| Part 1Objective:This experiment through the establishment of subcutaneous ovarian cancer Balb/C nude mice transplantation tumor model,to observe lichongshengsuiYin(LCSSY)effective component of the change of the ovarian cancer microenvironment of invasion and metastasis related factors,and explore the effective constituents in pharmacological rushed in pulp drink a key link in the process of invasion and metastasis of ovarian cancer and related molecular mechanisms.Method:To establish the model of ovarian cancer in Balb/C nude mice,and randomly divided into model group,bevacizumab group,Chinese medicine high dose.Chinese medicine dose and Chinese medicine low-dose group.Observe the tumor growth of nude mice after drug in18 days after each drug intervention,in which every two days measurement volume of tumors,draw the growth trend,the mice were euthanized at last after dosing,the tumors had weighed,using RT-PCR detection of tumors of TNF-??IL-1?mRNA expression;By immunohistochemical method to detect tumors of VEGF?MMP9?IL-1?HIF-1;Wesrern Blot method anayslis of Takl?p-tak1?IKK??p-IKK??IKB??MMP9?TNF-??IL1? differences of proteinResults:1.LCSSY effective component of different dose groups all can inhibit the growth of ovarian cancer?compared with the blank group were significantly different(P<0.05).2.LCSSY effective component of different dose groups all can effectively reduce tumor tissue TNF-??IL-1? mRNA expression and that inhibition effect in quantity-effect correlation(P<0.05),but the bevacizumab group and LCSSY high dose group of IL-1? mRNA expression did not see obvious difference(P>0.05).3.Compared with model group,LCSSY effective component of different dose groups can inhibit ovarian the expression of VEGF,MMP9?IL-1?HIF-1 protein,and significant difference(P<0.05)?4.Compared with model group,LCSSY effective component of different dose groups made lower the ovarian cancer tissue of Tak1?p-tak1?IKK??p-IKK??IKB??MMP9?TNF??IL-1? protein expression,which LCSSY effective component to Takl not seen obvious effect(p>0.05),but the effect on p-tak1 has changed,the quantity-effect(p<0.05);Drugs between various components inhibit of IKK??p-IKK??IKB expression,the quantity-effect(p<0.05);To TNF??IL1??MMP9 down-regulated expression,but LCSSY effective component of different dose groups effect of MMP9 no significant difference(P>0.05),and which to TNF??IL-1? have a quantity effect(P<0.05).Conclusion:LCSSY effective component can inhibit the growth of ovarian cancer,its mechanism may be inhibiting factors related to the invasion and metastasis microenvironment surrounding:matrix metalloproteinases(MMP9);inflammatory related factors(TNF?,,IL1,IL-6);hypoxia inducing factor(HIF-1);and its related pathways activated factor(Tak1,p-Tak1,IKK alpha,p-IKK alpha,IKB alpha)expression,and then inhibit ovarian cancer angiogenesis growth factors(VEGF)expression,as well as the expression of related pathways,Ultimately affect the invasion and metastasis of ovarian cancer.Part 2Objective: Cultivation of ovarian cancer cell SK0V3 ? in under the action of different doses of LCSSY effective component,observe the cell proliferation and apoptosis,invasion and metastasis and angiogenesis of change,verification principle LCSSY effective component can control ovarian cancer cell SKOV3 ability of invasion and metastasis.Method : In vitro cultivation of ovarian cancer SKOV3 cells,and preparation the drug-containing serum of LCSSY effective component,random divided into LCSSY high,medium and low dose groups,cultivation of chicken embryo villus allantois membrane vascular model,were randomly divided into LCSSY high,medium and low dose groups?model group and bevacizumab group,observation group drugs of chicken embryos villi allantoic membrane angiogenesis morphology change;MMT method to detect ovarian cancer SKOV3 cells in different drug-containing serum drug dose 24 h and 48 h after proliferation inhibition rate;Application of scratch experiment,Transwell little room to observe each drug effect on ovarian cancer SKOV3 cell migration and invasion ability;the drug-containing serum of LCSSY effective component and bevacizumab drug effect after ovarian cancer SKOV3 cells supernatant of the expression of VEGF,MMP-9,IL-6,IL-l,TNF-a.Results:1.After effective of LCSSY effective component,New blood vessels visible in each group after 1,2 levels of the number of blood vessels is to some extent reduce and statistically significant(P < 0.05);LCSSY effective components in each dose effect of inhibiting angiogenesis is still lower than bevacizumab group(P < 0.05);LCSSY dosage group 1,2 levels of blood vessels in low and high dose group compared with the number of significantly reduced(P < 0.05)?2.Different doses of LCSSY effective component effect on ovarian cancer SKOV3 cells after 24 h and 48 h cell proliferation inhibition rate,theresults showed that with the extension of treatment time,the cell survival rate and drug dose negatively correlated,with time dependent(P < 0.05)?3.Groups of drug effect after 24 h, scratch almost healed completely blank group,effective constituents in the bevacizumab group and LCSSY cell scratch healing ability to reduce the high dose group,with the increase of the time,the scratch area of no decreased significantly(P < 0.05)?4.Blank group of cells is dyed area is large,cell number was significantly increased,and the migration of bevacizumab group and LCSSY effective components in each dose group role in invasion and metastasis significantly reduced(P < 0.05);The migration ability of bevacizumab group > LCSSY effective components of high dose group > LCSSY effective components in the dose group > LCSSY effective components of low dose group?5.Compared with the blank group,after each cell supernatant on the expression of VEGF,MMP-9, IL-6,IL-1,TNF-a were significantly lower,the VEGF,TNF,IL-6,IL-1 expression level positively related with drug components dose(P < 0.05),in which the MMP-9 was no obvious difference in each dose(P > 0.05)?Conclusion: LCSSY effective component can inhibit ovarian cancer cell proliferation and apoptosis,invasion and metastasis ability,its proliferation inhibition rate of ovarian cancer SKOV3 cells was dose dependent;May be associated with the change of inflammatory factor and tumor necrosis factor,further inhibiting vascular growth factor VEGF expression,eventually inhibit ovarian cancer cell invasion and metastasis ?... |
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