| A new angiogenesis inhibitor,endostatin,was found by O'Reilly in 1997. It is a 20 KD C-terminal fragment of collagen XVIII isolated from cultures of mouse haemangioendothelioma (EOMA) that specifically inhibits endothelial proliferation,tumor growth and metastasis in vivo. Multiple cycles of therapy with endostatin block tumor growth without any acquired drug resistance and relevant toxicity. The molecular weight of recombinant human endostatin(rhES) is about 18.5KD,PI is 9.3,the amino acid sequence of rhES is similar to mouse's,and they have 85% common sequence. We focus on inserting human endostatin gene into genome of pichia.pastoris.GSH5 and confirming it's secretion of soluble rhES,and we the inhibitory effect;of rhES on growth and metastasis of mouse lung adenocarcinoma LA795 in mice T739.1 The expression and purification of human endostatin (hES) in pichia.pastoris.GSH5 and its biological activity analysis in vitroThe recombinant plasmid pPIC9/hES was constructed by our own laboratory. BgLII,SaCI and SaLI were used to linearize the recombinant plasmids,and the linearized plasmids were transformed into pichia.pastoris.GS\\5 to integrate the hES gene into genome with LiCl transformation method. The Mut phenotype of transformants were confirmed,To induce expression of rhES methanolwas added to final concentration 1%,the expression of rhES was confirmed by using SDS-PAGE and Western-blot assay. A high expression clone was chose to express rhES,the purified protein was acquired by using ultrafiltration concentration,70% (NH?2SO4 precopitation and heparin affinity chromatography methods (Hitrap.Heparin.HP). To investigate whether the purified protein could inhibit proliferation of human endothelial cell line ECV-304 cells in response to stimulation by bFGF we use MTT method.The results demonstrate the clone we got could highly express soluble rhES with good antigen activity,and its inhibitory effect on proliferation of human endothelial cell line ECV-304 cells in response to stimulation by bFGF was confirmed2 The construction of mice lung adenocarcinoma LA795 animal model and to investigate rhES's inhibitory effect on tumor growth and metastasis of lung adenocarcinoma LA795 in mice T739.LA795 cells were inoculated subcutaneously into the dorsa of 30 T739mice (4xl05 cells/mouse),and the mice were randomized into two groups. A clone could highly express the recombinant human endostatinwas selected,the rhES was purified by using ultrafiltration,(NH4)2SO4 precipitation and heparin affinity chromatography methods ( heparin.sepharose.Ctr6B ). The mice accepted therapy after 6 days of inoculation (the first group was given rhES 20mg/kg/d,and the second group was given equal volume PBS 0.3ml/kg/d,for 14 days). The volume of tumors in different groups was measured,the metastasis of tumors in liver,spleen,lung and the survival time of mice were observed. We treat the data with statistics mathod.We successfullygot 30 mice lung adenocarcinoma LA795 animal model and randomized them into two groups. Purified rhES was acquired to be used in treatment. The results show the tumors of rhES group grow slowly,the volume of tumors before and after treatment were 0.509+5.120E-02cm3and 2.073+0.202cm3;meanwhile the tumors of PBS group grow rapidly,the volume of tumors before and after treatment were 0.491 + 8.174E-02cm3 and 6. 761 + 0.208cm3. the volume of tumors in the two groups has significant difference (p<0.001) (the data were analyzed with repeated measurement analysis of variance by using spss.10.0 statistics software).The pathology shows there were widespread necrosis in tumors of the rhES group;but not in PBS group. There was no obvious metastasis in lungs,spleens and livers of mice in rhES group,but there were scattered metastasis of tumor in mice lungs of PBS group. The two groups have different survival time (37+ 3days),(28+2days),(p<0.01) .We constructed the clone which could highly express recombinant human endostatin by using gene recombination technology. Then we purified the rhES and confirm...
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