純中藥制劑_卡寧_對(duì)肝癌細(xì)胞的作用.kdh

1、2009年1月第6卷第1期·論著·中國(guó)醫(yī)藥導(dǎo)報(bào)CHINA MEDICAL HERALD中藥誘導(dǎo)細(xì)胞凋亡是中醫(yī)藥抗腫瘤的分子機(jī)制之一。從中醫(yī)藥角度闡述中藥誘導(dǎo)腫瘤細(xì)胞凋亡的機(jī)制有助于中醫(yī)腫瘤的臨床和科研工作的深入。已發(fā)現(xiàn)許多基因與細(xì)胞凋亡有關(guān),有凋亡抑制基因,如bcl-2;凋亡誘導(dǎo)基因,如p53等。腫瘤是一種基因病,與腫瘤發(fā)生相關(guān)的基因也可因陰陽分為兩大類,即癌基因與抑癌基因。細(xì)胞中的癌基因或抑癌基因原本是調(diào)控正常分化的正負(fù)因素。從中醫(yī)理論而言,癌基因?qū)訇?抑癌基因?qū)訇?“陰生陽長(zhǎng),陽殺陰藏”。1材料與方法1.1材料純中藥制劑“卡寧”對(duì)肝癌細(xì)胞的作用毛小玲,潘素瀅,張大鵬(廣

2、州醫(yī)學(xué)院第一附屬醫(yī)院,廣東廣州510120摘要目的:觀察抗癌中藥“卡寧”對(duì)體外培養(yǎng)的肝癌細(xì)胞株BEL-7402細(xì)胞的凋亡抑制基因bcl-2的影響,探討其抗腫瘤治療的作用機(jī)制。方法:取體外培養(yǎng)的對(duì)數(shù)生長(zhǎng)期BEL-7402細(xì)胞,隨機(jī)分為4組:對(duì)照組,5-氟尿嘧啶組,含藥“卡寧”血清1組(10%含藥血清組和含藥“卡寧”血清2組(15%含藥血清組;采用MTT 比色法觀察不同濃度含藥血清對(duì)體外培養(yǎng)的人肝癌細(xì)胞株BEL-7402細(xì)胞的抑制能力,PCR 檢測(cè)其對(duì)凋亡抑制基因bcl-2的影響。結(jié)果:PCR 結(jié)果顯示,“卡寧”1、2組bcl-2的活性明顯低于對(duì)照組(P =0.038;MTT 結(jié)果顯示,細(xì)胞生長(zhǎng)抑

3、制率隨含藥血清作用時(shí)間及濃度的增加而增大(P <0.05,15%含藥血清72h 的作用最強(qiáng)。結(jié)論:對(duì)腫瘤細(xì)胞bcl-2的抑制可能是抗癌中藥“卡寧”誘導(dǎo)腫瘤細(xì)胞凋亡作用機(jī)制的一部分。關(guān)鍵詞bcl-2;中草藥;肝臟腫瘤中圖分類號(hào)R735.7文獻(xiàn)標(biāo)識(shí)碼A文章編號(hào)1673-7210(200901(a -014-03Effect of chinese medicine "Caning"on hepatic cancer cellsMAO Xiao-ling,PAN Su-ying,ZHANG Da-peng(The First Affiliated Hospital of Gu

4、angzhou Medical College,Guangzhou 510120,ChinaAbstractObjective:To observe the effects of anticancer chinese medicine "Caning"on the anti-apoptosis gene bcl-2of in vitro cultured human hepatic cancer cell line BEL-7402cells,and investigates its anticancer therapeutic mechanism.Methods:In v

5、itro cultured BEL-7402cells in a logarithmic growth phase were randomly divided into 4groups:control group,5-Fluorouracil treated group,serum group 1containing "Caning"(10%pastille serumand serum group 2contain -ing "Caning"(15%pastille serum;then MTT assay was employed to detect

6、 the inhibitory ability of serums containing dif -ferent concentrations of "Caning"to in vitro cultured human hepatic cancer cell line BEL-7402cells,and PCR was used to detect the effect of "Caning"on the anti-apoptosis gene bcl-2.Results:Results of in vitro experiment suggested

7、that,the bcl-2gene activity in serum group 1and 2containing "Caning"was significantly lower than that of control group (P =0.038;result of MTT assay indicated that the cell growth inhibitory rate was increased along with the increase of the action time and concentration of serum containing

8、 "Caning"(P <0.05,and the 15%pastille serum showed the strongest inhibitory effect at 72h.Conclusion:"Caning"could significantly inhibit the growth of hepatic cancer cells partly by inhibiting the anti-apoptosis gene bcl-2.Key wordsbcl-2;Chinese medicine;Hepatic cancer基金項(xiàng)目廣東省中

9、醫(yī)藥管理局科技項(xiàng)目(編號(hào):1040105。作者簡(jiǎn)介毛小玲(1951-,主任醫(yī)師,主要從事中醫(yī)腫瘤、中醫(yī)婦科臨床與實(shí)驗(yàn)研究。142009年1月第6卷第1期·論著·MTT 比色法用含0.25%胰蛋白酶和0.02%EDTA 的消化液消化對(duì)數(shù)生長(zhǎng)期的BEL-7402細(xì)胞,用含20%胎牛血清的DMEM 培養(yǎng)基調(diào)成2.5×104/ml 的單細(xì)胞懸液,96孔細(xì)胞培養(yǎng)板中每孔加入100l ,待細(xì)胞貼壁6h 后加入含藥血清,終濃度分別為10%及15%。每一濃度設(shè)3個(gè)復(fù)孔,并設(shè)對(duì)照組。分別培養(yǎng)24、48和72h 后,每孔加入0.5%MTT 20l ,繼續(xù)培養(yǎng)4h ,1000r/min

10、 離心10min ,去上清后每孔加入DMSO 100l ,結(jié)晶溶解后酶標(biāo)儀570nm 波長(zhǎng)處測(cè)定吸光度值,并計(jì)算抑制率,實(shí)驗(yàn)重復(fù)3次。由于斷裂點(diǎn)位置不同,所擴(kuò)增的片段長(zhǎng)度波動(dòng)在300700bp 。水作空白對(duì)照。1.3統(tǒng)計(jì)學(xué)方法數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x ±s 表示,計(jì)量資料比較采用t 檢驗(yàn),計(jì)數(shù)資料比較采用采用2檢驗(yàn),所用數(shù)據(jù)均由SPSS 軟件進(jìn)行統(tǒng)計(jì)學(xué)處理,P <0.05表示差異有統(tǒng)計(jì)學(xué)意義。2結(jié)果2.1細(xì)胞形態(tài)學(xué)觀察含藥血清對(duì)BEL-7402細(xì)胞形態(tài)的影響為:空白對(duì)照組細(xì)胞生長(zhǎng)良好,細(xì)胞呈圓形,飽滿,折光性強(qiáng),胞質(zhì)透明,細(xì)胞核大,核膜清晰。含藥血清培養(yǎng)20h 后可見部

11、分細(xì)胞皺縮,胞質(zhì)折光性差、深暗,培養(yǎng)48h 后皺縮更多,可見大量細(xì)胞碎片。2.2MTT 檢測(cè)結(jié)果不同濃度含藥“卡寧”血清作用2472h 后的BEL-7402細(xì)胞生長(zhǎng)抑制率見表1。含藥血清可有效抑制BEL-7402細(xì)胞的生長(zhǎng)增殖,其抑制率隨時(shí)間的延長(zhǎng)和濃度的增加而增大(P <0.05,15%含藥血清72h 的抑制作用最強(qiáng)。表1含藥血清對(duì)BEL-7402細(xì)胞的生長(zhǎng)抑制率(x ±s ,%Table 1Growth inhibitory rate of BEL-7402cell treated by pastille serum(x ±s ,%與10%及15%含藥血清組比較,

12、#P <0.05;與10%含藥血清組24、48h 比較,P <0.05;與15%含藥血清組24、48h 比較,*P <0.052.3含藥中藥作用72h 后BEL-7402細(xì)胞bcl-2表達(dá)情況“卡寧”作用前細(xì)胞bcl-2陽性率為43.14%,10%含藥血清作用72h 后,bcl-2表達(dá)降低為20.57%;15%含藥血清作用后,bcl-2表達(dá)由40.12%降低為7.78%;而5-FU 組bcl-2的表達(dá)則由45.06%降低為30.67%(表2。15%含藥血清72h 的作用最強(qiáng)(P <0.05?！翱▽帯?、2組bcl-2的活性明顯低于對(duì)照組(P =0.038。第二輪PCR

13、產(chǎn)物電泳圖見圖1。表2含藥血清作用72h BEL-7402細(xì)胞bcl-2的表達(dá)(%Table 2bcl-2gene expression of BEL-7402cellafter 72hours treated by pastille serum (%與10%含藥血清組0h 比較,P <0.05;與15%含藥血清組0h 比較,*P <0.05;與10%及15%含藥血清組72h 比較,#P <0.05圖1第二輪PCR 產(chǎn)物電泳圖1、2:患者甲治療前后擴(kuò)增產(chǎn)物;3、4:患者乙治療前后擴(kuò)增產(chǎn)物;5、6:健康對(duì)照;M :分子量Marker箭頭所示為陽性片段3討論腫瘤的產(chǎn)生是由于機(jī)體正

14、虛邪實(shí)的結(jié)果。正氣不足主要是氣虛和血虛,邪實(shí)主要是血瘀、痰凝等,所以,醫(yī)宗必讀說:“積之成者,正氣不足,而后邪氣踞之?！币虼酸槍?duì)腫瘤病機(jī)正虛邪踞的特點(diǎn),治療原則應(yīng)是扶正祛邪?；隗w外誘導(dǎo)腫瘤細(xì)胞凋亡,從中藥中篩選新的天然抗癌藥和研究中藥抗癌的分子機(jī)制,對(duì)于中藥走向世界和實(shí)現(xiàn)中藥現(xiàn)代化有重要的意義。本課題組實(shí)驗(yàn)表明,傳統(tǒng)中藥甘草體外可有效地選擇性誘導(dǎo)幾種人腫瘤細(xì)胞凋亡,莪術(shù)活血化瘀,是具有臨床應(yīng)用價(jià)值和良好開發(fā)前景的天然細(xì)胞凋亡誘導(dǎo)劑。bcl-2是bcl-2凋亡抑制基因家族中的一員。它能抑制抑制率(%=1-用藥組平均吸光度值對(duì)照組平均吸光度值×100%對(duì)照組10%含藥血清組15%含藥血

15、清組5-FU 組分組24h 48h10.78±1.65#72h032.58±5.1751.12±4.21*15.09±2.82#對(duì)照組10%含藥血清組15%含藥血清組5-FU 組72h 41.23#152009年1月第6卷第1期·短篇論著·中國(guó)醫(yī)藥導(dǎo)報(bào)CHINA MEDICAL HERALD基金項(xiàng)目牡丹江醫(yī)學(xué)院科研項(xiàng)目(B200512作者簡(jiǎn)介吳宜艷(1963-,女,教授,主要從事藥理學(xué)教學(xué)及科研工作。蓽茇為胡椒科植物蓽茇Piper longum L.的未成熟果穗,是中、蒙、藏醫(yī)習(xí)慣用藥,功能溫中散寒,行氣止痛。臨床用于胃脘痛、腹瀉、腳

16、氣、目盲、齲齒痛,蒙、藏醫(yī)還用于治療冠心病,隨著中藥化學(xué)藥理等方面的深入研究,拓寬了臨床用藥,保證了臨床用藥的科學(xué)性和可靠性。目前蓽茇的藥理研究發(fā)蓽茇有效成分提取及抗炎作用的研究吳宜艷1,楊志2,劉廣勤2,孫浩巖1,朱文全1,于春艷1(1.牡丹江醫(yī)學(xué)院,黑龍江牡丹江157011;2.牡丹江醫(yī)學(xué)院紅旗醫(yī)院,黑龍江牡丹江157011摘要目的:對(duì)蓽茇有效成分進(jìn)行提取,觀察其對(duì)二甲苯致小鼠耳廓炎癥的作用。方法:采用溶劑法提取蓽茇有效成分,石油醚、乙酸乙酯萃取分離,得到三部分藥液。采用二甲苯引起小鼠耳廓腫脹致炎造模方法,觀察并記錄小鼠耳廓腫脹程度。結(jié)果:服用蓽茇的乙酸乙酯萃取物的小鼠,左右耳腫脹程度與地

17、塞米松對(duì)照品接近。結(jié)論:蓽茇提取物具有明顯的抗炎作用。關(guān)鍵詞蓽茇;提取物;抗炎中圖分類號(hào)R965文獻(xiàn)標(biāo)識(shí)碼A 文章編號(hào)1673-7210(200901(a -016-02Study on extraction of effective composition of Piper longum and its anti-in -flammatory effectsWU Yi-yan 1,YANG Zhi 2,LIU Guang-qin 2,SUN Hao-yan 1,ZHU Wen-quan 1,YU Chun-yan 1(1.Mudanjiang Medical University ,Muda

18、njiang 157011,China;2.Hongqi Hospital of Mudanjiang Medical University,Mudanjiang 157011,ChinaAbstractObjective :To study the extraction of effective composition of Piper longum,and to observe the effects on the inflammation of auricles induced by dimethylbenzine in mice.Methods :Solvent extraction

19、was used for the extraction of effective composition of Piper longum,we had got three parts of medical solution by petroleum ether and ethyl acetate ex -traction.The inflammation of auricle induced by dimethylbenzine in mice was used and mouse ear swelling was observed.Results :Swelling degree of ri

20、ght and left ears in mice which was given ethyl acetate extraction were similar to that was given dexamethasone.Conclusion :Piper longum extract has significant anti-inflammatory effect.Key wordsPiper longum ;Extraction ;Anti-inflammatory細(xì)胞凋亡,使細(xì)胞長(zhǎng)期存活,促使細(xì)胞內(nèi)某些癌基因活化,產(chǎn)生惡性細(xì)胞克隆。分化差、體積大的肝癌細(xì)胞中bcl-2基因表達(dá)增強(qiáng)。本實(shí)

21、驗(yàn)結(jié)果顯示,10%含藥血清作用24h 后,細(xì)胞抑制率為(14.44±3.20%,并隨含藥血清濃度的增加而增大,15%含藥血清作用24h 后細(xì)胞抑制率為(32.56±4.36%。隨著作用時(shí)間的延長(zhǎng),抑制作用增強(qiáng),15%含藥血清作用48h 后達(dá)(42.67±4.18%,而5-FU 組達(dá)(10.78±1.65%(P <0.05。癌基因bcl-2表達(dá)也有減少的趨勢(shì),15%血清作用72h 使bcl-2表達(dá)率從40.12%下降到7.78%,而5-FU 仍達(dá)30.67%,這也許是“卡寧”誘導(dǎo)肝癌細(xì)胞凋亡的機(jī)制。原發(fā)性肝癌在中國(guó)的發(fā)病率較高,約占世界總數(shù)的40%,其惡性程度高,自然生存期短,明確診斷時(shí)大多為中、晚期,缺乏有效的治療方法,總體5年生存率較低。所以,綜合治療成為肝癌的主要治療手段,包括介入治療