The Experimental Study On Lung Cancer Angiogenesis And Apoptotic Related Gene Expression Of Ling-Qi Capsule

Objective:(1) To observe the inhibitive effect of herb serum containing LQC on the proliferation of human lung adenocarcinoma in A549 cell line.(2) To observe the apoptotic induction and the effect of expression on apoptotic related gene(Bcl-2,C-myc,Bax,Survivin).(3) To observe the effect of herb serum containing LQC on cell morphology and cell cycle in cell line A549.(4) To observe the inhibitive effect of LQC on metastatic tumor and lung metastasis of Lewis tumor-bearing mice.(5) To approach the inhibitive effect of lung cancer angiogenesis and the effect of expression related ngiogenesis factor of LQC.Methods:(1)The effects of the contained herb-serum on the sensitivity and cell growth curve of A549 cells were observed with MTT assay.(2) The cell morphological effect of LQC on tumor cell with cell culture in vitro were observed with Wright-Giemsa staining and inverted microscope. To detect the cell cycle with flow cytometry in different time.And the the apoptotic induction of herb serum containing LQC was observed with AnnexinV/P staining.(3) RT-PCRwas used to measure the expression on apoptotic related gene(Bcl-2,C-myc,Bax,Survivin) by herb serum containing LQC.(4) The metastatic tumor model of tumor-bearing mice was established with Lewis cell line, the inhibition rate of metastasis and metastatic tumor were detected after treatment with LQC.(5) Immunohistochemistry was carried out to confirm the expression of angiogenesis factor(MMP-2,MMP-9,bFGF,COX-2)after treatment of tumor-bearing mice with LQC. Spectrophotometer was used to detected the iNOS content of tumor-bearing mice blood-serum after treatment with LQC.(6) The morphological change of metastatic tumor was detected with electron microscope and light microscope.Results:(1) The herb serum containing LQC showed growth inhibiting visibly to human lung adenocarcinoma in cell line A549.and there were dose-dependent and time-dependent manner in 24-72h. Compared with that of blank group, there was a significant difference(P<0.05, 0.01). The optimal action time of herb serum containing LQC on cell was in 71 hours.the inhibition rate was 34.07%.the inhibition rate of herb serum containing LQC attenuated in 96 hours.(2) There were some cells morphological change of herb serum containing LQC treating the A549 cell line in 24-48 hours. such as cell volume bcame small, cell became irregular and apart of cells fall off. The cell numbers of A549 reduced materially in 72hours, and some cell began to confluence, swelling, disruption and necrosis; When the herb serum containing LQC treated the A549 line in 72 hours, the cellstraining became deep, cell body began to diminute and round, some cell nuclears concentrated and showed purple black. Some cell nuclears became vague fairly after Wright-Giemsa straining.(3) The herb serum containing LQC could induce the arrest of A549 P cells in G0/G1 phase, and influence on the regulation point of G1/S, and showed dose-dependent and time-dependent manner. The herb serum containing LQC could induce A549 apoptosis and differentiation by down-regulated the expression of Bcl-2,survivin and up-regulated the expression of bax,C-myc.The apoptosis rate of A549 cell was 32.85% in 72 hours, There was statistically different compared with that of blank group(P<0.01). Moreover, showed classic DNA ladder in 180～200bp.(4) LQC could inhibite the tumor growth of tumor-bearing mice of Lewis lung cancer, The inhibition rate were 35.64%, 29.23%, 21.03%, respectively.LQC also showed markedly synergism and attenuation together with CYT. Meanwhile, LQC could inhibite the lung cancerometastasis and down-regulate the expression of vascular growth factor(bFGF,COX-2,MMP-9), could inhibit angiogenesis of lung cancer by reducing iNOS content of blood serum in mice.SoLQC had the anti-netustasis of lung cancer.Conclusions:(1) LQC could inhibite the proliferation of A549 cell line with lung cancer significantly, showed markedly growth inhibiting action in the dose-dependent manner during the time.(2)LQC could influence on cell cycle of A549 cell line, and could induce the aresst of A549 cells in G1/G0 phase and inhibite the synthesis and copy of DNA by influencing on G1/S control point of cell cycle.Meanwhile, apoptotic induction also play a role in it by influencing on expression on apoptotic related gene.(3) LQC could showed markedly inhibitory action on tumor-bearing mice of Lewis lung cancer, and could inhibite the tumor growth effectively.(4) LQC could inhibite the lung cancerometastasis effectively by influencing on the expression of vascular growth factor, Further inhibite lung cancer angiogenesis.