Studies On The Production Of Recombinant Human Tumor Related Apoptosis Inducing Ligand And Its Antitumor Effect

Tumor related apoptosis inducing ligand can selectively induced apoptosis in various human tumor cell lines.But it has no toxicity to normal cells.Since 1995,Wiley search the homologization conservative seqence of TNF family molecular in EST(expressed sequence tag),he found the TRAIL (TNF-related apoptosiinducing ligand).TRAIL is a typicalâ…¡transmembrane protein,C-terminatio extracellular domain(ECD)is high homologization with CD95L and TNF,it can induced some kinds of tumour cell apoptosis quickly,but it can do little influence for normal cell.So,TRAIL was considered a extreme potent anti-tumor factor.We found the TRAIL can influence 80.7% uterine cervix cancer cell ME180 apoptosis,incubation malignancy tumor cell strain Jurkat or Ag8,after 6 hour,above 80% tumor cell dead in our sreach.More surprising is 90% mortality rate or more high in incubation the TRAIL with neurospongioma cell.All these evidence suggestion that TRAIL is quite futuer,more safely anti-tumor factor. TRAIL,one kind of anti-tumor factor is more and more obvious.TNF(tumor necrosis factor)family had three molecular could induce cell apoptosis.They are TNF-α,FasL and TRAIL(TNF-related apoptosis-inducing ligand).Although TNF-αand FasL could induce cell apoptosis both,but TNF-αcould active nuclear factor(NF)NF-κB and creat severe Inflammatory reaction,and FasL could induce fatal hepatic injury.TNF-αand FasL's poison and adverse reaction limited their effort in tumor therpy greatly.TRAIL was found recent years as a new member in TNF family.Compare with FasL and TNF-α,TRAIL's most different;â‘ TNF-αand FasL expressed in most actived immune cell,but TRAIL expressed most normal cell continuum.â‘¡It can induce tumor cell apoptosis most powerful,but it had no effact to normal cell.â‘¢It has more anti-tumor spectram than FasL.â‘£It had little active effort to nuclear factor NF-κB,even if systemic administration,that had no severe effort inflammation effort even though TNF-α.Because of this,TRAIL was considered a kind of anti-tumor factor safty and more potent.TRAIL,also be known Apo-2L,general reside most tissue.TRAIL especially reside in spleen,parastata glandulosa and lung,but brain,liver and didymus didn't transcript the TRAIL factor.In heterogenesis cell,such as retrogression leukomonocyte strain K299,paristhmion T cell is especially abundant.Burkitt lymphadenoma Raji cell strain abundence is low.Moreover the fresh abstraction peripheral blood leukomonocyte's transtript number is very low or no transtript TRAIL mRNA copy.Scientist study the TRAIL,recombination express and physico-chemical property analysis,indented some kinds of cell apoptosis study and research,combination amino acids sequence analysis,scientist found;human TRAIL whole molecule consist of 281 amino acids,isoelectric point is 7.63. According to protein chemisty theory deduce dissolvability molecular is 114-281 amino acids,but in vivo didn't find the dissolvability molecular be enzyme cutting from icellular membrane,it seems that amino acids sequence dosen's have metalloprotease enzyme cut site.nodeoxidize SDS-PAGE analysis dissolvability molecular,molecule quality is 24 ku,assembly 48 ku dimeride,trimer is assembly 66ku.TRAIL is a typicalâ…¡transmembrane protein,it has not signal peptide at it's N-terminatio(intracellular region).Amino acids sequence 15-40 is hydrophobic region that can form transmembrane construction.C-terminatio(entracellular region)is identical with TNF and FasL,strongly conservation,that could form typitalβ-plate sheet structure andβ-chain sandwich.Even though dissolvability TRAIL recombination molecular is achieve from 114-181 and 95-281 amino acids, but eukaryon express total length TRAIL mRNA didn't achieve reconstruction. Alliance use anti-tumor factor and TRAIL,not only decrease poision and adverse reaction but also increase sensitivity on TRAIL therapy.This provide Rationale of the alliance use anti-tumor factor and TRAIL.Our research make use of gene recombination technique,though RT-PCR, from human activing lympholeukocyte amplify TNF-related apoptosi inducing ligand,We created a full human TRAIL expression vector pPICZαbased on the vector pPICZα,Then expressed TRAIL in the expression system.Identify the recombinant antibody,evaluate the advantage of Picbia pastoris expression system to express full human TRAIL.In order to improve these situations,studies were done as follows;â‘ Constuct expression vector pPICZα-hTRAIL and transforme it into Pichia pastoris via electroporation.Obtain rhTRAIL secreted into the culture supernatant by the Pichia pastoris;â‘¡Study the large-scale fermentation process of rhTRAIL in the New Brunswick Scientific bioflow 5000 fermentor,Obtain plenty of rhTRAIL for the experimental study of bioactivity;â‘¢In cell experiments,we found that rhTRAIL rapidly induced apoptosis of cancer cells;â‘£Design and construct a mouse model of breast cancer,the result indicate that rhTRAIL have inhibitory effect to the growth of mouse breast cancer cells. 1.Construction of full human TRAIL express system(1)RT-PCR amplify human TRAIL geneThis experiment make use of gene recombination technique.On the basis of NCBI announce the TRAIL mTRAIL,to design primer.Making use of RT-PCR ample TRAIL sequence.We achieve human complete TRAIL gene.RT-PCR production purify,and recombine with pMD 18-T,Construction of full human clone expression system.(2)Construction of full human TRAIL expression vector pPICZαThis experiment make use of gene recombination technique.,On the basis of vehicle pPICZαsequence design primer,leading in Xhoâ… and EcoRâ… enzyme cut site in sequence,amplify the cDNA,digesting with Xhoâ… and EcoRâ… and combination express pPICZα.(3)Identification of the full human TRAIL expression systemCloned the human TRAIL cDNA into expression vector pPICZα.Analyzed the recombinant protein by Western blot after transformation.Analyzed the purified recombinant TRAIL by western blot,the full human antibody of 140 KD molecular weight appeared.These results indicated that the functional soluble TRAIL could be induced in methylotropic yeast expression system.The cloning and expression of full human TRAIL indicated an important role for improved Pichia pastoris expression system in the cloning,screening and expression of full human TNF-related apoptosis-inducing ligand.2.Studies on large-scale fermentation and purification process of rhTRAIL(1)Studies on large-scale fermentation process of rhTRAILPichia pastoris has many advantages as a kind of expression host,and it's very suitable for large-scale expression of the extraneous proteins.So after identifying the bioactivities of the rhTRAIL,we explored the large-scale fermentation process of rhTRAIL and found that the best pH is pH4.0±0.2,DO between 25%～30% and the supply speed of methanol is 8.5ml/h/L initial fermentation volume.(2)A new method to purify rhTRAIL at large-scaleThen the eluate could be purified by SP Sepharose XL on pH4.0.This method overcame the shortcomings of the affinity chromatography and anion chromatography.3.The Apoptosis effect of rhTRAIL on tumor cell(1)Cell culturingThe different tumor cell was cultured with RPMI-1640 medium which contained 10% calf serum,100U/ml penicillin and 100mg/ml phytomycin,the incubator condition was 37℃,5% CO₂.(2)The morph character of apoptotic cellTwelve hours after the SKBr-3 and A-375 cell was administer by rhTRAIL, cells began to generate change of morphology;minification of cell volume, concentration of endochylema,augmentation of density,agglutination of nuclear chromatin,Integrity of cell membrane,which were much different from control groups.And cells which had the same morph character increased with time and concentration of effect of rhTRAIL.(3)MTT resultsThe growth inhibiting rate of rhTRAIL to SKBr-3 and A-375 cell increased and toxic effect enhanced with the concentration of rhTRAIL from 0.325μg/ml to 10.0μg/ml.(4)The detection results of cell apoptosisâ‘ The results of TUNEL Apoptotic index of control groups were 7.26±1.73%,while the Apoptotic index of positive groups were 15.36±3.21%,27.34±2.53% and 43.68±3.51% when the concentrations of rhTRAIL were 0.25μg/ml,0.5μg/ml and 1.0μg/ml, respectively,which were correlated with concentration and were significantly different from control groups(p＜0.05).â‘¡The results of FACSThe FACS results shoued that the cell cycle proceeding was inhibited,the cell population in S phase was decreased which increased in G1 phase after the cells were administered with rhTRAIL(0.25,0.5,1.0μg/ml).The G0/G1 were 37.41±0.42,45.26±0.32,60.67±0.34 and 69.10±0.38,respectively.There were significant differences between the three groups(P＜0.05).The apoptosis rates were 6.68%,24.59%,28.91%,respectively.4.restriction effect of rhTRAIL to mice mastitis strain D2F2(1)group division and administrationThe mice mastitis models was built using D2F2,every mouse was inoculated 0.2ml(1×10⁶)D2F2 cell to right lower limb under covering and they were divided five groups(control groups;fifteen and positive groups;ten)according to weight on the second day.Rats were done with abdominal operation,control group were infused 0.2ml/10g weight,while the low-dose,medium,high groups received purified rhTRAIL of(2.5mg/kg,5.0mg/kg,10.0mg/kg),they were given once a day for fifteen days,the positive group was administerd cyclophosphamide 30.0mg/kg once a day for fifteen days.All rats were killed after fifteen days,the results of the tumor weight,tumor volume and restriction effect showed that the rats weight and volume of low-dose, medium,high groups were few to the control group and the restriction effect was significantly great to control group(P＜0.01).(2)The pathologic change of tumor tissueThe results of the rats tumor tissue of every groups after HE dying showed that necrotis appearing in the tumor tissues,cytoplasm appearing erythrophil and cellular structure necrosis.Meantime the fact that necrosis was not only in the middle but also in the surface of the tumor demonstrated that cellular necrosis is due to cell apoptosis.(3)The ultramicrostructure changement of tumor tissueIt could see that microvilli on the surface of tumor cell disappeared and connect of cell decreased,disappeared,cell nucleus pyknosed,heterochromatin in the nucleus aggregated,the gap of the cell widen,chondriosome engorged from the ion electron microscope(appendix 6).Meantime tumor cells had apoptic changement which had laree area and most area didn't have normal tumor cells.The results demonstrated that the constructed expression vector was successfully,rhTRAIL can be got by large-scale fermentation and purification,the results of cell experiment and rats experiment showed that rhTRAIL could induced cell apoptosis.The result indicate our recombination vector is success.We obtained rhTRAIL through large scale fermentation and purification process.The results of cell experiment and animal experiment provided the potential to prepare therapeutic tumor cell and cell apoptosis.