| PART ONE EFFECTS OF RH-ENDOSTATIN (YH-16) ON PROLIFERATION OF MOUSE HEMANGIOENDOTHELIOMA CELL LINE EOMAObjective: To investigate the inhibitory effects of rh-endostatin (YH-16) on proliferation of mouse hemangioendothelioma cell line (EOMA) in vitro and the mechanism, and compared with Triamcinolone Acetonide, Pingyangmycin (PYM), interferon alpha 2a (INFα-2a), and investigate the combined effects on the growth of EOMA cells in vitro.Methods:①By MTT assay:the IR of EOMA cells managed with different concentration YH-16 for 24h,48h and 72h were compared to get an optimum concentration.②Compared inhibitory effects of YH-16 with Triamcinolone Acetonide,PYM and INFα-2a.③MTT assay was also used to detect the combined effect of any two of YH-16, Triamcinolone Acetonide and PYM. (Jin's equation was used to evaluate the efficacy of drug combination).④The changes in apoptosis and Caspase-3 protein expression were examined by flow cytometry (FCM) after 48h YH-16 managing EOMA.Results:①YH-16 with different concentration had significant inhibitory effects on proliferation of EOMA cells at 24h, 48h and 72h (P<0.01),and IR depended on the concentration.②The inhibitory rate (IR) of YH-16 (200μg/mL)was higher than YH-16 (50μg/mL,100μg/mL) at every time (P < 0.05) , but it didn't make sense to compare with YH-16(400μg/mL , 800μg/mL) (P > 0.05) , so 200μg/mL was the optimum.The optimum inhibiting concentration of PYM was为150μg/mL. The optimum inhibiting concentration of Triamcinolone Acetonide was为250μg/mL. The optimum inhibiting concentration of INFα-2a was 5×105 u/mL.③The inhibitory effects from strong to weak:PYM>YH-16>Triamcinolone Acetonide>INFα-2a.④Q value of YH-16 combined with Triamcinolone Acetonide or Pingyangmycin was lower than 0.85 ,suggesting the combined effect is antagonistic,Q value of Triamcinolone Acetonide combined with Pingyangmycin was between 0.85 and 1.15,suggesting the combined effect is additive.⑤The apoptosis rate of EOMA cells managed with YH-16 for 48h was also concentration -dependant.The expression of Caspase 3 increased in every concentration (P<0.01).Conclutions: YH-16 can effectively inhibit the growth of EOMA cells. It's stronger than Triamcinolone Acetonide ,interferon alpha 2a,but weaker than Pingyangmycin. YH-16 works through inducing apoptosis and Caspase-3 participates.YH-16 can be antagonistic to Triamcinolone Acetonide and PYM's inhibition of EOMA proliferation, while the effect of Triamcinolone Acetonide combined with PYM is additive. PART TWO ESTABLISHMENT OF KASABACH-MERRITT SYNDROME MODEL IN NUDE MOUSEObjective: To establish Kasabach-Merritt syndrome model in nude mouse and evaluate the significance of the model by observation of growth and development of the hemangioma in nude mouse.Methods:①Inoculation of 1×106 EOMA cells in 100uL PBS was injected into the subcutaneous tissues of the right flank of 8 female beige nude mice. Eight control mice were injected with 100uL PBS at the same site.②The tumors were measured every other day using tissue calipers. Tumor volume was determined using the formula (width)2×length×0.52.③Blood routine examination: blood samples were obtained by cutting off tails on day 1 before injection of EOMA cells, day 6, day 13 and day 20 after injection of EOMA cells.④Before mice were sacrificed, tumor-bearing mice were examined with CDFI (Color Doppler Flow Imaging).⑤Mice were sacrificed by cervical dislocation when difficulty with ambulation and lethargy occurred. Hearts, lungs, livers, kidney, right femurs, spleens and tumors were collected at autopsy and weighed. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and then stained with H&E for routine histological examination.⑥For bone marrow smears, 100uL PBS was made through a needle into medullary cavity of left femurs. A drop of bone marrow was then deposited on a slide and drawn across. Bone marrow smears were then stained with Wright-Giemsa stain. Megakaryocytes of each marrow smear were counted and classified according to morphologic characteristic by microscope.Results:①Hemangiomas developed in the subcutaneous tissue in 100% of the 8 mice injected with EOMA cells, whereas the 8 mice inoculated with PBS did not show any abnormal features at the site of injection. In EOMA-injected mice, hemangiomas developed rapidly, and typically, the tumor volume had enhanced about 30 times within 2 weeks after injection. All tumor-bearing mice appeared skin pallor. 2 tumor-bearing mice appeared purpura. 4 tumor-bearing mice had skin ulcer and bled because of tumour developed rapidly. The weight of tumor-bearing mice had no difference compared to controls(P>0.05).②As the hemangioma increased in size, the tumor-bearing mice developed thrombocytopenia and anemia. RBC count, hemoglobin concentration, hematocrit and platelet count decreased as the tumor size increased(compared to before innoculation,P<0.01). WBC count, MCV, MCH, MCHC had no difference compared to before innoculation (P>0.05).③Bone marrow examination revealed the megakaryocyte quantity of tumor-bearing mices had no difference compared to control(P>0.05), so did megakaryoblast,promegakaryocyt, granular megakaryocyte and naked nucleous megakaryocyte. But the number of thrombacytogenous megakaryocyte decreased obviously (P<0.01).④ultrasonic inspection showed tumor was located in subcutaneous, which had clear margin and regular shape.The tumor presented as a nonhomogeneous low echo lesion, which separated by high echo strip.CDFI showed venous signals or partially arterial flow with low velocity,and notable morphologic changes under compression.⑤The pathological result showed one mouse could be observed visually having hemorrhagic focus on the surface of heart, kidney and liver. Microscopic examination showed the mouse had bleeding focus in the kidney and liver parenchyma, in the alveolar cavity and between cardiac muscular fibers which had gotten thicker. There was not any anomaly in heart, lung, liver, kidney and bone by naked-eye observation. The pathological section showed metastasis was not found. Observed tumor by naked eyes, the skin was blue-purple and the visceral surface was wine and there was an intact capsule of mass and clear demarcation between the tumor and the periphery. Observed tumor with Microscope, intratumor which had many different sizes of cyst cavities was spongy structure and cyst cavities were full of red cells and the wall of Cyst getting thicker was structured by tumor cells. The nuclei of tumor cells were big and dyeing deeply. Nuclear division could be seen.Conclutions: The Kasabach-Merritt syndrome model in nude mouse was successfully constructed by local hypodermic inoculation. The rate of tumor formation in nude mice was 100%.The clinical features of this model were similar to Kasabach-Merritt syndrome, such as hemangiomas developed rapidly, platelet count decreased progressively, microangiopathic anemia, coagulation disorder. The model of Kasabach-Merritt syndrome in nude mouse was a good carrier for clinical and fundamental researches about hemangioma. PART THREE IN VIVO STUDY ON TREATMENT FOR KASABACH-MERRITT SYNDROME WITH RH-ENDOSTATIN (YH-16)Objective: To observe efficacy of intra-tumor injection of rh-endostatin to treat mouse Kasabach-Merritt syndrome and compare with triamcinolone acetonide,pingyangmycin.Methods:①Divided randomly 40 female nude mice into 5 groups: YH-16 group, PYM group, triamcinolone acetonide group, PBS group and no tumor group. Inoculation of 1×106 EOMA cells in 100uL PBS was injected into the subcutaneous tissues of the right flank of 8 female beige nude mice. Eight control mice were injected with 100uL PBS at the same site. Tumor-bearing mice were treated with intra-tumor injection every 3 days after tumor could be visible to the naked eyes.②The tumors were measured every other day using tissue calipers. Tumor volume was determined using the formula (width)2×length×0.52.③Blood routine examination: blood samples were obtained by cutting off tails on day 6, day 13 and day 20 after injection of EOMA cell.④Mice were sacrificed by cervical dislocation when difficulty with ambulation and lethargy occurred. Spleens and tumors were collected at autopsy and weighed. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and then stained with H&E for routine histological examination.⑤For bone marrow smears, 100uL PBS was made through a needle into medullary cavity of left femurs. A drop of bone marrow was then deposited on a slide and drawn across. Bone marrow smears were then stained with Wright-Giemsa stain. Megakaryocytes of each marrow smear were counted and classified according to morphologic characteristic by microscope.Results:①The volume of tumor in PYM group on day 3 to 7 after inoculation of EOMA cells made no difference to PBS group(P>0.05). But the difference was significant (P<0.05) on day 9 after inoculation and was very significant (P<0.01) on day 11 to 21. The volume of tumor in YH-16 group on day 3 to 9 after inoculation made no difference to PBS group(P>0.05). But the difference was very significant (P<0.01) on day 11 to 21. The volume of tumor in triamcinolone acetonide group on day 3 to 13 after inoculation made no difference to PBS group(P>0.05). But the difference was significant (P<0.05) on day 15 and was very significant (P<0.01) on day 17 to 21. The weight of mice in triamcinolone acetonide group on day 5 to 21 after inoculation had a very significant difference as compared with no tumor group (P<0.01) and the curve of weight presented downtrend as time went on. The weights of mice in YH-16 group, PYM group and PBS group were not affected and increased as time went on ,which made no difference to no tumor group (P>0.05).②RBC count, hemoglobin concentration, hematocrit and platelet count of nude mice in PYM group made no difference to no tumor group at every time after inoculation (P>0.05). RBC count, hemoglobin concentration, hematocrit and platelet count of nude mice in YH-16 group made no difference to no tumor group on day 6 after inoculation (P>0.05), but had a significant difference as compared with no tumor group on day 13 (P<0.05) and had a very significant difference on day 20 (P<0.01). RBC count, hemoglobin concentration, hematocrit and platelet count of nude mice in triamcinolone acetonide group made no difference to no tumor group on day 6 after inoculation (P>0.05), but had a very significant difference as compared with no tumor group on day 13 and day 20 (P<0.01).③Bone marrow examination revealed the megakaryocyte quantity of tumor-bearing groups had no difference compared to no tumor group (P>0.05), so did megakaryoblast, promegakaryocyt, granular megakaryocyte and naked nucleous megakaryocyte. Compared the tumor-bearing groups with each other, the difference was not significant (P > 0.05). The percentage of thrombacytogenous megakaryocyte in PBS group decreased obviously as compared with no tumor group (P < 0.05). The percentage of thrombacytogenous megakaryocyte in YH-16 group, PYM group and triamcinolone acetonide group made no difference to no tumor group (P>0.05).④The weight of tumors in YH-16 group, PYM group and triamcinolone acetonide group had a very significant difference as compared respectively with PBS group (P < 0.01). Compared the tumor-bearing groups with each other, the difference was significant (P<0.05). The weight of spleens in YH-16 group, PYM group, triamcinolone acetonide group and no tumor group had a very significant difference as compared with PBS group (P<0.01). No difference had been found between PYM group and no tumor group (P > 0.05). Compared respectively YH-16 group and triamcinolone acetonide group with no tumor group, the difference was significant (P < 0.05). Microscopic examination showed the number and the diameter of cyst cavities decreased obviously and the number of tumor cells in which apoptotic bodies could be seen also decreased in medication group.Conclutions: The development of Kasabach-Merritt syndrome could be inhibited obviously by intra-tumor injection of YH-16 and PYM. The efficacy of triamcinolone acetonide on Kasabach-Merritt syndrome was much poorer than that of YH-16 and PYM.
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