| 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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