| Objective:Viral myocarditis refers to infection caused by a variety of in order to myocardial cell degeneration and necrosis, interstitial inflammatory cell infiltration and fibrous exudation for cardiac disease, major changes occur. Its pathogenicity to Coxsackie virus group B virus (CVB) is the main, and in the six CVB serotypes in, CVB3 Anopheles strong mind, therefore, the current principal for the establishment of viral infection in CVB3 myocarditis model to carry out the research, the subject of viral myocarditis to choose research subjects as a main reason is that this disease has a high self-harm, such as children and adults of all ages can be involved stage disease, acute period, and into a dilated the possibility of cardiomyopathy at the same time, can also be caused by small-scale outbreaks, and viral myocarditis in recent years the incidence of the rise is, therefore, against CVB3 viral myocarditis treatment mechanism appears imminent.With that in mind, this research will establish a CVB3 mycocarditis cell infection model, through certain molecular biological technologies like the improved suppression subtractive hybridization (SSH), in order to explore the genetic expressive change of the chemotatic factors such as CXCL12?Sash1?small inducible cytokine A2?ribosomal protein S4, which are located in the attacked host cell and in the treatment group that use the method of Chinese Herbal boosting qi and enlivening blood formula (YiQi HuoXue Chinese Herbal Compound). The objective is to release the regulating function and the mode of Chinese Herbal boosting qi and enlivening blood formula to VMC, so as to further explore its biological effect and significance in the treatment of VMC, and to provide new theoretical basis and experimental basis. The research will also go further in showing that boosting qi and enlivening blood is a proven formula for treatment of VMC. Material and method:1.Research Materials1.1 Experimental Animals Newborn 2-3d wistar rats (provided by LiaoNing Chinese Medical College Department of Experimental Animals)1.2 Experimental Medicine and Reagents High Glucose DMEM(Gibco , USA) , Specialize fetal bovine serum(HaoYang Biological, TianJin), Penicillin (North China Pharmaceutical Co., Ltd., Shijiazhuang), Streptomycin sulfate (Merro Pharma, Dalian),Sodium bicarbonate (Tianjin Amino Acid Company, Tianjin),MTT(Sigma, USA), DMSO(Sigma, USA), Trypsin(Invitrogen, USA), PBS(Boster, WuHan). PFP (Santa, USA), ANT (Santa, USA), a-MHC(Abcam, USA), SABC immunohistochemistry (peroxidase)Kit (Boster, Wuhan), Antigen retrieval solution (Boster, Wuhan), Chromogenic DAB kit (Boster, Wuhan), Triton X-100(Solarbio, Beijing).2.Research Methods2.1 Yiqihuoxue cytotoxicity determination of compound Chinese medicine :Recovery from Hela cells by passage culture bottles after inoculation with 0.2ml of each hole to 96 hole cell culture plate, the home 37?, 5% carbon dioxide incubator to cultivate, when Hela cells were adherent monolayer growth, the measurement Yiqihuoxue largest non-toxic dose of Chinese medicine compound. DMEM culture medium with the traditional Chinese medicine compound Yiqihuoxue injection started from the stock solution concentration of the preparation 11. Each hole 0.2ml, each concentration for 4-hole complex, plus an additional four control normal growth and pore fluid. In 24h, 48h observation of cell growth state, a reference hole normal cells to the cytopathic effect is not caused by the largest concentration of drug dilution largest concentration of non-toxic (TD0).2.2 CVB3 Virulence Titration: In the 96-well plate dispose of the mono-layer Hella cell growth and wash each well twice with the HBSS solution. Using 2%fetal bovine serum as suspension, add ten concentrations of CVB3 virus diluted from 10-1 to 10-8. Each well with 0.1ml. On the 96 well plate, each concentration will be placed into four wells and four additional wells as a control. Each solution will be kept at 0.1ml and placed into a 5% Carbon Dioxide incubator and kept at 37oC. Cellular pathology will be observed at 24h and 48h. observation cell lesions.2.3 Yiqihuoxue Chinese herbal compound on CVB3-induced myocardial cell death inhibition (MTT method) :Logarithmic phase from myocardial cells, 0.2ml of each hole by adding half the volume of the concentration of tissue infections myocytes CVB3 virus adsorption 2h. Suction disposable culture medium containing the virus, containing 2% fetal bovine serum to the maintenance of gently washing liquid on it, adding the largest non-toxic drug dosage from the initial dilution of the concentration of 8 Yiqihuoxue Chinese herbal compound containing the growth solution, 0.2 per hole ml, each concentration for 4-hole complex, a separate zero-hole. In 48h, each hole by adding 0.18ml DMEM culture medium, then add 0.02ml MTT solution into incubator to continue to foster 4h, carefully suction hole disposable culture medium, each hole by adding 0.15ml DMSO, the oscillation oscillator home 8min, in enzyme-linked immunosorbent assay detector 490 nm of the hole measured absorbance (OD).2.4 the influence of YiQi HuoXue Chinese Herbal Compound to the genetic expression of Viral myocarditis,(VMC) chemotatic factors such as CXCL12, Sash1, MCP-1, ribosomal protein S4: through the improved suppression subtractive hybridization (SSH), the rat cardiac muscle cell CVB3 infection group and the gene that has a differential expression in YiQi HuoXue Chinese Herbal Compound group will be demeshed. The result wil be proved through FL RT-PCR.3 Experimental Results:3.1 Determination of Boosting Qi and Enlivening Blood Formula cytotoxicity: the maximum non-toxic dose for the formula is 7.813mg/ml.3.2 CVB3 virulence titration: the infection rate of 50% cultured CVB3 virus cells is 1×10-4.5. 3.3 Boosting qi and enlivening blood formula inhibition of myocardial cell death from CVB3 virus(MTT method):use of maximum non-toxic dose boosting qi and enlivening blood formula on post-attack myocardial cells, had the largest absorption value, and in comparison with other dilutions there was a clear difference, p<0.001. This shows that the maximum non-toxic dose of boosting qi and enlivening blood formula has a clear inhibitory effect on CVB3 induced myocardial cell death.3.4the influence of YiQi HuoXue Chinese Herbal Compound to the genetic expression of Viral myocarditis,(VMC) chemotatic factors such as CXCL12, Sash1, MCP-1, ribosomal protein S4: The results of SSH showed that genetic expression of CXCL12, Sash1, MCP-1, ribosomal protein S4 in treatment group were higher than that in CVB3 infection group. The results of fluorescent RT-PCR agreed with that of SSH.Conclusion:1.To primary neonatal rat cardiac myocytes in vitro methods, the establishment of the myocardial cells attack CVB3 infection model.2.Through observing the influence of YiQi HuoXue Chinese Herbal Compound to the genetic expression of Viral myocarditis,(VMC) chemotatic factors such as CXCL12, Sash1, MCP-1, ribosomal protein S4, the multi-functional, multistrata, multi-targeting effect of YiQi HuoXue Chinese Herbal Compound have been confirmed.3.The experiments further confirmed Yiqihuoxue VMC is an effective treatment of the law, and medicine Yiqihuoxue VMC compound is an effective prescription treatment, suggesting that this treatment in the VMC has broad application prospects. |
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