| Background and PurposeTumor has great threaten on human health. Currently, there were a total of10million new cancer worldwide, and7million people died from cancer. Among which,1/6of them occurred in China, and malignant tumor has become the second reason of death in the world. Glioma accounted40%-60%of the brain tumor, with the annual incidence of5-10/105. Due to the unclear etiology of glioma, this cancer has the aspects of high proliferation rate, high histopathological differentiation and wide infiltrative growth. The main treatment methods included surgery, radiotherapy and chemotherapy. Sometimes, the immunotherapy and TCM methods are used for treatment. Although the modern neuroimaging and tumor tracer technology have gain great development in the diagnosis of glioma, the microsurgical therapy could not ensure the recovery of patients, and therefore, the recurrence rate also remains a high level and the treatment methods is not so good.The average survival time of glioma cancer after treatment could not reach12monthes, and the2years survival time was10%. Although many researches has been conducted on the researches on the glioma cancer, the treatment methods for glioma cancer are still considered the challenge worldwide. Using of anti-glioma drug is usually the main treatment method during the treatment. Although previous studies have gain a certain efficacy on glioma treatment, tumor cell selection also shows poor, and immunosuppresion, drug resistance and adverse effects are usually existed. Currently, there are great improvement in researches on basic and clinic medicine research, and the main development direction is to invent targeting tumor cell drug. Therefore, a new type of targeting therapy for tumor, immunotoxin therapy, is developed.Immunotoxin is coupling by killer clips and guiding cell. The killer clips are biological poison proteins, which are from plants or microorganism, and are also named enzyme catalysis type toxins. The immunotoxin could kill cell after breaking into cell cytoplasm in theory. In theory, the immunotoxin should have the function of bonding tumor cell, and the activity of immunotoxin could not be effected after bonding with killer clips. The guiding cell could specific recognize the tumor cell, and would not attack the normal cell but only for tumor cell. Currently, the momoclonal antibody is the main guiding cell. However, due to the great amount of antibody molecular weight, this cell could not penetrate the center of solid tumor, so the researchers began to change the antibody. Although the single chain antibody could keep all "the characteristics of original antibody, the antibodies are easy to aggregation. The single chain antibody could not become the ideal guide molecules due to ignoring the interaction between heavy and light chains.In2007, a paper published in nature biotechnology showed:a CDR is taken out from heavy and light chains (complementarity-determining regions), and the middle part of guiding clips are bonding through a framework area. This paper indicated this clip could remain the specific characteristics of original antibody. Therefore, we would like to establish a new fusion engineering polypeptide with the aspects of low molecular, high penetrability and high specificity. In this research, the killing clips could inhibit the DT388of exotoxin of Corynebacterium diphtheria synthetized by eukaryotic cell protein. LCDR1-HFR2-HCDR3composed by LCDR1, HFR2and HCDR3is regarded as guiding cell, and it could specifically distinguish the U87monoclonal antibody. The gene engineering method was used to establish a new recombinant plasmid-pGEX-IT-87, and this plasmid was amplified by transferring to JM109, and the protein was expressed by BL21. By affinity chromatography separation and purification fusion peptides, we successfully established a new drug for treatment of glioma-immunotoxin’IT-87’. Raji was used as controls, the kill specificity and efficacy of IT-87were observed in our study. The tumor-burdened moldel was used to explore the targeting and killing efficacy by using SCID, so as to provide scientific basis and strategy for further studies on glioma. All statistical analysis was used by SPSS13.0.First part:Establishing, expression and purification of IT-87Objective:1. Gaining purpose gene fragments, including DT388gene and VLCDR1-VHFR2-VHCDR3gene, establishing the recombinant plasmid-pGEX-IT-87.2. Amplifying recombinant plasmid-pGEX-IT-87, transferring into Escherichia coli-BL21, and establishing expression strains.3. Expressing fused polypeptide, and performing isolation and purification of fused polypeptide to develop IT-87.Methods:1. Gaining purpose gene fragment-DT388: Double enzyme cleavage method was used for DT388, and agarose gel electrophoresis method was used for isolating DT388. We recycle, identify and gain the DT388.2. Extracting and identifying the cloning vector plasmid-pGEX-T: The bacteria liquid of pGEX-T was extracted, the plasmid was extracted by kit, and quality of plasmid was detected by plasmid agarose gel electrophoresis.3. Establishing pGEX-DT388: Double enzyme cleavage method was used for pGEX-T, and agarose gel electrophoresis method was used for cloning vector1. The pGEX-T was recycled and identified to combine DT388, and the pGEX-DT388was gained.4. Gaining VLCDR1-VHFR2-VHCDR3: Double enzyme cleavage method was used for pGEX-T of VLCDR1-VHFR2-VHCDR3, and agarose gel electrophoresis method was used for isolating VLCDR1-VHFR2-VHCDR3. We recycled, identified and gained the VLCDR1-VHFR2-VHCDR3.5. Establishing pGEX-IT-87: Double enzyme cleavage method was used for pGEX-DT388, and agarose gel electrophoresis method was used for pGEX-DT388. The pGEX-DT388was recycled and identified to combine VLCDR1-VHFR2-VHCDR3, and gain pGEX-IT-87.6. Amplifying the pGEX-IT-87: The JM109was prepared, and the pGEX-IT-87was transferred into clone bacteria, and we performed the positive detection for clone bacteria.7. pGEX-IT-87was transferred into expression host bacteria: We prepared the competent expression BL21, and positive clone bacteria was transferred to BL21.8. Expression, purification and identification of the IT-87: (1).The active positive expression bacteria strains were inoculated to culture medium; We induced expression of protein, collected thallus, ultrasound broken bacteria and protein, and purified the protein by NiNTA agarose.(2).Detection of the concentration of protein (BCA kit)(3). Detection of fusion protein:SDS-PAGE was used to detect fusion protein, and the molecular weight was analyzed.9. Amplifying experiment: We amplified the culture of expression bacteria, induced the expression of protein, collect thallus, ultrasound broken bacteria and protein, purified the protein by NiNTA agarose, and stored the bacteria in frozen for future experiment.Results:1. Double enzyme cleavage method was used for pGEX-T of carrier plasmid of DT388, and agarose gel electrophoresis method was used for isolating target gene. The target gene segments were recycled and identified to gain targeting gene segment1. The same method was used for pGEX-T of targeting gene segment2to gain the gene segment.2. Double enzyme cleavage method was used for pGEX-T, and agarose gel electrophoresis method was also used to recycle and identify the pGEX-T to bond DT388to gain pGEX-DT388, and then it was bonded to VLCDR1-VHFR2-VHCDR3to establish the pGEX-IT-87.3. pGEX-IT-87was cloned by clone bacteria JM109, and transferred into BL21and amount of inclusion body was induced by IPTG. The inclusion bodies were discharged by ultrasound cracking bacteria, and were purified by Ni affinity chromatography column. By dilution, ultrafiltration and concentration, we gained a certain high purify fusion protein (IT-87). The concentration of fusion protein was1.3mg/ml. Conclusion:By double enzyme cleavage method for pGEX-T, the targeting gene was isolated by agarose gel electrophoresis method, and the gene segment1was gained by recycling and identification. The same method was used to gain the targeting gene2(VLCDR1-VHFR2-VHCDR3). The pGEX-IT-87was gained by a certain methods. The cloned JM109was transferred into BL21, and a great amount of inclusion bodies was produced by IPTG. The ultrasound cracking bacteria released inclusion bodies, and Ni affinity chromatography column was used to purify. By dilution, ultrafiltration and concentration, we gained a certain high purify fusion protein (IT-87), which could be used for further studies.Second part:Vitro study on the effect of targeting therapy by IT-87Objective:Raji was used as controls, we observed the target activity and effect of IT-87on U87MG tumor cell in vitro.Methods:The cells in exponential phase were inoculated in plate with24holes. We added lOOul IT-87with the range of10-9to10-6M into plate with24holes. After culturing24hours, we added100μl AO and PI to dye. The living cells and dead cells were observed by fluorescence microscope with490nm, and fluorescence microscope with545nm could only observe the dead cells. The living cells and dead cells were counted.5views were gained in each hole, and the living cells of100cells were counted in each view. The survival rate was calculated by living cells divided by total cells. SPSS13.0software was used in this study, and factorial design was used to calculate all data. P<0.05was regarded as significant difference.Results:After dying for U87MG and Raji tumor cells by AO and PI, the cells were observed by fluorescence microscope, and living and dead cells were counted. When the concentration≥10-7M, the IT-87killed about90%of the U87MG cells. Inversely, IT-87could only kill a few Raji cells when the concentration was at the highest concentration of10-6M. After statistical analysis, the result showed that there was interaction between cells and concentration(F=1329.298, P=0.000), and there was main effect among several concentrations(F=1419.347, P=0.000), there was difference between the two cells(F=9524.386, P=0.000). There was difference among the four IT-87concentrations added in U87cells(F=1590.179, P=0.000), and there was difference in comparison of every two concentrations(all P=0.000). There was difference among the four IT-87concentrations added in Raji cells(F=12.769, P=0.000), there was difference among the concentration of10"6M and other concentrations(all P=0.000), and there was not difference among other concentrations(P>0.05). There was not difference between the two types of cells in the concentration of10’9M, but there was difference between the two cells among other concentrations(all P=0.000). It shoued that the killing effect of IT-87to the two type of cells was weak when the concentration was very low(10"9M), there was not difference between the two cells(P>0.05); But in other concentrations, IT-87had great killing effect in U-87MG cell, and the killing effect increased when the concentrations raised, almost all U-87MG cells were killed at the concentration of10"6M. Inversely, the killing effect of IT-87to Raji cells was weak, it only killed a little cells at very higt concentration(10-6M), there was great difference compared with U-87MG cell(P=0.000).Conclusion:In vitro cell killing experiment, the killing effect of IT-87to the two types of cells was weak when the concentration was very low(10-9M), there was not difference between them(P>0.05); While at other concentrations, the killing effect of IT-87to U-87MG cells was prominent(F=1590.179, P=0.000), the killing effect increased when the concentrations of IT-87raised, almost all U-87MG cells were killed at the concentration of10"6M; The killing effect of IT-87to Raji cells was weak, it only killed a little cells at very higt concentration1016M), there was great difference compared with U-87MG cell(F=9524.386,P=0.000), IT-87showed effective targeting and killing for U87MG.Third part:Vivo study on the effect of targeting therapy by IT-87Objective:1. SCID was used to establish U87MG and Raji tumor-burdened model.2. By Raji as control group, we observed inhibiting effect of IT-87on the growth of U87MG3. By body tracer technology, we observed the targeting specificity of IT-87for the U87MG tumor.Methods:1. Groups:A total of30mice at the age of5weeks were collected, and they were divided into three groups, including control group (n=12), treatment group (n=12) and imaging group (n=12).2. Establishment of tumor-burdened model:100ul Raji and100ul U87MG cell suspension with the concentration of5x107/ml were subcutaneously injected into the two sides of armpit in SCID mouse.3. Killing experiment of tumor-burdened model:(1) Bolting dosage of drugs:By vitro experiment results,9mice were divided into3groups (named group A、B、C, every group n=3), we performed three dosages of drugs for the three groups respectively (150ug,300ug and750ug; IP), the period of experiment was10days. As a result, we found the effect of group with dosage of300ug showed to be good, and the dosage was moderate and reaction of mouse was slight. Therefore, we performed formal experimental research by the dosage of300ug.(2) After injecting for8days, a total of30mouse were divided into control group (n=12), treatment group (n=12) and imaging group (n=6). The mice were injected by IT-87, with300μg for10times. The control group was injected with lOmM PBS+0.2M NaCI. After treatment, mice were back into the SPF stage of animal house. We measured the maximum diameter and the minimum diameter of the tumors with sliding caliper before injecting IT-87or PBS at the day10th14th、18th、22th、26th after injecting tumor cells, and calculated the tumor volume according to formula V=4/3πx(d/2)2x(D/2) every ttime, d=minimum diameter, D=maximum diameter, then we drew the curve of tumor volume growth.(3) After treatment, dislocated cervical spine surgery was used to kill mouse, then decoherenced the tumors from the mice and weighted them and record.4. Body tracer technology was used for establish subcutaneous tumor-burdened model with SCID: The FITC was used to record IT-87. After injecting for tumor cells in imaging group for8days, IT-87with FITC were injected into peritoneum, each mice was injected with150ug. NightOWL II LB983imaging system was used to observe the changes of fluorescence at the time of immediate,1h,2h,3h,4h and6h, and understand distribution of FITC-IT-87in mouse.5. SPSS13.0software was used in this study, All datas were measured repeatedly3times. Repeated measure ANOVA was used as the statistical method of mice tumor volume growth comparison; T-test was used as the statistical method of weight of mice tumor comparison, P<0.05was regarded as significant difference.Results:1. The U87MG and Raji tumor-burdened model were established successfully by SCID mouse. 2. The curve of mice tumor volume growth:SPSS13.0software was used, and repeated measure ANOVA was used as the statistical method. The result showed that there was interaction between U87MG cells and time (F=1948.941, P=0.000), there was main effect among the time points(F=10061.468, P=0.000), and there was difference between treatment group and control group of U87MG(F=10022.335, P=0.000). There was difference among the time points of U87MG cells(F=1376.517, P=0.000), there was difference among the time points of control group(P=0.000), and there was difference among the time points except day10th(P=0.000). The tumor volume of U87MG treatment group and control group increased when the time went on, but from the day14th(measured second time), The tumor volume of treatment group increased slowly than the control group’s, it illustrated that IT-87had great depressant effect on U87MG tumor volume increasing. Another statistical result showed that there was not interaction between Raji cancer and time(F=2.016, P=0.153), there was main effect among the time points(F=5262.131, P=0.000), and there was not difference between treatment group and control group of Raji cancer(F=2.031, P=0.168). There was difference among the time points of Raji treatment group(F=1409.127, P=0.000), there was difference among the time points of control group(F=15007.188, P=0.000), and there was not difference among the time points in the two groups(P>0.05). The tumor volume of Raji treatment group and control group increased when the time went on, the tumor volume increasing speed of the two groups was identical, it illustrated that IT-87had not depressant effect on Raji tumor volume increasing.3. The mouse were injected with IT-87with300μm for10times. The controls were injected with lOmM PBS+0.2M NaCI (pH7.4). As a result, IT-87could significant inhibit the growth of U87MG tumor (The average weight=1.46±0.67 and4.29±0.87g, respectively, t=8.883, p<0.0001); It showed that IT-87had killing effect for U87MG cancer. While there was no significant difference between experiment and control group regarding the weight of Raji tumor (The average weight=4.03±1.33and4.10±1.05g; t=0.138,p=0.892, p>0.05). It showed that IT-87had not killing effect for Raji cancer.4. After injection of IT-87among imaging group (n=6), the imaging results showed IT-87penatrated into U87tumor after injection of150μg IT-87for1-2hours. Then the IT-87gradually concentrated in the center of tumor. After6hours injection, all the drugs concentrated the whole tumor. Finally, IT-87could be found inside or around the tumor. Inversely, there was no IT-87inside or around the Raji cancer. It illustrated that IT-87had targeting effect on U87MG cancer.Conclusion:The experimental result showed that there was difference between treatment group and control group of U87MG in tumor volume increasing(F=10022.335, P=0.000), videlicetly, IT-87had great depressant effect on U87MG tumor volume increasing; But there was not difference between treatment group and control group of Raji cancer(F=2.031, P=0.168), videlicetly, IT-87had not depressant effect on Raji tumor volume increasing. IT-87could significant inhibit the growth of U87MG tumor (The average weight=1.46±0.67and4.29±0.87g, respectively, t=8.883, p<0.0001); It showed that IT-87had killing effect for U87MG cancer. While there was no significant difference between treatment and control group regarding the weight of Raji tumor (The average weight=4.03±1.33and4.10±1.05g; t=0.138,p=0.892, p>0.05), it showed that IT-87had not killing effect for Raji cancer. After injection of IT-87among imaging group(150μg [IP]/mouse), the imaging results showed IT-87gradually concentrated in the center of U87MG cancer after injection for3-4hours; While there was not IT-87inside or around the Raji cancer. It illustrated that IT-87 had a great effect on inhibiting the growth of U87MG cancer, and IT-87had a significantly targeting killing specificity, which suggested there might be potential future clinical treatment. |
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