| The ovarian cancer is a kind of disease seriously threatening the women's health and life; its death rate ranks No. 1 among gynecologic malignant tumors. Since the ovarian cancer hides itself well and there are no specific early diagnosis methods, when seeing a doctor, most patients have fallen into the later period of disease. The likely relapse caused by incompleteness of surgery of advanced ovarian cancer and drug resistance makes the 5-year survival rate of ovarian cancer patients about 20%-30%; therefore, seeking for new therapies is the key to improve the survival rate of ovarian cancer patients.With the rapid development of genetic engineering technology and completion of Human Genome Project, the understanding of occurrednce and development process of tumors is more and more deep, and the molecular targeted therapy against malignant tumors shows an attracting future. The recombinant toxin therapy of tumor is a kind of new therapy emerged following the development of tumor targeted therapy in recent years. The recombinant toxin refers to the powerful toxin protein generated by some bacteria and animals or plants; these toxin proteins link with antibodies or some cytokines, through gene recombination or bonded with short peptides, to kill the new combined molecule consisted of cells with specific antigen or receptor on surface. cell targetability and cell killing, has the function to identify the surface receptor of target cell and kill it. It is capable of generating specific killing ability towards some cancer cells, finding the tumor cells directly like "missile" inside the body, killing them while causing no or little damage to the normal body cells. At present, the biotherapy of tumor has become the 4th tumor therapy method, following surgery, radiotherapy and chemotherapy. As more and more surface molecules of tumor cells are found and used as target cells for tumor targeted therapy; as the "bio-missile" of tumor therapy, the recombinant toxin gains ground in the tumor targeted therapy day by day, becoming an important means in the tumor biotherapy.According to the biologic characteristics of ovarian cancer, the new approach using targeted therapy of recombinant toxin in ovarian cancer therapy is now becoming the hot point significantly attracting researchers' attention. The research in recent years shows that in the primary lesion and metastatic lesion of ovarian cancer patients, the urokinase-type plasminogen activator system (uPA/uPAR) has a high expression, and its expression level is closely concerned with patient's prognosis, and in the normal tissues, there is no or only little expression.The urokinase-type plasminogen activator (uPA) is a kind of serine protease.lt exists first in the form of single-chain urokinase-type plasminogen (pro-uPA); after catalyzed by plasmin, cathepsin, etc.,the broken peptide bond K158-I159 forms chain A and chain B, becoming activated double-chain uPA.The chain A of uPA contains connecting locations with urokinase-type plasminogen activator receptor (uPAR);after effecttive combination between uPA and uPAR on the surface of cell,the fibrinolytic system is activated,the extracellular matrix and basilar membrane are degraded, and the signal transmission function inside the cell is activated. Much data shows that uPA/uPAR system plays an important role in the infiltration and metastasis of ovarian cancer; therefore, using uPA/uPAR system as the new target of ovarian cancer therapy has solid theoretical foundation, providing a new thought for the therapy of ovarian cancer.For the selection of effectors, we choose the small molecule toxin, melittin, as the targeting drug bonded with specific target of uPA/uPAR system. The melittin accounts for 50% of dry weight of bee venom, being the main action matter of bee venom; melittin is a polypeptide consisted of 26 amino acid residues; 3 lysines and 2 arginine residues of the molecule make it become a strong base peptide; in the neutral aqueous solution, as monomer, melittin exists in the random coiled structure; as the pH value and ionic strength increase, melittin performs the self-association, forming the quadpolymer structure ofαhelix; additionally, in different solutions, the angles between a helix structural area and helix of melittin are different, first 21 amino acids in the a helix structure is polar, locating at the surface of helix, and the non-polar amino acids locate at the other side of helix. This amphiphilic property is the characteristic of membrane-bound peptidases and transmembrane helix in membrane proteins. It decides that melittin can be dissolved in water and bound with membrane naturally, and subsequently dissolving the cells, without going through cell internalization. Therefore, melittin can be used to replace chain B of uPA to play the anti-tumor effect. The main principles for the functioning of melittin are to enhance the permeability of cell membrane, and to promote the leakage of cell contents and the cell lyses. The traditional toxins are mostly formed by toxin molecules acting upon the contents inside the cell; they have to enter the cytoplasm to carry out proper biologic functions, and this disadvantage limits its clinic application. The molecular weight of melittin is small, in comparison with other toxins with higher molecular weights, such as ricin toxin, diphtheria toxin, etc., and its immunogenicity is low, unlikely causing anaphylactic responses, which give it more research and application value. Therefore, in this experiment, the uPA/uPAR system is taken as the new target acted by therapeutic drug against ovarian cancer. By using the characteristic that uPA breaks at peptide bond K158-I159 and pro-uPA transforms into activated double-chain, the chain A with the function to bond receptor is kept completely, and chain B is replaced with melittin. When the chain A of uPA is targetedly bonded with uPAR on the surface of cell of ovarian cancer, the melittin on chain B is released to kill the tumor cell directly. This process is done completely compliant with the structural change characteristics of uPA action under the physiological conditions. Therefore, the playing of rhuPA_a-melittin is guaranteed. Based on these, this research has mainly completed the following jobs:(1)build the rhuPA_a-melittin-pPICZαC eukaryon expression carrier and rhuPA_a-X-pPICZαC eukaryon expression carrier, which can replace the chain B of uPA freely; after transforming rhuPA_a-melittin-pPICZαC into pichia pastoris X-33, sieve out the strains with stable and effective secretion expression. After SDS-PAGE and Western Blot identifications, the target protein is rhuPA_a-melittin; (2)in the 80L fermentation scale, the large-scale preparation and purification processes of rhuPA_a-melittin are established;(3)observe the growth inhibition of rhuPA_a-melittin against human ovarian cancer cells SKOV3; (4)preliminarily explore the action mechanism of rhuPA_a-melittin against human ovarian cancer cells SK0V3.1.Screening and identification of stable and effective secretion expression rhuPA_a-melittin pichia pastoris strain1)Build the rhuPA_a-X-pPICZαC eukaryon expression carrier, which can replace the chain B of uPA freely, and rhuPA_a-melittin-pPICZαC eukaryon expression carrier. Design the specific primers, to fish the huPA_a gene from human ovarian cancer tissue, introduce the restriction enzyme cutting sites Xhol and ECORI and a part of yeastα-factor signal peptide with expression primer PCR, to recombine the PCR product to plasmid pPICZαC with same enzyme cutting;after corresponding enzyme cutting identification and sequencing proving, linearize the recombinant carrier and transform the pichia pastoris strain X-33.2)Screening of recombinant pichia pastoris strain:Apply the transformed pichia pastoris onto the YPD agar plate with Zeocin; after 72h cultivation under 28℃,select the clone of transforming yeast with Zeocin resistance, extract its genome DNA as template, and perform the identification with expression primer as PCR.3)Expression and identification of rhuPA_a-melittinTake the positive transformant after PCR identification, cultivate and amplify in shaker under 28℃, induce the expression with 0.5% methanol, after continuous separation of fermentation supernatant solution through centrifuge, Mono S cation column chromatography and reversed water partition column chromatography, the components at SDS-PAGE Commassie Blue Fast Staining single band are obtained. The supernatant protein of fermentation broth is analyzed and identified with Western blot method.2. Establishment of large-scale fermentation and purification processes of rhuPA_a-melittinThe pichia pastoris is a kind of single-cell eukaryote, as the expression host; with its inherent advantages, it is very suitable to produce exogenous protein by large-scale fermentation. This research explores the process of large-scale rhuPA_a-melittin fermentation expression with 80 L fermenter. It emphasizes the factors significantly influencing the fermentation, such as pH value of fermentation broth, components of fermentation medium, Dissolved Oxygen degree (DO value), methanol replenishment speed, initial biomass, fermentation time, etc. Based on results, the optimization conditions are: FM21 fermentation medium with 0.5% peptone, pH6.5, induce the expression at about 190g /L of thallus density, the final methanol replenishment speed is 8.5ml/h/L volume of initial broth, DO 20%～30%. After induction for 12 hours, the content of rhuPA_a-melittin in broth can be 480mg/L After large-volume continuous centrifuge, cation exchange chromatography and reversed water partition column chromatography and low-temperature rotary evaporation, rhuPA_a-melittin can be 320mg/L,over 95% in purity is obtained from supernatant, and the recovery rate is 67%.3.Study on growth inhibition of rhuPA_a-melittin against human ovarian cancer cell strainFirstly, identify the location and expression of uPAR in human ovarian caner cell strain SKOV3. Extract the total RNA of human ovarian caner cell strain SKOV3, and design the specific primer, identify with RT-PCR method and cell immunol fluorence staining inspection; the results show that there is a high expression of uPAR in human ovarian caner cell strain SKOV3 used in this experiment. With MTT method, plot the growth curves of uPAR antagonism group and rhuPA_a-melittin group against human ovarian caner cell strain SKOV3 for consecutive 7 days under the conditions of same rhuPA_a-melittin final concentration, the growth curve of SKOV3 cells 48 hours after adding ruPA_a-melittin of various concentrations, and relationship between growth inhibition effect of uPAR antagonism group and rhuPA_a-melittin group against human ovarian caner cell strain SKOV3 and drug concentration; the results show that by adding various concentrations of rhuPA_a-melittin, the inhibition effect against SKOV3 improves significantly as the concentration increases. It illustrates that the inhibition effect of rhuPA_a-melittin against SKOV3 is subject to an obvious quantity-effect relationship. The IC50 value is 8.82±0.75, which indicates that the recombinant immunotoxin rhuPA_a-melittin built in this research can be uPAR bonded specifically, and targetedly kill the ovarian cancer cells efficiently. Additionally, the minimum effective dose is screened out as 2-4μg/ml. 4.Preliminarily discussion of action mechanism of rhuPA_a-melittin against growth of human ovarian cancer cell strainIn this experiment, after observing the action of rhuPA_a-melittin of various concentrations directly with phase contrast microscope for 48 hours, we find it has an obvious inhibition effect against the growth of SKOV3 cells; as the concentration gradient increases, the inhibition effects also improves significantly; the majority of cells become round, the volume gets smaller, the cytoplasm granulating is obvious, the refraction of cell gets poor, and the color is dark. The majority of cells show wall separation; when the concentration reaches 32.0μg/ml, many cell structures become incomplete, the cell membrane breaks, some cells break and aggregate, the light transmission property is poor, and some cells show necrosis. The influence of rhuPA_a-melittin of various concentrations upon SKOV3 cell ultrastructure is observed under transmission electron microscope. The cells show obvious irregularity, part of cells crimp, the microvilli get fewer, the nucleus gets smaller, the mitochondrion in cytoplasm shows vacuole formation, the euchromatin reduces, and the heterochromatin inside the cell condenses in block shape, showing typical apoptosis symptoms. For the 32.0μg/ml group cell, a small part of them show changes same with above-mentioned 2 groups, and the majority of cells show cell cavitation, cell membrane breakage, and organelle overflow, symptoms of cytoclasis. The above results indicate that in certain concentration range, the apoptosis could be the action mechanism of inhibiting growth of ovarian cancer cells; and when the concentration is very high, its action mechanism towards ovarian cancer cells could be direct necrosis.With the flow cytometry, the influence of rhuPA_a-melittin upon proliferation period of SKOV3 cells has obvious dose dependent relationship. As the dose increases, the ratio of cells of G1 cell cycle decreases continuously; finally, the majority of cells stay at S cell cycle; at the same time, the percentage of DNA hypodiploid peaks (Sub-GI) ahead of G1 cycle also increases significantly, positive correlated to the action dose of rhuPA_a-melittin. It indicates that rhuPA_a-melittin may inhibit the growth of ovarian cancer cell by inducing apoptosis. The DNA electrophoresis results show that after 4,8,16μg/ml rhuPA_a-melittin treatment, there are relatively obvious step-shaped strands on 3 lanes; and it is not the case for control group and 2μg/ml lane. When detecting the expression of bcl-2 in SKOV3 cell after adding rhuPA_a-melittin of various concentrations for 48 hours with immunohistochemistry staining method, the expression decreases significantly, showing weak positive compared to undosed group; on the contrary, the expression of bax enhances significantly. The characteristics of bcl-2/bax expression show that the mechanism of apoptosis induction of rhuPA_a-melittin towards ovarian cancer cell SKOV3 may be related to the regulation of apoptosis path of mitochondrion.In general, this experiment builds the rhuPA_a-melittin-pPICZαC eukaryon expression carrier and rhuPA_a-X-pPICZαC eukaryon expression carrier, which can replace the chain B of uPA freely; screen out the rhuPA_a-melittin strains with stable and effective secretion expression; optimize the fermentation conditions with 80 L fermentation fermenter, and draw up new large-scale preparation and purification method of rhuPA_a-melittin; observe the growth inhibition of rhuPA_a-melittin against human ovarian cancer cells SKOV3, and preliminarily explore the its action mechanism. Taking uPA/uPAR system as a new platform, this experiment explores a new drug for ovarian cancer, and opens up a brand new design thought and path for the ovarian cancer therapy, enjoying important scientific and practical value. |
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