇, 谢渭芬, 等. 苦参碱对肝癌细胞端粒酶活性调控及细胞周期的影响. 第二军医大学学报, 2002, 23(5):498-500
8 孟志强, 于尔辛, 宋明志, 等. 健脾理气中药对肝癌端粒酶活性的影响[J]. 中国中医药基础医学杂志, 2000, 6(1):23-26
9 叶炯贤, 叶红军, 杜意平, 等. 刺五加叶皂甙对肝癌癌基因 表 达 的 调 节 作 用 [J]. 中 华 实 验 外 科 杂 志 ,2000, 17(5):426-427
10 孙靖璟, 周信达, 刘银坤, 等. 丹参对肝癌转移复发防治作用的研究. 中国中西医结合杂志, 1999, 19(5):292-295
11 王代树, 施学东, 梁云燕, 等. 中药白龙与 HMBA 对人肝癌细胞周期 CK1-P16 的调控. 中国中西医结合杂志, 2001, 21(10):763-765
12 刘连新, 姜洪池, 朱安龙, 等. 三氧化二砷对肝癌细胞凋亡 的 诱 导 及 机 理 探 讨 [J]. 中 华 医 学 杂 志 , 2001, 81(24):1526-1527
13 蓝惠玲, 王昌俊, 刘友章, 等. 健脾化淤中药逆转人肝癌细胞恶性表型作用研究. 中医药通报, 2005, 4(2):41-45
14 神代正道. 小柴胡汤によゐ肝癌细胞增殖抑制效果. とくに癌细胞のァボト-シス诱导について[J]. 现代东洋医学, 1995, 16(1):134
15 吴理茂, 赵一, 王勤, 等. 青蒿琥酯治疗肝癌的机理初探.中国中医基础医学杂志, 2002, 8(8):593-594
16 王晓素, 刘成, 刘平, 等. 养阴方对实验性肝癌诱癌过程的影响[J]. 中西医结合肝病杂志, 1995, 5(1):25
17 王晓明, 毛佳, 陈翌阳, 等. 实验性肝癌前期病变的研究及中药复方861对其的影响. 临床和实验医学杂志, 2003, 2(4):218-221
18 屠华成, 高林瑞, 孔令琪, 等. 健脾理气中药对大鼠肝脏癌 前 病 变 阻 断 作 用 的 实 验 研 究 [J]. 肿 瘤 , 1989, 9(1):31-32
19 吴万垠, 钱耕荪, 于尔辛. 健脾益气合剂阻抑小鼠 HBV协同AFB1致肝癌的机制. 华人消化杂志, 1998, 6(7):594
20 张慧珠, 杨林, 任雷鸣, 等. 姜黄素与阿霉素合用对 KB及 KBV200 细胞的杀伤作用. 中国药理学通报, 2001, 17(6):702-704
21 吴国土, 林建忠, 黄自强. 症痛散的抗癌作用与抗肿瘤药合用的协同作用实验研究. 世界肿瘤杂志, 2003, 2(3):209-211
22 王宝成, 郭军, 狄剑, 等. 榄香烯乳剂与肿瘤多药耐药的基础研究.中国肿瘤临床, 1996, 23(2):143-146
23 胡凯文, 陈倩义. 中药活性成分抗耐药肿瘤细胞体外筛选研究. 中国医药学报, 1998, 13(2):10
24 肖正明, 赵联合, 邱军, 等. 黄芪水提物对人肝癌细胞和瘤鼠免疫细胞的影响. 山东中医药大学学报, 2004, 28(2):136-139
25 闫智勇, 王再谟, 张天娥, 等. 癌肿宁对荷肝癌(H22)小鼠的抑瘤作用及其对 IL-2 和 NK 细胞活性的影响. 中西医结合肝病杂志, 1999, 9(2):24-26
26 高艳景, 袁孟彪, 辛华, 等. 大蒜素抑制 EGF 上调人肝癌细胞中 EGFR 表达的研究[J]. 山东中医药大学学报, 2000, 24(3):231
27 高艳景, 袁孟彪, 辛华, 等. 大蒜素抑制人肝癌细胞中VEGFmRNA 表达的研究. 中国药理学通报, 2001, 17(5):531-533
28 于尔辛. 中医治疗癌肿的思路[J]. 肿瘤, 2001, 21(6):419
2 段富津, 《方剂学》, 上海科技出版社出版, 1995, 6, 第一版
3 刘福英, 吕占军. 实验动物学. 北京: 中国农业科技出版社, 1997:180-182
4 李仪奎. 中药血清药理学实验方法的若干问题. 中药新药与临床药理, 1999, 10(2):95-98
5 蒙一纯, 丁霞, 本长恩, 等. 中药血清药理应用研究进展北京中医药大学学报. 1999, 4(22):15-16
6 刘建文主编. 药理实验方法学: 新技术与新方法. 北京:化学工业出版社, 2002:18-19
7 周明眉, 杨奎, 姜平远, 等. 中药血清药理学的方法研究-含药血清低温保存和血清灭活的影响.中药药理与临床, 1999, 15(2):44
8 黄馨慧, 罗明志, 弃浩, 等龙胆苦苷等6种中草药提取物对 SMMC-7721 人肝癌细胞增殖的影响. 西北药学杂志, 2004, 19(4)
9 Mosman T. Rapid colorimetric assay for cellar growth and survival: application proliferation and cytotoxic by assay [J]. Immune Methods, 1983, 55-65
10 Koike K, Tsutsumi T, Fujie H, et al. Molecular mechanism of hepatocarcinogenesiss. Oncology, 2002, 62:29-7
11 陈洪, 细胞凋亡与肝癌研究进展, 临床肿瘤学杂志, 1999, 4(1):67-70
12 郭强, 刘娜, 张倩. 凋亡基因在恶性肿瘤中的研究进展.山西中医学院学报, 2006, 7(1):25-27
13 Power C. Fanning N. Redmond HP. Cellular apoptosisand organ injury in sepsis: a review.Shock, 2002, 18(3):197-211
14 陈江, 黄文方, 卢贤瑜. 肝癌细胞凋亡基因调控的研究进展. 国外医学临床生物化学与检验学册, 2005, 26(10):715-717
15 邹劲林, 刘富元, 李艳芳, 等. p27和bcl-2与肿瘤[J].中国医学文摘·肿瘤学, 2001, 15(4):347-34
16 熊丽, 陆坚, 聂广, 等. 肝癌的分子发病学及其中医药防治机制探讨. 中西医结合肝病杂志, 2006, 16(1)
17 王筠, 张军平. 中药血清药理学实验方法研究概况. 天津中医药, 2005, 22(1):86-88
18 刘平. 关于中药血清药理学的若干思考. 中国中西医结合志, 1999, 19(5):263-266
19 薛洁, 谢默林, 中药血清药理学的方法学研究近况. 中草药, 2003, 34(6):9-11
20 余黎, 王坚, 朱荃. 中药血清药理学研究中血清处理方法的探讨[J]. 南京中医药大学学报, 2002, 18(7):222
21 肖月升, 耿建芳, 杨瑞合, 等. 初论“中心辨证”. 时珍国医国药2006, 17(6):1062-1063
1 王修杰, 袁淑兰, 王朝俊, 等. 丹参酮抗小鼠肝癌细胞作用和机理的初步研究. 中华肿瘤杂志, 1996, 18(6):412
2 李杰, 孙桂芝, 朴炳奎, 等. 中药肝康冲剂提取诱导人肝癌细胞系 BEL-7402 细胞凋亡的实验研究. 中国肿瘤生物治疗杂志, 1997, 4(3):234
3 张燕军, 夏天, 赵建斌, 等. 苦参碱对 SMMC-7721 细胞系的诱导分化作用. 第四军医大学学报, 1998, 19(3):340
4 黄炜, 黄济群, 张东方, 等. 18β-甘草次酸和甘草酸对人肝癌细胞增殖的抑制和诱导分化作用. 中国中医药科技, 2002, 9(2):92-93
5 徐学军, 周子成, 罗元辉, 等. β-榄香烯诱导人肝癌细胞株 SMMC-7721 凋亡的研究[J]. 第三军医大学学报, 1999, 21(4):268-271
6 徐瑞峰, 赵健雄, 程卫东, 等. 扶正抑瘤颗粒对小鼠肝癌H22 细胞凋亡及 Fas、FasL 表达的影响. 新中医, 2004, 36(12):65-67
7 陈忠伟, 林勇, 谢渭芬, 等. 苦参碱对肝癌细胞端粒酶活性调控及细胞周期的影响. 第二军医大学学报, 2002, 23(5):498-500
8 孟志强, 于尔辛, 宋明志, 等. 健脾理气中药对肝癌端粒酶活性的影响[J]. 中国中医药基础医学杂志, 2000, 6(1):23-26
9 叶炯贤, 叶红军, 杜意平, 等. 刺五加叶皂甙对肝癌癌基因 表 达 的 调 节 作 用 [J]. 中 华 实 验 外 科 杂 志 ,2000, 17(5):426-427
10 孙靖璟, 周信达, 刘银坤, 等. 丹参对肝癌转移复发防治作用的研究. 中国中西医结合杂志, 1999, 19(5):292-295
11 王代树, 施学东, 梁云燕, 等. 中药白龙与 HMBA 对人肝癌细胞周期 CK1-P16 的调控. 中国中西医结合杂志, 2001, 21(10):763-765
12 刘连新, 姜洪池, 朱安龙, 等. 三氧化二砷对肝癌细胞凋亡 的 诱 导 及 机 理 探 讨 [J]. 中 华 医 学 杂 志 , 2001, 81(24):1526-1527
13 蓝惠玲, 王昌俊, 刘友章, 等. 健脾化淤中药逆转人肝癌细胞恶性表型作用研究. 中医药通报, 2005, 4(2):41-45
14 神代正道. 小柴胡汤によゐ肝癌细胞增殖抑制效果. とくに癌细胞のァボト-シス诱导について[J]. 现代东洋医学, 1995, 16(1):134
15 吴理茂, 赵一, 王勤, 等. 青蒿琥酯治疗肝癌的机理初探.中国中医基础医学杂志, 2002, 8(8):593-594
16 王晓素, 刘成, 刘平, 等. 养阴方对实验性肝癌诱癌过程的影响[J]. 中西医结合肝病杂志, 1995, 5(1):25
17 王晓明, 毛佳, 陈翌阳, 等. 实验性肝癌前期病变的研究及中药复方861对其的影响. 临床和实验医学杂志, 2003, 2(4):218-221
18 屠华成, 高林瑞, 孔令琪, 等. 健脾理气中药对大鼠肝脏癌 前 病 变 阻 断 作 用 的 实 验 研 究 [J]. 肿 瘤 , 1989, 9(1):31-32
19 吴万垠, 钱耕荪, 于尔辛. 健脾益气合剂阻抑小鼠 HBV协同AFB1致肝癌的机制. 华人消化杂志, 1998, 6(7):594
20 张慧珠, 杨林, 任雷鸣, 等. 姜黄素与阿霉素合用对 KB及 KBV200 细胞的杀伤作用. 中国药理学通报, 2001, 17(6):702-704
21 吴国土, 林建忠, 黄自强. 症痛散的抗癌作用与抗肿瘤药合用的协同作用实验研究. 世界肿瘤杂志, 2003, 2(3):209-211
22 王宝成, 郭军, 狄剑, 等. 榄香烯乳剂与肿瘤多药耐药的基础研究.中国肿瘤临床, 1996, 23(2):143-146
23 胡凯文, 陈倩义. 中药活性成分抗耐药肿瘤细胞体外筛选研究. 中国医药学报, 1998, 13(2):10
24 肖正明, 赵联合, 邱军, 等. 黄芪水提物对人肝癌细胞和瘤鼠免疫细胞的影响. 山东中医药大学学报, 2004, 28(2):136-139
25 闫智勇, 王再谟, 张天娥, 等. 癌肿宁对荷肝癌(H22)小鼠的抑瘤作用及其对 IL-2 和 NK 细胞活性的影响. 中西医结合肝病杂志, 1999, 9(2):24-26
26 高艳景, 袁孟彪, 辛华, 等. 大蒜素抑制 EGF 上调人肝癌细胞中 EGFR 表达的研究[J]. 山东中医药大学学报, 2000, 24(3):231
27 高艳景, 袁孟彪, 辛华, 等. 大蒜素抑制人肝癌细胞中VEGFmRNA 表达的研究. 中国药理学通报, 2001, 17(5):531-533
28 于尔辛. 中医治疗癌肿的思路[J]. 肿瘤, 2001, 21(6):419