Experimental Research Of Bletilla Striata Microspheres

Purpose:This study was to separation and purification of Bletilla Striata polysaccharide with modified method. Material and method:Crude polysaccharide was prepared by ethanol precipitation followed the60℃water extraction, deproteinized with Savage method, petroleum ether defatted and activated carbon bleaching. It was further isolated by ion-exchange chromatography on DE-52column and gel filtration on Sephadex G-100column and obtained purified Bletilla Striata polysaccharide. Result:The Bletilla Striata polysaccharide was odorless and tasteless powder and confirmed to be homogeneous components by DE-52column and gel filtration on Sephadex G-100column chromatography, the mean yield ratio was (1.9+0.2)%. Conclusion:The method of separation and purification of Bletilla Striata polysaccharide simple and convenient, stable and feasible. The extracted Bletilla Striata polysaccharide was available for preparation of Bletilla Striata microsphere. Purpose:This study was to investigate the effect of anti-tumor Bletilla Striata polysaccharide in vitro. Matetial and method:Human gastric cancer cells (sgc), ovarian cancer cells (A2780) and hepatocarcinoma cells (HepG2) were cultured and treated with different doses of Bletilla Striata polysaccharide processing in vitro. The cell growth and proliferation of the three tumor cells was assayed by MTT method and the cell cycle was measured by flow cytometry after treatment in the three tumor cells with Bletilla Striata polysaccharide in different dose. Result:The proliferation of human gastric cancer cells, human ovarian cancer cells and liver cancer cells were inhibited by Bletilla Striata polysaccharide with time-and dose-dependent manner. It showed that human gastric cancer cells were increased in G0/G1phase, no significant changed of cells in S phase cells and G2/M phase cells decreased. The human ovariancancer cells increased in S-phase, the G2/M phase cells decreased and no significant changes in G0/G1phase cells. The hepatocarcinoma cells increased in the G0/G1phase cells, no significant changed in S phase cells and G2/M phase cells decreased by Flow cytometry after cultured with Bletilla Striata polysaccharide in200ug/ml for72h. Conclusion:Bletilla Striata polysaccharide can play an anti-tumor effect by inhibiting the growth and proliferation of human gastric cancer cell, human ovarian cancer cell and hepatocarcinoma cell and cell cycle arrest. Purpose:This study was to evaluate characterization of Bletilla Striata microspheres(BSMs) made from Bletilla Striata polysaccharide with a modified separation and purification method. Material and method:The microsphere was prepared with emulsification-cool condensation-chemical cross-linking method. Single factor and orthogonal experiment was performed to optimize the preparation process. The microsphere size was determined by a laser particle size analyzer, Dispersibility, suspensibility, settling time, water swelling ratio, catheter injection performance and dissolution time were observed. Result:The size and yield ratio of microsphere was affected by water-oil ratio and concentration of Bletilla striata polysaccharide. An unstable suspension formed in saline and contrast agent with a good dispersion. The deposition time was proportional to particle size. Microspheres swelled faster in saline than in contrast agent, but there was no difference of swelling ratio in different pH. The microspheres were easily passed through5F catheter. After absorbing water, the microspheres with diameter greater than400um were difficult passed through the3F micro-catheter. About6weeks the particles melted completely in vitro. Conclusion:Preparation of BSMs with a modified method is meet the characteristics of embolic agents. Purpose:This study was to evaluate the biocompatibility of Bletilla Striata microspheres(BSMs). Material and method:After thermal test of BSMs, the growth and proliferation of mouse fibroblasts and human umbilical vein endothelial cells were observed by MTT assay after treated with BSMs processing in vitro. The blood compatibility of BSMs was evaluated. The inflammatory reaction of the muscle tissue in Japanese rabbit was evaluated at series time when planted into the back of the rabbit. After embolization in the right renal artery of Japanese white rabbit, the arterial wall changes were evaluated. Result:BSMs played no effect on the growth and proliferation of mouse fibroblasts and human umbilical vein endothelial cells. It showed good blood compatibility, red cell morphology was normal and hemolytic was not seen. It was normal by the view of eyes and low level of tissue inflammation by the detected of pathology in the site of back of rabbit after BSMs planted into at series time. Inflammatory reaction was also slight in renal arterial wall after embolization with Bletilla Striata microspheres. The hepatic and renal function recovered to normal level in short time after embolization in right renal artery with BSMs. Conclusion: It showed good compatibility of BSMs and can be used as the medium-term embolic agent in clinic. The experiment research of embolization in Japanese white rabbit renal artery with BSMs was seen in the Master degree of dissertation in Song Song-lin in our interventional department. Purpose:This study was to evaluate the embolic effect of Bletilla Striata microspheres (BSMs) in VX2liver transplanted hepatocarcinoma. Material and method:VX2tumor was grown in the left lobe of liver of15Japanese rabbits. The rabbits were divided randomly into3groups of5animals each on the basis of treatment type:group A, which underwent hepatic arterial infusion of distilled water and group B which received hepatic arterial infusion of lipiodol+doxorubicin, group C received BSMs(200um), respectively. All animals were received MRI scanning of abdominal before and2weeks after interventional therapy. Tumor volume (Vpre, Vpost) was calculated by the formula V=0.5×a×b2(a:maximum longitudinal diameter, b:maximum transverse diameter) and tumor growth rate (GR, GR=[Vpost—Vpre]/Vpre×100%) were calculate simultaneously. After2weeks of therapy, the DSA examination was performed again to evaluate the status of recanalization and collateral circulation formation. The animals were euthanized to harvest specimens (including tumor and adjacent normal liver tissue). Pathology examination was performed to observe the morphology, the distribution of embolic agents. Immunohistochemical staining was further performed to evaluate apoptosis-related factors (bax, bcl-2) and tumor vascular endothelial cell growth factor (VEGF). Apoptosis of tumor cells was also evaluated by in situ terminal transferase labeling (TUNEL) method. Result:After14days of interventional therapy in VX2transplanted carcinoma, the differences in tumor growth ratio, apoptotic ratio (AI), apoptosis-related factors bax, bcl-2and VEGF between groups B and C were no significant (p>0.05). However, there were significant statistical differences between group B or C and group A (P<0.05). In BSMs group, it was noted that no tumor blood vessels recanalization and collateral vascular formation in postoperative hepatic angiography. Tumor presented with complete necrosis in gross-view, and fibrous non-structure organization tissue instead of tumor structure in microscopic view and seen the BSMs. Conclusion:BSMs is full compliance with the requirement of interventional procedures and is an ideal embolic agent. It is perfect effect in embolization therapy in VX2liver transplanted carcinoma. Purpose:This study was to explore the feasibility of establishing uterine fibroids animal model in Japanese white rabbit and evaluate the feasibility of interventional embolization in the animal model with Bletilla Striata microspheres(BSMs). Material and method:20female Japanese white rabbits were divided randomly into2groups, the control group of5rabbits which underwent intramuscular saline injection posterior thigh, the experimental group of15rabbits which received Estrogen and progestogen intramuscular injection posterior thigh to establish uterine fibroids model. It remained10rabbits in experimental group after20weeks then were divided randomly into2groups of5animals each, group A underwent internal iliac arterial infusion of BSMs(200um) and group B which received PVAs infusion in internal iliac artery. The animals were sacrificed the next day after interventional embolization, gross pathology observed the morphology and distribution of the embolic agent, the estrogen receptor (ER) and progesterone receptor (PR) were observed by immunohistochemical staining. Result:It established10uterine fibroids models successfully and the success rate was66.7%. The uterus appeared dull, swelling obviously and formatted nodule by gross pathology observation. Uterine smooth muscle cells arranged in disorder, thickening in smooth muscle, part of the uterus showed a tumor-like hyperplasia by microscopic observation in experimental group. There were significant statistical differences(P<0.05) between control group and experimental group of the ER and PR in the local site of uterus. After interventional embolization, uterine showed no obvious necrosis, BSMs and thrombosis appeared in the small arteries. Conclusion:It can establish uterine fibroids model successfully in Japanese white rabbit combined with estrogen and progestogen intramuscular injection and the model can maintain the biological characteristics of human uterine fibroids. The application of BSMs in interventional embolization in the Japanese white rabbits uterine fibroids model is feasible.