The Effect Of CD31, Angiopoietin-2 And Laminin5γ2 In Tumor Vasculogenic Mimicry

part oneThe Relationship of Vasculogenic Mimicry Capability and Expressionof Angiopoietin-2, Laminin5γ2 and CD31 in Gastric Carcinoma,Embryonal Rhabdomyosarcoma and Melanoma CellsObjective: The study was to investigate the capability of vasculogenic mimicry formation by gastric carcinoma, embryonal rhabdomyosarcoma and melanoma cells in vitro, and detected the expression of Angiopoietin-2, Laminin5γ2 and CD31 protein in tumour cells, and revealed it's effect in the vasculogenic mimicry signal transduction.Methods: The vasculogenic mimicry emerging was observed in poorly differentiated gastric carcinoma, embryonal rhabdomyosarcoma and melanoma cells by three-dimensional cultures, electronmicroscope and specific stain. Then examined the Ang2, LN-5y2 and CD31 expression in mimicry vessels by immunohistochemistry.Result: (1) Human poorly differentiated gastric carcinoma cells(BGC823) formed distinctive loop or networks after 8 days of three-dimensional cultures on thin layer type I collogen matrix. The tumour cells dilapsused matrix and formed lumen structure after 14 days with three-dimensional cultures in thick layer collagen I matrix.(2) Human rhabdomyosarcoma cells(RD) formed distinctive branch,loop or networks after 13 days cultures on thin layer three-dimensional type I collogen matrix. After RD were cultured 14 days in thick layer type I collagen matrix,the tumour cells dilapsused matrix and formed lumen structure.(3) The mice melanoma cells(B16) formed loop or networks after it grew for 10 days on thin layer three-dimensional type I collogen matrix, A large of cell fragments emerged in matrix. B16 cells invasive grown and formed lumen structure after 14 days of three-dimensional cultures in thick layer type I collagen matrix.(4) Electron microscopic observation showed that tumour cells grew well and formed mimicry lumen of blood vessel structure.(5) Apoptosis detection in situ found there's no apoptotic cells in human umbilical vein endothelial cells(HUVECs) grown in monolayer culture, on thin layer three-dimensional collogen gels, a large of B16 cells and HUVECs which distributed in the networks apoptosised.(6) The expression of CD31 was detected using immunohistochemistry in BGC823, RD and B16 cells in monolayer cultures, the positive stain enhanced in loop and network structure after it were cultured on thin layer three-dimensional collogen for 5-7 days. CD31 was positive in the lumen wall that tumour cells formed in thick layer collagen I matrix.(7) The expression of Ang2 was detected in BGC823,RD and B16 cells in monolayer cultures, the positive stain enhanced in loop and network structure after it were cultured on thin layer three-dimensional collogen for 5-7 days. Ang2 was positive in the lumen wall that tumour cells formed in thick layer collagen I matrix.(8) The expression of LN-5y2 was detected in BGC823 and B16 cells in monolayer cultures, but RD cells didn't express LN-5y2. The positive stain of LN-5y2 enhanced in loop and tubular network structure after BGC823 and B16 cells cultured on thin layer three-dimensional collogen for 5-7 days,but the RD cells hadn't expressed until 13 days later. LN-5y2 was positive in the lumen wall which tumour cells formed in thick layer collagen I matrix.(9) For antibody inhibition experiments, antibody to Ang2 could inhibit the formation of tubular network in B16 three-dimensional cultures, and the expression of LN-5y2 degrade inthat progress.(10) For antibody inhibition experiments, anti-LN-5y2 chain antibody could inhibit the formation of tubular network in B16 three-dimensional cultures.(11) The observations that antibody to CD31 couldn't inhibit the formation of tubular network in B16 three-dimensional culture.Conclusion: (1) Human poorly differentiated gastric carcinoma, embryonal rhabdomyosarcoma and mice melanoma cells can form vasculogenic mimicry in vitro.(2) High aggressive tumour can simulate as endothelial cells and express endothelial specific antigen in growth.(3) The experiment reveal that LN-5y2 is an important regulator of vasculogenic mimicry, and the vasculogenic mimicry can be abrogated by using inhibitors of LN-5y2.(4) Ang2 can promote the formation of vasculogenic mimicry by mediating the up-regulation of LN-5y2.(5) Three-dimensional culture can induce tumour cells' growth and differentiation.(6) The process of tubular network formation imply that vasculogenic mimicry is relate with apoptosis.part twoInvestigation of the Rleation between Expression of Angiopoietin-2,Laminin5y2 and CD31 in Human Gastric Carcinoma,Rhabdomyosarcoma Tissues and Vasculogenic MimicryObjective: The study was to detect the expression of Angiopoietin-2, Laminin5y2 and CD31 protein in human poorly differentiated gastric carcinoma and rhabdomyosarcoma tissues, and investigate it's effect in the vasculogenic mimicry signal transduction.Methods: Human poorly differentiated gastric carcinoma and rhabdomyosarcoma paraffin imbedding tissues were collected. All cases were observed by serial section and HE and PAS stain, and detected the expression of Ang2, LN-5y2 and CD31 protein in the cases by immunohistochemistry.Result: (1) The exhibition rates of vasculogenic mimicry were 26.1%(6/23) in poorly differentiated gastric carcinomas, and 69.2%(9/13) in rhabdomyosarcomas. The vasculogenic mimicry exhibited in every type of rhabdomyosarcomas.(2) The total expression rate of LN-5y2 was 60.9% (14/23) in poorly differentiated gastric carcinomas. The expression of LN-5y2 in gastric carcinomas with VM was significantly higher than that group without VM(P<0.05).(3) The total expression rate of Ang2 was 87.0% (20/23) in poorly differentiated gastric carcinomas. The expression of Ang2 in gastric carcinomas with VM was significantly higher than that group without VM(P<0.05).(4) The total expression rate of CD31 was 52.2% (12/23) in poorly differentiated gastric carcinomas. The expression of CD31 showed undifferentiated between the group of gastric carcinomas with VM and the group without VM(F>0.05).(5) The expression of CD31, Ang2 and LN-5y2 showed undifferentiated betweendifferent depth groups of invasion (P>0.05).(6) Coexpression rate of Ang2 and LN-5y2 was 52.2%, there was statistically significant correlation(r=0.261, P=0.031).(7) Coexpression rate of LN-5y2 and CD31 was 26.1%, there wasn't statistically significant correlation(r=-0.289, P=0.067).(8) Coexpression rate of Ang2 and CD31 was 47.8%, there was statistically significant correlation(r=0.348, P=0.026).(9) The total expression rate of LN-5y2 was 76.9% (10/13) in rhabdomyosarcomas. The expression was regional distinct.(10) The total expression rate of Ang2 was 92.3% (12/13) in rhabdomyosarcomas, the positive stain was situated in tumour cell's kytoplasm, vascular endothelial cell and vessel wall.(11) The total expression rate of CD31 was 84.6% (11/13) in rhabdomyosarcomas, the positive stain was situated in tumour cell's kytoplasm, vascular endothelial cell and smooth muscle cell of vessel wall. Endothelial-like cell which was concluded capillary and small vessel endothelial cell by HE stain didn't express CD31, but some atypical endothelial-like cell expressed or didn't express CD31.Conclusion: (1) The formation of vasculogenic mimicry is observed in both of human poorly differentiated gastric carcinoma and rhabdomyosarcoma tissues.(2) The overexpression of LN-5y2 is detected in both of human poorly differentiated gastric carcinoma and rhabdomyosarcoma tissues, the formation of vasculogenic mimicry is significantly correlated with the expression of LN-5y2.(3) The overexpression of Ang2 is detected in both of human poorly differentiated gastric carcinoma and rhabdomyosarcoma tissues, the expression of Ang2 is significantly correlated with the expression of LN-5y2 and the formation of vasculogenic mimicry.(4) The overexpression of CD31 is detected in both of human poorly differentiated gastric carcinoma and rhabdomyosarcoma tissues, the expression of CD31 is significantly correlated with the expression of Ang2 and is not correlated with the formation of vasculogenic mimicry.