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Preparation and Evaluation of Recombinant Targeted Anti-tumor Immunotxoin EGF-Linker-TCS

Author: LiYongMei
Tutor: LuoRen
School: First Military Medical University
Course: Traditional Chinese Medicine
Keywords: Trichosanthin Epidermal growth factor Targeted therapy Immunotoxin
CLC: R730.5
Type: PhD thesis
Year: 2007
Downloads: 267
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Abstract


BackgroundTumor is such a kind of diseases that seriously threaten human health. To find effective anti-tumor drugs and methods is an important research project in world medicine field. Research indicates that many Chinese traditional medicines have anti-tumor effects, but the components of them are complicated and their anti-tumor effects are caused by many components and many action pathways. So fully understanding the anti-tumor mechanism and tumor targeted site is important to accelerate the development of anti-tumor Chinese traditional medicines. Although some progress has been achieved since recent 50 years, chemotherapy、radiotherapy and surgical operation are still the main method for tumor therapy. There are obvious reactions of toxicity in treatment because the selectivity of existing chemotherapy and radiotherapy drugs is low and these drugs injure normal cells when they kill tumor cells. As a new generation of tumor therapy method , immunotoxin therapy is different with traditional chemotherapy. There is relatively high concentration of immunotoxin therapy drugs in tumor location and the retention time of these drugs is longer than that of chemotherapy drugs. Because the killing activity of immunotoxin drugs on tumor cells is strong and the toxicity of these drugs on normal cells is none or little, immunotoxin therapy is an important way to improve curing effect on tumor. Called "biomissile", immunotoxins are representative drugs in tumor-targeted therapy, and are one of hotspots in immunology research. Diverse uses of immunotoxins are constructed these years. Immunotoxins are special cell-killed molecular, composed of molecular of target and molecular of toxicity. The molecular of target is composed of antibody、cytokines and hormones, and toxins are derived from bacteria or plants. The molecular of target can take toxins to tumor focus and toxins can kill tumor cells but few normal cells. Research indicates that the target character of immunotoxins makes toxins easier to bind tumor cells, and to improve rate of killing tumor cells by toxins, and to lower toxicity effects on normal cells.Trichosanthin( TCS ) is such a kind of alkaline protein that is extracted from a plant of bryony, and is type I ribosome inactivating protein. TCS can be mainly used in the treatment of odinopoeia, extrauterine pregnancy, hydatidiform mole and choriocarcinoma. TCS can also inreversiblely inactivate ribosome and inhibit protein synthesis, and its applications include anti-virus and in anti-tumor and other fields. TCS can inhibit HIV proliferation in HIV-infected immunity inactivated cells. It is also found that in cultured cells TCS can obviously inhibit many viruses such as encephalitis B virus, hepatitis B virus, measles virus, herpes simplex virus and vesicular stomatitis virus and this inhibition effect is positively related to TCS concentration. However, the side-effect and toxicity of TCS in clinic limit its further popularizing and application, so we may consider how to make the most of the anti-tumor effects of TCS on the concentration with the anaphylactic reaction not induced, and we should find a kind of material which can kill tumor cells mostly cooperated with TCS.Exceptional high expressions of epidermal growth factor receptors( EGFR ) are observed in many malignant tumors, and are closely-related with malignant biology behaviors in tumor cells and poor prognosis in tumor patients. EGFR has been the important therapy target for EGFR-positive tumors. The EGFR-targeted therapies are mainly composed of two methods, monoclonal antibody and small molecular kinase antagonist. The main mechanisms of anti-tumor effects, which is undergoing widely experiments and clinical tests, mainly include cell cycle retardation effect, apoptosis promotion effect, anti tumor infiltration and migration effect, anti angiogenesis, more sensitive to cytotoxicity of chemotherapy and radiotherapy. On the other hand, as antigen, human EGFR can excite special immunity reaction in mouse, and there are also cross reactions between human EGFR antibodies and mouse homologous EGFR, through which we can cure EGFR-positive tumors, So far, little obvious EGFR-related toxicity in EGFR-targeted therapy has been observed, and it is perhaps because remarkable controlled low EGFR expression in normal cells and relative independence of EGFR pathway signal in normal cells.In this respect, we used Chinese traditional medicine TCS as toxin, and used EGF as carrier, to prepare recombinant immunotoxin EGF-TCS, and evaluate its targeted anti-tumor effects. In these works we carried useful explorations in anti-tumor therapy using Chinese traditional medicines, and it is important for promoting development and modernization of Chinese traditional medicines.Objective1. The ingredient of TCS was complicated, and the steps of purification were complicated and the recovery rate was low. Currently TCS in expression vectors constructed was low expressed and expensive, which imposes difficulty for further research. We hope to obtain a method, which is effectively, stably, simply and cheaply, to prepare protein TCS, EGF-TCS and EGF-Linker-TCS with high purity by gene engineering.2. We detected the EGF receptors on normal human liver cell LO-2, breast cancer cell MCF-7 and stomach adenocarcinoma cell BCG-823, and studied the feasibility of EGF as carrier of targeted anti-tumor Immunotoxins.3. We evaluated targeted anti-tumor effects of TCS, EGF-TCS and EGF-Linker-TCS anti-tumor effects in vitro through calculating half inhibition concentration( IC50) and selection index of TCS, EGF-TCS and EGF-Linker-TCS for normal human liver cell LO-2, hepatoma cell BEL-7402, breast tumor cell MCF-7 and stomach adenocarcinoma cell BCG-823 and researching on cell apoptosis selectively induced by TCS, EGF-TCS and EGF-Linker-TCS.4. We carried research on tumor growth inhibition rate and liver migration in constructed hepatoma animal model. And we evaluated anti-tumor effects of EGF-Linker-TCS in vivo.Methods1. Cloning, Expression and Purification of recombinant fusion protein TCS、EGF-TCS、EGF-linker-TCSThe genome of Kuowei was extracted, and PCR was used to amplify TCS gene. The plasmid pQE30/TCS was constructed. The EGF gene was amplified by PCR , and was inserted into pQE30/TCS, and the plasmid pQE30/EGF-TCS was constructed. We considered that adding linker to the middle of fusion protein perhaps could make each of two proteins keep its own conformation better and keep its own activity better. Then EGF-Linker gene was amplified and was inserted to pQE30/TCS and the prokaryotic expression plasmid pQE30/EGF-Linker-TCS was constructed. The plasmid pQE30/TCS, plasmid pQE30/EGF-TCS and plasmid pQE30/EGF-Linker-TCS were sequenced .The three recombinant plasmids was transformed into bacteria M15. After induced, recombinant protein TCS,EGF-TCS and EGF-Linker-TCS were expressed in engineering bacteria M15, the expression rate of total bacteria protein which was analyzed by SDS-PAGE were examined. After identified the recombinant plasmid pQE30/TCS、pQE30/EGF-TCS、pQE30/EGF-Linker-TCS were transformed into M15. Single bacteria was selected and inoculated into LB culture medium which contained 50ug/ml ampicillin. The bacteria was cultured in the shaker and when the OD reached 0.6, IPTG was added to induce recombinant protein expression for 3 hours and the bacteria sample was analyzed by SDS-PAGE. Then the bacteria were broken by ultrasonic and the supernatants were recovered after centrifuged. The purity of recombinant TCS、EGF-TCS、EGF-Linker-TCS was examined by Ni-NTA Agrose affinity chromatography.2. Detection of EGF receptor on normal cells and tumor cellsBecause the targeted point of the fusion protein composed of EGF and TCS is EGF receptors, we used flow cytometry to carry EGF receptor detection on normal cells and tumor cells.3. Detection of the 50% inhibitory concentration ( IC50) and the selectivity index of purified TCS, EGF-TCS and EGF-Linker-TCS on tumor cells and normal cellWe detected the effects of purified TCS, EGF-TCS and EGF-linker-TCS on tumor cells and normal cell by crystal violet method. We also detected the 50% inhibitory concentration ( IC50 ) and the selectivity index. The killing rate was calculated with the following formula: The killing rate (%)=[(A value in control- A value in test)/A value in control]×100%.The inhibitory rate was calculated and through which IC50 was calculated by origin 6.0. Then the selectivity index was calculated on basis of IC50:The selectivity index=IC50(normal cell)/IC50(tumor cell).4. Observation of apoptosis of cells induced by EGF-TCS and EGF-linker-TCS through flow cytometryEGF-TCS and EGF-Linker-TCS could bind tumor cells through EGF receptors. TCS could inactivate ribosome and inhibit protein synthesis to make tumor cells die. Perhaps there were other mechanisms to lead eukaryotic cells death. We used flow cytometry to observe apoptosis of LO-2、BEL-7402、MCF-7 and BGC-823 induced by EGF-TCS and EGF-Llinker-TCS.5. Observation of the effects of EGF-Linker-TCS on BEL-7402 by electric microscopeTo make further observation on mechanism of cell apoptosis induced by fusion protein composed of EGF and TCS, we used electric microscope to observe the effects of EGF-Linker-TCS on hepatoma cell BEL-7402.6. Anti-tumor experiment in naked mice tumor model by EGF-Linker-TCSThe hepatoma cell BEL-7402 was inoculated subcutaneously on right armpit in naked mice and EGF-linker-TCS was injected intravenously (100ug/kg,50ug/kg,25ug/kg) to mice for therapy after 6 days of inocluation. There was control group and mice were killed after 2 days of drug withdrawal, and the tumors were weighed and the tumor inhibitory rate was calculated. The livers were taken to calculate tumor migration rate. Tumor tissues were fixed by 10% formaldehyde for histology and vWA immunohistochemistry experiments.Results1. Construction of recombinant fusion protein TCS、EGF-TCS、EGF-Linker-TCS The plasmid pQE30/TCS, plasmid pQE30/EGF-TCS and plasmid pQE30/EGF-Linker-TCS were sequenced,and the determined sequence was identical with the designed one. The three recombinant plasmids was transformed into M15. After induced, recombinant protein TCS, EGF-TCS and EGF-Linker-TCS were expressed in engineering bacteria M15 with soluble form and the expression rate reached 30% of total bacteria protein which was analyzed by SDS-PAGE. The recombinant TCS could occur immune response with special antibody specially which was analyzed by western blot. The purity of recombinant TCS、EGF-TCS、EGF-Linker-TCS was more than 95% after purified by Ni-NTA Agrose affinity chromatography.2. Detection of EGF receptor on normal liver cells and tumor cellswe used flow cytometry to carry EGF receptor detection on normal liver cells and tumor cells. The results: the EGF receptor expression rate was 5.51% in normal liver cell LO-2, and was 72.33% in hepatoma cell BEL-7402, and was 63.92% in breast tumor cell MCF-7, and was 70.41% in stomach adenocarcinoma cell BGC-823. The quantity of EGF receptors are much more than that of normal cells.3. Detection of the 50% inhibitory concentration ( IC50) and the selectivity index of purified TCS, EGF-TCS and EGF-linker-TCS on tumor cells and normal cellThe results: IC50 of TCS was 39.70 ug/ml、33.37 ug/ml、36.53 ug/ml、41.52 ug/ml for LO-2、BEL-7402、MCF-7 and BGC-823 respectively, and the selectivity index of TCS was 1.19、1.09、0.96 for BEL-7402、MCF-7 and BGC-823 respectively;IC50 of EGF-TCS was 115.34 ug/ml、8.90 ug/ml、13.75 ug/ml、15.36 ug/ml for LO-2、BEL-7402、MCF-7 and BGC-823 respectively, and the selectivity index of EGF-TCS was 12.96、8.39、7.51 for BEL-7402、MCF-7 and BGC-823 respectively;IC50 of EGF-Linker-TCS was 113.99 ug/ml、6.98 ug/ml、11.97 ug/ml、 13.53 ug/ml for LO-2、BEL-7402、MCF-7 and BGC-823 respectively, and the selectivity index was 16.33、9.52、8.43 for BEL-7402、MCF-7 and BGC-823 respectively. The results of experiment accorded with our design. The IC50 of EGF-TCS and EGF-linker-TCS for tumor cells was much less than that of TCS, and the IC50 of EGF-TCS and EGF-linker-TCS for normal cells was very close to that of TCS. The selectivity index of EGF-TCS and EGF-Linker-TCS was obviously higher than that of TCS, and was both greater than 2 and these two proteins were fit for the demand of targeted drugs. The IC50 of EGF-linker-TCS for tumor cells was a little less than that of EGF-TCS and the selectivity index of EGF-Linker-TCS was a little higher than that of EGF-TCS.4. The apoptosis effect on tumor cells induced by EGF-TCS and EGF-Linker-TCSThe apoptosis effect on breast tumor cell MCF-7 , stomach adenocarcinoma cell BCG-823 and hepatoma cell BEL-7402 induced by EGF-TCS in the concentration of 5μg/ml was detected by flow cytometry, and the apoptosis rate was 35.61%、39.59% and 50.57% respectively. EGF-TCS in the same concentration could induce little apoptosis on normal liver cell LO-2 and the apoptosis rate was 8.99%;The apoptosis effect on breast tumor cell MCF-7,stomach adenocarcinoma cell BCG-823 and hepatoma cell BEL-7402 induced by EGF-Linker-TCS in the concentration of 5μg/ml was detected by flowcytomy, and the apoptosis rate was 36.67%, 42.56%, and 58.56%, respectively. EGF-Linker-TCS in the same concentration could induce little apoptosis on normal liver cell LO-2 and the apoptosis rate was 8.08%.5. Observation of the effects of EGF-Linker-TCS on hepatoma cell BEL-7402 by electric microscopeIt was observed by electric microscope that the cell nucleolus was clear and intact, and there were abundant mitochondria and endoplasmic reticulum in control group; some characters of cell apoptosis in various phase appeared such as chromatin concentration, non-regular nucleolus, vacuolar endoplasmic reticulum and apoptosis body in EGF-Linker-TCS group.6. Tumor inhibition test by EGF-Linker-TCS in naked mouse tumor modelTo further observe the anti-tumor effect of recombinant toxin, we carried tumor inhibition test in vivo in naked mouse tumor model. The result illustrated that EGF-Linker-TCS could inhibit tumor growth markedly. The inhibition rate was 71.3%、60.87% and 45.22%, for high dosage group, middle dosage group and low dosage group respectively. Variance analysis illustrated that EGF-Linker-TCS could markedly inhibit the growth of naked mouse tumors,variance analysis had significant difference( F=8.712 P=0.006 );and We also observed the inhibition effect on tumor migration by EGF-Linker-TCS. After anatomizing naked mouse and checking liver, we found single white focus caused by migration of tumor transplant to liver. The number of liver migration focus in each naked mice was calculated, revealed that EGF-Linker-TCS could markedly inhibit liver migration of tumor transplant in naked mouse,Variance analysis showed significant difference( F=8.744 P=0.002 ) Immunohistochemistry experiment showed that EGF-Linker-TCS could makedly inhibit tumor angiogenesis. There were no visible blood vessels in tumor tissues in high dosage group and there were fewer blood vessels in tumor tissues in middle and low dosage group than those in control group, which revealed that perhaps EGF-Linker-TCS inhibited tumor growth and migration by inhibiting tumor angiogenesis.ConclusionsAfter the study, we can make some conclusions as below:1. We find a method, which is effectively, stably, simply and cheaply to prepare fusion protein TCS, EGF-TCS and EGF-Linker-TCS with high purity by gene engineering. The development of TCS lays a foundation of further study on the relationship between structure and the function of it.2. EGF receptors are over-expressed in many tumor cells. EGF can bind to these over-expressed EGF receptors specially and can be used as the target part of immunotoxins.3. We carried the research about targeted toxicity of TCS, EGF-TCS and EGF-Linker-TCS on tumor cells in vitro and proved that EGF-TCS and EGF-Linker-TCS had good selectivity and relatively higher cyto-toxicity on tumor cells with no selectivity on normal cells. The selectivity index of EGF-TCS and EGF-Linker-TCS for tumor cells is more than 2, and the cyto-toxicity of the two proteins on normal cells was less than that of TCS.4. Immunotoxin EGF-Linker-TCS inhibited tumor growth and liver migration markedly, which illustrated that the anti-tumor effect of EGF-Linker-TCS was evident in naked mouse tumor model.

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