| Objective:To study the effects and mechanisms on vasculogenic mimicry(VM) of tumor-bearing nude mice intervened by XiaotanSanjie Recipe(XTSJR),based on the model of human gastric carcinoma subcutaneously transplanted in nude mice. A special double staining method and electron microscope technique were applied to observe the morphology of VM. Expressions of MMP-2(matrix metalloproteinase 2),MMP-9 and LN-5γ2(laminin 5 gamma 2 chain, LN-5γ2) were detected by immunohistochemical method.Methods:1. The tumor-bearing mice model was established by subcutaneously inoculating with xenografts of MKN-45 human gastric carcinoma into the right armpit of 30 BALB/c nude mice.2. After modeling, the tumor-bearing mice were randomly divided into normal saline(NS)group, XTSJR group and doxycycline hyclate(DH)group, ten in each. Then all the mice were administered with corresponding medicine by gastrogavage for 4 weeks as follows: the mice in NS group were given isotonic saline 0.4ml once a day, those in XTSJR group were given XTSJR 0.4ml once a day, and the mice in DH group were given doxycycline 0.4 ml once a day. The growth situation of tumor, the life status and the body weight change of the mice were regularly observed.3. The experiment was terminated on the 4th week. The mice were sacrificed, then the tumor mass were weighed and the tumor inhibition rate was calculated.4. Immunohistochemical staining of CD34 and PAS special double histochemical staining were used to study if there were VM in the model of MKN-45 human gastric carcinoma subcutaneously transplanted in nude mice. The amount of VM in tumor was counted. And scanning electron microscope was applied to observe the morphology of VM. 5. Immunohistochemical method was used to detect the expression of MMP-2, MMP-9 and LN-5γ2.Results:1. We duplicated the model of MKN-45 human gastric carcinoma subcutaneously transplanted in nude mice successfully. The rate of tumor formation was 100%. 7 days after modeling, the lump could be touched in the right armpit subcutaeous of the tumor-bearing mice and increased gradually. The mice emerged failure symptoms of weight loss, athrepsy, lassitude, less eating and slow action.2. The impact of XiaotanSanjie Recipe on the growth of the tumor-bearing mice: The tumor inhibition rate was 30.0% in DH group and 43.5% in XTSJR group. Compared with NS group, average tumor weight of DH group and XTSJR group was significantly lower(P<0.01); The difference between XTSJR group and DH group had a statistical significance(P<0.05).3. It was confirmed by Immunohistochemical staining of CD34 and PAS special double histochemical staining that there was vasculogenic mimicry in the model of MKN-45 human gastric carcinoma subcutaneously transplanted in nude mice, which was the formation of fluid-conducting channels by highly invasive tumor cells and was identified by the presence of PAS-positive and CD34-negative loops lined by tumour cells. Scanning electron microscope observation also showed that tumor cells grew well and formed mimicry lumen of blood vessel structure.4. The impact of XiaotanSanjie Recipe on vasculogenic mimicry of the tumor-bearing nude mice: Compared with NS group, the amount of VM in DH group and XTSJR group was significantly less(P<0.01); The difference between DH group and XTSJR group was significant(P<0.01).5. The impact of XiaotanSanjie Recipe on the expression of MMP-2 protein: Compared with NS group, the MMP-2 expression in DH group and XTSJR group was significantly decreased, the difference had a statistical significance(P<0.01); The difference between DH group and XTSJR group was significant(P<0.01). 6. The impact of XiaotanSanjie Recipe on the expression of MMP-9 protein: Compared with NS group, the MMP-9 expression in DH group and XTSJR group was significantly decreased, the difference had a statistical significance(P<0.01); The difference between DH group and XTSJR group was significant(P<0.01).7. The impact of XiaotanSanjie Recipe on the expression of LN-5γ2 protein: Compared with NS group, the LN-5γ2 expression in DH group and XTSJR group was significantly decreased, the difference had a statistical significance(P<0.01); The difference between DH group and XTSJR group was significant(P<0.01).Conclusion:1. XiaotanSanjie Recipe can obviously inhibit tumor growth of the model of MKN-45 human gastric carcinoma subcutaneously transplanted in nude mice.2. There was vasculogenic mimicry in the model of MKN-45 human gastric carcinoma subcutaneously transplanted in nude mice.3. XiaotanSanjie Recipe can inhibit the formation of VM in xenografted tumor of nude mice. The mechanism may be due to down-regulation the expressions of MMP-2, MMP-9 and LN-5γ2.
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