| Lung cancer has become the leading cause of cancer death in the late twentieth century, at present the incidence is on the rise. Tumor metastasis is the most dangerous stage of tumor occurrence and evolution. Clinically, more than 60% of lung cancer patients were found metastasis when detected. Therefore, strengthen the research of lung cancer and its metastasis is imperative. This paper is mainly divided into two parts of theory discussion and vitro cell experiment. The theoretical part reviewed the mechanism research progress on tumor metastasis from modern medicine and traditional Chinese medicine, and deeply discussed the pathogenesis and treatment of malignant tumor and its metastasis based on collateral disease theory. Think of malignant tumor formation and metastasis of the main pathogenesis evolution process is:pathogen by gas network to the blood vessels, causing phlegm and blood stasis, cancer toxin condensed form tumor. If the hidden poison is further stimulated, then can cause metastases occur sequentially, from light to heavy. Therefore we put forward the collateral disease thinking into malignant tumors and metastasis. Yiqiyangyin meridians detoxification method and Jiawei Shengmaiyin were reasoned at the same time. The vitro cell experiment part mainly studied the effect of serum containing Jiawei Shengmaiyin on H460 in proliferation, cell cycle, migration, adhesion, and the intervention effect of epithelial mesenchymal transition and its molecular mechanism. Objective:Study the effect of serum containing Jiawei Shengmaiyin on H460 cell proliferation, migration, adhesion, cycle, and detect the expression of epithelial marker E-cad and mesenchymal marker Vimentin, N-cad by using serum pharmacology method. Discuss whether Jiawei Shengmaiyin can inhibit the occurrence of EMT and the metastase of NSCLC. Method:The Jiawei Shengmaiyin was concentrated to contain certain drug content supplemented. Gavaged the rabbits and took blood to make medicated serum and blank serum, then filtered and packaged for preparation. The experiment was divided into 10%,5%,2.5%and 10%medicated serum group and blank serum group. Cultured H460 cells in vitro, detected the influence of different medicated serum on H460 proliferation by MTT; detected the influence of different medicated serum on H460 cell cycle by flow cytometry; observed the influence of different medicated serum on H460 cell migration by scratch experiment preliminary; detected the influence of different medicated serum on non small cell lung cancer cells migration by Transwell technique (it was found that H460 cells cannot pass through the 8mmTranswell membrane, so used the non small cell lung cancer A549 cells instead); observed the effect of medicated serum on H460 cell adhesion by using Matrigel to simulate vivo basement membrane; detected E-cad and Vimentin protein change of H460 cells with medicated serum by Western blot; detected Vimentin and N-cad gene expression on H460 cells with medicated serum by RT-PCR.Results:1. The serum containing Jiawei Shengmaiyin has some effect on H460 cells inhibiting proliferation in the range of 24h, shows significant difference with the blank serum group (P<0.01).5% and 10% medicated serum groups are slightly better than the positive control DDP group, but all serum groups almost shows no cell proliferation inhibition effect in 48h.2. The flow cytometry results show that, compared with blank serum, all medicated serum group of cells in S phase percentage has increased after different concentrations of medicated serum acts H460 cells for 24h, and there is a certain dose effect relationship,10% medicated serum group S phase rate up to 61.1%. Medicated serum group cells mainly concentrate in the Go-Gi phase in 48h, and the DDP group continues to be concentrated in the S phase, with S phase cells 61.6%.3. The wound scratch assay shows, different concentrations of serum treated H460 cells for 24h and 48h, medicated serum group scratch distance is larger than the blank serum group, with a concentration and time dependence. The above preliminary illustrate that Jiawei Shengmaiyin has inhibitory effect on H460 cell migration.4. Transwell experiment shows that, with the increase concentrations of medicated serum, the number of A549 cells migrated to the lower surface of the membrane is reduced, and has significant difference with the blank serum group (P<0.01).5% and 10% medicated serum group migration rate is only 25.64% and 13.31%.5. Adhesion experiment found, serum containing Jiawei Shengmaiyin treated H460 cells for 24h and 48h, compared with the blank control group, medicated serum group decreases cell adhesion rate, and has significant difference with the blank serum group (P<0.01).24h adhesion rates is 93.51%,90.15%,82.47%(2.5%,5%,10% medicated serum), the 48h adhesion is 76.23%,68.33%,53.33%. The higher the medicated serum concentration is, the lower the adhesion rate is, and the 48h adhesion rate is lower than 24h.6. Western blot experiment shows, with the increase concentrations of medicated serum, the protein E-cad increases and Vimentin decreases, and has significant difference with the blank serum group (P<0.01).It also presents a concentration relationship, with 10% medicated serum group gets the highest E-cad protein and the lowest Vimentin protein.7. RT-PCR experiment results shows, for a certain time, with the increase concentration of medicated serum, the gene expression of interstitial type molecular Vimentin and N-cad decreased, compared with the blank serum control group, except 48h 2.5% medicated serum group had statistical difference (P<0.05), the other medicated serum groups are significantly different (P<0.01). For the same serum concentration, with the prolongation of time, the gene expression of Vimentin and N-cad decreased, and mesenchymal marker Vimentin and N-cad expression has a negative correlation with medicated serum concentration and action time.Conclucions:1.Jiawei Shengmaiyin has certain inhibitory effect on the proliferation of H460 cells, combined with flow cytometry detection results, the inhibitory effect on H460 cells of 24h mainly through S phase arrest to achieve, shows no proliferation inhibition in 48h.2. Jiawei Shengmaiyin has inhibitory effect on H460 cells in migration and heterogeneous adhesion.3. Jiawei Shengmaiyin inhibits the metastasis of H460 cells by inhibiting the occurrence of EMT. |
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