Objective To evaluate the long-term toxicity of endostatin injection to Beagle dogs,and to provide safety evidence for clinical experiments.Methods Twenty-four pure-blood Beagle dogs were assigned to four groups averagely with six dogs for each group,and the sex ratio was 1∶1.The animals in three of four groups received endostatin injection at low(1.6mg/kg),medium(6.3mg/kg)and high(25.0mg/kg)dosage,respectively,and the animals in the other group acted as control.The Beagle dogs were injected intravenously daily for up to 3 months with the exception of the dogs in control group.The indicators of ordinary toxic response,weight,electrocardiogram and hematology were collected,and the blood biochemical analysis,routine urinalysis and histopathologic examination were also performed.Results Only ordinary toxic responses such as anorexia and laxness were observed from l dog in high dose group,and simultaneously disappeared in 4 days.The weight of all Beagle dogs studied increased in a normal rate.No significant difference was found in each group of dogs on heart rate,electrocardiogram and indicators of urinalysis.No evident changes were observed in dogs on hematological and biochemical indexes.The pathological chances in different levels were observed in a dog's liver of low dosage group and a dog's kidneys of blank control group,but such changes were regarded as the spontaneous pathological changes,not related to the application of endostatin injection.Conclusion The non-toxic dose of endostatin injection on Beagle dogs was estimated to be 6.3 mg/kg under the present conditions.

Perepherial blood lymphocytes from normal donors were isolated by Haemonetice V50 Ampheresis System, and incubated in RPMI 1640 contained 1000U/ml rIL-2 and 10%AB serum at 37癈, 5%CO2. Five days later, these cells were harvested and tested their LAK activity. Then 10 patients with malignant metastases were treated by transfer of allo-LAK cells. After 2 and 6 months, the PEL from patients were seperated for the test of response to IL-2, proliferation to ConA and production of IL-2. The result showed that no significantly reducing of these value after patients being treated by Allo-LAK cells. Combined with clinical observation and pathological biopsy, it was concluded that GVHD couldn't be caused by transfer 6f allogenic LAK cells.

Objective: To highly express rh-Endostatin from Pichia pastoris and purify it to homogeneity. Methods: Constructed Pichia pastoris X33/pLW202 was amplified and inoculated to ferment media. The supernatant of the strain was collected after induction. Through ultrafiltration, affinity and gel chromatography, rh-Endostatin with high purication was obtained. Results: 60mg/L rh-Endostatin was obstained from supernatant. HPLC showed its purity was above 98%. Conclusion: High level expression of secreted rh-Endostatin has been successfully achieved in Pichia pastoris expression system.

Aim To explore inhibitory effects and mechanism of engineering endostatin on growth and metastasis of melanoma cells in mice. Methods Melanoma cells(2× 106/mouse)were inoculated sabcutaneously to C57BL/6 mice. After tumorigenesis,endostatin(8mg/kg.d)was administrated to tumor-bearing mice,once a day ,twenty-one in all.Dietetic state and weight change of the tumor-bearing mice were observed and tumorous sige was measured during administration of endostatin. On 26thday,the tumor-bearing mice were sacrificed,subcutaneously tumorous weight was weighed and brain,lung ,liver,spleen and kidney were excised and sections were made to supply the pathological examination. Results Area under curve in the endostatin-treated group was obviously less than that in tamor control group(P∨ 0.01). Pathological study revealed that lavge areal necrosis arose in tumor and newborn cappillaries around the tumor disapeared. Conclusion Endostatin possosses strongly inhibitory effects on growth and metastasis of mouse melanoma and formation of newborn capillaries around tumor.