The Influence Of Ganoderma Lucidumepithelial On The Nude Mice’sub-Skin Xenograft Of Human Ovarian Cancer

Objective: Ovarian cancer is one of the common gynecologicalmalignancy. Its incidence rate is the second only to the uterine cervixcarcinoma and endometrial carcinoma.But the mortality rate is the first for alltypes of gynecological tumors.Its five year survival rate is only about20-30%,which poses a serious threat to the life of women.Etiology of ovarian cancer isunclear.Due to the lack early diagnosis method,the most of them reachedadvanced when discoveried. It relies mainly on cytoreductive surgery andchemotherapy for treating method.But duing to chemotherapy resistance,ovarian cancer’s recurrence and metastasis lead to treatment failure, or evendeath. Therefore, looking for effective inhibition of tumor growth and thestudy of its antitumor mechanism has become the development direction of thetreatment of ovarian cancer.Traditional Chinese medicine attracted theattention of domestic and foreign research scholars for its low toxicity andhigh efficiency advantages. Extracting antitumor component from herbalbecomes effective the important way of anticancer agent. Ganoderma lucidumhas very high medicinal value as thousands of years history of Chinesetraditional medicinal materials. Through decades of scientific research ofmodern pharmacology, confirmed that Ganoderma lucidum has notablecurativeto in to enhance human immunity, anti-tumor, protecting liver,protecting nerve, regulating GLU and blood lipid, anti-inflammatory andanalgesic and so on. Ganoderma lucidum spores is the fungus germ cells.It isejected from Ganoderma lucidum at maturity. It is the essence of ganodermalucidum with all the genetic substance. Its effective component higher75times than Ganoderma lucidum.At present, studies have shown that spores ofGanoderma lucidum has antitumor effect breast cancer, cervical cancer,leukemia, liver cancer, stomach cancer and other diseases. Ganoderma lucidum spore has the effect of antagonistic cyclophosphamide toxicity inmice. As a kind of traditional Chinese medicine,it has been widely used inmalignant tumor patients.Studies have shown that Ganoderma lucidumanti-tumor effects on ovarian cancer cells such as inhibit proliferation,adhesion, migration, invasion and induce apoptosis. And Ganoderma lucidumenhances chemosensitivity of epithelial ovarian cancer cells to cisplatin evenreverses resistance to cisplatin in chemoresistance cell line.But on ovariancancer in vivo experiments has not reported.The experiment is to study thebroken Ganoderma lucidum spore and paclitaxel effect of subcutaneousovarian cancer in nude mice. To provide certain theory basisim for theGanoderma lucidum spore therapy of ovarian cancer.Methods:1Cell culture: Culture of ovarian serous papillaryadenocarcinoma cell line SKOV₃conventionally, for the construction ofsubcutaneous ovarian cancer model in nude mice.2Human ovarian epithelial cancer subcutaneous model of construction innude mice:To digest logarithmic growth phase of SKOV₃cells bytrypsin,10%fetal bovine serum termination of digestion, disperse the cellsfully and centrifuge,2000r/min （r=8cm）5min,discard supernatant, join inPBS, blow well and count, again centrifuge, using PBS dilute into2.5×107/ml single cell suspension, in sterile conditions inoculation on every nude micedorsal proximal hindlimb of subcutaneous of0.2ml. They were randomlydivided into four groups （control group, broken Ganoderma lucidum sporepowder group, Paclitaxel injection group, Ganoderma lucidum spore powderplus paclitaxel combination group）after7days, experiment.3Preparation of Ganoderma lucidum: The Ganoderma lucidum used inthis experiment was purchased from Energene Naturals Inc in Toronto. A stocksolution of75mg/ml was prepared by adding water to the power and thenboiling for5min. After a brief centrifugation, the supernatant was collected.The stock solution was stored at4℃. Preparation of Paclitaxel injection:diluted paclitaxel into3mg/ml with0.9%sodium chloride injection,immediately use. 4Treatment methods: Broken Ganoderma lucidum spore powder groupgavage500mg/kg/d, gavage,for4weeks. Paclitaxel injection group30mg/kg/w, intraperitoneal injection, for4times. The combination group gavage500mg/kg/d broken Ganoderma lucidum spore powder, and30mg/kg/wPaclitaxel intraperitoneal injection. The control group gavage the same volumeof0.9%sodium chloride injection every day, intraperitoneal injection thesame volume of0.9%sodium chloride injection every day.5Experiment of tumor suppression: On the day of1,7,14,21,28days, toobserve the nude spirit, activities and stool, to weight and measurement thexenograft’s the longest diameter （a） and the shortest diameter （b）. Accordingto V （mm3）=πa b²/6obtain xenograft volume. According to （1treatmentgroup tumor volume/controls tumor volume）×100%calculate the tumorinhibition rate. After finished treatment for24hours, executing the nude mice,stripping tumor tissue, measuring its volume and weight.6HE staining: A portion of the tumor tissue （about the size of5×5×1mm³） with4%paraformaldehyde fixed observation of transplantation tumor,cytologic morphology.7Immunohistochemistry: Taking part of xenograft （about the size of5×5×1mm³） in4%paraformaldehyde.To determinate the expression of Bcl-2with immunohistochemical technique.8Q-PCR: Taking part of xenograft in-80℃refrigerator. To determinatethe expression of miRNA200a with Q-PCR.9The statistical method: To process experiment data with SPSS13.0statistical software. Measurement data is described withx±s. The compareof multisamplet mean with the single factor variance analysis, P <0.05fordifferences with statistical significance. The compare of the ranked data withKruskal-Wallis, P <0.05for differences with statistical significance.Results:1The nude mice body weight of four groups has no significantdifference. The difference is not statistically significant （P>0.05）. Weobserve the nude mice without diarrhea, dispirited spirit, slow etc.Transplantation tumor volume of the first day ofhad no significant difference, the difference was not statistically significant. Transplantation tumor volumeof the seventh, fourteenth, Twenty-first days of medication groups less thanthe control group, but the difference was not statistically significant.transplantation tumor volume of the28day of medication group less than thecontrol group, the difference was statistically significant. The tumor inhibitionrate of Paclitaxel injection group and combination group increasing trend fromthe seventh day. The tumor inhibition rate of Ganoderma lucidum sporepowder group rise from the fourteenth day. The tumor inhibition rate ofcombined treatment group greater than simple drug groupon the day oftwenty-eighth. The weight of transplantation tumor of medication group wereless than that of the control group, the difference was statistically significant（P <0.05）.2The effect of broken Ganoderma lucidum spore powder on the quantityof BCL-2: The expression of Bcl-2of drug treatment group is lower than thecontrol group, the difference is statistically significant.（P<0.05）3The effect of broken Ganoderma lucidum spore powder on the quantityof miRNA200a: The expression of miRNA200a of drug treatment group islower than the control group, the difference is statistically significan（P<0.05）.Conclusions:1The volume and weight of transplantation tumor oftwenty-eighth day of broken Ganoderma lucidum spore powder group is lessthan the control group’s, and the difference has statistical significance. Itshowed that Ganoderma lucidum spore powder has the anti-tumor effect.2The expression of Bcl-2of the broken Ganoderma lucidum sporepowder group is lower than the control group, the difference is statisticallysignificant. Suggesting that the broken Ganoderma lucidum spore powder playanti-tumor effect through inhibition the expression of Bcl-2probably.3The expression of miRNA200a of the broken Ganoderma lucidumspore powder group is lower than the control group, the difference isstatistically significant. Suggesting that the broken Ganoderma lucidum sporepowder play anti-tumor effect through inhibition the expression of miRNA200a probably.