| ObjectiveBy comparison with 5-fluorouracil,observe the influence of Hypericum japonicum Thunb extract on the effect of tumor growth,immune function and quality of life of the tumor-bearing mice to study the anti-hepatocarcinoma effect and toxicity reducing and efficacy enhancing effects of chemotherapy. Using real-time fluorescent PCR gene chip technology to study the tumor drug resistance and drug metabolism related gene expression on the liver tissue of H22 hepatoma transplanted subcutaneously in tumor-bearing mice, screening differentially expressed genes and analysis of its significance。Methods1.The experimental group of single use the Hypericum japonicum Thunb:Health clean female ICR mice, establish H22 hepatoma-bearing mice transplantation tumor model. The tumor mouse models were randomly divided into eight groups:the model group, administered with physiological saline by intragastric infusion;the chemotherapy group, given 5-fluorouracil by intraperitoneal injection; the Hypericum japonicum Thunb water and 95% ethanol extract three different doses groups, administered with the extract by intragastric infusion;2.The experimental group of Hypericum japonicum Thunb extract combined with chemotherapy:Health clean female ICR mice, establish models and were randomly divided into six groups:the model group, administered with physiological saline by intragastric infusion;the chemotherapy group, given 5-fluorouracil by intraperitoneal injection; Hypericum japonicum Thunb extract effective dose group(Based on the single use of Hypericum japonicum experimental, filtering out the highest inhibition rate of Hypericum japonicum Thunb extract dose for the effective dose), administered with the extract by intragastric infusion;the combination of Hypericum japonicum Thunb extract effective dose with 5-fluorouracil groups,given the extract and 5-fluorouracil together.Then added a normal control group,given physiological saline by intragastric infusion.1 times a day, after 10 days of continuous drug withdrawal.Take the weight and killed after pick the blood, take the liver, tumors, spleen and thymus weight. Calculate weight change, inhibition rate of tumor, spleen index and thymus index, count the white blood cell,general preparation of tumor histopathology of HE staining,light microscopic observation on the pathological changes of tumor tissue.The statistical analysis of the data was done with the statistical package of SPSS 16.0.3.Detection of the tumor drug resistance and metabolism related gene expression:take the mouse liver tissue of the normal group, the model group, the 5-fluorouracil group, the extract effective dose group, the combination administration group,extraction of RNA and reverse transcriptase into cDNA, Using real-time fluorescent PCR gene chip detection the fluorescence intensity of the 84 tumor drug resistance and drug metabolism related gene, calculation of the gene expression difference degree. The data analysis usingΔΔACt method.Results1.Weight shows growth trend after given Hypericum japonicum Thunb treatment.The weight gain of given Hypericum japonicum Thunb significantly higher than 5-FU group (P< 0.01),the Hypericum japonicum Thunb water extract high, medium and low dose groups are higher than the model group,and the combination administration group significantly higher than 5-FU group (P<0.01).The mouse activity, mental status, food intake, mortality of low dose group of water extract and middle dose group of alcohol extract,compared with the model group and 5-fluorouracil group showed some advantages2. Average tumor weight of the treatment groups lower than the model group (P<0.05), 5-FU group significantly lower than Hypericum japonicum Thunb extract groups (P< 0.01).Tumor inhibition rate of the water extracts groups is 18.25%～43.27%,the alcohol extract groups is 15.07%～40.98%. Average tumor weight of the combination administration group significantly lower than single use of the extract group and slightly higher than single 5-FU group.3.The index of thymus and spleen of the all extract treatment groups significantly higher than 5-FU group (P<0.01), the 5-FU group lower than the model group.The splenic index of all extract treatment groups significantly higher than the model group. The index of combination administration group significantly higher than single 5-FU group(P<0.01)。4. Peripheral white blood cell count of single use of the extract treated group is significantly higher than 5-FU group (P<0.01), combination administration group slightly higher than single 5-FU group.5. Light microscopic observation revealed tumor cell of model group visible shaped clear, core paste, nucleolus visible, more pathological nuclear division; the 5-FU group visible large tumor cell necrosis, a large number of apoptotic cells; the extract treated group Visible necrosis of tumor cells less than the chemotherapy group, combination administration group visible clear large tumor cell necrosis.6. PCR gene microarray detection gene expression of tumor-bearing mouse liver tissue of the normal group, the model group, the 5-fluorouracil group, the extract effective dose group, the combination administration group, filters out large number of differentially expressed genes of cancer drug resistance and drug metabolism related gene, includes classic drug resistant gene MDRandMRP, and the drug metabolism gene CYP families and GST.Model group compared to the normal have 30 differentially expressed genes;5-FU group compared with the model group 10 differentially expressed genes; the Hypericum japonicum Thunb water extract low-dose group compared with the model group 7 differentially expressed genes; the combination group compared with the model group have 10 differentially expressed genes.Conclusions1.Hypericum japonicum Thunb on H22 hepatoma transplanted subcutaneously in tumor-bearing mice show different degrees of inhibition, increase tumor-bearing mice body weight, improve the quality of life and enhance immune function, synergistic effect of fluorouracil is not obvious, but there is a certain degree of attenuation of 5-fluorouracil, can act as an anti-cancer drug for in-depth study.2. PCR gene chip technique screening differentially expressed genes of drug resistance and metabolism related gene of liver tissues, indicates that normal tissues and tumor tissues in certain resistance gene changes have a correlation, for the study on the resistance mechanism provide a new research thought. Hypericum japonicum Thunb effect on gene expression provide a basis for research molecular biological mechanisms of anti-hepatocarcinoma. |
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