Study On The Isolation And Purification Of Recombinant Human Lactoferrin And Its Anti-Tumor Activity

Uterine cervix cancer is one of the frequent malignant tumors in the department of gynecology. Each year,more than 20,0000 died because of this disease. In developing countries, the uterine cervix cancer is the first one in gynecological tumors of women; however, we don't know the etiopathogenisis clearly. The therapy of uterine cervix cancer mostly uses chemosynthesis drugs at present, but their side effects and multidrug resistance become frequent during treatment. Naturally occurring drugs have been paid more attention by researcher for their low side-effect and no-drug-resistance. So screening naturally occurring drugs of anti-uterine cervix cancer becomes the investigative hotspot of pharmacology field.Human lactoferrin (hLF) is glycosylated protein which belongs to the family of iron-binding protein. It possesses sorts of physiological functions, such as bacteriostasis; viral infection; adjust the ferric balance; promote the ferric transmission and absorption; regulate the immune function; strengthen the disease resistance; The recently researches also proved that the hLF could inhibit the human tumor growth,prevent and cure tumor. But there is no report about the anti-Uterine cervix cancer effection of the recombinant human lactoferrin (rhLF).The ion exchange and metal-chelating were used to extract the rhLF from the milk of the transgenic goat. After the dialysis and glesiccation, it was used to the Hela cells cultured in vitro. We investigated the mechanism of anti-Uterine cervix cancer effection and the anti-tumor activity of rhLF, such results were obtained:(1) The ion exchange and metal-chelating were used, respectively; the eluent was freeze-dried to get the protein. Using the SDS-PAGE electrophoresis to definite and the yield were 30% and 65%, respectively.(2) The effect of rhLF to the Hela cell proliferation were detected by the MTT. The result showed that the rhLF could significantly restrain the growth of the Hela cells. When the rhLF quantity was 100mg/ml, the inhibition ratio achieved 73.25% after cultivated 48h.(3) The results of HE staining,Hoechst staining and TUNEL staining all showed that the rhLF has the effect to induce the Hela cells apoptosis.(4) The analysis of cell cycle displayed that the amount of the Hela cells in the period of G2/M were increased and the apoptotic peak was showed up evidently. (5) The p53 gene and PCNA were detected using the immunal histochemistry; the results showed that rhLF could inhibit the protein expression of these two genes.