【作者】 王金烁；

【导师】 杨瑞合；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2007， 硕士

【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。更多还原

【Abstract】 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).Results1 the result in vitro1.1 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 vivo2.1 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.Conclusion1 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.更多还原