活血行气方药物血清对人胃癌SGC-7901细胞株作用的实验研究
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摘要
目的:胃癌在世界范围内和我国均是最常见的恶性肿瘤之一。根据世界卫生组织公布的全球统计报告,世界胃癌年发病率为13.86/10万人,仅次于肺癌居第二位。我国是胃癌的高发区,每年我国新发现40万胃癌患者,占世界胃癌发病人数的42%。中国胃癌死亡率是欧美发达国家的4-8倍。胃癌是当前危害我国人民身体健康的重大疾病。对于肿瘤的治疗方法大多采用手术配合放射疗法和化学疗法的治疗手段,但多数抗肿瘤药物缺乏选择作用,肿瘤患者在接受放射、化学疗法杀伤肿瘤细胞的同时,对机体正常组织和细胞也有相当大的损伤。常会出现如骨髓造血功能抑制、消化道反应、免疫功能低下、肝脏及肾脏等组织的损害等。目前已经有大量实验研究表明,许多中药及中药复方具有抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡、改善机体免疫力、诱导肿瘤细胞分化、抑制肿瘤细胞基因表达、抑制肿瘤细胞转移、逆转多药耐药、抗突变抗氧化等作用。中医对于胃癌的发病,认为多是由于先天不足、正气亏虚,导致外邪侵袭;或是由于饮食不节、导致脾胃损伤;或是由于情志失调、气机不畅;或是由于劳逸失当、导致气滞血瘀等因素,最终可造成或加重脾胃损伤,脾失健运,胃失和降,湿聚生痰,痰阻血脉,导致血行不畅,进而化生瘀毒,阻于胃脘。日久逐渐形成积聚,进一步导致机体脏腑功能失调,气机运行失畅,体内津液代谢紊乱,最后瘀血、邪毒、痰浊相互搏结而发病。在治疗上多采用健脾以运化水谷、化生气血,活血化瘀,化痰散结,清热解毒、调肝补肾、益气养阴、扶正祛邪等,提高机体的抗病能力,改善症状,控制肿瘤生长、转移和侵袭。纵观活血行气方,具有活血祛瘀、行气止痛的功效,能解气分之郁结,活血而不耗血,祛瘀又能生新,起到气血兼顾,符合“气行则血行”之治疗原则。对于改善胃癌中晚期肿胀、腹满、疼痛等症状有一定的作用。通过此实验,探讨中药药物血清抗胃癌作用机制,为国药治疗胃癌提供有力依据。
材料与方法:实验以人胃癌SGC-7901细胞株为实验对象,对人胃癌SGC-7901细胞株进行体外细胞培养,通过中药血清药理学方法研究活血行气方药物血清对人胃癌SGC-7901细胞株作用机制。体外细胞培养是指从动物活体体内取出组织,于模拟体内生理环境等特定的体外条件,进行孵育培养,使之生存并生长。血清药理学方法是指将中药或中药复方经口给动物灌服一定时间后采集动物血液,分离血清,用含有药物成分的血清进行体外实验的实验方法。实验选择SD大鼠30只,雌雄各半,体重180g-220g,中药煎剂1ml/100g大鼠,中药汤剂灌胃给药,2ml/次,每日两次给药,连续3天;对照组、西药组予生理盐水等量等次灌胃3天。分别在末次给药后2小时采血,戊巴比妥钠麻醉,无菌条件下进行腹主动脉取血,把同种条件药物血清混匀、过滤,56℃,30min灭活,-20℃保存备用。
实验随机分为正常血清对照组(对照组) ,西药组,中药组,中西医结合组。其中,对照组为大鼠血清加入RPMI1640培养液,浓度10%;西药组用含10%顺铂组大鼠血清RPMI1640培养液配制,DDP的终浓度为5ul/ml(小),10ul/ml(中),20ul/ml(大)三组;中药组为大鼠药物血清加入RPMI1640培养液,浓度分别为5%(小)、10%(中)、20%(大)三组;中西医结合组为含中药浓度10%+ DDP终浓度20ul/ml组成。
实验首先采用MTT比色法测定人胃癌SGC-7901细胞增殖抑制率,MTT比色法作为一种检测细胞存活和生长的方法,其检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲瓒并沉积在细胞中,而死细胞无此功能。二甲基亚砜能溶解细胞中的甲瓒,用酶联免疫检测仪在570nm波长处测定其光吸收值,可间接反映活细胞数量。在一定细胞数范围内,MTT结晶形成的量与细胞数成正比。经药物处理后的细胞株经HE染色后,制成光学显微镜标本;药物处理后人胃癌SGC-7901细胞株经固定、脱水、包埋等程序后制成透射电镜标本,通过光镜、透射电镜观察经活血行气方药物血清处理后的人胃癌SGC-7901细胞株的形态学变化;通过DNA琼脂凝胶电泳法检测人胃癌SGC-7901细胞凋亡梯带分析及流式细胞化学方法研究人胃癌SGC-7901细胞在活血行气方药物血清影响下细胞形态学变化、细胞凋亡定性定量分析,测定细胞凋亡率。
本实验通过蛋白质印迹(Western blot)法测定经活血行气方药物血清处理后的SGC-7901人胃癌细胞株P53、Bcl-2基因蛋白表达水平的变化,对经过药物处理后的细胞进行细胞蛋白质的提取,蛋白质的SDS-PAGE电泳,蛋白质转膜、抗体杂交、化学发光显影定影后,图像分析。
本实验中,对药物处理后细胞进行固定、脱水、复染等程序后进行酶细胞化学染色,通过酶细胞化学染色方法观察活血行气方药物血清对人胃癌SGC-7901细胞株Mg~2+-ATP酶、G-6-P酶活性的影响。
结果:
1.经活血行气方药物血清作用人胃癌SGC-7901细胞株24h后,实验结果显示,中药小剂量组未见明显抑制作用,其余各组均表现出不同程度的抑制作用,中药中剂量组的抑制率为17.24%,中药大剂量组的抑制率为24.76%,中西医结合组的抑制率为42.97%;经活血行气方药物血清作用人胃癌SGC-7901细胞株48h后,中药小剂量组出现抑制作用,其抑制率为21.79%,中药中剂量组的抑制率为34.95%,中药大剂量组的抑制率为48.26%,各药物血清组随着作用时间的延长抑制效果更明显,抑制效应呈时间依赖性,以中西医结合组药学血清作用48h组抑制作用最佳,抑制率达51.09%。
2.HE染色后,光镜下可见,对照组细胞贴壁生长,呈长梭形,大小比较均匀,核卵圆形居中,胞质清亮透明。药物血清组部分细胞由长梭形变成圆形,细胞核不规则,胞浆浓缩,染色质边集,浓缩药物血清组细胞。透射电镜下可见,经药物血清处理的人胃癌SGC-7901细胞变圆,微绒毛及突起明显减少,伪足消失,线粒体空泡化,核染色质凝集,可见调亡小体。凋亡细胞发生率与药物浓度呈正相关。DNA琼脂凝胶电泳检测可见活血行气方药物血清能诱导SGC-7901人胃癌细胞形成凝胶电泳特异梯状带,以流式细胞仪定量分析活血行气方药物血清作用48h后,在G1峰前出现凋亡峰中药组凋亡率为9.87±2.13%,西药组的凋亡率为48.24±1.32%,中西医结合组的凋亡率为47.65±2.47%。
3. P53基因是目前被最为广泛和深入研究的抑瘤基因之一,也是迄今为止所发现的突变频率最高和突变范围最广泛的抑癌基因,P53基因突变是胃癌发生发展的重要始动因素之一。Bcl-2基因是一种癌基因,其表达的Bcl-2蛋白是控制线粒体中致凋亡因子释放的主要调节因子,在维持细胞生理性分化、发育和细胞数量的动态平衡中具有重要作用。经蛋白质印迹(Western blot)法测定人胃癌SGC-7901细胞株经活血行气方药物血清处理后,P53、Bcl-2基因表达的变化。结果显示,对照组中P53、Bcl-2蛋白为中等表达,与对照组相比,中药组及中西医结合组中P53蛋白表达增强,Bcl-2蛋白表达减弱(P<0.05)。
4.经药物血清处理后,在光镜下见对照组Mg~2+-ATPase主要定位在细胞膜的内侧,黑色酶反应颗粒大小均匀,密度大,呈线状规则排列;G-6-Pase主要定位在细胞质内,酶反应颗粒粗大,排列密集,呈融合状;与对照组比较,各组Mg~2+-ATPase和G-6-Pase反应颗粒变小,数量减少,密度减低,有的为阴性。提示酶活性明显下降。
结论:
1.活血行气方药物血清对人胃癌SGC-7901细胞株在体外具有抑制增殖的作用。
2活血行气方药物血清对人胃癌SGC-7901细胞株具有诱导细胞凋亡的作用。
3.活血行气方药物血清可上调人胃癌SGC-7901细胞株P53基因蛋白表达水平,下调Bcl-2基因蛋白表达水平。活血行气方药物血清诱导人胃癌SGC-7901细胞株凋亡的机制可能是通过上调促凋亡基因P53表达,下调癌基因Bcl-2的表达实现的。
4.活血行气方药物血清具有降低人胃癌SGC-7901细胞株Mg~2+-ATP酶、G-6-P酶活性的作用。
Purpose:Gastric cancer is one of the most common malignant tumor in the world and china. According to the reporting and statistics of global by the world health organization, The gastric cancer incidence is 13.86/10 million, after lung canner to second place. Our country is hige occurrence of gastric cancer, the occurrence ratio is 42% of the world,about 40 million stomach cancer patients each year. Stomach cancer mortality rate is 4- 8 times of the european countries. Most treatments for cancer, surgery with radiotherapy and chemotherapy treatment, but most of the anticancer drugs lack of selection, cancer patients undergoing radiation and chemotherapy kill cancer cells while normal tissues and cells of the body also has considerable damage. Such as the inhibition of bone marrow, gastrointestinal reactions, immune function, liver and kidney tissue damage and so on often occurring.At present, there have been a lot of experimental studies show that,the traditional Chinese medicine has effects on generation inhibition of tumor and inducing apoptosis, improve the immunity of the body, induced the tumor cells differentiated, inhibition of tumor cell gene expression, inhibition of tumor cell metastasis, multidrug resistance reversal, anti-mutant antioxidant,and so on. Gastric cancer attack for Traditional Chinese medicine,that more is due to congenital deficiency and the vital qi deficiency, led to invasion of the exogenous evils,or because of improper diet.Improper diet including immoderate diet、insanitary diet、particular diet,led to damage of spleen and stomach; Or because of emotional disorders,led to unsmooth flow of qi;or because of overstrain and overease,led to stagnation of qi and blood stasis. eventually can cause and aggravate damage of spleen and stomach,the function of transforming and transportation the food and water about spleen discharge,stomach can,t in charging sending dowm, dampness aggregate phlegm, phlegm obstruct blood vessels, causes poor of blood transportation, and then metaplasia stagnat and Poison,and blocking the stomach. Falling gradually accumulate. Further lead to disfunction of the viscera, unsmooth flow of qi, the body fluid metabolism, at last the bleeding, cult poison, phlegm together and gastric cancer attacked. Traditional Chinese medicine in the treatment of gastric cancer,often strengthened the function of transforming and transportation the food and water about spleen,transforming qi and blood, strengthened blood circulation and clear stagnant blood. disperse Sputum,clearing hot and detoxification, conditioning and benefit Liver-kidney. Benefit qi and yin, assistance vital qi and Get rid of evil qi.Improve the body's resistance to disease, improve symptoms, control of tumor growth, metastasis and invasion. The Houxuesingqifang strengthened blood circulation and clear stagnant blood,transportation qi and stop pain,free qi flow,strengthened blood vessels,but consumption blood. Removed blood stasis can help growth again. Consistent of the principle of treatment about qi promotes generation of blood,qicompels blood to circulate. Advanced gastric cancer to weaken distension and depression or pain sensation in the epigastrium and other symptoms . Through this experiment, discussion the mechanism of Tranditional Chinese medicines anti-cancer by serum , provide a strong basis for traditional Chinese medicine treatment of gastric cancer .
Material and method:With the chinese herbal medicine serum pharmacology method,To research the mechanism of Huoxuexingqi formula in treating Human Stomach Cell Line SGC-7901. The chinese herbal medicine serum pharmacology refers to the Chinese herbal medicine or traditional Chinese medicine formula by mouth fed to experimental animals ,after a certain time , experimenter collecting blood,and separation of serum, then the serum containing pharmaceutical ingredients for in vitro experimental methods. The chinese herbal medicine serum pharmacological method is the more commonly used method by experimenter. My experiment was chosen for thirdty SD rats, include male and female, weight about 180g to 220g, through the stomach way to medicine decoction of traditional Chinese herbal medicine, 2 ml each, twice daily,three days continuous through the stomach way to medicine decoction of traditional Chinese herbal medicine.Then the control group and the western medicine group for saline. The dose is same to traditional Chinese herbal medicine, 2ml each, twice daily,three days continuous, Collecting blood at 2 hours after the last for drug.using pentobarbital sodium narcoticing, blood was collected by abdominal aorta under nobacteria conditions,.mixing the serum under the same conditions, filtering, 56℃, Inactivated 30 Minutes,then ,the serum is prepared , preservation under -20℃. After cell culture success ,the cells grew into the monolayer ,the experiment beginning.
The experiment randomly divided into control group, western medicine group, traditional Chinese medicine group,and traditional Chinese medicine combinating western medicine group.The western medicine group including the concentration of DDP are 5ul/ml(small),10ul/ml(Medium) and 20ul/ml(large) , the traditional Chinese medicine group including the concentration of the serum are 5%(small),10%(Medium) and 20%(large). The traditional Chinese medicine combinating western medicine group is concentration of DDP 20ul/ml plus 10% serum .
At first ,determined the proliferation rate of the human stomach cell line SGC–7901 by MTT test. MTT as a detection method for cell survival and growth, and its detection principle for the mitochondria in living cells by exogenous succinate dehydrogenase MTT can restore the blue-violet water-insoluble crystalline formazan and deposited in the cell , death cell is no such function. DMSO can dissolve formazan in the cells, enzyme-linked immunosorbent detector was measured at 570nm wavelength of light absorption value can reflect the number of living cells. In a certain range of cells, MTT crystals formed is proportional to the quantity and cell number. The human stomach cell line SGC–7901 after drug treatment by HE staining, Made of optical microscope specimens,The human stomach cell line SGC–7901 after drug treatment by fixation, dehydration, embedding, etc. made into TEM samples.With light microscope and transmission electron microscope,to observe the morphological change of the Human Stomach Cell Line SGC-7901 after dealing by drug serum, qualitative and quantitative analysis of apoptosis ,measured the rate of cell apoptosis. P53 gene is currently the most extensive and in-depth study of the tumor suppressor gene, is one of the mutations detected so far and the most frequently mutated tumor suppressor genes most widely, P53 gene mutation is the development of gastric cancer is one important factor in initiating . Bcl-2 gene is an oncogene, the expression of Bcl-2 protein is to control the release of mitochondrial factors in apoptosis of the main regulatory factor in the maintenance of cellular differentiation, growth and cell numbers has an important role in the dynamic balance.To observe change of the Human Stomach Cell Line SGC-7901 gene express level of P53 and Bcl-2 by immumo blot(western blot) after Huoxuexingqi Formula treatmented. First,Cell protein extract,after drug treamented, SDS-PAGE electrophoresis of proteins, transmembrane protein, antibody hybridization, chemiluminescence development snapshot, the image analysis.,after drug treatment cells were fixed, dehydrated, stained and other procedures for enzymatic staining.
To detect the change of Mg~2+-ATPase and G-6-Pase activity of the Human Stomach Cell Line SGC-7901 after Huoxuexingqi Formula treatmented .
Results:
1. By drug treatmented Within 24h, the effect of the drug serum of each group was differently obvious except in low concentration. The inhibition rate of traditional Chinese medicine medium group is 17.24%, the inhibition rate of traditional Chinese medicine in high dose group was 24.76% , the inhibition rate of traditional Chinese medicine combinating western medicine group is 42.97%; By the Serum of Huoxuexingqi Formula treatmented within 48h, low dose group showed inhibitory effect of traditional Chinese medicine, and its inhibition rate was 21.79%, the inhibition rate of traditional Chinese medicine in the medium dose group was 34.95%, inhibition rate of traditional Chinese medicine in high dose group was 48.26%.which was more obvious with the time - ratio , whose inhibition effects was time-dependent.the inhibition ratio of Group DDP+Huoxuexingqi approaching to 51.09%,the effect is the best.
2. After HE staining, uder light microscope, the growth of control group cell adhesion, fusiform, more uniform size of nuclear oval centered, clear and transparent cytoplasm. the shape of the cells changed into round or oval shape, irregular nucleus, cytoplasm condensation, chromatin margination, drug serum concentration cells.The chromatin concentrated, under the transmission electron microscope, shows that the serum treatment by drugs in human gastric cancer SGC-7901 cells became round, microvilli, and protrusions significantly reduced pseudopod disappear, mitochondrial vacuolization, nuclear chromatin condensation, apoptotic bodies were seen. With the DNA abstraction of cells and agarose gel electrophoresis,appearance“ladder strip”. Quantitative analysis by flow cytometry blood circulation of drug serum 48h later ,the apoptotic peak before the G1 peak ,apoptotic rate of the Medicines was 9.87±2.13%, western group was 48.24±1.32%, Chinese and Western Medicine combined group was 47.65±2.47%.
3. determination the change of P53, Bcl-2 gene expression by Western blot method for the Human Stomach Cell Line SGC-7901 by serum of Huoxuexingqi formula.The results showed that the control group P53, Bcl-2 protein expression was moderate, compared with the control group, combining Chinese and Western medicine group and the group increased expression of P53 protein, Bcl-2 protein expression was decreased.Drug serum of Huoxuexingqi formula can up-regulate p53 ,down-regulate Bcl-2 and achieve the suppression of Human Stomach Cell Line SGC-7901.
4. The serum treatment, the control group in the light microscope, Mg2 +-ATPase was mainly located in the inner cell membrane, enzymatic black uniform particle size, density, linear rule was ordered; G-6-Pase mainly localized in the cytoplasm the enzyme reaction particles thick, densely arranged, was fusion-like; with the control group,the response granules of Mg~2+-ATPase and G-6-Pase in the Human Stomach Cell Line SGC-7901 became smaller after the treatment of the Huoxuexingqi formula, the amount of the granules became fewer, the density was lower.
Conclusion:
1. The serum of Huoxuexingqi formula had effects on generation inhibition of Human Stomach Cell Line SGC-7901.
2. The serum of Huoxuexingqi formula can inducing apoptosis.
3. Drug serum of Huoxuexingqi formula can up-regulate p53 ,down-regulate bcl-2 and achieve the suppression of Human Stomach Cell Line SGC-7901. The serum of Huoxuexingqi formula make Human Stomach Cell Line SGC-7901 apoptosis may be through increased expression of apoptotic gene P53, decreased Bcl-2 oncogene expression achieved. 4. The activity of the Mg~2+-ATPase and G-6-Pase decreased after using the Huoxuexingqi formula.
材料与方法:实验以人胃癌SGC-7901细胞株为实验对象,对人胃癌SGC-7901细胞株进行体外细胞培养,通过中药血清药理学方法研究活血行气方药物血清对人胃癌SGC-7901细胞株作用机制。体外细胞培养是指从动物活体体内取出组织,于模拟体内生理环境等特定的体外条件,进行孵育培养,使之生存并生长。血清药理学方法是指将中药或中药复方经口给动物灌服一定时间后采集动物血液,分离血清,用含有药物成分的血清进行体外实验的实验方法。实验选择SD大鼠30只,雌雄各半,体重180g-220g,中药煎剂1ml/100g大鼠,中药汤剂灌胃给药,2ml/次,每日两次给药,连续3天;对照组、西药组予生理盐水等量等次灌胃3天。分别在末次给药后2小时采血,戊巴比妥钠麻醉,无菌条件下进行腹主动脉取血,把同种条件药物血清混匀、过滤,56℃,30min灭活,-20℃保存备用。
实验随机分为正常血清对照组(对照组) ,西药组,中药组,中西医结合组。其中,对照组为大鼠血清加入RPMI1640培养液,浓度10%;西药组用含10%顺铂组大鼠血清RPMI1640培养液配制,DDP的终浓度为5ul/ml(小),10ul/ml(中),20ul/ml(大)三组;中药组为大鼠药物血清加入RPMI1640培养液,浓度分别为5%(小)、10%(中)、20%(大)三组;中西医结合组为含中药浓度10%+ DDP终浓度20ul/ml组成。
实验首先采用MTT比色法测定人胃癌SGC-7901细胞增殖抑制率,MTT比色法作为一种检测细胞存活和生长的方法,其检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲瓒并沉积在细胞中,而死细胞无此功能。二甲基亚砜能溶解细胞中的甲瓒,用酶联免疫检测仪在570nm波长处测定其光吸收值,可间接反映活细胞数量。在一定细胞数范围内,MTT结晶形成的量与细胞数成正比。经药物处理后的细胞株经HE染色后,制成光学显微镜标本;药物处理后人胃癌SGC-7901细胞株经固定、脱水、包埋等程序后制成透射电镜标本,通过光镜、透射电镜观察经活血行气方药物血清处理后的人胃癌SGC-7901细胞株的形态学变化;通过DNA琼脂凝胶电泳法检测人胃癌SGC-7901细胞凋亡梯带分析及流式细胞化学方法研究人胃癌SGC-7901细胞在活血行气方药物血清影响下细胞形态学变化、细胞凋亡定性定量分析,测定细胞凋亡率。
本实验通过蛋白质印迹(Western blot)法测定经活血行气方药物血清处理后的SGC-7901人胃癌细胞株P53、Bcl-2基因蛋白表达水平的变化,对经过药物处理后的细胞进行细胞蛋白质的提取,蛋白质的SDS-PAGE电泳,蛋白质转膜、抗体杂交、化学发光显影定影后,图像分析。
本实验中,对药物处理后细胞进行固定、脱水、复染等程序后进行酶细胞化学染色,通过酶细胞化学染色方法观察活血行气方药物血清对人胃癌SGC-7901细胞株Mg~2+-ATP酶、G-6-P酶活性的影响。
结果:
1.经活血行气方药物血清作用人胃癌SGC-7901细胞株24h后,实验结果显示,中药小剂量组未见明显抑制作用,其余各组均表现出不同程度的抑制作用,中药中剂量组的抑制率为17.24%,中药大剂量组的抑制率为24.76%,中西医结合组的抑制率为42.97%;经活血行气方药物血清作用人胃癌SGC-7901细胞株48h后,中药小剂量组出现抑制作用,其抑制率为21.79%,中药中剂量组的抑制率为34.95%,中药大剂量组的抑制率为48.26%,各药物血清组随着作用时间的延长抑制效果更明显,抑制效应呈时间依赖性,以中西医结合组药学血清作用48h组抑制作用最佳,抑制率达51.09%。
2.HE染色后,光镜下可见,对照组细胞贴壁生长,呈长梭形,大小比较均匀,核卵圆形居中,胞质清亮透明。药物血清组部分细胞由长梭形变成圆形,细胞核不规则,胞浆浓缩,染色质边集,浓缩药物血清组细胞。透射电镜下可见,经药物血清处理的人胃癌SGC-7901细胞变圆,微绒毛及突起明显减少,伪足消失,线粒体空泡化,核染色质凝集,可见调亡小体。凋亡细胞发生率与药物浓度呈正相关。DNA琼脂凝胶电泳检测可见活血行气方药物血清能诱导SGC-7901人胃癌细胞形成凝胶电泳特异梯状带,以流式细胞仪定量分析活血行气方药物血清作用48h后,在G1峰前出现凋亡峰中药组凋亡率为9.87±2.13%,西药组的凋亡率为48.24±1.32%,中西医结合组的凋亡率为47.65±2.47%。
3. P53基因是目前被最为广泛和深入研究的抑瘤基因之一,也是迄今为止所发现的突变频率最高和突变范围最广泛的抑癌基因,P53基因突变是胃癌发生发展的重要始动因素之一。Bcl-2基因是一种癌基因,其表达的Bcl-2蛋白是控制线粒体中致凋亡因子释放的主要调节因子,在维持细胞生理性分化、发育和细胞数量的动态平衡中具有重要作用。经蛋白质印迹(Western blot)法测定人胃癌SGC-7901细胞株经活血行气方药物血清处理后,P53、Bcl-2基因表达的变化。结果显示,对照组中P53、Bcl-2蛋白为中等表达,与对照组相比,中药组及中西医结合组中P53蛋白表达增强,Bcl-2蛋白表达减弱(P<0.05)。
4.经药物血清处理后,在光镜下见对照组Mg~2+-ATPase主要定位在细胞膜的内侧,黑色酶反应颗粒大小均匀,密度大,呈线状规则排列;G-6-Pase主要定位在细胞质内,酶反应颗粒粗大,排列密集,呈融合状;与对照组比较,各组Mg~2+-ATPase和G-6-Pase反应颗粒变小,数量减少,密度减低,有的为阴性。提示酶活性明显下降。
结论:
1.活血行气方药物血清对人胃癌SGC-7901细胞株在体外具有抑制增殖的作用。
2活血行气方药物血清对人胃癌SGC-7901细胞株具有诱导细胞凋亡的作用。
3.活血行气方药物血清可上调人胃癌SGC-7901细胞株P53基因蛋白表达水平,下调Bcl-2基因蛋白表达水平。活血行气方药物血清诱导人胃癌SGC-7901细胞株凋亡的机制可能是通过上调促凋亡基因P53表达,下调癌基因Bcl-2的表达实现的。
4.活血行气方药物血清具有降低人胃癌SGC-7901细胞株Mg~2+-ATP酶、G-6-P酶活性的作用。
Purpose:Gastric cancer is one of the most common malignant tumor in the world and china. According to the reporting and statistics of global by the world health organization, The gastric cancer incidence is 13.86/10 million, after lung canner to second place. Our country is hige occurrence of gastric cancer, the occurrence ratio is 42% of the world,about 40 million stomach cancer patients each year. Stomach cancer mortality rate is 4- 8 times of the european countries. Most treatments for cancer, surgery with radiotherapy and chemotherapy treatment, but most of the anticancer drugs lack of selection, cancer patients undergoing radiation and chemotherapy kill cancer cells while normal tissues and cells of the body also has considerable damage. Such as the inhibition of bone marrow, gastrointestinal reactions, immune function, liver and kidney tissue damage and so on often occurring.At present, there have been a lot of experimental studies show that,the traditional Chinese medicine has effects on generation inhibition of tumor and inducing apoptosis, improve the immunity of the body, induced the tumor cells differentiated, inhibition of tumor cell gene expression, inhibition of tumor cell metastasis, multidrug resistance reversal, anti-mutant antioxidant,and so on. Gastric cancer attack for Traditional Chinese medicine,that more is due to congenital deficiency and the vital qi deficiency, led to invasion of the exogenous evils,or because of improper diet.Improper diet including immoderate diet、insanitary diet、particular diet,led to damage of spleen and stomach; Or because of emotional disorders,led to unsmooth flow of qi;or because of overstrain and overease,led to stagnation of qi and blood stasis. eventually can cause and aggravate damage of spleen and stomach,the function of transforming and transportation the food and water about spleen discharge,stomach can,t in charging sending dowm, dampness aggregate phlegm, phlegm obstruct blood vessels, causes poor of blood transportation, and then metaplasia stagnat and Poison,and blocking the stomach. Falling gradually accumulate. Further lead to disfunction of the viscera, unsmooth flow of qi, the body fluid metabolism, at last the bleeding, cult poison, phlegm together and gastric cancer attacked. Traditional Chinese medicine in the treatment of gastric cancer,often strengthened the function of transforming and transportation the food and water about spleen,transforming qi and blood, strengthened blood circulation and clear stagnant blood. disperse Sputum,clearing hot and detoxification, conditioning and benefit Liver-kidney. Benefit qi and yin, assistance vital qi and Get rid of evil qi.Improve the body's resistance to disease, improve symptoms, control of tumor growth, metastasis and invasion. The Houxuesingqifang strengthened blood circulation and clear stagnant blood,transportation qi and stop pain,free qi flow,strengthened blood vessels,but consumption blood. Removed blood stasis can help growth again. Consistent of the principle of treatment about qi promotes generation of blood,qicompels blood to circulate. Advanced gastric cancer to weaken distension and depression or pain sensation in the epigastrium and other symptoms . Through this experiment, discussion the mechanism of Tranditional Chinese medicines anti-cancer by serum , provide a strong basis for traditional Chinese medicine treatment of gastric cancer .
Material and method:With the chinese herbal medicine serum pharmacology method,To research the mechanism of Huoxuexingqi formula in treating Human Stomach Cell Line SGC-7901. The chinese herbal medicine serum pharmacology refers to the Chinese herbal medicine or traditional Chinese medicine formula by mouth fed to experimental animals ,after a certain time , experimenter collecting blood,and separation of serum, then the serum containing pharmaceutical ingredients for in vitro experimental methods. The chinese herbal medicine serum pharmacological method is the more commonly used method by experimenter. My experiment was chosen for thirdty SD rats, include male and female, weight about 180g to 220g, through the stomach way to medicine decoction of traditional Chinese herbal medicine, 2 ml each, twice daily,three days continuous through the stomach way to medicine decoction of traditional Chinese herbal medicine.Then the control group and the western medicine group for saline. The dose is same to traditional Chinese herbal medicine, 2ml each, twice daily,three days continuous, Collecting blood at 2 hours after the last for drug.using pentobarbital sodium narcoticing, blood was collected by abdominal aorta under nobacteria conditions,.mixing the serum under the same conditions, filtering, 56℃, Inactivated 30 Minutes,then ,the serum is prepared , preservation under -20℃. After cell culture success ,the cells grew into the monolayer ,the experiment beginning.
The experiment randomly divided into control group, western medicine group, traditional Chinese medicine group,and traditional Chinese medicine combinating western medicine group.The western medicine group including the concentration of DDP are 5ul/ml(small),10ul/ml(Medium) and 20ul/ml(large) , the traditional Chinese medicine group including the concentration of the serum are 5%(small),10%(Medium) and 20%(large). The traditional Chinese medicine combinating western medicine group is concentration of DDP 20ul/ml plus 10% serum .
At first ,determined the proliferation rate of the human stomach cell line SGC–7901 by MTT test. MTT as a detection method for cell survival and growth, and its detection principle for the mitochondria in living cells by exogenous succinate dehydrogenase MTT can restore the blue-violet water-insoluble crystalline formazan and deposited in the cell , death cell is no such function. DMSO can dissolve formazan in the cells, enzyme-linked immunosorbent detector was measured at 570nm wavelength of light absorption value can reflect the number of living cells. In a certain range of cells, MTT crystals formed is proportional to the quantity and cell number. The human stomach cell line SGC–7901 after drug treatment by HE staining, Made of optical microscope specimens,The human stomach cell line SGC–7901 after drug treatment by fixation, dehydration, embedding, etc. made into TEM samples.With light microscope and transmission electron microscope,to observe the morphological change of the Human Stomach Cell Line SGC-7901 after dealing by drug serum, qualitative and quantitative analysis of apoptosis ,measured the rate of cell apoptosis. P53 gene is currently the most extensive and in-depth study of the tumor suppressor gene, is one of the mutations detected so far and the most frequently mutated tumor suppressor genes most widely, P53 gene mutation is the development of gastric cancer is one important factor in initiating . Bcl-2 gene is an oncogene, the expression of Bcl-2 protein is to control the release of mitochondrial factors in apoptosis of the main regulatory factor in the maintenance of cellular differentiation, growth and cell numbers has an important role in the dynamic balance.To observe change of the Human Stomach Cell Line SGC-7901 gene express level of P53 and Bcl-2 by immumo blot(western blot) after Huoxuexingqi Formula treatmented. First,Cell protein extract,after drug treamented, SDS-PAGE electrophoresis of proteins, transmembrane protein, antibody hybridization, chemiluminescence development snapshot, the image analysis.,after drug treatment cells were fixed, dehydrated, stained and other procedures for enzymatic staining.
To detect the change of Mg~2+-ATPase and G-6-Pase activity of the Human Stomach Cell Line SGC-7901 after Huoxuexingqi Formula treatmented .
Results:
1. By drug treatmented Within 24h, the effect of the drug serum of each group was differently obvious except in low concentration. The inhibition rate of traditional Chinese medicine medium group is 17.24%, the inhibition rate of traditional Chinese medicine in high dose group was 24.76% , the inhibition rate of traditional Chinese medicine combinating western medicine group is 42.97%; By the Serum of Huoxuexingqi Formula treatmented within 48h, low dose group showed inhibitory effect of traditional Chinese medicine, and its inhibition rate was 21.79%, the inhibition rate of traditional Chinese medicine in the medium dose group was 34.95%, inhibition rate of traditional Chinese medicine in high dose group was 48.26%.which was more obvious with the time - ratio , whose inhibition effects was time-dependent.the inhibition ratio of Group DDP+Huoxuexingqi approaching to 51.09%,the effect is the best.
2. After HE staining, uder light microscope, the growth of control group cell adhesion, fusiform, more uniform size of nuclear oval centered, clear and transparent cytoplasm. the shape of the cells changed into round or oval shape, irregular nucleus, cytoplasm condensation, chromatin margination, drug serum concentration cells.The chromatin concentrated, under the transmission electron microscope, shows that the serum treatment by drugs in human gastric cancer SGC-7901 cells became round, microvilli, and protrusions significantly reduced pseudopod disappear, mitochondrial vacuolization, nuclear chromatin condensation, apoptotic bodies were seen. With the DNA abstraction of cells and agarose gel electrophoresis,appearance“ladder strip”. Quantitative analysis by flow cytometry blood circulation of drug serum 48h later ,the apoptotic peak before the G1 peak ,apoptotic rate of the Medicines was 9.87±2.13%, western group was 48.24±1.32%, Chinese and Western Medicine combined group was 47.65±2.47%.
3. determination the change of P53, Bcl-2 gene expression by Western blot method for the Human Stomach Cell Line SGC-7901 by serum of Huoxuexingqi formula.The results showed that the control group P53, Bcl-2 protein expression was moderate, compared with the control group, combining Chinese and Western medicine group and the group increased expression of P53 protein, Bcl-2 protein expression was decreased.Drug serum of Huoxuexingqi formula can up-regulate p53 ,down-regulate Bcl-2 and achieve the suppression of Human Stomach Cell Line SGC-7901.
4. The serum treatment, the control group in the light microscope, Mg2 +-ATPase was mainly located in the inner cell membrane, enzymatic black uniform particle size, density, linear rule was ordered; G-6-Pase mainly localized in the cytoplasm the enzyme reaction particles thick, densely arranged, was fusion-like; with the control group,the response granules of Mg~2+-ATPase and G-6-Pase in the Human Stomach Cell Line SGC-7901 became smaller after the treatment of the Huoxuexingqi formula, the amount of the granules became fewer, the density was lower.
Conclusion:
1. The serum of Huoxuexingqi formula had effects on generation inhibition of Human Stomach Cell Line SGC-7901.
2. The serum of Huoxuexingqi formula can inducing apoptosis.
3. Drug serum of Huoxuexingqi formula can up-regulate p53 ,down-regulate bcl-2 and achieve the suppression of Human Stomach Cell Line SGC-7901. The serum of Huoxuexingqi formula make Human Stomach Cell Line SGC-7901 apoptosis may be through increased expression of apoptotic gene P53, decreased Bcl-2 oncogene expression achieved. 4. The activity of the Mg~2+-ATPase and G-6-Pase decreased after using the Huoxuexingqi formula.
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